Article

Antagonistic effects of dual PTS-catalysed phosphorylation on the Bacillus subtilis transcriptional activator LevR

May 1998
Molecular Microbiology 28(2):293-303

May 1998
28(2):293-303

DOI:10.1046/j.1365-2958.1998.00781.x

Source
PubMed

Authors:

Isabelle Martin-Verstraete

Institut Pasteur and Université de Paris

Jörg Stülke

Georg-August-Universität Göttingen

Anne Galinier

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LevR, which controls the expression of the levoperon of Bacillus subtilis, is a regulatory protein containing an N-terminal domain similar to the NifA/NtrC transcriptional activator family and a C-terminal domain similar to the regulatory part of bacterial anti-terminators, such as BgIG and LicT. Here, we demonstrate that the activity of LevR is regulated by two phosphoenolpyruvate (PEP)-dependent phosphorylation reactions catalysed by the phosphotransferase system (PTS), a transport system for sugars, polyols and other sugar derivatives. The two general components of the PTS, enzyme I and HPr, and the two soluble, sugar-specific proteins of the lev-PTS, LevD and LevE, form a signal transduction chain allowing the PEP-dependent phosphorylation of LevR, presumably at His-869. This phosphorylation seems to inhibit LevR activity and probably regulates the induction of the lev operon. Mutants in which His-869 of LevR has been replaced with a non-phosphorylatable alanine residue exhibited constitutive expression from the lev promoter, as do levD or levE mutants. In contrast, PEP-dependent phosphorylation of LevR in the presence of only the general components of the PTS, enzyme I and HPr, regulates LevR activity positively. This phosphorylation most probably occurs at His-585. Mutants in which His-585 has been replaced with an alanine had lost stimulation of LevR activity and PEP-dependent phosphorylation by enzyme I and HPr. This second phosphorylation of LevR at His-585 is presumed to play a role in carbon catabolite repression.

Caractérisation de l'opéron métabolique fru2 de Streptococcus agalactiae : phylogénie, induction, et régulation

Thesis

Full-text available

Dec 2015

Kevin Patron

Streptococcus agalactiae est la première cause d’infections néonatales, et est aussi un pathogène émergent chez l’adulte immunodéprimé. L’objectif de ce travail de thèse a été de caractériser l’opéron métabolique fru2 de S. agalactiae (i) en étudiant sa phylogénie, (ii) en identifiant ses inducteurs, et (iii) en élaborant son schéma de régulation. Cet opéron est composé de 7 gènes qui codent un activateur transcriptionnel de la famille DeoR-like (Fru2R), un transporteur PTS (PTSFru2), et trois enzymes qui sont potentiellement impliquées dans la voie non oxydative des pentoses phosphates. Nous avons mis en évidence que cet opéron avait été acquis au cours de l’évolution, et n’était présent que chez les souches de complexes clonaux responsables d’infections chez l’adulte immunodéprimé et la personne âgée. Nous avons ensuite montré que certains milieux complexes, sources de carbone, et liquides biologiques humains permettaient l’activation de cet opéron. Le rôle et fonctionnement de la protéine Fru2R ont été caractérisés (i) en montrant son rôle d’activateur transcriptionnel, (ii) en identifiant les acides aminés essentiels à son activité, et (iii) en démontrant sa capacité à se fixer au niveau de la région promotrice de fru2. Nous avons ensuite décrit le rôle des protéines du PTSFru2 dans la régulation de l’opéron fru2 (i) en caractérisant l’impact d’une délétion du PTSFru2 sur la régulation de l’opéron fru2, (ii) en identifiant les acides aminés essentiels à l’activité des protéines EIIAFru2 et EIIBFru2, et (iii) en révélant l’interaction physique de EIIBFru2 avec Fru2R. Enfin, nous avons montré l’implication de la répression catabolique, plus particulièrement de CcpA, dans la régulation de cet opéron. Les données obtenues ont permis d’élaborer un schéma de régulation de l’opéron fru2 en fonction de la source de carbone disponible.

PTS regulation domain-containing transcriptional activator CelR and sigma factor σ 54 control cellobiose utilization in Clostridium acetobutylicum

Article

Dec 2015
MOL MICROBIOL

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domain (PRD)-containing enhancer binding proteins (EBPs) are an important class of σ(54) -interacting transcriptional activators. Although PRD-containing EBPs are present in many Firmicutes, most of their regulatory functions remain unclear. In this study, the transcriptional regulons of about 50 PRD-containing EBPs in diverse Firmicutes species are reconstructed by using a comparative genomic approach, which contain the genes associated with utilization of β-glucosides, fructose/levan, mannose/glucose, pentitols, and glucosamine/fructosamine. We then present experimental evidence that the cel operon involved in cellobiose utilization is directly regulated by CelR and σ(54) (SigL) in Clostridium acetobutylicum. The predicted three CelR-binding sites and σ(54) promoter elements upstream of the cel operon are verified by in vitro binding assays. We show that CelR has an ATPase activity which is strongly stimulated by the presence of DNA containing the CelR-binding sites. Moreover, mutations in any one of the three CelR-binding sites significantly decreased the cel promoter activity probably due to the need for all three DNA sites for maximal ATPase activity of CelR. It is suggested that CelR is regulated by PTS-mediated phosphorylation at His-551 and His-829, which exerts a positive effect and an inhibitory effect, respectively, on the CelR activity. This article is protected by copyright. All rights reserved.

Interaction with Enzyme IIB Mpo (EIIB Mpo ) and Phosphorylation by Phosphorylated EIIB Mpo Exert Antagonistic Effects on the Transcriptional Activator ManR of Listeria monocytogenes

Article

Full-text available

Apr 2015
J BACTERIOL

Unlabelled: Listeriae take up glucose and mannose predominantly through a mannose class phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS(Man)), whose three components are encoded by the manLMN genes. The expression of these genes is controlled by ManR, a LevR-type transcription activator containing two PTS regulation domains (PRDs) and two PTS-like domains (enzyme IIA(Man) [EIIA(Man)]- and EIIB(Gat)-like). We demonstrate here that in Listeria monocytogenes, ManR is activated via the phosphorylation of His585 in the EIIA(Man)-like domain by the general PTS components enzyme I and HPr. We also show that ManR is regulated by the PTS(Mpo) and that EIIB(Mpo) plays a dual role in ManR regulation. First, yeast two-hybrid experiments revealed that unphosphorylated EIIB(Mpo) interacts with the two C-terminal domains of ManR (EIIB(Gat)-like and PRD2) and that this interaction is required for ManR activity. Second, in the absence of glucose/mannose, phosphorylated EIIB(Mpo) (P∼EIIB(Mpo)) inhibits ManR activity by phosphorylating His871 in PRD2. The presence of glucose/mannose causes the dephosphorylation of P∼EIIB(Mpo) and P∼PRD2 of ManR, which together lead to the induction of the manLMN operon. Complementation of a ΔmanR mutant with various manR alleles confirmed the antagonistic effects of PTS-catalyzed phosphorylation at the two different histidine residues of ManR. Deletion of manR prevented not only the expression of the manLMN operon but also glucose-mediated repression of virulence gene expression; however, repression by other carbohydrates was unaffected. Interestingly, the expression of manLMN in Listeria innocua was reported to require not only ManR but also the Crp-like transcription activator Lin0142. Unlike Lin0142, the L. monocytogenes homologue, Lmo0095, is not required for manLMN expression; its absence rather stimulates man expression. Importance: Listeria monocytogenes is a human pathogen causing the foodborne disease listeriosis. The expression of most virulence genes is controlled by the transcription activator PrfA. Its activity is strongly repressed by carbohydrates, including glucose, which is transported into L. monocytogenes mainly via a mannose/glucose-specific phosphotransferase system (PTS(Man)). Expression of the man operon is regulated by the transcription activator ManR, the activity of which is controlled by a second, low-efficiency PTS of the mannose family, which functions as glucose sensor. Here we demonstrate that the EIIB(Mpo) component plays a dual role in ManR regulation: it inactivates ManR by phosphorylating its His871 residue and stimulates ManR by interacting with its two C-terminal domains.

Expression of the phosphotransferase system (PTS) both mediates and is mediated by Mlc regulation in Escherichia coli

Article

Jul 1999
MOL MICROBIOL

Jacqueline Plumbridge

The ptsHIcrr operon encodes the cytoplasmic components of the phosphotransferase system (PTS). It is expressed from two major promoters, of which the upstream promoter has previously been shown to be induced by glucose and to be dependent upon cAMP/CAP. This promoter is now shown to be repressed by Mlc. Mlc is a transcriptional regulator controlling, among others, the gene ptsG, encoding EIICBGlc, the glucose-specific transporter of the PTS. Transcription of ptsH p0 and ptsG are subject to the same regulatory pattern. In addition to induction by glucose and repression by Mlc, mutations in ptsHIcrr, which interrupt the PEP-dependent phosphate transfer through the soluble components of the PTS, lead to high expression of both ptsH and ptsG, while mutations inactivating EIIBCGlc are non-inducible. Mutations in mlc lead to high constitutive expression and are dominant, implying that Mlc is the ultimate regulator of ptsHI and ptsG expression. Growth on other PTS sugars, besides glucose, also induces ptsH and ptsG expression, suggesting that the target of Mlc regulation is the PTS. However, induction by these other sugars is only observed in the presence of ptsG+, thus confirming the importance of glucose and EIICBGlc in the regulation of the PTS. The ptsG22 mutation, although negative for glucose transport, shows a weak positive regulatory phenotype. The mutation has been sequenced and its effect on regulation investigated.

Studies of the Listeria monocytogenes Cellobiose Transport Components and Their Impact on Virulence Gene Repression

Article

Jul 2019

Background: Many bacteria transport cellobiose via a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). In Listeria monocytogenes, two pairs of soluble PTS components (EIIACel1/EIIBCel1 and EIIACel2/EIIBCel2) and the permease EIICCel1 were suggested to contribute to cellobiose uptake. Interestingly, utilization of several carbohydrates, including cellobiose, strongly represses virulence gene expression by inhibiting PrfA, the virulence gene activator. Results: The LevR-like transcription regulator CelR activates expression of the cellobiose-induced PTS operons celB1-celC1-celA1, celB2-celA2, and the EIIC-encoding monocistronic celC2. Phosphorylation by P∼His-HPr at His550 activates CelR, whereas phosphorylation by P∼EIIBCel1 or P∼EIIBCel2 at His823 inhibits it. Replacement of His823 with Ala or deletion of both celA or celB genes caused constitutive CelR regulon expression. Mutants lacking EIICCel1, CelR or both EIIACel exhibitedslow cellobiose consumption. Deletion of celC1 or celR prevented virulence gene repression by the disaccharide, but not by glucose and fructose. Surprisingly, deletion of both celA genes caused virulence gene repression even during growth on non-repressing carbohydrates. No cellobiose-related phenotype was found for the celC2 mutant. Conclusion: The two EIIA/BCel pairs are similarly efficient as phosphoryl donors in EIICCel1-catalyzed cellobiose transport and CelR regulation. The permanent virulence gene repression in the celA double mutant further supports a role of PTSCel components in PrfA regulation.

Regulation of the Bacillus subtilis mannitol operon activator MtlR by EIIB domain

Article

Sep 2012

Houda Zouiyed

The Bacillus subtilis mtl operon encodes the enzymes necessary for mannitol utilization. Its expression is controlled by MtlR, a transcriptional activator belonging to the DeoR family. MtlR contains a HTH domain followed by two PTS regulation domains (PRDs), an EIIBGat domain and an EIIAMtl-like domain.The general mechanism of the regulation of MtlR activity is based on its phosphorylation by components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). The phosphorylation of EIIBGat on cysteine 419 by P~EIIAMtl has a major negative effect on the activity of MtlR. The absence of EIIAMtl in a mtlF mutant therefore leads to constitutively active MtlR.In this study a new mechanism of MtlR regulation based on the interaction of the PTS component EIIBMtl with MtlR is presented.We observed that the deletion of the entire mtlAFD operon or of mtlF and only the 3’-part of mtlA (encoding the EIIBMtl domain) abolishes the constitutive MtlR activity of the mtlF mutant, suggesting that MtlR activity depends on functional EIIBMtl. By carrying out yeast two-hybrid experiments we could establish a direct, specific and bidirectional interaction between EIIBMtl and the EIIBGatEIIAMtl-like part of MtlR.Complementation of the above mutants was possible with entire MtlA, but not with the EIIBMtl domain. EIIBMtl is normally fused to the membrane protein EIICMtl; we therefore fused EIIBMtl to another membrane, which indeed restored MtlR function in the absence of EIICMtl. The EIICMtl domain is therefore not essential for the interaction between EIIBMtl and MtlR; it is rather the vicinity of the membrane which is required for the activation of MtlR.A regulation model of MtlR activity is proposed. In this model, the MtlR-mediated induction of the mtlAFD operon requires the phosphorylation of PRDII by P~His-HPr and the dephosphorylation of EIIBGat by EIIAMtl. The presence of unphosphorylated EIIBMtl, which prevails when the inducer mannitol is present, is also required. Under these conditions unphosphorylated EIIBMtl sequesters MtlR dephosphoryled on cysteine 419, but phosphorylated at His-342, to the membrane thereby activating the transcription activator, which leads to increased expression of the mtlAFD operon.

