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The secG deletion mutation of Escherichia coli is suppressed by expression of a novel regulatory gene of Bacillus subtilis
Abstract
SecG, a membrane component of E. coli protein translocase, stimulates the translocation of proteins across the cell membrane through the cycle of topology inversion, which is coupled to the membrane-insertion and deinsertion cycle of SecA [Nishiyama et al. (1996) Cell 85, 71-81]. A gene of B. subtilis able to suppress the cold-sensitive phenotype of the secG deletion mutant of E. coli was cloned and found to encode a novel regulatory protein, ScgR. Similarity search revealed homology with known proteins such as GlnR of B. subtilis. Plasmid-encoded ScgR stimulated protein translocation in the deletion mutant. ScgR increased the proportion of cardiolipin at the expense of phosphatidylglycerol, but did not affect the composition of other lipid components of the cell, suggesting that the increased cardiolipin level compensates for the SecG function and thereby stimulates protein translocation.
Supplementary resource (1)
... This molecule increases the hydrophobicity and viscosity of the cell membrane by changing its composition [57]. These changes had been related to an increase in survival in the high osmotic environment of B. subtilis [58], as well as to nonspecific resistance to antibiotics and antimicrobial peptides, by decreasing the permeability of the cell envelope [59,60]. ...
Bacteria of the Bacillus cereus group are environmental Gram-positive spore-forming bacteria ubiquitously distributed. Despite the high degree of genetic similarity among the different strains, they show strong phenotypic variability, from mammal or entomopathogen strains to soil-dwelling saprophytes, and from psychrophylic to thermotolerant strains. Most of the phenotypes are linked to the presence of large plasmids that encode for diverse toxins. However, other processes, like mutation or recombination, also participate in shaping the evolution and population structure of these bacteria. Here we review different aspects of the evolution of this group.
... Increased CL synthesis would change the composition of the cell membrane, increasing the hydrophobicity and viscosity of this membrane [54], which could have varied phenotypic effects. Increased CL levels stimulate protein translocation across the cell membrane in B. subtilis [55] and E. coli [56], and are important for high osmolarity survival in B. subtilis [57] and S. aureus [58]. High CL concentrations decrease cell envelope permeability, affecting nonspecific antibiotic resistance in E. coli [59], resistance to organic solvents [60], daptomycin resistance in S. aureus [61] and Enterococcus faecalis [62], and resistance to antimicrobial peptides such as enterococcal AS-48 [63] and platelet microbicidal peptide tPMP-1 [64]. ...
Background
The Bacillus cereus sensu lato group currently includes seven species (B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis and B. cytotoxicus) that recent phylogenetic and phylogenomic analyses suggest are likely a single species, despite their varied phenotypes. Although horizontal gene transfer and insertion-deletion events are clearly important for promoting divergence among these genomes, recent studies have demonstrated that a major basis for phenotypic diversity in these organisms may be differential regulation of the highly similar gene content shared by these organisms. To explore this hypothesis, we used an in silico approach to evaluate the relationship of pathogenic potential and the divergence of the SigB-dependent general stress response within the B. cereus sensu lato group, since SigB has been demonstrated to support pathogenesis in Bacillus, Listeria and Staphylococcus species.
Results
During the divergence of these organisms from a common “SigB-less” ancestor, the placement of SigB promoters at varied locations in the B. cereus sensu lato genomes predict alternative structures for the SigB regulon in different organisms. Predicted promoter changes suggesting differential transcriptional control of a common gene pool predominate over evidence of indels or horizontal gene transfer for explaining SigB regulon divergence.
Conclusions
Four lineages of the SigB regulon have arisen that encompass different gene contents and suggest different strategies for supporting pathogenesis. This is consistent with the hypothesis that divergence within the B. cereus sensu lato group rests in part on alternative strategies for regulation of a common gene pool.
... The ⌬secG ::kan mutant accumulates precursor proteins and is unable to grow at low temperatures . The cold-sensitive phenotype of the mutant is suppressed when the level of acidic phospholipid is increased by the overexpression of E. coli pgsA (Kontinen and Tokuda, 1995) or Bacillus subtilis scgR (Kontinen et al., 1996). The mechanism underlying the suppression by an increase in the acidic phospholipid level is not fully understood. ...
