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Signal Transduction Pathways Involving Protein Phosphorylation in Prokaryotes
Abstract and Figures
Bacteria are capable of sensing a wide variety of environmental signals,
including changes in chemical concentrations, the presence of a host organism, or variation in physical parameters such as temperature, osmolarity,
viscosity, or light. One response to changing conditions is to move to a more
"favorable" locale; changes in locomotive behavior can be observed less than
one second after a change in chemical composition of the medium. Another
possible course of action is to adapt the cell to the new environment, either by
changing enzyme activity or by altering expression of specific genes or groups
of genes. The bacterium may use the modified enzymes or new gene products
to adjust to its surroundings temporarily, or to establish a new long-term state
(e.g. the sporulation response to starvation).
Figures - uploaded by Melvin Isaac Simon
Author content
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Content may be subject to copyright.
... Therefore, we selected these six genes for further experiments. Sequence comparison between several Gram-negative bacteria shows that these PAS-domain-containing genes are homologs of the aer gene [29] ( Figure 1B). We named aer2, aer3, aer4, aer5, aer6, and aer7. ...
... We named aer2, aer3, aer4, aer5, aer6, and aer7. quence comparison between several Gram-negative bacteria shows that these PAS-domain-containing genes are homologs of the aer gene [29] ( Figure 1B). We named aer2, aer3, aer4, aer5, aer6, and aer7. ...
Bacterial chemotaxis is the phenomenon in which bacteria migrate toward a more favorable niche in response to chemical cues in the environment. The methyl-accepting chemotaxis proteins (MCPs) are the principal sensory receptors of the bacterial chemotaxis system. Aerotaxis is a special form of chemotaxis in which oxygen serves as the signaling molecule; the process is dependent on the aerotaxis receptors (Aer) containing the Per-Arnt-Sim (PAS) domain. Over 40 MCPs are annotated on the genome of Vibrio cholerae; however, little is known about their functions. We investigated six MCPs containing the PAS domain in V. cholerae El Tor C6706, namely aer2, aer3, aer4, aer5, aer6, and aer7. Deletion analyses of each aer homolog gene indicated that these Aer receptors are involved in aerotaxis, chemotaxis, biofilm formation, and intestinal colonization. Swarming motility assay indicated that the aer2 gene was responsible for sensing the oxygen gradient independent of the other five homologs. When bile salts and mucin were used as chemoattractants, each Aer receptor influenced the chemotaxis differently. Biofilm formation was enhanced by overexpression of the aer6 and aer7 genes. Moreover, deletion of the aer2 gene resulted in better bacterial colonization of the mutant in adult mice; however, virulence gene expression was unaffected. These data suggest distinct roles for different Aer homologs in V. cholerae physiology.
... This work largely began with research into E. coli chemotaxis signaling (Figure 2A). E. coli bacteria perform a biased random walk of forward "runs" that are separated by random "tumbles" in order to produce a net motion toward nutrients and away from repellents [16][17][18][19]. As the bacteria swim, they respond to changes in their surrounding nutrient concentrations by changing their tumbling likelihood, but they then adapt to these changes over the next few minutes to allow them to accurately respond to additional changes. ...
Biochemical reaction networks perform a variety of signal processing functions, one of which is computing the integrals of signal values. This is often used in integral feedback control, where it enables a system's output to respond to changing inputs, but to then return exactly back to some pre-determined setpoint value afterward. To gain a deeper understanding of how biochemical networks are able to both integrate signals and perform integral feedback control, we investigated these abilities for several simple reaction networks. We found imperfect overlap between these categories, with some networks able to perform both tasks, some able to perform integration but not integral feedback control, and some the other way around. Nevertheless, networks that could either integrate or perform integral feedback control shared key elements. In particular, they included a chemical species that was neutrally stable in the open loop system (no feedback), meaning that this species does not have a unique stable steady-state concentration. Neutral stability could arise from zeroth order decay reactions, binding to a partner that was produced at a constant rate (which occurs in antithetic control), or through a long chain of covalent cycles. Mathematically, it arose from rate equations for the reaction network that were underdetermined when evaluated at steady-state.
... The canonical bEBPs are regulated through either phosphorylation, ligand binding, or protein-protein interaction. The bEBPs that are part of two-component systems are regulated through phosphorylation of the conserved aspartate present in the N-terminal REC domain through the cognate histidine kinase (22)(23)(24)(25). The EBPs regulated through ligand binding harbor effector binding domains such as GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) and PAS (Per-Arnt-Sim) at the N terminus (12,25). ...