Regulation of the mannitol utilization genes in Bacillus subtilis

Thesis

Full-text available

Jan 2013

Kambiz Morabbi Heravi

Bacillus subtilis takes up mannitol by a phosphoenolpyruvate-dependent phosphotransferase system (PTS). The mannitol utilization system is encoded by the mtlAFD operon consisting of mtlA (encoding membrane-bound EIICBMtl), mtlF (encoding phosphocarrier EIIAMtl), and mtlD (encoding mannitol 1-phosphate dehydrogenase). This operon is activated by MtlR whose coding gene is located approx. 14.4 kb downstream of the operon. The regulation of the mannitol utilization genes in B. subtilis was studied by fusion of the promoters of mtlAFD (PmtlA) and mtlR (PmtlR) to lacZ as a reporter gene. Both the PmtlA and PmtlR were inducible by mannitol and glucitol, while glucose reduced their activities. The promoter strength of PmtlA was about 4.5-fold higher than that of PmtlR. Identification of the transcription start sites of PmtlA and PmtlR revealed that both of these promoters contain a sigma A-type promoter structure. The promoter -35 and -10 boxes in PmtlA were TTGTAT and TAACAT and in PmtlR TTGATT and TATATT, respectively. Catabolite responsive elements (cre) were detected in the sequences of PmtlA and PmtlR overlapping the -10 boxes. Shortening the mRNA 5untranslated region (5UTR) increased the PmtlA activity, whereas PmtlR activity was decreased by shortening of its mRNA 5UTR. Alignment of the -35 upstream sequences of PmtlA and PmtlR revealed the putative MtlR binding site. This sequence comprised a similar incomplete inverted repeat in both the PmtlA and PmtlR sequences (TTGNCACAN4TGTGNCAA). This sequence was encompassed by two 11 bp distal and proximal flanking sequences. Construction of PmtlA-PlicB hybrid promoters and shortening of the 5-end of PmtlA indicated the probable boundaries of putative MtlR binding site in PmtlA. Increasing the distance between the putative MtlR binding site and -35 box lowered the PmtlA maximal activity, although PmtlA remained inducible by mannitol. PmtlA became inactive by disruption of the TTGNCACAN4TGTGNCAA sequence. In contrast, manipulation of the distal and proximal flanking sequences only reduced the maximal activity of PmtlA, whereas PmtlA remained highly inducible. These flanking sequences contained AT-rich repeats similar to the consensus sequence of alpha CTD binding sites. Regulation of PmtlA and PmtlR was investigated by deletion of mtlAF, mtlF, mtlD, and mtlR. Deletion of the mtlAF genes rendered PmtlA and PmtlR constitutive showing the inhibitory effect of EIICBMtl and EIIAMtl (PTS transporter components) on MtlR in the absence of mannitol. The constitutive activity of PmtlA was increased by the deletion of mtlF. In contrast, the deletion of mtlAFD showed a significant reduction in the PmtlA constitutive activity. Disruption of mtlD made B. subtilis sensitive to mannitol in a way that addition of mannitol or glucitol to the bacterial culture ended in cell lysis. Besides, PmtlA and PmtlR were similarly induced by glucitol and mannitol in a mtlD::erm mutant. Also, deletion of mtlR rendered PmtlA and PmtlR uninducible by mannitol or glucitol. In contrast, deletion of the glucitol utilization genes had no influence on the inducibility of PmtlA or PmtlR by glucitol. The PmtlA activity was drastically reduced in ptsH-H15A (HPr-H15A) mutant similar to the delta mtlR mutant. The mutation of histidine 289 in the PRDI domain of MtlR to alanine reduced the activity of PmtlA, whereas the PmtlA activity in the mtlR H230A mutant was almost similar to wild type. In contrast, mutation of the PRDII domain of MtlR to H342D mainly relieved PmtlA from glucose repression. Moreover, MtlR double mutant H342D C419A which was produced in E. coli was shown to be active in vitro. These results represent the positive regulation of MtlR via phosphorylation of the PRDII domain by HPr(H15~P). Also, dephosphorylation of the domains EIIBGat- and EIIAMtl-like of MtlR by EIIAMtl and EIICBMtl transporter components causes activation. The PmtlA activity was repressed in the presence of glucose and fructose, while sucrose and mannose had no influence on the PmtlA activity. Therefore, catabolite repression of PmtlA and PmtlR were studied by CcpA-dependent carbon catabolite repression mutants, such as ptsH-S46A, delta crh, delta hprK, and delta ccpA. Induction of PmtlA and PmtlR in these mutants did not result in a complete loss of catabolite repression. Therefore, the catabolite responsive elements (cre sites) of PmtlA and PmtlR were investigated. Using a constitutive promoter, PgroE, it was shown that the cre sites of PmtlA and PmtlR were weakly functional. In contrast, deletion of the glucose PTS transporter, encoded by ptsG, resulted in a complete loss of glucose repression in PmtlA and PmtlR. Thus, the main glucose repression of mannitol PTS function at the posttranslational level in a HPr-mediated manner via MtlR-H342 and at transcriptional level by CcpA-dependent carbon catabolite repression.

Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

Article

Full-text available

May 2015
INFECT IMMUN

Whole genome sequencing analysis of ∼ 800 strains of group A Streptococcus (GAS) found that the gene encoding multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. Results revealed that Mga is phosphorylated at histidines in vivo. Using phospho- and non-phosphomimetic substitutions at conserved PRD histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at histidines, H207 and H273 of PRD-1, and H327 of PRD-2, are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation mimicking alanine substitutions at H273 and H327 relieved the inhibition and the mutant strains exhibited wild type phenotype. The opposing regulatory profiles observed for phosphorylation and non-phosphorylation mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited wild type virulence phenotype in mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay foundation towards understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer advantage to the pathogen and contribute to M59 GAS pathogenesis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

Article

Full-text available

Jun 2014
MICROBIOL MOL BIOL R

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.

Transcription regulators controlled by interaction with enzyme IIB components of the phosphoenolpyruvate: Sugar phosphotransferase system.

Article

Jan 2013
Biochim Biophys Acta

Numerous bacteria possess transcription activators and antiterminators composed of regulatory domains phosphorylated by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). These domains, called PTS regulation domains (PRDs), usually contain two conserved histidines as potential phosphorylation sites. While antiterminators possess two PRDs with four phosphorylation sites, transcription activators contain two PRDs plus two regulatory domains resembling PTS components (EIIA and EIIB). The activity of these transcription regulators is controlled by up to five phosphorylations catalyzed by PTS proteins. Phosphorylation by the general PTS components EI and HPr is usually essential for the activity of PRD-containing transcription regulators, whereas phosphorylation by the sugar-specific components EIIA or EIIB lowers their activity. For a specific regulator, for example the Bacillus subtilis mtl operon activator MtlR, the functional phosphorylation sites can be different in other bacteria and consequently the detailed mode of regulation varies. Some of these transcription regulators are also controlled by interaction with a sugar-specific EIIB PTS component. The EIIBs are frequently fused to the membrane-spanning EIIC and EIIB-mediated membrane sequestration is sometimes crucial for the control of a transcription regulator. This is also true for the Escherichia coli repressor Mlc, which does not contain a PRD but nevertheless interacts with the EIIB domain of the glucose-specific PTS. In addition, some PRD-containing transcription activators interact with a distinct EIIB protein located in the cytoplasm. The phosphorylation state of the EIIB components, which changes in response to the presence or absence of the corresponding carbon source, affects their interaction with transcription regulators.

Enzyme I and HPr from Lactobacillus casei: their role in sugar transport, carbon catabolite repression and inducer exclusion

Article

May 2000
MOL MICROBIOL

We have cloned and sequenced the Lactobacillus casei ptsH and ptsI genes, which encode enzyme I and HPr, respectively, the general components of the phosphoenolpyruvate–carbohydrate phosphotransferase system (PTS). Northern blot analysis revealed that these two genes are organized in a single-transcriptional unit whose expression is partially induced. The PTS plays an important role in sugar transport in L. casei, as was confirmed by constructing enzyme I-deficient L. casei mutants, which were unable to ferment a large number of carbohydrates (fructose, mannose, mannitol, sorbose, sorbitol, amygdaline, arbutine, salicine, cellobiose, lactose, tagatose, trehalose and turanose). Phosphorylation of HPr at Ser-46 is assumed to be important for the regulation of sugar metabolism in Gram-positive bacteria. L. casei ptsH mutants were constructed in which phosphorylation of HPr at Ser-46 was either prevented or diminished (replacement of Ser-46 of HPr with Ala or Thr respectively). In a third mutant, Ile-47 of HPr was replaced with a threonine, which was assumed to reduce the affinity of P–Ser–HPr for its target protein CcpA. The ptsH mutants exhibited a less pronounced lag phase during diauxic growth in a mixture of glucose and lactose, two PTS sugars, and diauxie was abolished when cells were cultured in a mixture of glucose and the non-PTS sugars ribose or maltose. The ptsH mutants synthesizing Ser-46–Ala or Ile-47–Thr mutant HPr were partly or completely relieved from carbon catabolite repression (CCR), suggesting that the P–Ser–HPr/CcpA-mediated mechanism of CCR is common to most low G+C Gram-positive bacteria. In addition, in the three constructed ptsH mutants, glucose had lost its inhibitory effect on maltose transport, providing for the first time in vivo evidence that P–Ser–HPr participates also in inducer exclusion.

Non-canonical food sources: bacterial metabolism of Maillard reaction products and its regulation ☆

Article

Oct 2023

Proteins are an important part of our regular diet. During food processing, their amino acid composition can be chemically altered by the reaction of free amino groups with sugars-a process termed glycation. The resulting Maillard reaction products (MRPs) have low bioavailability and thus predominantly end up in the colon where they encounter our gut microbiota. In the following review, we summarize bacterial strategies to efficiently metabolize these non-canonical amino acids. A particular focus will be on the complex regulatory mechanisms that allow a tightly controlled expression of metabolic genes to successfully occupy the ecological niches that result from the chemical diversity of MRPs.

Functional and structural diversification of incomplete phosphotransferase system in cellulose-degrading clostridia

Article

Mar 2023
ISME J

Carbohydrate utilization is critical to microbial survival. The phosphotransferase system (PTS) is a well-documented microbial system with a prominent role in carbohydrate metabolism, which can transport carbohydrates through forming a phosphorylation cascade and regulate metabolism by protein phosphorylation or interactions in model strains. However, those PTS-mediated regulated mechanisms have been underexplored in non-model prokaryotes. Here, we performed massive genome mining for PTS components in nearly 15,000 prokaryotic genomes from 4,293 species and revealed a high prevalence of incomplete PTSs in prokaryotes with no association to microbial phylogeny. Among these incomplete PTS carriers, a group of lignocellulose degrading clostridia was identified to have lost PTS sugar transporters and carry a substitution of the conserved histidine residue in the core PTS component, HPr (histidine-phosphorylatable phosphocarrier). Ruminiclostridium cellulolyticum was then selected as a representative to interrogate the function of incomplete PTS components in carbohydrate metabolism. Inactivation of the HPr homolog reduced rather than increased carbohydrate utilization as previously indicated. In addition to regulating distinct transcriptional profiles, PTS associated CcpA (Catabolite Control Protein A) homologs diverged from previously described CcpA with varied metabolic relevance and distinct DNA binding motifs. Furthermore, the DNA binding of CcpA homologs is independent of HPr homolog, which is determined by structural changes at the interface of CcpA homologs, rather than in HPr homolog. These data concordantly support functional and structural diversification of PTS components in metabolic regulation and bring novel understanding of regulatory mechanisms of incomplete PTSs in cellulose-degrading clostridia.

Exceptionally versatile take II: post-translational modifications of lysine and their impact on bacterial physiology

Article

Full-text available

Feb 2022

Among the 22 proteinogenic amino acids, lysine sticks out due to its unparalleled chemical diversity of post-translational modifications. This results in a wide range of possibilities to influence protein function and hence modulate cellular physiology. Concomitantly, lysine derivatives form a metabolic reservoir that can confer selective advantages to those organisms that can utilize it. In this review, we provide examples of selected lysine modifications and describe their role in bacterial physiology.

Carbon Catabolite Repression on the Rgg2/3 Quorum Sensing System in Streptococcus pyogenes is Mediated by PTS Man and Mga

Article

Full-text available

Jan 2022
MOL MICROBIOL

Streptococcus pyogenes, also known as Group A Streptococcus or GAS, is a human-restricted pathogen causing a diverse array of infections. The ability to adapt to different niches requires GAS to adjust gene expression in response to environmental cues. We previously identified the abundance of biometals and carbohydrates led to natural induction of the Rgg2/3 cell-cell communication system (quorum sensing, QS). Here we determined the mechanism by which the Rgg2/3 QS system is stimulated exclusively by mannose and repressed by glucose, a phenomenon known as carbon catabolite repression (CCR). Instead of CcpA, the primary mediator of CCR in Gram-positive bacteria, CCR of Rgg2/3 requires the PRD-containing transcriptional regulator Mga. Deletion of Mga led to carbohydrate-independent activation of Rgg2/3 by down-regulating rgg3, the QS repressor. Through phosphoablative and phosphomimetic substitutions within Mga PRDs, we demonstrated that selective phosphorylation of PRD1 conferred repression of the Rgg2/3 system. Moreover, given the carbohydrate specificity mediating Mga-dependent governance over Rgg2/3, we tested mannose-specific PTS components and found the EIIA/B subunit ManL was required for Mga-dependent repression. These findings provide newfound connections between PTSMan, Mga, and QS, and further demonstrate that Mga is a central regulatory nexus for integrating nutritional status and virulence.

PRD-Containing Virulence Regulators (PCVRs) in Pathogenic Bacteria

Article

Full-text available

Oct 2021

Bacterial pathogens rely on a complex network of regulatory proteins to adapt to hostile and nutrient-limiting host environments. The phosphoenolpyruvate phosphotransferase system (PTS) is a conserved pathway in bacteria that couples transport of sugars with phosphorylation to monitor host carbohydrate availability. A family of structurally homologous PTS-regulatory-domain-containing virulence regulators (PCVRs) has been recognized in divergent bacterial pathogens, including Streptococcus pyogenes Mga and Bacillus anthracis AtxA. These paradigm PCVRs undergo phosphorylation, potentially via the PTS, which impacts their dimerization and their activity. Recent work with predicted PCVRs from Streptococcus pneumoniae (MgaSpn) and Enterococcus faecalis (MafR) suggest they interact with DNA like nucleoid-associating proteins. Yet, Mga binds to promoter sequences as a homo-dimeric transcription factor, suggesting a bi-modal interaction with DNA. High-resolution crystal structures of 3 PCVRs have validated the domain structure, but also raised additional questions such as how ubiquitous are PCVRs, is PTS-mediated histidine phosphorylation via potential PCVRs widespread, do specific sugars signal through PCVRs, and do PCVRs interact with DNA both as transcription factors and nucleoid-associating proteins? Here, we will review known and putative PCVRs based on key domain and functional characteristics and consider their roles as both transcription factors and possibly chromatin-structuring proteins.

Metabolic engineering of Thermoanaerobacterium aotearoense SCUT27 for glucose and cellobiose co-utilization by identification and overexpression of the endogenous cellobiose operon

Article

Jan 2021
BIOCHEM ENG J

Cellobiose is abundant in partial hydrolysis of lignocellulosic hydrolysates for the weak activity of β-glucosidase, the rate-limiting step in the enzymatic hydrolysis. Thermoanaerobacterium aotearoense SCUT27 termed SCUT27 has broad substrate spectrums and can co-utilize glucose and xylose, xylose and cellobiose. However, SCUT27 can’t co-utilize glucose and cellobiose due to carbon catabolite repression (CCR), which couldn’t be eliminated after ccpA knockout. The ability of co-utilizing glucose and cellobiose is a desirable feature of SCUT27 for industrial application. Four operons related to cellobiose utilization had been explored in SCUT27, named as Cel1, Cel2, Cel3 and Cel4. RT-PCR results showed that Cel2 operon is the only intact operon including integral cellobiose PTS transport elements and glycoside hydrolase, which is very important for cellobiose utilization. The transcription of Cel2 was regulated by σ⁵⁴-dependent transcription activator CelRcel2 (V518_2450). With sigma factor 54 (σ⁵⁴) deletion, the transcription strength of Cel2 decreased significantly. By replacing the σ⁵⁴-dependent promoter with the strong promoter of gene adhE (V518_0444), gene cluster bglpcel2EIIABCcel2 (V518_2444-2449) could be easily overexpressed and SCUT27/ΔcelRcel²/padhE obtained the ability to co-utilize glucose and cellobiose, which was a potential candidate for ethanol or lactic acid fermentation from partial hydrolysis of lignocellulosic hydrolysates.

