Journal of Bacteriology

The energy converting complex of the sodium-driven flagellar motor in bacteria comprises two proteins, PomA and PomB, whose transmembrane regions form ion conducting channels and is called the stator complex. The transmembrane protein PomB is attached to the cell wall by its periplasmic region and has a plug segment following the transmembrane helix to prevent ion flux. PomB ({Delta}41-120), which lacks the periplasmic region from E41 to K120 immediately following its transmembrane region shows similar motility as that of wild-type PomB. In this study, three deletion mutants after the plug region, PomB ({Delta}61-120), PomB ({Delta}61-140), and PomB ({Delta}71-150), were generated and those deletion mutants were examined for their functionality. PomB ({Delta}61-120) conferred similar motility as that of the wild-type protein, whereas the other two mutants showed almost no motility in soft agar plate; however, we observed some swimming cells with speed similar to that of the wild-type cells. To observe dominance of wild-type proteins, we introduced the two PomB mutants into a wild-type strain, and its ability to swim was not affected by the mutants. Then, we purified the mutant PomAB complexes to confirm the stator formation. When we introduced the PomB mutations in the plug region, the reduced motility by the deletion was rescued, suggesting that the stator was activated. Our results indicate that the deletion prevents stator from transformation to an active form; however, the linker and plug regions from E41 to S150 are not essential for the motor function of PomB but are important for its regulation. IMPORTANCEThe stator complex of flagella consists of PomA and PomB proteins and interacts with the rotor complex. PomB has a peptidoglycan binding (PGB) domain to fix the stator for generation of torque. PomB is attached to the cell wall only when the stator is activated by interaction between the cytoplasmic region of PomA and the rotor protein FliG. The flexible linker of PomB, which is a naturally unfolded region, is flanked by the peptidoglycan-binding (PGB) domain and transmembrane region. The plug region, which interacts with the periplasmic loops of PomA to prevent activation of the stator, is located next to its transmembrane region. In this study, we reveal the role of the flexible linker in activation of the stator complex.

Pseudomonas aeruginosa utilizes dual flagellar stator systems for motility; and dual stator bacteria possess auxiliary flagellar rotor ring components in their periplasm. In P. aeruginosa MotAB and MotCD comprise the dual stator system and MotY is the auxiliary periplasmic ring component. We investigated motility of strains and their isogenic {Delta}motY derivatives which expressed chimeric MotB/MotD periplasmic domains and characterized differences in motility behaviors. We found in general motility is severely impaired in strains carrying motY deletions and which express C-terminal periplasmic regions of MotD, compared with strains expressing MotB C-terminal counterparts. Motility in soft agar is slightly increased in strains expressing N-terminal MotB transmembrane domains and MotD C-terminal periplasmic plug, PGB, and extensions, but motility is severely impaired in {Delta}motY strains. Addition of the extended 24-residue C-terminus of MotB to the C-terminus of MotD does not significantly affect either motility or compensate for the deleterious effect of {Delta}motY mutation, but does significantly increase motility in motY+ backgrounds. The soft agar motility results for organisms with wild type MotAB stators was not different from those with MotAB stators carrying 24 residue MotB C-terminal deletions; however, motility of these mutants was significantly lower in {Delta}motY mutants compared to {Delta}motY mutants expressing wild type MotAB stators. We discuss contributions of stator functional domains to motility and the effects of {Delta}motY deletion. Lastly, we speculate on possible mechanistic roles for the two stator plug types based on thermodynamic considerations related to differences in composition of the hydrophobic surfaces of the two amphipathic helices. IMPORTANCEPseudomonas aeruginosa uses two torque-generating stators, MotAB and MotCD, to drive flagellar rotation. Dual stator bacteria have additional rotor components; in P. aeruginosa, this component has been identified as MotY. This study investigates the interaction between the C-terminal plug and periplasmic regions of the MotB and MotD components of the MotAB and MotCD stator complexes with MotY. Motility assays of periplasmic chimeric strains expressing variants with MotB and MotD C-terminal plug and peptidoglycan binding domains reveal an enhanced sensitivity of MotD C-terminus to MotY deletions. These data suggest that critical interactions, either direct or indirect, must take place between the basal body MotY ring complex and MotD periplasmic regions for proper function of the MotCD stator in flagellar motility.