SecB maintains the structures of a subset of precursor proteins competent for translocation across the Escherichia coli cytoplasmic membrane. SecG, a membrane component of the translocation machinery, stimulates protein translocation by undergoing the cycle of membrane topology inversion. Null mutants of secB and secG are unable to form isolated colonies on rich medium and at low temperature respectively. A 3.2 kb DNA fragment carrying the secB-gpsA region on a multicopy plasmid was found to suppress the null mutation of either gene. However, subcloning of the DNA fragment revealed that secB is not involved in the suppression of either mutation. Instead, gpsA located downstream from the secB gene was found to be responsible for the suppression of both mutations. The activity of the gpsA-encoded sn-glycerol-3-phosphate dehydrogenase, which is involved in phospholipid synthesis, was significantly lower in the secB null mutant than in the wild type, presumably because of a polar effect. Suppression of the secB null mutation required the wild-type level of GpsA activity. In contrast, overexpression of the enzyme was essential for suppression of the secG null mutation. Moreover, the gpsA-dependent suppression of the secG null mutation occurred only on rich medium, i.e. not on minimal medium. These results indicate that the SecB function is dispensable even in rich medium, and further demonstrate that overexpression of enzymes involved in phospholipid synthesis partly compensates for the SecG function.
... Overexpression of TnrA. Plasmid pTNR14 was constructed by cloning the BsaJI-AseI DNA fragment from pJVK75 (14) containing the tnrA coding sequence into the EcoRV site of pBluescriptSKϪ (Stratagene). The EcoRI-HindIII tnrA DNA fragment from pTNR14 was cloned into pET23ϩ (Novagen, Inc.) to give plasmid pTNR16. ...
Transcription of the Bacillus subtilis nrgAB promoter is activated during nitrogen-limited growth by the TnrA protein. A common inverted repeat, TGTNAN7TNACA (TnrA site), is centered 49 to 51 bp upstream of the transcriptional start sites for the TnrA-regulated nrgAB, gabP P2, and nas promoters. Oligonucleotide-directed mutagenesis of the nrgAB promoter region showed that conserved nucleotides within the TnrA site, the A+T-rich region between the two TnrA half-sites, and an upstream A tract are all required for high-level activation of nrgAB expression. Mutations that alter the relative distance between the two half-sites of the nrgAB TnrA site abolish nitrogen regulation of nrgAB expression. Spacer mutations that change the relative distance between the TnrA site and -35 region of the nrgAB promoter reveal that activation of nrgAB expression occurs only when the TnrA site is located 49 to 51 bp upstream of the transcriptional start site. Mutational analysis of the conserved nucleotides in the gabP P2 TnrA site showed that this sequence is also required for nitrogen-regulated gabP P2 expression. The TnrA protein, expressed in an overproducing Escherichia coli strain, had a 625-fold-higher affinity for the wild-type nrgAB promoter DNA than for a mutated nrgAB promoter DNA fragment that is unable to activate nrgAB expression in vivo. These results indicate that the proposed TnrA site functions as the binding site for the TnrA protein. TnrA was found to activate nrgAB expression during late exponential growth in nutrient sporulation medium containing glucose, suggesting that cells become nitrogen limited during growth in this medium.
... Several genes were identified previously as high-copy suppressors of the cold sensitivity of KN370. The Bacillus subtilis pgsA and scgR genes were both identified by selection of coldresistant transformants from a plasmid-borne genomic library (26,27); the E. coli pgsA and gpsA genes encoded on highcopy-number plasmids also suppressed the cold-sensitive phe- FIG. 5. Effect of glpD26 on growth. Strains were grown on LB plates at 20°C. ...
SecG is an auxiliary protein in the Sec-dependent protein export pathway of Escherichia coli. Although the precise function of SecG is unknown, it stimulates translocation activity and has been postulated to enhance
the membrane insertion-deinsertion cycle of SecA. Deletion of secG was initially reported to result in a severe export defect and cold sensitivity. Later results demonstrated that both of
these phenotypes were strain dependent, and it was proposed that an additional mutation was required for manifestation of
the cold-sensitive phenotype. The results presented here demonstrate that the cold-sensitive secG deletion strain also contains a mutation in glpR that causes constitutive expression of the glp regulon. Introduction of both the glpRmutation and the secG deletion into a wild-type strain background produced a cold-sensitive phenotype, confirming the hypothesis that a second
mutation (glpR) contributes to the cold-sensitive phenotype of secG deletion strains. It was speculated that the glpR mutation causes an intracellular depletion of glycerol-3-phosphate due to constitutive synthesis of GlpD and subsequent channeling
of glycerol-3-phosphate into metabolic pathways. In support of this hypothesis, it was demonstrated that addition of glycerol-3-phosphate
to the growth medium ameliorated the cold sensitivity, as did introduction of a glpD mutation. This depletion of glycerol-3-phosphate is predicted to limit phospholipid biosynthesis, causing an imbalance in
the levels of membrane phospholipids. It is hypothesized that this state of phospholipid imbalance imparts a dependence on
SecG for proper function or stabilization of the translocation apparatus.