... In B. subtilis, Spo0A and Spo0F share similar response regulator receiver domains (residues 6 -116 in Spo0F and 6 -120 in Spo0A), and both proteins interact with the phosphotransfer protein Spo0B using conserved secondary structure [12,[23][24][25][26]. The residues of B. subtilis Spo0F and Spo0A that are important for signal transduction were previously identified and characterized [4,12,13,[27][28][29][30]. We aligned the amino acid sequence of the B. subtilis Spo0A ( Figure 2) and Spo0F receiver domains ( Figure S2) to C. difficile Spo0A to predict orthologous functional residues. ...
Clostridioides difficile is an anaerobic, Gram-positive pathogen that is responsible for C. difficile infection (CDI). To survive in the environment and spread to new hosts, C. difficile must form metabolically dormant spores. The formation of spores requires activation of the transcription factor Spo0A, which is the master regulator of sporulation in all endospore-forming bacteria. Though the sporulation initiation pathway has been delineated in the Bacilli, including the model spore-former Bacillus subtilis, the direct regulators of Spo0A in C. difficile remain undefined. C. difficile Spo0A shares highly conserved protein interaction regions with the B. subtilis sporulation proteins Spo0F and Spo0A, although many of the interacting factors present in B. subtilis are not encoded in C. difficile. To determine if comparable Spo0A residues are important for C. difficile sporulation initiation, site-directed mutagenesis was performed at conserved receiver domain residues and the effects on sporulation were examined. Mutation of residues important for homodimerization and interaction with positive and negative regulators of B. subtilis Spo0A and Spo0F impacted C. difficile Spo0A function. The data also demonstrated that mutation of many additional conserved residues altered C. difficile Spo0A activity, even when the corresponding Bacillus interacting proteins are not apparent in the C. difficile genome. Finally, the conserved aspartate residue at position 56 of C. difficile Spo0A was determined to be the phosphorylation site that is necessary for Spo0A activation. The finding that Spo0A interacting motifs maintain functionality suggests that C. difficile Spo0A interacts with yet unidentified proteins that regulate its activity and control spore formation
... The canonical bEBPs are regulated through either phosphorylation, ligand binding, or protein-protein interaction. The bEBPs that are part of two-component systems are regulated through phosphorylation of the conserved aspartate present in the N-terminal REC domain through the cognate histidine kinase (22)(23)(24)(25). The EBPs regulated through ligand binding harbor effector binding domains such as GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) and PAS (Per-Arnt-Sim) at the N terminus (12,25). ...
Diverse sigma factors associate with the RNA polymerase (RNAP) core enzyme to initiate transcription of specific target genes in bacteria. σ54-Mediated transcription uses AAA+ activators that utilize their ATPase activity for transcription initiation. FleQ is a σ54-dependent master transcriptional regulator of flagellar genes in Pseudomonas aeruginosa. The ATPase activity of FleQ is regulated via a P-loop ATPase, FleN, through protein-protein interaction. We report a high-resolution crystal structure of the AAA+ domain of FleQ in complex with antiactivator FleN. The data reveal that FleN allosterically prevents ATP binding to FleQ. Furthermore, FleN remodels the region of FleQ essential for engagement with σ54 for transcription initiation. Disruption of the conserved protein-protein interface, by mutation, shows motility and transcription defects in vivo and multiflagellate phenotype. Our study provides a detailed mechanism used by monoflagellate bacteria to fine-tune the expression of flagellar genes to form and maintain a single flagellum.
In gram-positive bacteria, phosphorylated arginine functions as a protein degradation signal in a similar manner as ubiquitin in eukaryotes. The protein-arginine phosphorylation is mediated by the McsAB complex, where McsB possesses kinase activity and McsA modulates McsB activity. Although mcsA and mcsB are regulated within the same operon, the role of McsA in kinase activity has not yet been clarified. In this study, we determined the molecular mechanism by which McsA regulates kinase activity. The crystal structure of the McsAB complex shows that McsA binds to the McsB kinase domain through a second zinc-coordination domain and the subsequent loop region. This binding activates McsB kinase activity by rearranging the catalytic site, preventing McsB self-assembly, and enhancing stoichiometric substrate binding. The first zinc-coordination and coiled-coil domains of McsA further activate McsB by reassembling the McsAB oligomer. These results demonstrate that McsA is the regulatory subunit for the reconstitution of the protein-arginine kinase holoenzyme. This study provides structural insight into how protein-arginine kinase directs the cellular protein degradation system.