The mannose phosphotransferase system (Man-PTS) - Mannose transporter and receptor for bacteriocins and bacteriophages

Article

Jul 2020
BBA-BIOMEMBRANES

Mannose transporters constitute a superfamily (Man-PTS) of the Phosphoenolpyruvate Carbohydrate Phosphotransferase System (PTS). The membrane complexes are homotrimers of protomers consisting of two subunits, IIC and IID. The two subunits without recognizable sequence similarity assume the same fold, and in the protomer are structurally related by a two fold pseudosymmetry axis parallel to membrane-plane (Liu et al. (2019) Cell Research 29 680). Two reentrant loops and two transmembrane helices of each subunit together form the N-terminal transport domain. Two three-helix bundles, one of each subunit, form the scaffold domain. The protomer is stabilized by a helix swap between these bundles. The two C-terminal helices of IIC mediate the interprotomer contacts. PTS occur in bacteria and archaea but not in eukaryotes. Man-PTS are abundant in Gram-positive bacteria living on carbohydrate rich mucosal surfaces. A subgroup of IICIID complexes serve as receptors for class IIa bacteriocins and as channel for the penetration of bacteriophage lambda DNA across the inner membrane. Some Man-PTS are associated with host-pathogen and -symbiont processes.

Complex Oligosaccharide Utilization Pathways in Lactobacillus

Article

Apr 2020
CURR ISSUES MOL BIOL

Lactobacillus is the bacterial genus that contains the highest number of characterized probiotics. Lactobacilli in general can utilize a great variety of carbohydrates. This characteristic is an essential trait for their survival in highly competitive environments such as the gastrointestinal tract of animals. In particular, the ability of some strains to utilize complex carbohydrates such as milk oligosaccharides as well as their precursor monosaccharides, confer upon lactobacilli a competitive advantage. For this reason, many of these carbohydrates are considered as prebiotics. Genome sequencing of many lactobacilli strains has revealed a great variety of genes involved in the metabolism of carbohydrates and some of them have already been characterized. In this review, the current knowledge at biochemical and genetic levels on the catabolic pathways of complex carbohydrates utilized by lactobacilli will be summarized.

Influence of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System on Toxin Gene Expression and Virulence in Bacillus anthracis

Article

Full-text available

Nov 2019
MOL MICROBIOL

AtxA, the master virulence gene regulator of Bacillus anthracis, is a PRD‐Containing Virulence Regulator (PCVR) as indicated by the crystal structure, post‐translational modifications, and activity of the protein. PCVRs are transcriptional regulators, named for PTS Regulatory Domains (PRDs) subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP‐PTS), and for their impact on virulence gene expression. Here we present data from experiments employing physiological, genetic, and biochemical approaches that support a model in which the PTS proteins HPr and Enzyme I (EI) are required for transcription of the atxA gene, rather than phosphorylation of AtxA. We show that atxA transcription is reduced 2.5‐fold in a mutant lacking HPr and EI, and that this change is sufficient to affect anthrax toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to parent levels, suggesting that phosphotransfer activity of HPr and EI is important for regulation of atxA. In a mouse model for anthrax, a HPr‐ EI‐ mutant was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS‐independent, promoter. Our data support a model in which HPr transfers a phosphate to an unidentified downstream transcriptional regulator to influence atxA gene transcription. AtxA is a critical virulence gene regulator of Bacillus anthracis. The crystal structure, post‐translational modifications, and activity of AtxA suggest that the protein is phosphorylated by the phosphoenolpyruvate phosphotransferase system (PEP‐PTS). We present data from physiological, genetic, and biochemical approaches supporting a model in which the PTS proteins Hpr and EI are required for transcription of the atxA gene, rather than phosphorylation of AtxA, and that at PTS mutant is attenuated for virulence.

Complex Oligosaccharide Utilization Pathways in Lactobacillus

Chapter

Jan 2019

Complex oligosaccharide utilization pathways in Lactobacillus

Chapter

Jan 2019

Lactobacillus is the bacterial genus that contains the highest number of characterized probiotics. Lactobacilli in general can utilize a great variety of carbohydrates. This characteristic is an essential trait for their survival in highly competitive environments such as the gastrointestinal tract of animals. In particular, the ability of some strains to utilize complex carbohydrates such as milk oligosaccharides as well as their precursor monosaccharides, confer upon lactobacilli a competitive advantage. For this reason, many of these carbohydrates are considered as prebiotics. Genome sequencing of many lactobacilli strains has revealed a great variety of genes involved in the metabolism of carbohydrates and some of them have already been characterized. In this chapter, the current knowledge at the biochemical and genetic levels of the catabolic pathways of complex carbohydrates utilized by lactobacilli will be summarized.

Kinase Inhibition Leads to Hormesis in a Dual Phosphorylation-Dephosphorylation Cycle

Article

Full-text available

Nov 2016
PLOS COMPUT BIOL

Many antimicrobial and anti-tumour drugs elicit hormetic responses characterised by low-dose stimulation and high-dose inhibition. While this can have profound consequences for human health, with low drug concentrations actually stimulating pathogen or tumour growth, the mechanistic understanding behind such responses is still lacking. We propose a novel, simple but general mechanism that could give rise to hormesis in systems where an inhibitor acts on an enzyme. At its core is one of the basic building blocks in intracellular signalling, the dual phosphorylation-dephosphorylation motif, found in diverse regulatory processes including control of cell proliferation and programmed cell death. Our analytically-derived conditions for observing hormesis provide clues as to why this mechanism has not been previously identified. Current mathematical models regularly make simplifying assumptions that lack empirical support but inadvertently preclude the observation of hormesis. In addition, due to the inherent population heterogeneities, the presence of hormesis is likely to be masked in empirical population-level studies. Therefore, examining hormetic responses at single-cell level coupled with improved mathematical models could substantially enhance detection and mechanistic understanding of hormesis.

Modulating Salmonella Typhimurium's Response to a Changing Environment through Bacterial Enhancer-Binding Proteins and the RpoN Regulon

Article

Full-text available

Aug 2016

Transcription sigma factors direct the selective binding of RNA polymerase holoenzyme (Eσ) to specific promoters. Two families of sigma factors determine promoter specificity, the σ70 (RpoD) family and the σ54 (RpoN) family. In transcription controlled by σ54, the Eσ54-promoter closed complex requires ATP hydrolysis by an associated bacterial enhancer-binding protein (bEBP) for the transition to open complex and transcription initiation. Given the wide host range of Salmonella enterica serovar Typhimurium, it is an excellent model system for investigating the roles of RpoN and its bEBPs in modulating the lifestyle of bacteria. The genome of S. Typhimurium encodes 13 known or predicted bEBPs, each responding to a unique intracellular or extracellular signal. While the regulons of most alternative sigma factors respond to a specific environmental or developmental signal, the RpoN regulon is very diverse, controlling genes for response to nitrogen limitation, nitric oxide stress, availability of alternative carbon sources, phage shock/envelope stress, toxic levels of zinc, nucleic acid damage, and other stressors. This review explores how bEBPs respond to environmental changes encountered by S. Typhimurium during transmission/infection and influence adaptation through control of transcription of different components of the S. Typhimurium RpoN regulon.

Signal Transduction by Trigger Enzymes: Bifunctional Enzymes and Transporters Controlling Gene Expression

Article

Full-text available

Mar 2010

Involvement of PTS in the regulatio of PrfA, a transcriptional activator of virulence genes in Listeria monocytogenes

Article

Full-text available

Dec 2006

Rana Herro

The activity of the Listeria monocytogenes transcription activator PrfA, which is required for the expression of several virulence genes, including the hemolysin-encoding hly, is inhibited by the presence of glucose, fructose and other rapidly metabolizable carbon sources. This inhibition is not mediated via the main carbon catabolite repression mechanism operative in Gram-positive bacteria, since inactivation of the catabolite control protein A (CcpA) did not prevent repression of virulence genes by the above sugars. We used a Bacillus subtilis strain (BUG1199) containing the prfA gene under control of an IPTG-inducible promoter and the lacZ reporter gene fused to the PrfA-activated L. monocytogens hly promoter to test whether the catabolite co-repressor P-Ser-HPr might be involved in PrfA regulation. Indeed, accumulation of P-Ser-HPr in an hprK mutant (hprKV267F) producing HPr kinase/phosphorylase (HprK/P) with normal kinase, but almost no phosphorylase activity strongly inhibited transcription activation by PrfA even in the absence of a repressing sugar. In response to the concentration of certain metabolites, the bifunctional HprK/P either phosphorylates HPr at Ser-46 (kinase function) or dephosphorylates P-Ser-HPr (phosphorylase function). Preventing the formation of P-Ser-HPr in the hprKV267F mutant by replacing Ser-46 in HPr with an alanine restored PrfA activity. In contrast, inactivation of crh, which encodes a HPr homologue that also becomes phosphorylated at Ser-46, did not enhance PrfA activity. PrfA in the hprKV267F mutant also remained inactive when the ccpA gene was mutated. In fact, disruption of ccpA in the hprK wild-type strain BUG1199 also led to the inactivation of PrfA, which is in agreement with previous findings that ccpA mutants contain large amounts of P-Ser-HPr similar to the hprKV267F mutant. It therefore seems that elevated concentrations of P-Ser-HPr due either to growth on rapidly metabolisable carbon sources or to specific mutations directly or indirectly inhibit PrfA activity. To carry out its catalytic function in sugar transport, HPr of the phosphotransferase system (PTS) is also phosphorylated by phosphoenolpyruvate and enzyme I at His-15. However, P-Ser-HPr is only very slowly phosphorylated by enzyme I, which probably accounts for PrfA inhibition. In agreement with this concept, disruption of the enzyme I- or HPr-encoding genes also strongly inhibited PrfA activity. PrfA activity therefore seems to depend on a fully functional PTS phosphorylation cascade.

Transcription of malP is subject to PTS-dependent regulation in Corynebacterium glutamicum

Article

Jul 2015
MICROBIOL-SGM

The Gram-positive Corynebacterium glutamicum co-metabolizes most carbon sources such as the PTS sugar glucose and the non-PTS sugar maltose. Maltose is taken up via the ABC-transporter MusEFGK2I and is further metabolized to glucose phosphate by amylomaltase MalQ, maltodextrin phosphorylase MalP, glucokinase Glk and phosophoglucomutase Pgm. Surprisingly, growth of C. glutamicum strains lacking the general PTS components EI or HPr was strongly impaired on the non-PTS sugar maltose. Complementation experiments showed that a functional PTS phosphorelay is required for optimal growth of C. glutamicum on maltose implying its involvement in the control of maltose metabolism and/or uptake. To identify the target of this PTS-dependent control transport measurements with 14C-labelled maltose, Northern Blot analyses and enzyme assays were performed. The activities of the maltose transporter and enzymes MalQ, Pgm, and GlK were not decreased in PTS-deficient C. glutamicum strains, which was corroborated by comparable transcript amounts of musE, musK and musG as well as of malQ in C. glutamicum ΔptsH and C. glutamicum WT. By contrast, MalP activity was significantly reduced and only residual amounts of malP transcripts were detected in C. glutamicum ΔptsH when compared to C. glutamicum WT. Promoter activity assays with the malP promoter in C. glutamicum ΔptsH and C. glutamicum WT confirmed that malP transcription is reduced in the PTS-deficient strain. Taken together, we here show for the first time a regulatory function of the PTS in C. glutamicum and identify malP transcription as its target.

Carbohydrate Utilization by Listeria monocytogenes and its Influence on Virulence Gene Expression

Chapter

Full-text available

Feb 2014

The Gram-positive bacterium Listeria monocytogenes possesses a large number of transport systems for sugars and sugar derivatives. The uptake of the triol glycerol occurs in an energy-independent process via facilitated diffusion. In contrast, ion-driven transporters, ATP binding cassette (ABC) transporters and the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) require energy for the carbohydrate uptake reaction. Rhamnose and several sugar-phosphates are taken up via ion-driven permeases. L. monocytogenes contains several ABC transporters one of which was found to transport maltose. However, most carbohydrates used by L. monocytogenes are transported by the PTS. Among others they include glucose, fructose, mannose, cellobiose, gentiobiose, trehalose, D-arabitol and several -glucoside type heterosides (salicin, arbutin, esculin, amygdalin). For many PTS the substrate has not yet been determined. Expression of several genes encoding PTS of unknown specificity, such as lmo1974-1968, was found to be upregulated during intracellular growth, suggesting that L. monocytogenes uses certain carbon sources transported by the PTS for intracellular proliferation. Certain carbohydrates taken up via the PTS strongly repress the expression of virulence genes. Virulence gene expression depends on the transcription activator PrfA (positive regulatory factor A). Its activity was found to be strongly inhibited by the presence of several PTS substrates. The underlying repression mechanism is not yet understood. It differs from common carbon catabolite repression, which in firmicutes is mediated by the PTS protein seryl-phosphorylated HPr bound to the catabolite control protein A. PrfA activity is probably regulated by a PTS component or a metabolic intermediate derived from an efficiently utilized carbohydrate.

Untersuchung des Einflusses von Phosphorylierung auf Struktur, Dynamik und Funktion des HPr-Proteins Erlangung des Doktorgrades

Article

RNA Recognition by Transcriptional Antiterminators of the BglG/SacY Family: Functional and Structural Comparison of the CAT Domain from SacY and LicT

Article

Full-text available

Nov 1999

Transcriptional antiterminators of the BglG/SacY family are regulatory proteins that mediate the induction of sugar metabolizing operons in Gram-positive and Gram-negative bacteria. Upon activation, these proteins bind to specific targets in nascent mRNAs, thereby preventing abortive dissociation of the RNA polymerase from the DNA template. We have previously characterized the RNA-binding domain of SacY from Bacillus subtilis and determined its three-dimensional structure by both NMR and crystallography. In the present study, we have characterized the paralogous domain from LicT and we present the first structural comparison between two BglG/SacY family members. Similar to SacY, the RNA-binding activity of LicT is contained within the 56 N-terminal amino acid residue fragment corresponding to the so-called co-antiterminator (CAT) domain. Surface plasmon resonance affinity measurements show that, compared to SacY-CAT, LicT-CAT binds more tightly and more specifically to its cognate RNA target, with a KD value of about 10−8 M. The crystal structure of LicT-CAT has been determined at 1.8 Å resolution and compared to that of SacY-CAT. Both molecules fold as symmetrical dimers, each monomer comprising a four-stranded antiparallel β-sheet that stacks against the β-sheet of the other monomer in a very conserved manner. Comparison of the proposed RNA-binding surfaces shows that many of the conserved atoms concentrate in a central region across one face of the CAT dimer, whereas variable elements are mostly found at the edges. Interestingly, the electrostatic potential maps calculated for the two molecules are quite different, except for the core of the RNA-binding site, which appears essentially neutral in both structures.

Salmonella Utilizes D-Glucosaminate via a Mannose Family Phosphotransferase System Permease and Associated Enzymes

Article

Full-text available

Aug 2013
J BACTERIOL

Salmonella enterica is a globally significant bacterial food-borne pathogen that utilizes a variety of carbon sources. We report here that Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) uses d-glucosaminate (2-amino-2-deoxy-d-gluconic acid) as a carbon and nitrogen source via a previously uncharacterized mannose family phosphotransferase system (PTS) permease, and we designate the genes encoding the permease dgaABCD (d-glucosaminate PTS permease components EIIA, EIIB, EIIC, and EIID). Two other genes in the dga operon (dgaE and dgaF) were required for wild-type growth of S. Typhimurium with d-glucosaminate. Transcription of dgaABCDEF was dependent on RpoN (σ54) and an RpoN-dependent activator gene we designate dgaR. Introduction of a plasmid bearing dgaABCDEF under the control of the lac promoter into Escherichia coli strains DH5α, BL21, and JM101 allowed these strains to grow on minimal medium containing d-glucosaminate as the sole carbon and nitrogen source. Biochemical and genetic data support a catabolic pathway in which d-glucosaminate, as it is transported across the cell membrane, is phosphorylated at the C-6 position by DgaABCD. DgaE converts the resulting d-glucosaminate-6-phosphate to 2-keto-3-deoxygluconate 6-phosphate (KDGP), which is subsequently cleaved by the aldolase DgaF to form glyceraldehyde-3-phosphate and pyruvate. DgaF catalyzes the same reaction as that catalyzed by Eda, a KDGP aldolase in the Entner-Doudoroff pathway, and the two enzymes can substitute for each other in their respective pathways. Examination of the Integrated Microbial Genomes database revealed that orthologs of the dga genes are largely restricted to certain enteric bacteria and a few species in the phylum Firmicutes.