... However, these attempts resulted in the cloning of the B. subtilis gene for phosphatidylglycerol synthetase, pgsA [160]. The same strategy identi¢ed the B. subtilis scgR, a gene that regulates the cardiolipin^phosphatidylglycerol ratio [161]. Anionic phospholipids are essential for protein translocation and SecA ATPase activity of the B. subtilis translocase [162]. ...
In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.
... Plasmids and lacZ fusions. pTNR4 was constructed by inserting an EcoRI-BsaBI DNA fragment from pJVK75 (22) containing the tnrA promoter region between the EcoRI and StuI sites of pMTL21P (7). pTNR6 contains the EcoRI-HindIII tnrA promoter DNA fragment from pTNR4 cloned into the lacZ transcriptional fusion vector pSFL7 (39). ...
Two Bacillus subtilis transcriptional factors, TnrA and GlnR, regulate gene expression in response to changes in nitrogen availability. These two
proteins have similar amino acid sequences in their DNA binding domains and bind to DNA sites (GlnR/TnrA sites) that have
the same consensus sequence. Expression of the tnrA gene was found to be activated by TnrA and repressed by GlnR. Mutational analysis demonstrated that a GlnR/TnrA site which
lies immediately upstream of the −35 region of the tnrA promoter is required for regulation of tnrA expression by both GlnR and TnrA. Expression of the glnRA operon, which contains two GlnR/TnrA binding sites (glnRAo1 and glnRAo2) in its promoter region, is repressed by both GlnR and TnrA. The glnRAo2 site, which overlaps the −35 region of the glnRA promoter, was shown to be required for regulation by both GlnR and TnrA, while the glnRAo1 site which lies upstream of the −35 promoter region is only involved in GlnR-mediated regulation. Examination of TnrA binding
to tnrA and glnRA promoter DNA in gel mobility shift experiments showed that TnrA bound with an equilibrium dissociation binding constant of
55 nM to the GlnR/TnrA site in the tnrA promoter region, while the affinities of TnrA for the two GlnR/TnrA sites in the glnRA promoter region were greater than 3 μM. These results demonstrate that GlnR and TnrA cross-regulate each other's expression
and that there are differences in their DNA-binding specificities.
Spore-forming bacteria are major players in life on Earth, and they are fascinating, as they adapt to many ecological niches. The spores are able to resist high temperatures, to dry out and to recover and germinate when outgrowth conditions become available. Spore-formers are mainly Gram-positive firmicute bacteria with low GC content; the Bacillus and Clostridia genera include prominent species. Bacillus subtilis is one of the most thoroughly studied bacteria, serving as a model to study sporulation and the regulatory networks controlling this differentiation system. B. subtilis is also a bacterium widely used for producing enzymes and metabolites of industrial importance. Among the spore-forming firmicutes, the Bacillus cereus group presents the most remarkable diversity. Frequently designated as “the Good, the Bad and the Ugly”, this bacterial group covers eight closely related species. They include the mammal pathogen Bacillus anthracis, responsible for anthrax disease, the insect pathogen Bacillus thuringiensis, used worldwide as a bioinsecticide to control pests of agricultural, forest and medical importance, and B. cereus senso stricto, an important food contaminant responsible for gastrointestinal disorders and, occasionally, more serious systemic diseases in immunecompromised individuals. The specific mammal toxicity of B. anthracis, the insecticidal activity of B. thuringiensis and the emetic activity of some B. cereus strains are due to plasmid genes. However, in addition to these specific traits, all these bacteria share chromosomal genes conferring the ability to colonize and to grow in abiotic and biotic environments. Interestingly, and in sharp contrast to these pathogens, a few B. cereus strains are used as probiotics in livestock animals and even, formerly, for humans, indicating the versatility of this group of bacteria. Among the pathogenic Clostridia, Clostridium botulinum and Clostridium perfringens are important sources of foodborne poisonings and infections, and the emergent opportunistic human pathogen Clostridium difficile is responsible for severe nosocomial intestinal infections. Due to the high resistance of the spores, to their adhesion properties and to their capacity to form biofilms, these anaerobic Clostridia and facultative anaerobic Bacilli are difficult to eliminate and have a direct economic impact upon the food industry and human health.This special issue of Research in Microbiology contains eight articles covering three main topics highlighted during the first BSPIT (Bactéries sporulantes pathogènes ou d'intérêt technologique) meeting which was held in France (Paris, July 6th and 7th, 2015) and hosted by the SFM (Société Française de Microbiologie), and which was mainly attended by French-speaking scientists.