The bacterial chemotaxis regulatory circuit mainly consists of coupling protein CheW, sensor histidine kinase CheA, and response regulator CheY. Most bacteria, such as Escherichia coli, have a single gene encoding each of these proteins. Interestingly, the Lyme disease pathogen, Borreliella burgdorferi, has multiple chemotaxis proteins, e.g., two CheA, three CheW, and three CheY proteins. The genes encoding these proteins mainly reside in two operons: cheW2-cheA1-cheB2-cheY2 (A-I) and cheA2-cheW3-cheX-cheY3 (A-II). Previous studies demonstrate that all the genes in A-II are essential for the chemotaxis of B. burgdorferi; however, the role of those genes in A-I remains unknown. This study aimed to fill this gap using the CheW2 gene, the first gene in A-I, as a surrogate. We first mapped the transcription start site of A-I upstream of cheW2 and identified a σ70-like promoter (PW2) and two binding sites (BS1 and BS2) of BosR, an unorthodox Fur/Per homolog. We then demonstrated that BosR binds to PW2 via BS1 and BS2 and that deletion of bosR significantly represses the expression of cheW2 and other genes in A-I, implying that BosR is a positive regulator of A-I. Deletion of cheW2 has no impact on the chemotaxis of B. burgdorferi in vitro but abrogates its ability to evade host adaptive immunity, because the mutant can establish systemic infection only in SCID mice and not in immunocompetent BALB/c mice. This report substantiates the previous proposition that A-I is not implicated in chemotaxis; rather, it may function as a signaling transduction pathway to regulate B. burgdorferi virulence gene expression.
Bacterial chemoreceptors regulate the cytosolic multidomain histidine kinase CheA through largely unknown mechanisms. Residue substitutions in the peptide linkers that connect the P4 kinase domain to the P3 dimerization and P5 regulatory domain affect CheA basal activity and activation. To understand the role that these linkers play in CheA activity, the P3-to-P4 linker (L3) and P4-to-P5 linker (L4) were extended and altered in variants of Thermotoga maritima (Tm) CheA. Flexible extensions of the L3 and L4 linkers in CheA-LV1 (linker variant 1) allowed for a well-folded kinase domain that retained wild-type (WT)-like binding affinities for nucleotide and normal interactions with the receptor-coupling protein CheW. However, CheA-LV1 autophosphorylation activity registered ∼50-fold lower compared to WT. Neither a WT nor LV1 dimer containing a single P4 domain could autophosphorylate the P1 substrate domain. Autophosphorylation activity was rescued in variants with extended L3 and L4 linkers that favor helical structure and heptad spacing. Autophosphorylation depended on linker spacing and flexibility and not on sequence. Pulse-dipolar electron-spin resonance (ESR) measurements with spin-labeled adenosine 5'-triphosphate (ATP) analogues indicated that CheA autophosphorylation activity inversely correlated with the proximity of the P4 domains within the dimers of the variants. Despite their separation in primary sequence and space, the L3 and L4 linkers also influence the mobility of the P1 substrate domains. In all, interactions of the P4 domains, as modulated by the L3 and L4 linkers, affect domain dynamics and autophosphorylation of CheA, thereby providing potential mechanisms for receptors to regulate the kinase.
Enzyme-manipulated hydrogelation based on self-assembly of small molecules is an attractive methodology for development of functional biomaterials. Upon the catalysis of enzymes, small-molecule precursors are converted into assemblable building blocks, which arrange into high-ordered nanofibers via non-covalent interactions at the molecular level, and further trap water to form hydrogels at the macroscopic level. Such approach has numerous advantages of region- and enantioselectivity, and mild reaction conditions for encapsulation of biomedications or cells that are fragile to environmental change. In addition to the common applications as drug reservoirs or cell scaffolds, the utilization of endogenous enzymes as stimuli to initiate self-assembly in the living cells and tissue is considered as an intelligent spatiotemporally controllable hydrogelation strategy for biomedical applications. The enzyme-instructed in situ self-assembly and hydrogelation can modulate the cell behavior, and even present therapeutic bioactivities, which provides a new perspective in the field of disease treatment. In this review, we categorize distinct enzymatic stimuli and elaborate substrate design, catalytic characteristics, and mechanisms of self-assembly and hydrogelation. The biomedical applications in drug delivery, tissue engineering, bioimaging, and in situ gelation-produced bioactivity are outlined. Advantages and limitations regarding the state-of-the-art enzyme-driven hydrogelation technologies and future perspectives are also discussed.