A phosphotransferase system permease is a novel component of CadC signaling in Salmonella enterica

Article

Oct 2012
FEMS MICROBIOL LETT

In Salmonella enterica serovar Typhimurium, the proteolytic cleavage of the membrane-bound transcriptional regulator CadC acts as a switch to activate genes of the lysine decarboxylase system in response to low pH and lysine signals. To identify the genetic factors required for the proteolytic activation of CadC, we performed genome-wide random mutagenesis. We report here that a phosphotransferase system (PTS) permease STM4538 acts as a positive modulator of CadC function. The transposon insertion in STM4538 reduces the expression of the CadC target operon cadBA under permissive conditions. In addition, a deletional inactivation of STM4538 in the wild-type background leads to the impaired proteolytic cleavage of CadC. We also show that only the low pH signal is involved in the proteolytic processing of CadC, but the lysine signal plays a role in the repression of the lysP gene encoding a lysine-specific permease, which negatively controls the expression of the cadBA operon. Our data suggest that the PTS permease STM4538 affects proteolytic processing, which is a necessary but not sufficient step for CadC activation, rendering CadC able to activate target genes. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Sites of positive and negative regulation in the Bacillus subtilis antiterminators LicT and SacY

Article

Sep 2001
MOL MICROBIOL

The Bacillus subtilis homologous transcriptional antiterminators LicT and SacY control the inducible expression of genes involved in aryl β-glucoside and sucrose utilization respectively. Their RNA-binding activity is carried by the N-terminal domain (CAT), and is regulated by two similar C-terminal domains (PRD1 and PRD2), which are the targets of phosphorylation reactions catalysed by the phosphoenolpyruvate: sugar phosphotransferase system (PTS). In the absence of the corresponding inducer, LicT is inactivated by BglP, the PTS permease (EII) specific for aryl β-glucosides, and SacY by SacX, a negative regulator homologous to the EII specific for sucrose. LicT, but not SacY, is also subject to a positive control by the general PTS components EI and HPr, which are thought to phosphorylate LicT in the absence of carbon catabolite repression. Construction of SacY/LicT hybrids and mutational analysis enabled the location of the sites of this positive regulation at the two phosphorylatable His207 and His269 within LicT-PRD2, and suggested that the presence of negative charges at these sites is sufficient for LicT activation in vivo. The BglP-mediated inhibition process was found to essentially involve His100 of LicT-PRD1, with His159 of the same domain playing a minor role in this regulation. In vitro experiments indicated that His100 could be phosphorylated directly by the general PTS proteins, this phosphorylation being stimulated by P∼BglP. We confirmed that, similarly, the corresponding conserved His99 residue in SacY is the major site of the negative control exerted by SacX on SacY activity. Thus, for both antiterminators, the EII-mediated inhibition process seems to rely primarily on the presence of a negative charge at the first conserved histidine of the PRD1.

The ��54 regulon (sigmulon) of Pseudomonas putida

Article

Dec 2003
ENVIRON MICROBIOL

Summaryσ54 is unique among the bacterial sigma factors. Besides not being related in sequence with the rest of such factors, its mechanism of transcription initiation is completely different and requires the participation of a transcription activator. In addition, whereas the rest of the alternative sigma factors use to be involved in transcription of somehow related biological functions, this is not the case for σ54 and many different and unrelated genes have been shown to be transcribed from σ54-dependent promoters, ranging from flagellation, to utilization of several different carbon and nitrogen sources, or alginate biosynthesis. These genes have been characterized in many different bacterial species and, only until recently with the arrival of complete genome sequences, we have been able to look at the σ54 functional role from a genomic perspective. Aided by computational methods, the σ54 regulon has been studied both in Escherichia coli, Salmonella typhimurium and several species of the Rhizobiaceae. Here we present the analysis of the σ54 regulon (sigmulon) in the complete genome of Pseudomonas putida KT2440. We have developed an improved method for the prediction of σ54-dependent promoters which combines the scores of σ54-RNAP target sequences and those of activator binding sites. In combination with other evidence obtained from the chromosomal context and the similarity with closely related bacteria, we have been able to predict more than 80% of the σ54-dependent promoters of P. putida with high confidence. Our analysis has revealed new functions for σ54 and, by means of comparative analysis with the previous studies, we have drawn a potential mechanism for the evolution of this regulatory system.

Transcriptional regulation of cellobiose utilization by PRD-domain containing Sigma54-dependent transcriptional activator (CelR) and catabolite control protein A (CcpA) in Bacillus thuringiensis

Article

Full-text available

Jan 2024

Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil bacteria. In bacteria, the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), encoded by the cel operon, is responsible for the transport and utilization of cellobiose. In this study, we analyzed the transcription and regulation of the cel operon in Bacillus thuringiensis (Bt). The cel operon is composed of five genes forming one transcription unit. β-Galactosidase assays revealed that cel operon transcription is induced by cellobiose, controlled by Sigma54, and positively regulated by CelR. The HTH-AAA⁺ domain of CelR recognized and specifically bound to three possible binding sites in the celA promoter region. CelR contains two PTS regulation domains (PRD1 and PRD2), which are separated by two PTS-like domains-the mannose transporter enzyme IIA component domain (EIIAMan) and the galactitol transporter enzyme IIB component domain (EIIBGat). Mutations of His-546 on the EIIAMan domain and Cys-682 on the EIIBGat domain resulted in decreased transcription of the cel operon, and mutations of His-839 on PRD2 increased transcription of the cel operon. Glucose repressed the transcription of the cel operon and catabolite control protein A (CcpA) positively regulated this process by binding the cel promoter. In the celABCDE and celR mutants, PTS activities were decreased, and cellobiose utilization was abolished, suggesting that the cel operon is essential for cellobiose utilization. Bt has been widely used as a biological pesticide. The metabolic properties of Bt are critical for fermentation. Nutrient utilization is also essential for the environmental adaptation of Bt. Glucose is the preferred energy source for many bacteria, and the presence of the phosphotransferase system allows bacteria to utilize other sugars in addition to glucose. Cellobiose utilization pathways have been of particular interest owing to their potential for developing alternative energy sources for bacteria. The data presented in this study improve our understanding of the transcription patterns of cel gene clusters. This will further help us to better understand how cellobiose is utilized for bacterial growth.

Nitrogen Source Utilization and Its Regulation

Chapter

Apr 2014

Trigger Enzymes: Coordination of Metabolism and Virulence Gene Expression

Chapter

Full-text available

Aug 2015

σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

Article

Full-text available

May 2019

The solventogenic C. beijerinckii DSM 6423, a microorganism that naturally produces isopropanol and butanol, was previously modified by random mutagenesis. In this work, one of the resulting mutants was characterized. This strain, selected with allyl alcohol and designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ54 sigma factor. Complementation with wild-type sigL fully restored solvent production and sugar assimilation and RT-qPCR analyses revealed its transcriptional control of several genes related to solventogensis, demonstrating the central role of σ54 in C. beijerinckii DSM 6423. Comparative genomics analysis suggested that this function is conserved at the species level, and this hypothesis was further confirmed through the deletion of sigL in the model strain C. beijerinckii NCIMB 8052.

σ 54 (σ L ) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

Preprint

Full-text available

Dec 2018

Microbial production of butanol and isopropanol, two high value-added chemicals, is naturally occurring in the solventogenic Clostridium beijerinckii DSM 6423. Despite its ancient discovery, the precise mechanisms controlling alcohol synthesis in this microorganism are poorly understood. In this work, an allyl alcohol tolerant strain obtained by random mutagenesis was characterized. This strain, designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 ( sigL ), which encodes the σ ⁵⁴ sigma factor. Complementation with the wild-type sigL gene fully restored solvent production and sugar assimilation, demonstrating that σ ⁵⁴ plays a central role in regulating these pathways in C. beijerinckii DSM 6423. Genomic comparison with other strains further revealed that these functions are probably conserved among the C. beijerinckii strains. The importance of σ ⁵⁴ in C. beijerinckii was further assessed by the characterization of a sigL deletion mutant of the model strain NCIMB 8052 obtained with a CRISPR/Cas9 tool. The resulting mutant exhibited phenotypic traits similar to the AA strain, and was subsequently complemented with the sigL gene from either the wild type or the AA strains. The results of this experiment confirmed the crucial role of σ ⁵⁴ in the regulation of both solventogenesis and sugar consumption pathways in C. beijerinckii .

Regulons and Protein‐Protein Interactions of PRD‐containing Bacillus anthracis Virulence Regulators Reveal Overlapping but Distinct Functions

Article

Dec 2017

Bacillus anthracis produces three regulators, AtxA, AcpA, and AcpB, which control virulence gene transcription and belong to an emerging class of regulators termed “PCVRs” (Phosphoenolpyruvate‐dependent phosphotransferase regulation Domain‐Containing Virulence Regulators). AtxA, named for its control of toxin gene expression, is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. Reports of PCVR activity suggest overlapping function. AcpA and AcpB independently positively control transcription of the capsule biosynthetic operon capBCADE, and culture conditions that enhance AtxA level or activity result in capBCADE transcription in strains lacking acpA and acpB. We used RNA‐Seq to assess the regulons of the paralogous regulators in strains constructed to express individual PCVRs at native levels. Plasmid and chromosome‐borne genes were PCVR controlled, with AtxA, AcpA, and AcpB having a ≥4‐fold effect on transcript levels of 145, 130, and 49 genes respectively. Several genes were coregulated by two or three PCVRs. We determined that AcpA and AcpB form homomultimers, as shown previously for AtxA, and we detected AtxA‐AcpA heteromultimers. In co‐expression experiments, AcpA activity was reduced by increased levels of AtxA. Our data show that the PCVRs have specific and overlapping activity, and that PCVR stoichiometry and potential heteromultimerization can influence target gene expression. This article is protected by copyright. All rights reserved.

Sophisticated Regulation of Transcriptional Factors by the Bacterial Phosphoenolpyruvate: Sugar Phosphotransferase System

Article

Feb 2017
J MOL BIOL

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a carbohydrate transport and phosphorylation system present in bacteria of all different phyla and in archaea. It is usually composed of three proteins or protein complexes, enzyme I, HPr and enzyme II, which are phosphorylated at histidine or cysteine residues. However, in many bacteria, HPr can also be phosphorylated at a serine residue. The PTS functions not only as carbohydrate transporter, but also regulates numerous cellular processes by either phosphorylating its target proteins or by interacting with them in a phosphorylation-dependent manner. The target proteins can be catabolic enzymes, transporters and signal transduction proteins, but are most frequently transcriptional regulators. In this review we will describe how PTS components interact with or phosphorylate proteins to regulate directly or indirectly the activity of transcriptional repressors, activators or antiterminators. We will briefly summarize the well-studied mechanism of carbon catabolite repression in firmicutes, where the transcriptional regulator CcpA needs to interact with seryl-phosphorylated HPr in order to be functional. We will present new results related to transcriptional activators and antiterminators containing specific PTS regulation domains (PRDs), which are the phosphorylation targets for three different types of PTS components. Moreover, we will discuss how the phosphorylation level of the PTS components precisely regulates the activity of target transcriptional regulators or antiterminators, with or without PRD, and how the availability of PTS substrates and thus the metabolic status of the cell is connected with various cellular processes, such as biofilm formation or virulence of certain pathogens.

Carbohydrate Uptake and Metabolism

Article

Jan 2002

The genes encoding the polysaccharide-hydrolyzing enzymes are often organized in an operon or regulon together with genes that encode the enzymes catalyzing the uptake of the extracellular hydrolytic products and the first intracellular steps in their catabolism. The Bacillus subtilis genome encodes about 23 secondary and 11 ATP-binding cassette (ABC) carbohydrate transporters. The major facilitator superfamily (MFS) comprises eight B. subtilis proteins of unknown carbohydrate specificity exhibiting significant similarity to the GalP/XylE subfamily. The genes encoding the polysaccharide-hydrolyzing enzymes are often organized in an operon or regulon together with genes that encode the enzymes catalyzing the uptake of the extracellular hydrolytic products and the first intracellular steps in their catabolism. Glycolysis is one of the most conserved metabolic pathways in living organisms. To conserve cellular resources, expression of most of the hundreds of carbohydrate catabolism genes is induced only when the corresponding carbohydrate is present in the growth medium. Expression of antiterminator-controlled genes or operons usually occurs from a constitutive promoter; transcription stops at a terminator located in the leader region of these genes and operons, providing very short transcripts. The carbohydrate transport systems operative in gram-positive and gram-negative bacteria are very similar and most likely developed early in evolution.

Regulation of the main Listeria monocytogenes glucose transporter and effects on virulence gene expression

Article

Apr 2011

Francine Moussan Aké

L. monocytogenes is a ubiquitous foodborne pathogenic Gram-positive bacterium, which can multiply in host cells and infect humans causing septicemia, spontaneous abortion and méningoencephalitis. This bacterium transports glucose via phosphoenolpyruvate:sugar phosphotransferase systems (PTS) and non-PTS permeases. Two major glucose-transporting PTSs belong to the mannose class. One is encoded by the manLMN (man) operon and the second by the mpoABCD (mpo) operon. One goal was to study the transport of glucose by the proteins encoded by these operons and to identify non-PTS glucose transporters. Growth studies in MM supplemented with glucose and glucose consumption assays with several mutants revealed that deletion of manL (encodes EIIABMan) or manM (encodes EIICMan) significantly slowed glucose utilization (3- to 4-fold) compared to the WT AML73 or EGDe strain. Deletion of mpoA (encodes EIIAMpo) had no significant effect on glucose utilization (same phenotype as the WT) whereas deletion of mpoB (encodes EIIBMpo) significantly slowed glucose utilization (4- to -5 fold). By using qRT-PCR, we show that expression of the man operon is induced by glucose, whereas the mpo operon is expressed constitutively. Nevertheless, deletion of mpoA causes constitutive man operon expression whereas deletion of mpoB inhibits it. The PTSMpo therefore functions as a constantly synthesized glucose sensor regulating man operon expression. Deletion of ptsI (encodes the general PTS component EI) also inhibits man expression and the ΔptsI mutant was most strongly impeded in glucose utilization. The residual glucose uptake probably owes to three GlcU-like non-PTS transporters. The successful heterelogous complementation of the E. coli LJ140 strain, wich is unable to transport glucose, suggests that the L. monocytogenes GlcU proteins, GlcU1, GlcU2 and GlcU3 (identified by sequences homology to GlcU proteins in other firmicutes) are indeed capable of transporting glucose.A potential role of PTS and non-PTS components in PrfA regulation was studied in the L. monocytogenes AML73 strain (contains a Phly-gus fusion) and in the ΔmanL, ΔmanM, ΔmpoB, ΔmpoA, ΔptsI, glcU mutants derived from it. For that purpose, I carried out β-D-glucuronidase activity tests with bacteria grown either in liquid or on solid medium and qRT-PCR experiments (expression of actA and hly genes). Interestingly, deletion of ptsI, manL, manM and mpoB caused elevated PrfA activity (2- to -14 fold) and elevated expression of virulence gene expression (actA and hly) in the ΔmanL, ΔmanM and ΔmpoB mutants was observed. Nevertheless, glcU inactivation and mpoA deletion had no effect on PrfA activity. The elevated PrfA activity disappeared when the prfA gene was also deleted in the ΔmanL, ΔmanM and ΔmpoB mutants, confirming that the stimulatory effect of the various mutations on virulence gene expression is PrfA-dependent. All mutants exhibiting elevated virulence gene expression contain no or only little unphosphorylated EIIABMan, which we therefore suspect to play a major role in glucose-mediated PrfA inhibition. The effect of the PTS mutations was also tested in in vitro host cells infection assays (Caco-2, Jeg-3 cells) and in an in vivo mouse model. Deletion of ptsI led to elevated infection of the host cells, which probably owes to the elevated synthesis of the InlA protein.