Introduction
Historical Outline
Protein Targeting to the Translocase
Signal Peptides
Co‐translational Protein Targeting
Post‐translational Protein Targeting
Converging Targeting Pathways
Translocase: A Multisubunit Integral Membrane Protein Complex
SecA
SecY
SecE
SecG
SecD, SecF, and YajC
Conserved Protein Translocases in Bacteria, Eukaryotes, and Archaea
Role of Lipids in Protein Translocation
Mechanism of Protein Translocation
ATP‐driven Translocation
Proton Motive Force‐driven Translocation
Dynamics of the Protein‐conducting Channel
Regulation of Protein Translocation
Outlook and Perspectives
Acknowledgments
Cardiolipin is an ever-present component of the energy-conserving inner membranes of bacteria and mitochondria. Its modulation of the structure and dynamism of the bilayer impacts on the activity of their resident proteins, as a number of studies have shown. Here we analyze the consequences cardiolipin has on the conformation, activity, and localization of the protein translocation machinery. Cardiolipin tightly associates with the SecYEG protein channel complex, whereupon it stabilizes the dimer, creates a high-affinity binding surface for the SecA ATPase, and stimulates ATP hydrolysis. In addition to the effects on the structure and function, the subcellular distribution of the complex is modified by the cardiolipin content of the membrane. Together, the results provide rare and comprehensive insights into the action of a phospholipid on an essential transport complex, which appears to be relevant to a broad range of energy-dependent reactions occurring at membranes.
To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside. SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity. The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory. The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation. Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation. In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered. Remarkably, non-bilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex. Optimal activity required a mixture of anionic and non-bilayer lipids at concentrations that correspond to concentrations found in the natural membrane.
The major route of protein translocation in bacteria is the so-called general secretion pathway (Sec-pathway). This route has been extensively studied in Escherichia coli and other bacteria. The movement of preproteins across the cytoplasmic membrane is mediated by a multimeric membrane protein complex called translocase. The core of the translocase consists of a proteinaceous channel formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG and the peripheral adenosine triphosphatase (ATPase) SecA as molecular motor. Many secretory proteins utilize the molecular chaperone SecB for targeting and stabilization of the unfolded state prior to translocation, while most nascent inner membrane proteins are targeted to the translocase by the signal recognition particle and its membrane receptor. Translocation is driven by ATP hydrolysis and the proton motive force. In the last decade, genetic and biochemical studies have provided detailed insights into the mechanism of preprotein translocation. Recent crystallographic studies on SecA, SecB and the SecYEG complex now provide knowledge about the structural features of the translocation process. Here, we will discuss the mechanistic and structural basis of the translocation of proteins across and the integration of membrane proteins into the cytoplasmic membrane.
In closing, it is important to distinguish what mer regulation is not. Thus, while it would seem quite appropriate for a heavy metal-responsive regulator, MerR is not a 'finger' or a 'fist,' nor is it apparently a 'zipper' or given to 'looping.' Further, its relationship with MerD cannot presently be understood, in the simple sense, to be similar to those of the so-called two-component regulators. Although mer is a relatively new model system used to study transcriptional regulation, work from several laboratories building on and extending the insights and techniques devised for the more classical regulated systems now reveal that the mer system has some remarkable properties. Currently, MerR is the only known prokaryotic activator which stably sequesters sigma-70 RNA polymerase at an inactive promoter. The 'in-the-spacer' binding position of MerR and its ligand-induced helix underwinding and hypersensitive induction kinetics are also novel among described prokaryotic activators. In general, the behavior of the gene products of the mer operon challenges the conventional wisdom about what happens to proteins whose sulfhydryl groups react with Hg(II); similarly, mer regulation requires some expansion of our models of how transcriptional activation works. The question of whether other systems (including the versions of the mer operon found in gram-positive bacteria and the related oxidative stress regulator of gram-negative bacteria) will prove to have similar properties is a subject of active enquiry.