Statement of Significance
: Hydrogel is a semi-solid soft material containing a large amount of water. Due to the features of adjustable flexibility, extremely porous architecture, and the high similarity of structure to natural extracellular matrices, the hydrogel has broad application prospects in biomedicine. In recent 20 years, enzyme-manipulated hydrogelation based on self-assembly of small molecules has developed rapidly as an attractive methodology for the construction of functional biomaterials. Upon the catalysis of enzymes, small-molecule precursors are converted into assemblable building blocks, which arrange into high-ordered nanofibers via non-covalent interactions at the molecular level, and further trap water to form hydrogels at the macroscopic level. This review summarized the characteristics of enzymatic hydrogel, as well as the traditional application and emerging prospect of enzyme-instructed self-assembly and hydrogelation.
The nucleotide sequence of Pseudomonas aeruginosa phoB was determined. The sequence data suggest that the PhoB polypeptide consists of 229 amino acid residues and has a predicted molecular weight of 25,708. In the regulatory region of the gene, a very well conserved phosphate box was found. The sequence data also predicted the presence of an open reading frame downstream of phoB, which could be phoR. The deduced amino acid sequence of phoB was significantly homologous to that of the Escherichia coli phoB gene product and to those of several known procaryotic transcriptional regulators such as PhoP, OmpR, VirG, Dye, NtrC, and AlgR.
A DNA fragment of Escherichia coli cloned on pBR322 elevated the production of alkaline phosphatase and phosphate-binding protein in a phoR phoM strain. Nucleotide sequence analysis and enzyme assays revealed that the DNA fragment contained the ackA gene, which codes for acetate kinase. A high gene dosage of ackA was needed to induce the production of alkaline phosphatase and phosphate-binding protein in this strain. Overexpression of ackA elevated the intracellular ATP concentration, an effect that might be related to activation of the phosphate regulon in the phoR phoM strain.
In both prokaryotes and eukaryotes methyl groups can be added to and removed from the carboxyl groups of proteins. Recent work has revealed that these reactions have a role in several behavioural phenomena. The nature of this role has been uncovered in one case--that of bacterial chemotaxis.
The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.
Expression of Escherichia coli outer-membrane porin proteins (OmpF and OmpC) is regulated by the osmolarity of the medium. EnvZ and OmpR, which are positive regulatory factors for the transcriptional osmotic regulation of the ompF and ompC genes, belong to a group of two-component regulatory factors that respond to a variety of environmental stimuli in bacteria. EnvZ-OmpR phosphotransfer was revealed to be involved in signal transduction in response to an osmotic stimulus, and to play a crucial physiological role in the consequent osmotic activation of the porin genes. Based on the various lines of experimental evidence, a model is proposed for the molecular mechanism underlying the osmotic regulation through phosphorylation of the activator (OmpR) by the membrane-located kinase (Env2).
An in vivo approach was taken to assess whether the phosphorylated state of the transcription activator OmpR was affected by changes in the osmolarity of the growth medium or by mutations in envZ, the gene encoding the inner membrane histidine kinase that phosphorylates OmpR. We present results that support the view that increased phosphorylation of OmpR is correlated with enhanced expression of ompC. The in vivo phosphorylation approach was also used to show that OmpR can be phosphorylated in an envZ null strain. This result indicates that phosphorylation cross talk can occur in vivo between OmpR and a kinase(s) that is functionally homologous to envZ.
An existing cpxA(Ts) mutant was resistant to amikacin at levels that inhibited completely the growth of a cpxA+ and a cpxA deletion strain and failed to grow as efficiently on exogenous proline. These properties are similar to those of mutants altered in a gene mapped to the cpxA locus and variously designated as ecfB, ssd, and eup. The amikacin resistance phenotype of the cpxA mutant was inseparable by recombination from the cpxA mutant phenotype (inability to grow at 41 degrees C without exogenous isoleucine and valine) and was recessive to the cpxA+ allele of a recombinant plasmid. Using methods that ensured independent mutations in the cpxA region of the chromosome, we isolated six new amikacin-resistant mutants following nitrosoguanidine mutagenesis. Three-factor crosses mapped the mutations to the cpxA locus. When transferred by P1 transduction to a cpxB11 Hfr strain, each of the mutations conferred the Tra- and Ilv- phenotypes characteristic of earlier cpxA mutants. Two of the new mutations led to a significantly impaired ability to utilize exogenous proline, and four led to partial resistance to colicin A. Two of the new cpxA alleles were recessive to the cpxA+ allele, and four were dominant, albeit to different degrees. On the basis of these data, we argue that cpxA, ecfB, eup, and ssd are all the same gene. We discuss the cellular function of the cpxA gene product in that light.