Trigger Enzymes: Coordination of Metabolism and Virulence Gene Expression

Article

Full-text available

Jul 2015

Trigger Enzymes: Coordination of Metabolism and Virulence Gene Expression, Page 1 of 2 Abstract Virulence gene expression serves two main functions, growth in/on the host, and the acquisition of nutrients. Therefore, it is obvious that nutrient availability is important to control expression of virulence genes. In any cell, enzymes are the components that are best informed about the availability of their respective substrates and products. It is thus not surprising that bacteria have evolved a variety of strategies to employ this information in the control of gene expression. Enzymes that have a second (so-called moonlighting) function in the regulation of gene expression are collectively referred to as trigger enzymes. Trigger enzymes may have a second activity as a direct regulatory protein that can bind specific DNA or RNA targets under particular conditions or they may affect the activity of transcription factors by covalent modification or direct protein-protein interaction. In this chapter, we provide an overview on these mechanisms and discuss the relevance of trigger enzymes for virulence gene expression in bacterial pathogens.

Regulation des Mannose-Operons in Bacillus subtilis

Thesis

Jan 2010

Tianqi Sun

Das Mannose-Operon in B. subtilis besteht aus drei Genen, manP, manA und yjdF, von denen aber nur manP und manA für die Verwertung der Mannose notwendig sind. Stromaufwärts und in derselben Orientierung wie das Mannose-Operon liegt das regulatorische Gen manR. Dieses codiert den Transkriptionsaktivator für die Expression des Mannose-Operons und des eigenen Gens, die beide durch Mannose induzierbar sind. Mit lacZ als Reportergen verursachte die Zugabe von Mannose eine 4-7-fache Erhöhung der Transkriptionsrate des manP-Promotors (PmanP), und eine circa 3-fache Erhöhung des manR-Promotors (PmanR). Durch "Primer Extension"-Experimente wurde der Transkriptionsstart von manPA-yjdF und manR identifiziert. Die aus diesem Transkriptionsstart abgeleiteten 35- und 10-Regionen der beiden Promotoren stimmen mit der Konsensussequenz von vegetativen sA abhängigen Promotoren überein. Eine Deletion von manR bewirkte ein nahezu vollständiges Abschalten beider Promotoren. Durch Deletionsanalyse wurde die Lage der Operatorsequenz von ManR zwischen den Basenpaaren -79 und -35 bezüglich des Transkriptionsstarts von manPA-yjdF und zwischen -81 und -35 bezüglich des Transkriptionsstarts von manR bestimmt. Ein Austausch dieser Region gegen eine entsprechende Region eines Mannitol-Operons führte dazu, dass die entsprechenden Hybridpromotoren nun durch Mannitol regulierbar wurden. Eine Deletion von manP (Mannose-Transporter) hatte eine konstitutive Expression von PmanP und PmanR zur Folge. Dagegen führte eine Deletion von manA (Mannose-6-Phosphat-Isomerase) zur Sensitivität gegen Mannose. Anhand von Aminosäurensequenz-Vergleichen wurden in ManR fünf Domänen postuliert, nämlich eine N-terminale DNA-Bindedomäne, zwei PRDs, eine PTS-EIIA- und eine PTS-EIIB-ähnliche Domäne am C-Terminus. Die Deletion der EIIA- und EIIB-ähnlichen Domänen führte zu einer konstitutiven Expression von PmanP und PmanR. Da die Deletion des Gens für den Mannose-Transporter ebenfalls zur Konstitutivität führte, wurde gefolgert, dass es eine Phosphatübertragung zwischen Transporter und Regulator gibt, die die Aktivität von ManR beeinflusst. In Analogie zu anderen PRD-Regulatoren sollte die PRDI in Abwesenheit von Mannose phosphoryliert vorliegen und in Anwesenheit von Mannose durch den Mannosetransporter dephosphoryliert werden. Tatsächlich spielt wie auch bei anderen PRD-Regulatoren die PEP-abhängige Phosphorylierung an His15 von HPr eine essentielle Rolle bei der Aktivierung von PmanP und PmanR. Außerdem unterliegen sowohl PmanP als auch PmanR der Kohlenstoff-Katabolitrepression (CCR). Aufgrund der Homologie von ManR zu anderen PRD-haltigen Transkriptionsaktivatoren kann man davon ausgehen, dass die PRDII in Abwesenheit von Glucose phosphoryliert und in Anwesenheit von Glucose dephosphoryliert ist. Der Mannose-Regulator ist demnach nur aktiv, wenn PRDI dephosphoryliert und PRDII phosphoryliert ist, also wenn nur Mannose ohne Glucose im Medium vorliegt. Genau dieses Verhalten wurde an den beiden Mannose-Promotoren beobachtet. Dies überlappt mit einem zweiten, für B. subtilis bekannten Mechanismus der Katabolitrepression, dessen zentrale Bestandteile der Repressor CcpA, HPr und HPr-Kinase sind. Bei einem Überschuss an Intermediaten der Glykolyse, insbesondere Fruktose-1,6-biphosphat wird die HPr-Kinase stimuliert, die ATP-abhängig HPr an Ser46 phosphoryliert. Dieses bindet an CcpA und der Komplex bindet schließlich an sogenannte cre-Sequenzen in entsprechenden CCR-regulierten Promotoren. Tatsächlich konnte eine cre-Sequenz im manR-Promotor identifiziert werden. Eine Mutation der Sequenz führte zu einer Erniedrigung der Katabolitrepression der Mannose-Promotoren. Eine geringe Erniedrigung der CCR wurde auch in der ptsH-S46A-Mutante und einer Deletionsmutante des zu HPr homologen crh beobachtet, aber erst in der ptsH-S46A Δcrh Doppelmutante war die CCR auf PmanP and PmanR stark reduziert. Dies zeigte, dass die beiden Proteine sich offensichtlich in der Funktion komplementieren. Zusammengefasst lässt sich sagen, dass durch diese Arbeit ein autoregulatorisches System für die Mannose-Verwertung in Bacillus subtilis bezüglich Induktion und Katabolitrepression weitgehend aufgeklärt wurde und aufgrund des starken manP Promotors eine Anwendung in der Konstruktion von Expressionsvektoren finden sollte.

A Mannose Family Phosphotransferase System Permease and Associated Enzymes Are Required for Utilization of Fructoselysine and Glucoselysine in Salmonella enterica Serovar Typhimurium

Article

Full-text available

Jul 2015
J BACTERIOL

Unlabelled: Salmonella enteric serovar Typhimurium, a major cause of food-borne illness, is capable of using a variety of carbon and nitrogen sources. Fructoselysine and glucoselysine are Maillard reaction products formed by the reaction of glucose or fructose, respectively, with the ε-amine group of lysine. We report here that S. Typhimurium utilizes fructoselysine and glucoselysine as carbon and nitrogen sources via a mannose family phosphotransferase (PTS) encoded by gfrABCD (glucoselysine/fructoselysine PTS components EIIA, EIIB, EIIC, and EIID; locus numbers STM14_5449 to STM14_5454 in S. Typhimurium 14028s). Genes coding for two predicted deglycases within the gfr operon, gfrE and gfrF, were required for growth with glucoselysine and fructoselysine, respectively. GfrF demonstrated fructoselysine-6-phosphate deglycase activity in a coupled enzyme assay. The biochemical and genetic analyses were consistent with a pathway in which fructoselysine and glucoselysine are phosphorylated at the C-6 position of the sugar by the GfrABCD PTS as they are transported across the membrane. The resulting fructoselysine-6-phosphate and glucoselysine-6-phosphate subsequently are cleaved by GfrF and GfrE to form lysine and glucose-6-phosphate or fructose-6-phosphate. Interestingly, although S. Typhimurium can use lysine derived from fructoselysine or glucoselysine as a sole nitrogen source, it cannot use exogenous lysine as a nitrogen source to support growth. Expression of gfrABCDEF was dependent on the alternative sigma factor RpoN (σ(54)) and an RpoN-dependent LevR-like activator, which we designated GfrR. Importance: Salmonella physiology has been studied intensively, but there is much we do not know regarding the repertoire of nutrients these bacteria are able to use for growth. This study shows that a previously uncharacterized PTS and associated enzymes function together to transport and catabolize fructoselysine and glucoselysine. Knowledge of the range of nutrients that Salmonella utilizes is important, as it could lead to the development of new strategies for reducing the load of Salmonella in food animals, thereby mitigating its entry into the human food supply.

The Second Paradigm for Activation of Transcription

Article

Feb 2005
PROG MOL BIOL TRANSL

Gene transcription is central to the development, differentiation, and adaptation of cells. Control of transcription requires the interplay of signaling pathways with the molecular machinery of transcription, the DNA-dependent RNA polymerase (RNAP) enzyme, regulatory proteins that act upon it, and the nucleic acid that is transcribed. The genetic tractability of bacteria, in particular Escherichia coli and Bacillus subtilis, and yeast has allowed rapid progress in elucidating the types of strategy used for the control of gene expression at the level of transcription. The RNAP is evolutionarily conserved in sequence, structure, and function from bacteria to humans. The simple (in terms of subunit composition) bacterial RNAP is an excellent model system to study the control of gene transcription. This chapter also describes the components of such a system and how they interact to allow regulation of RNAP activity at the level of the DNA opening event (i.e., open complex formation) necessary for trancription initiation.

The bacterial phosphoenolpyruvate: Sugar phosphotransferase system (PTS): An interface between energy and signal transduction

Article

Jun 2012

Bernhard Erni

The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) mediates the uptake and phosphorylation of carbohydrates, and is involved in signal transduction. It comprises two general phosphotransferase proteins (EI and HPr) and a—species dependent—variable number of sugar-specific enzyme II complexes (IIA, IIB, IIC). EI and HPr transfer phosphoryl groups from PEP to the IIA units. IIA and IIB sequentially transfer phosphates to the sugar, which is translocated by the IIC unit. The ratio of phosphorylated to non-phosphorylated IIA and IIB varies with transport activity, and the phosphorylation state of some of the IIA and IIB serves as signal input for regulation of catabolite repression, intermediate metabolism, gene expression and chemotaxis in response to the availability of carbohydrates and PEP (glycolytic activity). PTS occur in about one-third of all eubacteria and in a few archaebacteria but not in animals and plants. Uniqueness and pleiotropic function make the PTS a potential target for anti-infectives. The PTS transporter for mannose is utilized as a gate for the penetration of bacteriophage lambda DNA across, and insertion of certain bacteriocins (small antimicrobial peptides) into the inner membrane. The PTS of Escherichia coli is in the focus of this review, but occasionally comparisons with other species are made. The topics are: History; Modular design of the E. coli PTS; Structure function and catalytic mechanism of the protein modules; Regulation of and by the PTS; The PTS in pathogenicity and virulence; Computational models; Metabolic engineering.

Functional Characterization of AtxA, the Bacillus anthracis Virulence Regulator

Article

May 2012

Troy G. Hammerstrom

Coordinated expression of virulence genes in Bacillus anthracis occurs via a multi-faceted signal transduction pathway that is dependent upon the AtxA protein. Intricate control of atxA gene transcription and AtxA protein function have become apparent from studies of AtxA-induced synthesis of the anthrax toxin proteins and the poly-D-glutamic acid capsule, two factors with important roles in B. anthracis pathogenesis. The amino-terminal region of the AtxA protein contains winged-helix (WH) and helix-turn-helix (HTH) motifs, structural features associated with DNA-binding. Using filter binding assays, I determined that AtxA interacted non-specifically at a low nanomolar affinity with a target promoter (Plef) and AtxA-independent promoters. AtxA also contains motifs associated with phosphoenolpyruvate: sugar phosphotransferase system (PTS) regulation. These PTS-regulated domains, PRD1 and PRD2, are within the central amino acid sequence. Specific histidines in the PRDs serve as sites of phosphorylation (H199 and H379). Phosphorylation of H199 increases AtxA activity; whereas, H379 phosphorylation decreases AtxA function. For my dissertation, I hypothesized that AtxA binds target promoters to activate transcription and that DNA-binding activity is regulated via structural changes within the PRDs and a carboxy-terminal EIIB-like motif that are induced by phosphorylation and ligand binding. I determined that AtxA has one large protease-inaccessible domain containing the PRDs and the carboxy-terminal end of the protein. These results suggest that AtxA has a domain that is distinct from the putative DNA-binding region of the protein. My data indicate that AtxA activity is associated with AtxA multimerization. Oligomeric AtxA was detected when co-affinity purification, non-denaturing gel electrophoresis, and bis(maleimido)hexane (BMH) cross-linking techniques were employed. I exploited the specificity of BMH for cysteine residues to show that AtxA was cross-linked at C402, implicating the carboxy-terminal EIIB-like region in protein-protein interactions. In addition, higher amounts of the cross-linked dimeric form of AtxA were observed when cells were cultured in conditions that promote toxin gene expression. Based on the results, I propose that AtxA multimerization requires the EIIB-like motif and multimerization of AtxA positively impacts function. I investigated the role of the PTS in the function of AtxA and the impact of phosphomimetic residues on AtxA multimerization. B. anthracis Enzyme I (EI) and HPr did not facilitate phosphorylation of AtxA in vitro. Moreover, markerless deletion of ptsHI in B. anthracis did not perturb AtxA function. Taken together, these results suggest that proteins other than the PTS phosphorylate AtxA. Point mutations mimicking phosphohistidine (H to D) and non-phosphorylated histidine (H to A) were tested for an impact on AtxA activity and multimerization. AtxA H199D, AtxA H199A, and AtxA H379A displayed multimerization phenotypes similar to that of the native protein, whereas AtxA H379D was not susceptible to BMH cross-linking or co-affinity purification with AtxA-His. These data suggest that phosphorylation of H379 may decrease AtxA activity by preventing AtxA multimerization. Overall, my data support the following model of AtxA function. AtxA binds to target gene promoters in an oligomeric state. AtxA activity is increased in response to the host-related signal bicarbonate/CO2 because this signal enhances AtxA multimerization. In contrast, AtxA activity is decreased by phosphorylation at H379 because multimerization is inhibited. Future studies will address the interplay between bicarbonate/CO2 signaling and phosphorylation on AtxA function.