Using localized mutagenesis of whole cells, we have isolated a temperature-sensitive UDP-N-acetylglucosamine acyltransferase mutant of Escherichia coli that loses all detectable acyltransferase activity and quickly dies after a shift from 30 to 42 degrees C. Acyltransferase activity and temperature resistance are restored by transforming the mutant with a hybrid plasmid containing the E. coli gene for UDP-GlcNAc acyltransferase (lpxA). In addition, a new assay has been developed for quantitating the amount of lipid A (the active component of endotoxin) in E. coli and related Gram-negative strains. Cells are labeled with 32Pi and extracted with chloroform/methanol/water (1:2:0.8, v/v) to remove glycerophospholipids. The residue is then hydrolyzed with 0.2 M HCl to liberate the "monophosphoryl" lipid A degradation products (Qureshi, N., Cotter, R. J. and Takayama, K. (1986) J. Microbiol. Methods 5, 65-77), each of which bears a single phosphate residue at position 4'. The amount of lipid A is normalized to the total amount of labeled glycerophospholipid present in the cells. The steady state ratio of lipid A to glycerophospholipid in wild-type cells is approximately 0.12. The lipid A content of the acyltransferase mutant is reduced 2-3-fold, and the rate of lipid A synthesis is reduced 10-fold when compared to wild-type after 60 min at 42 degrees C. These results provide physiological evidence that UDP-N-acetylglucosamine acyltransferase is the major committed step for lipid A biosynthesis in E. coli and that lipid A is an essential molecule.
Although the antibiotic thiostrepton is best known as an inhibitor of protein synthesis, it also, at extremely low concentrations (< 10(-9) M), induces the expression of a regulon of unknown function in certain Streptomyces species. Here, we report the purification of a Streptomyces lividans thiostrepton-induced transcriptional activator protein, TipAL, whose N-terminus is similar to a family of eubacterial regulatory proteins represented by MerR. TipAL was first purified from induced cultures of S.lividans as a factor which bound to and activated transcription from its own promoter. The tipAL gene was overexpressed in Escherichia coli and TipAL protein purified in a single step using a thiostrepton affinity column. Thiostrepton enhanced binding of TipAL to the promoter and catalysed specific transcription in vitro. TipAS, a second gene product of the same open reading frame consisting of the C-terminal domain of TipAL, is apparently translated using its own in-frame initiation site. Since it is produced in large molar excess relative to TipAL after induction and also binds thiostrepton, it may competitively modulate transcriptional activation.
The Escherichia coli cytoplasmic membrane protein, p12, stimulates the protein translocation activity reconstituted with SecY, SecE and SecA. The gene encoding p12, which is located at 69 min on the E. coli chromosome, was deleted to examine the role of p12 in protein translocation in vivo. The deletion strain exhibited cold-sensitive growth. Pulse-chase experiments revealed that precursors of outer membrane protein A, maltose binding protein and beta-lactamase accumulated at 20 degrees C but not at 37 degrees C. The deletion strain harboring a plasmid which carries the gene encoding p12 under the control of the araBAD promoter was able to grow in the cold when p12 was expressed with the addition of arabinose. Furthermore, the accumulated precursors were rapidly processed to the mature forms upon the expression of p12. Immunoblot analysis revealed the steady-state accumulation of precursor proteins at 20 degrees C, whereas the accumulation was only marginal at 37 degrees C, indicating that the function of p12 is more critical at 20 degrees C than at 37 degrees C. Finally, proteoliposomes were reconstituted with or without p12 to demonstrate that the stimulation of the activity by p12 increases with a decrease in temperature. From these results, we concluded that p12 is directly involved in protein translocation in E. coli and plays a critical role in the cold. We propose the more systematic name, SecG, for p12.
A cytoplasmic membrane protein, p12, of Escherichia coli was discovered as a new factor that stimulates the protein translocation activity reconstituted with SecA, SecY, and SecE (Nishiyama, K., Mizushima, S., and Tokuda, H. (1993) EMBO J. 12, 3409-3415). Direct involvement of p12 in protein translocation was subsequently demonstrated in vivo by genetic studies, and the name SecG has been proposed for p12 (Nishiyama, K., Hanada, M., and Tokuda, H. (1994) EMBO J. 13, 3272-3277). To elucidate the role of SecG in protein translocation and to characterize the translocation apparatus comprising these four Sec proteins, the activity of reconstituted proteoliposomes was examined in detail as a function of the amount of each component. SecG markedly stimulated the translocation activity over wide ranges of amounts of the other three Sec proteins, indicating that none of the other three Sec proteins substitutes for the SecG function. Detailed kinetic analyses indicated that the activity of proteoliposomes was dependent on the amount of the SecY-SecE complex when SecG was absent and the amount of the SecY.SecE.SecG complex when the proteoliposomes contained SecG. The translocation activity of the latter complex was significantly higher than that of the former one. Binding of SecA to liposomes appreciably increased when they contained both SecY and SecE, whereas the further presence of SecG did not enhance the binding. On the other hand, the ATPase activity of SecA, which was dependent on proOmpA and SecY.SecE-containing proteoliposomes, was significantly enhanced when the proteoliposomes contained SecG. Taken together, these results indicate that SecG enhances the translocation activity of the apparatus after the step of SecA targeting to SecY.SecE.
A novel factor, which is a membrane component of the protein translocation machinery of Escherichia coli, was discovered. This factor was found in the trichloracetic acid-soluble fraction of solubilized cytoplasmic membrane. The factor was purified to homogeneity by ion exchange column chromatographies and found to be a hydrophobic protein with a molecular mass of approximately 12 kDa. The factor caused > 20-fold stimulation of the protein translocation when it was reconstituted into proteoliposomes together with SecE and SecY. SecE, SecY, SecA and ATP were essential for the factor-dependent stimulation of the activity. The factor stimulated the translocation of all three precursor proteins examined, including authentic proOmpA. Stimulation of the translocation of proOmpF-Lpp, a model presecretory protein, was especially remarkable, since no translocation was observed unless proteoliposomes were reconstituted with the factor. Partial amino acid sequence of the purified factor was determined. An antibody raised against a synthetic peptide of this sequence inhibited the protein translocation into everted membrane vesicles, indicating that the factor is playing an important role in protein translocation into membrane vesicles. The partial amino acid sequence was found to coincide with that deduced from the reported DNA sequence of the upstream region of the leuU gene. Cloning and sequencing of the upstream region revealed the presence of a new open reading frame, which encodes a hydrophobic protein of 11.4 kDa. We propose that the factor is a general component of the protein translocation machinery of E. coli.
E. coli preprotein translocase comprises SecA and SecY/E/G complex. SecA delivers the preprotein to the putative protein-conducting channel formed by SecY/E by undergoing ATP-driven cycles of membrane insertion and deinsertion. SecG renders the translocase highly efficient. An antibody raised against the C-terminal region of SecG inhibits preprotein translocation into everted membrane vesicles despite the exposure of this region to the inside of membrane vesicles in the absence of preprotein translocation. When preprotein translocation was started with ATP and then blocked by the inhibition of ATP hydrolysis, the C-terminal region was exposed to the outside of membrane vesicles. Another region of SecG showed a change in membrane sidedness upon preprotein translocation, indicating that SecG undergoes topology inversion. This topology inversion was tightly coupled to the SecG function and linked with the insertion-deinsertion cycle of SecA.
The strictly anaerobic Gram-negative beer spoilage bacteria Megasphaera cerevisiae, Pectinatus cerevisiiphilus and P. frisingensis were subjected to cellular fatty acid analysis, employing acid- and base-catalysed cleavage, gas chromatography and mass spectrometry. M. cerevisiae contained 12:0, 16:0, 16:1, 18:1, 17:cyc, 19:cyc, 12:0(3OH), 14:0(3OH)) as the main fatty acids, and alk-1-enyl chains instead of acyl chains were detected to a considerable extent (14% of total fatty acids), indicating the presence of plasmalogens. The fatty acid pattern of M. cerevisiae was almost identical to that of M. elsdenii, the only species previously assigned to this genus. P. cerevisiiphilus and P. frisingensis yielded fatty acids that were heavily dominated by odd-numbered chains; 11:0, 15:0, 17:1, 18:cyc and 13:0(3OH) were the main fatty acids detected in both species. Alk-1-enyl chains with similar chain lengths were also found. Both Pectinatus species contained six different 3-hydroxy fatty acids with chain lengths between 11 and 15 carbons, 13:0(3OH) being dominant and the others accounting for generally less than 1% of total fatty acids. Among the minor components, an unsaturated 3-hydroxy fatty acid was detected which was shown to be 13:1(3OH). In addition, fatty acid analysis was shown to be applicable to detection of bacterial contamination of beer.
A method has been described for the isolation of DNA from micro-organisms which yields stable, biologically active, highly polymerized preparations relatively free from protein and RNA. Alternative methods of cell disruption and DNA isolation have been described and compared. DNA capable of transforming homologous strains has been used to test various steps in the procedure and preparations have been obtained possessing high specific activities. Representative samples have been characterized for their thermal stability and sedimentation behaviour.