The bacterial phosphotransferase system: a perfect link of sugar transport and signal transduction

Chapter

Full-text available

Mar 2004

Bacteria transport and concomitantly phosphorylate several sugars via the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PTS). This allows the introduction of these sugars into the glycolytic pathway at low cost and provides, thus, an advantage as compared to the use of other sugar transport systems. Four of the PTS proteins are involved in phosphotransfer from phosphoenolpyruvate to the incoming sugar. The phosphorylation state of these proteins can, thus, indicate the sugar supply, and different PTS proteins are indeed involved in different signal transduction strategies that link the availability of sugars and the physiological state of the cell to the activity of transport proteins, metabolic enzymes, and transcriptional regulators. Moreover, there are PTS-related proteins that are exclusively devoted to regulatory purposes. The general physiological scenarios (input information, regulatory output) of many regulatory events caused by PTS proteins are conserved in a broad range of bacteria. However, their actual molecular mechanisms may differ substantially.

Loss of protein kinase-catalyzed phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, by mutation of the ptsH gene confers catabolite repression resistance to several catabolic genes of Bacillus subtilis

Article

Full-text available

Jun 1994

In gram-positive bacteria, HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), is phosphorylated by an ATP-dependent, metabolite-activated protein kinase on seryl residue 46. In a Bacillus subtilis mutant strain in which Ser-46 of HPr was replaced with a nonphosphorylatable alanyl residue (ptsH1 mutation), synthesis of gluconate kinase, glucitol dehydrogenase, mannitol-1-P dehydrogenase and the mannitol-specific PTS permease was completely relieved from repression by glucose, fructose, or mannitol, whereas synthesis of inositol dehydrogenase was partially relieved from catabolite repression and synthesis of alpha-glucosidase and glycerol kinase was still subject to catabolite repression. When the S46A mutation in HPr was reverted to give S46 wild-type HPr, expression of gluconate kinase and glucitol dehydrogenase regained full sensitivity to repression by PTS sugars. These results suggest that phosphorylation of HPr at Ser-46 is directly or indirectly involve

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: Molecular cloning and nucleotide sequence of the Staphylococcus carnosus ptsI gene and expression and complementation studies of the gene product

Article

Full-text available

May 1992

A digoxigenin-labeled DNA probe that was complementary to the gene ptsH and the beginning of the gene ptsI was used to clone a 3.2-kb HincII-BamHI restriction fragment containing the complete ptsI gene of Staphylococcus carnosus. The restriction fragment was cloned in the antisense orientation to the lac promoter in the low-copy-number vector pSU18. The nucleotide sequences of the ptsI gene, which encodes enzyme I (EC 2.7.3.9), and the corresponding flanking regions were determined. The primary translation product, derived from the nucleotide sequence, consists of 574 amino acids and has a calculated molecular weight of 63,369. Amino acid sequence comparison showed 47% similarity to enzyme I of Escherichia coli and 37% similarity to the enzyme I domain of the multiphosphoryl transfer protein of Rhodobacter capsulatus. The histidinyl residue at position 191 could be identified as the probable phosphoenolpyruvate-dependent phosphorylation site of enzyme I of S. carnosus because of sequence homologies with the peptide sequences of enzyme I-active sites of Enterococcus faecalis and Lactococcus lactis. Several in vivo and in vitro complementation studies with the enzyme I ptsI genes of S. carnosus and the E. coli ptsI mutant JLT2 were carried out. The generation times and interaction between enzyme I with histidine-containing protein from gram-positive and gram-negative bacteria were measured in a phosphoryl group transfer test.

Induction of levansucrase in Bacillus subtilis: An antitermination mechanism negatively controlled by the phosphotransferase system

Article

Full-text available

Mar 1990

The target of the induction by sucrose of the levansucrase gene is a transcription terminator (sacRt) located upstream from the coding sequence, sacB. The two-gene locus sacX-sacY (formerly sacS) and the ptsI gene were previously shown to be involved in this induction. ptsI encodes enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. SacX is strongly homologous to sucrose-specific phosphotransferase system-dependent permeases. SacY is a positive regulator of sacB. Here we show that SacY is probably an antiterminator interacting directly with sacRt, since in Escherichia coli the presence of the sacY gene stimulates the expression of a reporter gene fused downstream from sacRt. Missense mutations affecting sacY were sequenced, and the sacB regulation was studied in isogenic strains carrying these mutations or in vitro-generated mutations affecting sacX, sacY, or ptsI. The phenotype of double mutants suggests a model in which SacX might be a sucrose sensor that would be phosphorylated by the phosphotransferase system and, in this state, could inhibit the SacY antiterminator. Exogenous sucrose, or a mutation inactivating the phosphotransferase system, would dephosphorylate SacX and allow antitermination at sacRt.

Site-directed mutagenesis with the ptsH gene of Bacillus subtilis. Isolation and characterization of heat-stable proteins altered at the ATP-dependent regulatory phosphorylation site

Article

Full-text available

Dec 1988

The codon for Ser-46 of the ptsH gene of Bacillus subtilis was modified by site-directed mutagenesis to the codons for Ala, Thr, Tyr, and Asp. The mutant genes were overexpressed, three of the corresponding proteins were purified to homogeneity with the exception for the Asp derivative, which could not be detected, although the gene had the desired nucleotide sequence. The phosphotransferase activity of the altered proteins was determined to be 20-35% of wild type activity, which correlates well with the slow phosphorylation of heat-stable protein (HPr) by enzyme I and phosphoenolpyruvate. The ATP-dependent HPr kinase, which previously was shown to be involved in the regulation of carbohydrate uptake of Gram-positive bacteria by covalent phosphorylation of Ser-46 of HPr, is entirely inactive toward the OH group of Thr-46 and Tyr-46 proteins. In addition, we constructed a strain of B. subtilis, where the altered gene coding for the Ala-46 derivative of HPr was introduced into the bacterial chromosome. The physiological properties of this mutant are described.

Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection

Article

Full-text available

Feb 1987
METHOD ENZYMOL

The method takes advantage of a strong biological selection against the original DNA template which is preferentially destroyed on transfection. The use of this spatial template can be combined with many of the previously described in vitro mutagenesis methods. What we describe here is not, therefore, a new procedure for site-directed mutagenesis but is, rather, the use of standard and well-established procedures in conjunction with an unusual template. This combination permits flexibility in the choice of mutagenesis techniques and makes possible the highly efficient recovery of mutants.

The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon

Article

Full-text available

Jan 1996

The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.

Two different mechanisms mediate catabolite repression of the Bacillus subtilis levanase operon

Article

Full-text available

Jan 1996

There are two levels of control of the expression of the levanase operon in Bacillus subtilis: induction by fructose, which involves a positive regulator, LevR, and the fructose phosphotransferase system encoded by this operon (lev-PTS), and a global regulation, catabolite repression. The LevR activator interacts with its target, the upstream activating sequence (UAS), to stimulate the transcription of the E sigma L complex bound at the "-12, -24" promoter. Levanase operon expression in the presence of glucose was tested in strains carrying a ccpA gene disruption or a ptsH1 mutation in which Ser-46 of HPr is replaced by Ala. In a levR+ inducible genetic background, the expression of the levanase operon was partially resistant to catabolite repression in both mutants, indicating that the CcpA repressor and the HPr-SerP protein are involved in the glucose control of this operon. In addition, a cis-acting catabolite-responsive element (CRE) of the levanase operon was identified and investigated by site-directed mutagenesis. The CRE sequence TGAAAACGCTT(a)ACA is located between positions -50 and -36 from the transcriptional start site, between the UAS and the -12, -24 promoter. However, in a background constitutive for levanase, neither HPr, CcpA, nor CRE is involved in glucose repression, suggesting the existence of a different pathway of glucose regulation. Using truncated LevR proteins, we showed that this CcpA-independent pathway required the presence of the domain of LevR (amino acids 411 to 689) homologous to the BglG family of bacterial antiterminators.

Purification and Characterization of Recombinant Human p50csk Protein Tyrosine Kinase from an Escherichia coli Expression System Overproducing the Bacterial Chaperones GroES and GroEL

Article

Full-text available

Mar 1995

An Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL was engineered and has been successfully used to produce large quantities of the recombinant human protein-tyrosine kinase p50csk. The co-overproduction of the two chaperones with p50csk results in increased solubility of the kinase and allows purification of milligram amounts of active enzyme. Analysis of the purified protein by SDS/polyacrylamide gel electrophoresis reveals a single band with an apparent molecular mass of 50 kDa, indicating that recombinant human p50csk has been purified to near homogeneity. The purified enzyme displays tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties, including in vitro substrate specificity and enzymatic characteristics of the enzyme, have been assessed and compared with those of members of the Src family of protein-tyrosine kinases. Results indicate that p50csk and p56lck have different substrate specificities and that p50csk and p60c-src have similar kinetic parameters. The successful production and purification of an enzymatically active form of p50csk will enable further characterization of this important kinase and allow clarification of its physiological role. In addition, the results suggest that the approach described may be generally applicable to improve the solubility of recombinant proteins which otherwise are produced in an insoluble form in E. coli.

New β-glucoside (bgl) genes in Bacillus subtilis: The bglP gene product has both transport and regulatory functions similar to those of BglF, its Escherichia coli homolog

Article

Full-text available

Apr 1995

The Bacillus subtilis sacY and sacT genes encode antiterminator proteins, similar to the Escherichia coli bglG gene product and required for transcription of sucrose metabolism genes. A Tn10 insertion into bglP (formerly sytA) has been previously identified as restoring sucrose utilization to a strain with deletions of both sacY and sacT. The nucleotide sequence of bglP showed a high degree of homology with the E. coli bglF gene (BglF is a beta-glucoside permease of the phosphotransferase system and also acts as a negative regulator of the BglG antiterminator). Complementation studies of an E. coli strain with a deletion of the bgl operon showed that BglP was a functional beta-glucoside permease. In B. subtilis, bglP complemented in trans both the bglP::Tn10 original insertion and a phenotypically similar bglP deletion. Disruption of licT abolished the suppressor phenotype in a bglP mutant. LicT is a recently identified third B. subtilis antiterminator of the BglG/SacY family. These observations indicated that BglP was also a negative regulator of LicT. Both LicT and BglP seem to be involved in the induction by beta-glucosides of an operon containing at least two genes, bglP itself and bglH, encoding a phospho-beta-glucosidase. Other beta-glucoside genes homologous to bglP and bglH have been recently described in B. subtilis. Thus, B. subtilis possesses several sets of beta-glucoside genes, like E. coli, but these genes do not appear to be cryptic.

Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli

Article

Full-text available

Nov 1993

Cellulolytic strains of Bacillus stearothermophilus were isolated from nature and screened for the presence of activities associated with the degradation of plant cell walls. One isolate (strain XL-65-6) which exhibited strong activities with 4-methylumbelliferyl-beta-D-glucopyranoside (MUG) and 4-methylumbelliferyl-beta-D-cellobiopyranoside (MUC) was used to construct a gene library in Escherichia coli. Clones degrading these model substrates were found to encode the cellobiose-specific genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Both MUG and MUC activities were present together, and both activities were lost concurrently during subcloning experiments. A functional E. coli ptsI gene was required for MUC and MUG activities (presumably a ptsH gene also). The DNA fragment from B. stearothermophilus contained four open reading frames which appear to form a cel operon. Intergenic stop codons for celA, celB, and celC overlapped the ribosomal binding sites of the respective downstream genes. Frameshift mutations or deletions in celA, celB, and celD were individually shown to result in a loss of MUC and MUG activities. On the basis of amino acid sequence homology and hydropathy plots of translated sequences, celA and celB were identified as encoding PTS enzyme II and celD was identified as encoding PTS enzyme III. These translated sequences were remarkably similar to their respective E. coli homologs for cellobiose transport. No reported sequences exhibited a high level of homology with the celC gene product. The predicted carboxy-terminal region for celC was similar to the corresponding region of E. coli celF, a phospho-beta-glucosidase. An incomplete regulatory gene (celR) and proposed promoter sequence were located 5' to the proposed cel operon. A stem-loop resembling a rho-independent terminator was present immediately downstream from celD. These results indicate that B. stearothermophilus XL-65-6 contains a cellobiose-specific PTS for cellobiose uptake. Similar systems may be present in other gram-positive bacteria.

LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family

Article

Full-text available

May 1996

Gene licS of Bacillus subtilis encodes an excreted Beta-1,3-1,4-endoglucanase necessary for lichenan utilization. Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family. Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family. The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t. The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two- to threefold. This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t. However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in beta-glucan utilization.

Transcriptional analysis of bglPH expression in Bacillus subtilis: Evidence for two distinct pathways mediating carbon catabolite repression

Article

Full-text available

Jun 1996

In Bacillus subtilis, aryl-beta-glucosides such as salicin and arbutin are catabolized by the gene products of bglP and bglH, encoding an enzyme II of the phosphoenolpyruvate sugar-phosphotransferase system and a phospho-beta-glucosidase, respectively. These two genes are transcribed from a single promoter. The presence of a transcript of about 4,000 nucleotides detected by Northern (RNA) blot analysis indicates that bglP and bglH are part of an operon. However, this transcript is only present when cells are grown in the presence of the inducing substrate, salicin. In the absence of the inducer, a transcript of about 110 nucleotides can be detected, suggesting that transcription terminates downstream of the promoter at a stable termination structure. Initiation of transcription is abolished in the presence of rapidly metabolized carbon sources. Catabolite repression of bglPH expression involves the trans-acting factors CcpA and HPr. In a ccpA mutant, transcription initiation is relieved from glucose repression. Furthermore, we report a catabolite responsive element-CcpA-independent form of catabolite repression requiring the ribonucleic antiterminator-terminator region, which is the target of antitermination, and the wild-type HPr protein of the phosphotransferase system. Evidence that the antitermination protein LicT is a crucial element for this type of regulation is provided.

In Vitro Reconstitution of Transcriptional Antitermination by the SacT and SacY Proteins of Bacillus subtilis

Article

Full-text available

Sep 1996

Expression of the sacPA and sacB genes of Bacillus subtilis is positively modulated by transcriptional regulatory proteins encoded by the sacT and sacY genes, respectively. Previous genetic studies led to the suggestion that SacT and SacY function as nascent mRNA binding proteins preventing early termination of transcription at terminators located in the leader regions of the corresponding genes. Here we report the overproduction, purification to near homogeneity, and characterization of the two antiterminators, SacT and SacY. Using mRNA band migration retardation assays and a reconstituted transcriptional antitermination system, the mRNA binding functions and antitermination activities of purified SacT and SacY are demonstrated under in vitro conditions. The results establish for the first time that members of the BglG family of antiterminators function in antitermination in the absence of other proteins in vitro. Purified SacT is shown to be phosphorylated by phosphoenolpyruvate in a phosphotransferase-catalyzed reaction dependent on Enzyme I and HPr. Unexpectedly, the purified SacT is shown to be functional in mRNA binding and in transcriptional antitermination independently of its phosphorylation state.