The biosynthesis of lipid A component was shown to be defective in a temperature-senitive firA mutant of Escherichia coli. Cells were biosynthetically labelled with [14C]acetate and incorporation of radioactivity into the glycerophospholipid compared to lipid A fractions was measured. The lipid A/glycerophospholipid biosynthesis ratio of the firA mutant at 37°C was approximately 50% and at the nonpermissive temperature of 42°C was less than 20% of that observed in the corresponding wild-type strain. Analysis of radiolabelled lipid A 4′-monophosphate derivatives and glycerophospholipids by thin-layer chromatography revealed that the firA mutant at 42°C elaborated an altered lipid A, and its phosphatidylglycerol content was low. The chemical composition of the extracted lipopolysaccharides differed significantly between the firA and the wild-type strain only in the proportion of hexadecanoic acid, which was minimal in the wild type grown at 37°C and 42°C and in firA lipopolysaccharide grown at 37°C. In the firA mutant lipopolysaccharide produced at 42°C, hexadecanoic acid was present in approximately every third molecule, attached to the hydroxyl group of the amide-linked (R)-3-hydroxytetradecanoic acid at the reducing glucosamine of lipid A. Inspection of dephosphorylated free lipid A preparations by laser-desorption mass spectrometry confirmed that significant amounts of heptaacyl lipid A was elaborated by the firA strain grown at 42°C.
Two general approaches have been used to define genetically the genes that encode components of the cellular protein export machinery. One of these strategies identifies mutations that confer a conditional-lethal, pleiotropic export defect (sec,secretion). The other identifies dominant suppressors of signal sequence mutations (prl,proteinlocalization). Subsequent characterization reveals that in at least three cases,prlA/secY,prlD/secA, andprlG/secE, both types of mutations are found within the same structural gene. This convergence is satisfying and provides compelling evidence for direct involvement of these gene products in the export process.
We have isolated and partially sequenced the gene coding for alpha-amylase (EC 3.2.1.1) from Bacillus amyloliquefaciens by molecular cloning in the plasmid pUB110 using Bacillus subtilis as a host. The nucleotide sequence of the NH2-terminal region of the cloned gene was determined and found to contain a 31-residue-long stretch of amino acids preceding the NH2-terminal sequence of the extracellular alpha-amylase. Within this sequence there is a 15-residue-long stretch of uncharged amino acids similar to that found at the NH2 terminus of other precursors to exported proteins. This "signal sequence" is probably removed in conjunction with the translocation of alpha-amylase through the cytoplasmic membrane. In vitro labeling of alpha-amylase with radioactive amino acids in a coupled transcription-translation system followed by partial sequencing established the exact location of the NH2 terminus of the alpha-amylase gene. The nucleotide sequence preceding the NH2 terminus has properties resembling the RNA-polymerase- and ribosome-binding sites found at the 5' terminus of many prokaryotic genes.
Reconstitution of the translocation machinery for secretory proteins from purified constituents was performed. SecY was solubilized from SecY/SecE-overproducing Escherichia coli cells and purified by chromatography on ion-exchange and size-exclusion columns. Proteoliposomes active in protein translocation were reconstituted from the purified preparations of SecY and SecE. The reconstituted translocation activity was SecA- and ATP-dependent. Although the purified preparations of SecY and SecE were still contaminated with minute amounts of other proteins, the elution profiles of SecY and SecE on column chromatographies coincided with the elution profiles of reconstituted translocation activity, indicating that SecY and SecE are the indispensable components in these preparations. We conclude that SecY, SecE, and SecA are essential components of the protein secretion machinery and that translocation activity can be reconstituted from only these three proteins and phospholipids.
Converging physiological, genetic, and biochemical studies have established the salient features of preprotein translocation across the plasma membrane of Escherichia coli. Translocation is catalyzed by two proteins, a soluble chaperone and a membrane-bound translocase. SecB, the major chaperone for export, forms a complex with preproteins. Complex formation inhibits side-reactions such as aggregation and misfolding and aids preprotein binding to the membrane surface. Translocase consists of functionally linked peripheral and integral membrane protein domains. SecA protein, the peripheral membrane domain of translocase, is the primary receptor for the SecB/preprotein complex. SecA hydrolyzes ATP, promoting cycles of translocation, preprotein release, Δµ~H+-dependent translocation, and rebinding of the preprotein. The membrane-embedded domain of translocase is the SecY/E protein. It has, as subunits, the SeeY and SecE polypeptides. The SecY/E protein stabilizes and activates SecA and participates in binding it to the membrane. SecA recognizes the leader domain of preproteins, whereas both SecA and SecB recognize the mature domain. Many proteins translocate without requiring SecB, and some proteins do not need translocase to assemble into the plasma membrane. Translocation is usually followed by endoproteolytic cleavage by leader peptidase. The availability of virtually every pure protein and cloned gene involved in the translocation process makes E. coli the premier organism for the study of translocation mechanisms.