The levanase operon of Bacillus subtilis expressed in Eschenchia coli can substitute for the mannose permease in mannose uptake and bacteriophage lambda infection

Article

Full-text available

Jan 1997

Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, a maltose- and maltodextrin-specific porin of the outer membrane. LamB also serves as a receptor for several other bacteriophages. Lambda DNA requires, in addition to LamB, the presence of two bacterial cytoplasmic integral membrane proteins for penetration, namely, the IIC(Man) and IID(Man) proteins of the E. coli mannose transporter, a member of the sugar-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS transporters for mannose of E. coli, for fructose of Bacillus subtilis, and for sorbose of Klebsiella pneumoniae were shown to be highly similar to each other but significantly different from other PTS transporters. These three enzyme II complexes are the only ones to possess distinct IIC and IID transmembrane proteins. In the present work, we show that the fructose-specific permease encoded by the levanase operon of B. subtilis is inducible by mannose and allows mannose uptake in B. subtilis as well as in E. coli. Moreover, we show that the B. subtilis permease can substitute for the E. coli mannose permease cytoplasmic membrane components for phage lambda infection. In contrast, a series of other bacteriophages, also using the LamB protein as a cell surface receptor, do not require the mannose transporter for infection.

Multiple Phosphorylation of SacY, a Bacillus subtilisTranscriptional Antiterminator Negatively Controlled by the Phosphotransferase System

Article

Full-text available

Aug 1997

The Bacillus subtilis SacY transcriptional antiterminator is a regulator involved in sucrose-promoted induction of the sacB gene. SacY activity is negatively controlled by enzyme I and HPr, the general energy coupling proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and by SacX, a membranal protein homologous to SacP, theB. subtilis sucrose-specific PTS-permease. Previous studies suggested that the negative control exerted by the PTS on bacterial antiterminators of the SacY family involves phosphoenolpyruvate-dependent phosphorylation by the sugar-specific PTS-permeases. However, data reported herein show direct phosphorylation of SacY by HPr(His∼P) with no requirement for SacX. Experiments were carried out to determine the phosphorylatable residues in SacY. In silico analyses of SacY and its homologues revealed the modular structure of these proteins as well as four conserved histidines within two homologous domains (here designated P1 and P2), present in 14 distinct mRNA- and DNA-binding bacterial transcriptional regulators. Single or multiple substitutions of these histidyl residues were introduced in SacY by site-directed mutagenesis, and their effects on phosphorylation and antitermination activity were examined. In vitro phosphorylation experiments showed that SacY was phosphorylated on three of the conserved histidines. Nevertheless, in vivo studies using cells bearing asacB′-lacZ reporter fusion, as well as SacY mutants lacking the phosphorylatable histidyls, revealed that only His-99 is directly involved in regulation of SacY antitermination activity.

Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system

Article

Mar 1989
Microbiol Rev

Milton H. Saier

The bacterial phosphotransferase system (PTS) functions in a variety of regulatory capacities. One of the best characterized of these is the process by which the PTS regulates inducer uptake and catabolite repression. Early genetic and physiological evidence supported a mechanism whereby the phosphorylation state of an enzyme of the PTS, the enzyme III specific for glucose (IIIGlc), allosterically inhibits the activities of a number of permeases and catabolic enzymes, the lactose, galactose, melibiose, and maltose permeases, as well as glycerol kinase. Extensive biochemical evidence now supports this model. Evidence is also available showing that substrate binding to those target proteins enhances their affinities for IIIGlc. In the case of the lactose permease, this positively cooperative interaction represents a well documented example of transmembrane signaling, demonstrated both in vivo and in vitro. Although the PTS-mediated regulation of cyclic AMP synthesis (catabolite repression) is not as well defined from a mechanistic standpoint, a model involving allosteric activation of adenylate cyclase by phospho-IIIGlc, together with the evidence supporting it, is presented. These regulatory mechanisms may prove to be operative in gram-positive as well as gram-negative bacteria, but the former organisms may have introduced variations on the theme by covalently attaching IIIGlc-like moieties to some of the target permeases and catabolic enzymes. It appears likely that the general process of PTS-catalyzed protein phosphorylation-dephosphorylation will prove to be important to the regulation of numerous bacterial physiological processes, including chemotaxis, intermediary metabolism, gene transcription, and virulence.

Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria.

Article

Jan 1985
Microbiol Rev

Cloning: A Laboratory Manual

Article

Jan 1982

Molecular cloning: A laboratory manual, second ed

Article

Jan 1989

Miller JH.. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY

Article

Jan 1972

J. H. Miller

Experiments In Molecular Genetics

Article

Jan 1972

Jeffrey H. Miller

Molecular Cloning: A Laboratory Manual

Chapter

Jan 1983

Cooperative and non-cooperative DNA binding modes of catabolite control protein CcpA from Bacillus megaterium result from sensing two different signals. J. Mol. Biol. 266: 665-676

Article

Jan 1997

DNA Sequencing With Chain Terminating Inhibitors

Article

Jan 1978

A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.

New vectors for high level expression of recombinant protein in bacteria

Article

Jun 1992

A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG (K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262-267) which has a glycine-rich "kinker" immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage efficiency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.

Regulation of the sacPA operon of Bacillus subtilis: Identification of phosphotransferase system components involved in SacT activity

Article

Jun 1992

The sacT gene which controls the sacPA operon of Bacillus subtilis encodes a polypeptide homologous to the B. subtilis SacY and the Escherichia coli BglG antiterminators. Expression of the sacT gene is shown to be constitutive. The DNA sequence upstream from sacP contains a palindromic sequence which functions as a transcriptional terminator. We have previously proposed that SacT acts as a transcriptional antiterminator, allowing transcription of the sacPA operon. In strains containing mutations inactivating ptsH or ptsI, the expression of sacPA and sacB is constitutive. In this work, we show that this constitutivity is due to a fully active SacY antiterminator. In the wild-type sacT+ strain or in the sacT30 mutant, SacT requires both enzyme I and HPr of the phosphotransferase system (PTS) for antitermination. It appears that the PTS exerts different effects on the sacB gene and the sacPA operon. The general proteins of the PTS are not required for the activity of SacY while they are necessary for SacT activity.

Mutagenesis of the Bacillus subtilis “−12, −24” promoter of the levanase operon and evidence for the existence of an upstream activating sequence

Article

Aug 1992

The levanase operon of Bacillus subtilis is controlled by RNA polymerase associated with sigma 54 factor and by the LevR activator that is homologous to the NifA/NtrC family of regulators. A "-12, -24" promoter is present at the appropriate distance from the transcription start site. The drastic down effect of base substitutions in the TGGCAC, TTGCA consensus sequence on the expression of the levanase operon confirmed the involvement of the "-12, -24" region in promoter function. Deletion derivatives of the upstream sequence of the operon promoter were constructed using translational levD'-'lacZ fusions and were integrated as single copies at the amyE locus of the B. subtilis chromosome. A cis-acting DNA sequence that is required for activation of the operon promoter by LevR was identified. This regulatory sequence is about 50 base-pairs long and is centered 125 base-pairs upstream from the transcription start site in a region containing a 16 base-pair palindromic structure. This region of dyad symmetry functions as a regulatory element when placed up to at least 600 base-pairs upstream from the "-12, -24" promoter, although the efficacy of activation is lowered. Thus, in common with most sigma 54-dependent promoters, an upstream activating sequence (UAS) is involved in the control of expression of the levanase operon. The isolation and characterization of eight mutations in the UAS region confirmed the importance of the palindromic structure in promoter activation. Moreover, the expression of the levanase operon was inhibited by placing the UAS in trans on a multicopy plasmid, probably through titration of the LevR polypeptide. In conclusion, the levanase promoter region can be divided into two regulatory sequences: the "-12, -24" promoter recognized by the sigma 54 RNA polymerase holoenzyme and the UAS, an inverted repeat sequence that is probably the LevR binding site.

The transcriptional regulator LevR of Bacillus subtilis has domains homologous to both σ54- and phosphotransferase system-dependent regulators

Article

Apr 1991

The regulatory gene levR of the levanase operon of Bacillus subtilis was cloned and sequenced. It encodes a polypeptide of Mr 106,064 with two domains homologous to members of two families of bacterial activators. One domain in LevR is homologous with one region of bacterial regulators including SacT and SacY of B. subtilis and BglG from Escherichia coli. Another domain of LevR is homologous to one part of the central domain of NifA and NtrC, which control nitrogen assimilation in Gram-negative bacteria. The levanase promoter contains two regions almost identical to the -12, -24 consensus regions present in sigma 54-dependent promoters. The expression of the levanase operon in E. coli was strongly dependent on sigma 54. Taken together, these results suggest that the operon is expressed from a -12, -24 promoter regulated by a sigma 54-like-dependent system in B. subtilis.

Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon

Article

Sep 1990

The levanase gene (sacC) of Bacillus subtilis is the distal gene of a fructose-inducible operon containing five genes. The complete nucleotide sequence of this operon was determined. The first four genes levD, levE, levF and levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of Escherichia coli. The levD and levE gene products are homologous to the N and C-terminal part of the enzyme IIIMan, respectively, whereas the levF and levG gene products have similarities with the enzymes IIMan. Surprisingly, the polypeptides encoded by the levD, levE, levF and levG genes are not involved in mannose uptake, but form a fructose phosphotransferase system in B. subtilis. This transport is dependent on the enzyme I of the phosphotransferase system (PTS) and is abolished by deletion of levF or levG and by mutations in either levD or levE. Four regulatory mutations (sacL) leading to constitutive expression of the lavanase operon were mapped using recombination experiments. Three of them were characterized at the molecular level and were located within levD and levE. The levD and levE gene products that form part of a fructose uptake PTS act as negative regulators of the operon. These two gene products may be involved in a PTS-mediated phosphorylation of a regulator, as in the bgl operon of E. coli.

The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators

Article

Aug 1990

The expression of the Bacillus subtilis sacPA operon is induced by sucrose. A DNA fragment containing the upstream region of this operon was cloned. This fragment contains a promoter from which the operon is expressed. This upstream region also contains a palindromic DNA sequence very similar to the transcriptional terminator which regulates the induction of the B. subtilis sacB gene. Of 37 nucleotides in a region partially overlapping the sacP palindromic sequence, 34 were identical to the corresponding region of the sacB gene. A similar motif is also present in the bgl operon of Escherichia coli. The sacT locus controlling sacPA expression had been identified by a single constitutive mutation sacT30 which mapped close to the sacPA operon. DNA fragments containing the sacT+ and sacT30 alleles were cloned and sequenced. The sacT gene product is very similar to the B. subtilis sacY and to the E. coli bglG gene products. The constitutive sacT30 mutation was identified. It corresponds to a Asp-96-to-Tyr missense mutation located in a highly conserved region in SacT and SacY. These results strongly suggest that sacT is a specific regulatory gene of the sacPA operon.

??-Glucoside Permease Represses the bgl Operon of Escherichia Coli by Phosphorylation of the Antiterminator Protein and also Interacts with Glucose-Specific Enzyme III, the Key Element in Catabolite Control

Article

Aug 1990

The beta-glucoside (bgl) operon of Escherichia coli is subject to both positive control by transcriptional termination/antitermination and negative control by the beta-glucoside-specific transport protein, an integral membrane protein known as enzyme IIBgl. Previous results led us to speculate that enzyme IIBgl exerts its negative control by phosphorylating and thereby inactivating the antiterminator protein, BglG. Specifically, our model postulated that the transport protein enzyme IIBgl exhibits protein-phosphotransferase activity in the absence of beta-glucosides. We now present biochemical evidence that the phosphorylation of protein BglG does indeed occur in vivo and that it is accompanied by the loss of antitermination activity. BglG persists in the phosphorylated state in the absence of beta-glucosides but is rapidly dephosphorylated when beta-glucosides become available for transport. Our data also suggested specific interactions between the beta-glucoside transport protein and the glucose-specific enzyme III (enzyme IIIGlc), a component of glucose transport and a key element in regulation of catabolite repression. These observations indicate that enzyme IIIGlc may, in conjunction with enzyme IIBgl, modulate the transport of beta-glucosides and the phosphorylation of the antiterminator protein. In the absence of both sugars, when the catabolite-controlled promoter of the operon is derepressed, enzyme IIIGlc may mediate tight repression of antitermination.

Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: Sugar phosphotransferase system

Article

Apr 1989
Microbiol Rev

Milton H. Saier

Protein phosphorylation regulates transcription of the β-glucoside utilization operon in E. coli

Article

Oct 1989
CELL

We have investigated the interaction between BglF and BglG, two proteins that regulate expression of the E. coli bgl operon. BglF is both a negative regulator of operon expression and a phosphotransferase involved in uptake of beta-glucosides. BglG is a positive regulator that functions as a transcriptional antiterminator. We show here that BglF is phosphorylated by the soluble components of the phosphotransferase system: Enzyme I, HPr, and the phosphate donor phosphoenolpyruvate. Phosphorylated BglF can then transfer phosphate either to beta-glucosides or to wild-type BglG. Mutant BglG derivatives, which give constitutive expression of the bgl operon, show little or no phosphorylation by BglF. Hence BglF exerts its negative effect on operon expression by phosphorylating BglG, blocking its action as an antiterminator. BglG is dephosphorylated only in the presence of both BglF and beta-glucosides. Based on these results, we propose the following mechanism: In the absence of beta-glucosides, BglG is phosphorylated by BglF and is inactive in antitermination. Addition of inducer stimulates BglF to dephosphorylate BglG, allowing BglG to function as a positive regulator of operon expression. Beta-Glucosides are then phosphorylated and transported into the cell by BglF.

Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase

Article

Jul 1987

The structural gene for the enzyme levanase of Bacillus subtilis (SacC) was cloned in Escherichia coli. The cloned gene was mapped by PBS1 transduction near the sacL locus on the B. subtilis chromosome, between leuA and aroD. Expression of the enzyme was demonstrated both in B. subtilis and in E. coli. The presence of sacC allowed E. coli to grow on sucrose as the sole carbon source. The complete nucleotide sequence of sacC was determined. It includes an open reading frame of 2,031 bp, coding for a protein with calculated molecular weight of 75,866 Da, including a putative signal peptide similar to precursors of secreted proteins found in Bacilli. The apparent molecular weight of purified levanase is 73 kDa. The sacC gene product was characterized in an in vitro system and in a minicell-producing strain of E. coli, confirming the existence of a precursor form of levanase of about 75 kDa. Comparison of the predicted aminoacid sequence of levanase with those of the two other known beta-D-fructofuranosidases of B. subtilis indicated a homology with sucrase, but not with levansucrase. A stronger homology was detected with the N-terminal region of yeast invertase, suggesting the existence of a common ancestor.

A bacterial gene involved in transcription antitermination: regulation at a Rho-independent terminator in the bgl operon of E. coli

Article

Aug 1987
CELL

We have investigated the mechanism of regulation of the bgl operon in Escherichia coli K-12. A regulatory region has been located downstream of the bgl promoter, revealing a 130 base leader containing a sequence from +64 to +112 characteristic of a rho-independent terminator. In vitro, over 90% of the transcripts initiated at the bgl promoter terminate within the leader, at the 3' end of the terminator. Transcriptional fusions containing the terminator require the bglC gene in trans for expression; fusions deleted for the sequence show bglC-independent expression. A mutation resulting in partially constitutive expression of the fusion maps within the terminator. We propose that the bglC gene product mediates positive regulation of the bgl operon by functioning as an antiterminator at the rho-independent terminator located within the leader.

Phosphoenolpyruvate:carbohydrate Phosphotransferase System of Bacteria

Article

Oct 1985
Microbiol Rev

Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the regulation of gene expression. By sensing the presence of PTS carbohydrates in the medium and adjusting the phosphorylation state of IIAGlc, cells can adapt quickly to changing conditions in the environment. In gram-positive bacteria, it has been demonstrated that HPr can be phosphorylated by ATP on a serine residue and this modification may perform a regulatory function.

An enzymatic method for [32P]phosphoenolpyruvate synthesis

Article

Feb 1983

A convenient method for the enzymatic synthesis of [32P]phosphoenolpyruvate from [γ-32P]ATP using partially pufified phosphoenolpyruvate carboxykinase from Escherichia coli is described. The synthesis was shown to convert essentially all the [γ-32P]ATP to [32P]phosphoenolpyruvate, which was subsequently separated from residual [γ-32P]ATP and by chromatography on AG-1-X8-bicarbonate resin.

Isoelectrophoretic separation and the detection of soluble proteins containing acid-labile phosphate: Use of the phosphoenolpyruvate: Sugar phosphotransferase system as a model system for N1-P-histidine- and N3-P-histidine-containing proteins

Article

Jun 1984

Procedures have been developed for the detection of acid-labile phosphorylations of proteins. The phosphoproteins were separated by native isoelectric focusing while maintaining the gel at about 0 degree C, and denaturing urea-Nonidet isoelectric focusing gels were adapted to run at -10 degrees C. The proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), HPr, which contains 1-P-histidine, and factor IIIglc and enzyme I, which contain 3-P-histidine when they are phosphorylated, were used to develop the conditions. Autoradiography of [32P]-labeled phosphoproteins was carried out on frozen gels which had not been acid fixed in order to avoid hydrolysis of the phosphohistidines . The frozen gels were subsequently fixed and stained, and reautoradiography revealed whether the phosphoproteins were acid stable or labile. In addition to the known proteins of the PTS, at least one other protein whose phosphorylation was dependent on enzyme I and HPr was found in Salmonella typhimurium and Escherichia coli [E.B. Waygood , and R.L. Mattoo (1983) Canad . J. Biochem. Cell Biol. 61, 150-153]. Initial experiments with rat tissues have demonstrated acid-labile phosphorylations in proteins which were either [gamma-32P]ATP or [32P]phosphoenolpyruvate dependent. The interconversion of phosphoenolpyruvate and ATP in crude extracts of bacterial cells was examined, and appropriate controls were found. Protein phosphorylation dependent upon phosphoenolpyruvate was much greater in S. typhimurium and E. coli than the corresponding ATP-dependent phosphorylation, while the opposite was found for rat tissues.

A Simple Procedure for the Synthesis of [32P]Phosphoenol Pyruvate via the Pyruvate Kinase Exchange Reaction at Equilibrium

Article

Nov 1983
Biochim Biophys Acta

A one step procedure is presented for the preparation of [32P]phosphoenolpyruvate from [gamma-32P]ATP using pyruvate kinase. The reaction is carried out at chemical equilibrium and involves only an exchange of isotope between ATP and phosphoenolpyruvate. The initial phosphoenolpyruvate/ATP ratio in the reaction mixture determines the degree of 32P incorporation into phosphoenolpyruvate when isotopic equilibrium is achieved.

Catabolite repression in Bacillus subtilis: A global regulatory mechanism for the Gram-positive bacteria?

Article

Mar 1995

Three components involved in catabolite repression (CR) of gene expression in Bacillus have been identified. The cis-acting catabolite responsive element (CRE), which is present in many genes encoding carbon catabolic enzymes in various species of the Gram-positive bacteria, mediates CR of several genes in Bacillus subtilis, Bacillus megaterium, and Staphylococcus xylosus. CR of most genes regulated via CRE is also affected by the trans-acting factors CcpA and HPr. Similarities between CcpA and Lac and Gal repressors suggest binding of CcpA to CRE. HPr, a component of the phosphoenolpyruvate:sugar phosphotransferase system, undergoes regulatory phosphorylation at a serine residue by a fructose-1,6-diphosphate-activated kinase. A mutant of HPr, which is not phosphorylatable at this position because of an exchange of serine to alanine, lacks CR of several catabolic activities. This mutant phenotype is similar to the one exhibited by a ccpA mutant. Direct protein-protein interaction between CcpA and HPr(Ser-P) was recently demonstrated and constitutes a link between metabolic activity and CR.

Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to catabolite repression in Gram-positive bacteria

Article

Apr 1995

CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram-positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA-his immobilized on a Ni-NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phospho-carrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His-15 in a PEP-dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-dependent reaction catalysed by a metabolite-activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl-phosphorylated HPr required the presence of fructose 1,6-bisphosphate. These findings suggest that carbon catabolite repression in Gram-positive bacteria is a protein kinase-triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P-ser-HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carbon catabolite repression and PTS transport activity.

Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis

Article

Jun 1995

Growth under conditions of salt stress has important effects on the synthesis of degradative enzymes in Bacillus subtilis. Salt stress strongly stimulates the expression of sacB, encoding levansucrase (about ninefold), and downregulates the expression of aprE, encoding alkaline protease (about sixfold). It is suggested that the DegS-DegU two-component system is involved in sensing salt stress. Moreover, it has been shown that the level of sacB expression strongly depends on the growth conditions; its expression level is about eightfold higher in cells grown on agar plates than in cells grown in liquid medium.

Interactions of Wild-type and Truncated LevR of Bacillus subtilis with the Upstream Activating Sequence of the Levanase Operon

Article

Sep 1994

Transcription of the levanase operon of Bacillus subtilis is controlled by LevR, an activator of the NifA/NtrC family of regulators. An upstream activating sequence (UAS) located in a 16 bp palindromic structure has previously been characterized. LevR was overproduced in B. subtilis and interaction between the activator and the UAS was demonstrated by gel shift and footprint experiments. The LevR protein specifically binds to the two-halves of the palindromic structure centered at -125 bases upstream from the transcriptional start site. In addition, footprint analysis suggests that LevR interacts with a third DNA region located at positions -90 to -80. To investigate the function of the different domains of the LevR activator, stop codons were introduced at various positions in the levR gene. The ability of the truncated LevR polypeptides to activate transcription, to respond to the inducer or to interact with the UAS was tested. The results obtained suggest that LevR is a multidomain protein. The amino-terminal part of the protein is required for DNA binding whereas the central domain allows the activation of transcription. The carboxy-terminal region is involved in the modulation of the LevR activity by the inducer.

Phosphoenolpyruvate: Carbohydrate phosphotransferase systems of bacteria

Article

Oct 1993
Microbiol Rev

The role of phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, in the regulation of carbon metabolism in gram-positive bacteria

Article

Jan 1993

HPr of the Gram-positive bacterial phosphotransferase system (PTS) can be phosphorylated by an ATP-dependent protein kinase on a serine residue or by PEP-dependent Enzyme 1 on a histidyl residue. Both phosphorylation events appear to influence the metabolism of non-PTS carbon sources. Catabolite repression of the gluconate (gnt) operon of B. subtilis appears to be regulated by the former phosphorylation event, while glycerol kinase appears to be regulated by the latter phosphorylation reaction. The extent of our understanding of these processes will be described.

Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a complex formed between CcpA and seryl-phosphorylated HPr

Article

Oct 1995

Catabolite repression of various Bacillus subtilis catabolic operons which carry a cis-acting catabolite-responsive element (CRE), such as the gnt operon, is mediated by CcpA, a protein belonging to the GalR-Lacl family of bacterial transcriptional repressors/activators, and the seryl-phosphorylated form of HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. Footprinting experiments revealed that the purified CcpA protein interacted with P-ser-HPr to cause specific protection of the gnt CRE against DNase I digestion. The specific recognition of the gnt CRE was confirmed by the results of footprinting experiments using mutant gnt CREs carrying one of the following base substitutions within the CRE consensus sequence: G to T at position +149 or C to T at position +154 (+1 is the gnt transcription initiation nucleotide). The two mutant CREs causing a partial relief from catabolite repression were not protected by the CcpA/P-ser-HPr complex in footprinting experiments. Based on these and previous findings, we propose a molecular mechanism underlying catabolite repression in B. subtilis mediated by CcpA and P-ser-HPr.

The Fructose Transporter of Bacillus subtilis Encoded by the Lev Operon Backbone Assignment and Secondary Structure of the IIBLev Subunit

Article

Jan 1997

The fructose transporter of the Bacillus subtilis phosphotransferase system consists of two membrane associated (IIA and IIB) and two transmembrane (IIC and IID) subunits [Martin-Verstraete, I., Débarbouillé, M., Klier, A. & Rapoport, G. (1990) J. Mol. Biol. 214, 657–671]. It mediates uptake by a mechanism which couples translocation to phosphorylation of the transported solute. The 18-kDa IIBLev subunit transfers phosphoryl groups from His9 of the IIA subunit to the sugar. The three-dimensional structure of IIBLev or similar proteins is not known. IIBLev was overexpressed in Escherichia coli and isotopically labelled with 13C/15N in H2O as well as in 70% D2O. 15N-edited NOESY, 13C-edited NOESY and 13C,15N triple-resonance experiments yielded a nearly complete assignment of the 1H, 13C and 15N resonances. Based on qualitative interpretation of NOE, scalar couplings, chemical shift values and amide exchange data, the secondary structure and topology of IIBLev was determined. IIBLev comprises six parallel β-strands, one antiparallel β-strand and 5 α-helices. The order of the major secondary-structure elements is (βα)5β (strand order 7651423). Assuming that the βαβ-motives form right-handed turn structures, helices αA and αB are packed to one face and helices αC, αD and αE to the opposite face of the parallel βsheet. His15 which is transiently phosphorylated during catalysis is located in the loop β1/αA of the topological switch point. The amino terminal (β/α)4 part of IIBLev has the same topology as phosphoglyceromutase (PGM; PDB entry 3pgm). Both proteins catalyze phosphoryltransfer reactions which proceed through phosphohistidine intermediates and they show a similar distribution of invariant residues in the topologically equivalent positions of their active sites. The protein fold of IIBLev has no similarity to any of the known structures of other phosphoenol pyruvate-dependent-carbohydrate-phosphotransferase-system proteins.

Protein Phosphorylation Chain of a Bacillus subtilis Fructose-Specific Phosphotransferase System and Its Participation in Regulation of the Expression of the lev Operon †

Article

Mar 1997

The proteins encoded by the fructose-inducible lev operon of Bacillus subtilis are components of a phosphotransferase system. They transport fructose by a mechanism which couples sugar uptake and phosphoenolpyruvate-dependent sugar phosphorylation. The complex transport system consists of two integral membrane proteins (LevF and LevG) and two soluble, hydrophilic proteins (LevD and LevE). The two soluble proteins from together with the general proteins of the phosphotransferase system, enzyme I and HPr, a protein phosphorylation chain which serves to phosphorylate fructose transported by LevF and LevG. We have synthesized modified LevD and LevE by fusing a His-tag to the N-terminus of each protein allowing rapid and efficient purification of the proteins. We determined His-9 in LevD and His-15 in LevE as the sites of PEP-dependent phosphorylation by isolating single, labeled peptides derived from 32P-labeled LevD, LevD(His)6, and LevE(His)6. The labeled peptides were subsequently analyzed by amino acid sequencing and mass spectroscopy. Mutations replacing the phosphorylatable histidyl residue in LevD with an alanyl residue and in LevE with a glutamate or aspartate were introduced in the levD and levE genes. These mutations caused strongly reduced fructose uptake via the lev-PTS. The mutant proteins were synthesized with a N-terminal His-tag and purified. Mutant LevD(His)6 was very slowly phosphorylated, whereas mutant LevE(His)6 was not phosphorylated at all. The corresponding levD and levE alleles were incorporated into the chromosome of a B. subtilis strain expressing the lacZ gene under control of the lev promoter. The mutations affecting the site of phosphorylation in either LevD or LevE were found to cause constitutive expression from the lev promoter of B. subtilis.

MicroReview antitermination of transcription of catabolic operons

Article

Mar 1997

Blanka Rutberg

Antitermination of transcription mediated by proteins interacting with mRNA sequences is described for nine operons/regulons. Eight of the systems are catabolic, while the ninth, the Klebsiella pneumoniae nas regulon, is involved in the assimilation of nitrate and nitrite. Six of the catabolic operons/regulons are found in Bacillus subtilis, one is found in Escherichia coli, and one in Pseudomonas aeruginosa. The antitermination system of five of the operons/regulons (E. coli blg, and sacPA, sacB, bgl, and lic from B. subtilis) are assigned to the bgl-sac family on the basis of extensive similarities with regard to antiterminator proteins and the sequences of the antiterminators. Other members of the bgl-sac family are the arb operon of Erwinia chrysanthemi and a presumed bgl operon of Lactococcus lactis. The antitermination systems of the other four operons/regulons (B. subtilis glp, B. subtilis hut, P. aeruginosa ami, and K. pneumoniae nas) seem to be unrelated both to the bgl-sac family and to each other. The antiterminator protein of the B. subtilis glp regulon has been found not only to cause antitermination but also to stabilize the resultant mRNA and to mediate glucose repression. If other antiterminator proteins, and antitermination factors, also prove to have additional functions, it will broaden the impact of antitermination as a means of controlling gene expression.

Cooperative and non-cooperative DNA binding modes of catabolite control protein CcpA from Bacillus megaterium result from sensing two different signals

Article

Apr 1997

Carbon catabolite repression (CCR) of several operons in Bacillus subtilis and Bacillus megaterium is mediated by the cis-acting cre sequence and trans-acting catabolite control protein (CcpA). We describe purification of CcpA from B. megaterium and its interaction with regulatory sequences from the xyl operon. Specific interaction of CcpA with cre as scored by DNase I footprints at concentrations similar to the in vivo situation requires the presence of effectors. We have found two molecular effectors for CcpA activity, which lead to different recognition modes of DNA. The heat-stable phosphotransfer protein HPr from the PTS sugar uptake system triggers non-cooperative binding of CcpA to cre when phosphorylated at Ser46 (HPr-Ser46-P). Glucose 6-phosphate (Glc-6-P) triggers cooperative binding of CcpA to cre and two auxiliary cre* sites, one of which overlaps the -35 box of the xyl promoter. Binding to cre* depends on the presence of the functional cre sequence. A mutation in cre abolishes carbon catabolite repression in vivo and binding of CcpA to cre and cre* in vitro, indicating looping of the intervening DNA. The two triggers are not simultaneously active. The acidity of the buffer determines which of them activates CcpA when both are present in vitro. Glc-6-P is preferred at pH values below 5.4, and HPr-Ser46-P is preferred at neutral pH. The Ccpa dimers present at neutral pH form tetramers and higher oligomers at pH 4.6, explaining cooperativity of binding to DNA. CcpA is the first member of the LacI/GalR family of regulators, for which oligomerization without the leucine zipper at the C terminus is demonstrated.

Antagonistic effects of dual PTS-catalysed phosphorylation on the Bacillus subtilis transcriptional activator LevR

Abstract

No full-text available

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