The ATPase activity of SecA is stimulated by E. coli plasma membrane vesicles bearing SecY protein and a precursor protein such as proOmpA. This activity is termed "translocation ATPase". Liposomes alone can also stimulate SecA ATPase, but membrane proteins block this stimulation in native inner membranes. We define the stimulation of SecA ATPase by lipid as "SecA/lipid ATPase". SecA/lipid ATPase, translocation ATPase, and translocation into inner membrane vesicles require acidic phospholipids, suggesting an underlying unity of mechanism. ProOmpA and ATP stabilize liposome-bound SecA. Full SecA/lipid ATPase activity and stability are also seen when a mixture of a leader peptide and either OmpA or maltose binding protein (MBP) are added instead of proOmpA, while neither the leader peptide alone nor OmpA or MBP suffice. Cytosolic proteins in conjuction with a leader peptide are less active in this reaction, indicating that liposome-bound SecA protein recognizes both leader and mature domains.
We have previously reconstituted the soluble phase of precursor protein translocation in vitro using purified proteins (the precursor proOmpA, the chaperone SecB, and the ATPase SecA) in addition to isolated inner membrane vesicles. We now report the isolation of the SecY/E protein, the integral membrane protein component of the E. coli preprotein translocase. The SecY/E protein, reconstituted into proteoliposomes, acts together with SecA protein to support translocation of proOmpA, the precursor form of outer membrane protein A. This translocation requires ATP and is strongly stimulated by the protonmotive force. The initial rates and the extents of translocation into either native membrane vesicles or proteoliposomes with pure SecY/E are comparable. The SecY/E protein consists of SecY, SecE, and an additional polypeptide. Antiserum against SecY immunoprecipitates all three components of the SecY/E protein.
The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features. An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation. Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site. One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites. The derived amino acid (aa) sequence of B. subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum. The B. subtilis and C. acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations. The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B. subtilis GS despite its lack of adenylylation.
The E. coli secG deletion mutant is unable to grow and is defective in protein translocation at low temperature. A gene of Bacillus subtilis, which is able to restore the growth of the deletion mutant at low temperature, was found as a multi-copy suppressor. Sequencing of this gene revealed significant homology to E. coli pgsA, which encodes phosphatidylglycerophosphate synthase, an enzyme involved in acidic phospholipid synthesis. A plasmid carrying E. coli pgsA also restored the growth of the deletion mutant. Furthermore, protein translocation in the deletion mutant was stimulated when it harbored a plasmid carrying pgsA. A possible mechanism underlying the pgsA-dependent suppression of the secG deletion mutation is discussed.
SecA, the peripheral subunit of E. coli preprotein translocase, alternates between a membrane inserted and a deinserted state as part of the catalytic cycle of preprotein translocation. When SecA is complexed with SecY/E and preprotein, ATP drives a profound conformational change, leading to membrane insertion of a 30 kDa domain of SecA. The inserted domain is protease-inaccessible from the cytosolic side of the membrane, but becomes accessible upon membrane disruption. Concomitant with 30 kDa domain insertion, approximately 20 aminoacyl residues of the preprotein are translocated. Additional ATP, which may be hydrolyzed at the second ATP site of SecA, releases the translocated preprotein and allows the 30 kDa domain to deinsert, whence it can exchange with cytosolic SecA. Thus, SecA is the mobile subunit of an integral membrane transporter, consuming ATP during both the insertion and deinsertion phases of its catalytic cycle while guiding preprotein segments across the membrane.
The protein translocation apparatus in Escherichia coli has been studied both genetically and biochemically. In vitro protein translocation systems involving everted membrane vesicles or reconstituted proteoliposomes have significantly contributed to biochemical clarification of the structure, mechanism and energetics of the apparatus. It is established that SecA, SecY and SecE are essential components, and play fundamental roles in the translocation reaction, and that both ATP and a proton motive force are required for the translocation. A new membrane factor, SecG, was found to participate in the formation of the apparatus, causing significant enhancement of the activity. SecD was found to play a role in the release of translocated proteins from the outer surface of the cytoplasmic membrane.
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