Pseudomonas aeruginosa is a well-known human pathogen that contributes significantly to chronic infections, particularly in cystic fibrosis (CF) patients. During this chronic infection, P. aeruginosa undergoes a phenotype change, the inactivation of mucA, which leads to the production of exopolysaccharide alginate, known as mucoid, a key virulence factor associated with biofilm formation. This mucoid phenotype allows the bacterium to persist in the lungs of CF patients for the duration of their lives. Previously, we identified ebselen oxide (EbO) as an inhibitor that suppresses alginate production in P. aeruginosa. In the current study, we synthesized a series of structural analogs based on EbO and ebselen (Eb) and evaluated their ability to inhibit alginate production. These analogs did have similar or lower inhibitory activity than EbO. The mechanism by which EbO inhibits alginate production remains unclear. We employed RNA sequencing analysis of P. aeruginosa treated with inhibitors and identified several candidate genes potentially involved in this inhibitory pathway. Interestingly, we observed that a transposon and in-frame deletion mutants of the candidate genes were defective for alginate production. These findings suggest there are additional requirements for optimal alginate production in conditions that mimic the CF lung beyond the algD-A operon. IMPORTANCEWhen bacteria encounter the correct conditions, they can dedicate their energy toward a specific function to maximize the function. One example is the low calcium response in Yersinia pestis in which the bacteria arrest growth when grown at 37 {degrees}C in the absence of calcium because it uses all its energy for type III secretion. Another example is production of alginate by P. aeruginosa in the lungs of CF patients that can lead to occlusion of the airways. In both cases, the dedicated use of energy toward type III secretion for Y. pestis and alginate biosynthesis for P. aeruginosa reduces the ability of the bacteria to multiply. In the lab, suppressors can be easily identified that restore bacteria growth. The suppressor mutations are often located in the operons that are up-regulated and thereby prevent the execution of the energetically costly process. While these results indicate these processes are energetically costly, we still do not understand how the bacteria dedicate their energy toward these processes over other cellular processes such as growth. Previously, we identified ebselen oxide (EbO) as an inhibitor that suppresses alginate production in P. aeruginosa, but chemical analogues fail to improve the inhibitory activity. We used RNA sequencing analysis of P. aeruginosa treated with inhibitors and identified several candidate genes potentially involved in this inhibitory pathway. Interestingly, we observed that a transposon and in-frame deletion mutants of these genes involved in redox reactions were defective for alginate production. These findings suggest there proteins may shunt energy for optimal alginate production in conditions that mimic the CF lung beyond the algD-A operon. Results from P. aeruginosa alginate production may inform how other bacteria can similarly focus energy toward specific cellular processes.

Sporulation is an adaptive response to starvation in bacteria that consists of a series of developmental changes in cellular morphology and physiology, leading to the formation of a highly resistant endospore. In Bacillus subtilis, there is an intricate developmental program which involves the precise coordination of gene expression and ongoing morphological changes to yield the mature spore. The histone-like protein HBsu is involved in proper spore packaging and compaction of the chromosomal DNA. Previously, we found that the acetylation of different lysine residues on HBsu impairs sporulation frequency and spore resistance properties. One mechanism by which HBsu influences the process of sporulation could be through the regulation of gene expression. To test this idea, we performed RT-qPCR to analyze gene expression throughout the sporulation process in wildtype and seven acetylation-mimicking (glutamine substitutions) mutant strains. Acetylation of HBsu at K41 increased the expression of key early and late sporulation genes, especially during the later stages. For example, overexpression of {sigma}F and {sigma}G drive expression of their regulon members at inappropriate times. These findings suggest that K41 acetylation activates gene expression and might represent an "on-off" switch for important regulatory factors as cells transition from early to late phases. The gene expression profiles of hbsK3Q, hbsK37Q, hbsK75Q, hbsK80Q, and hbsK86Q mutants were largely unchanged, but did have significant reductions of key late sporulation proteins, which could explain the observed defects in spore resistance properties. We propose that acetylation of HBsu at specific sites directly regulates gene expression during sporulation and this is required for proper timing and coordination.

Type IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

Fis (Factor for Inversion Stimulation) is a global regulator that is highly expressed during exponential growth and undetectable in stationary growth. Quorum sensing (QS) is a global regulatory mechanism that controls gene expression in response to cell density and growth phase. In V. parahaemolyticus, a marine species and a significant human pathogen, the QS regulatory sRNAs, Qrr1 to Qrr5, negatively regulate the high cell density QS master regulator OpaR. OpaR is a positive regulator of capsule polysaccharide (CPS) formation required for biofilm formation and a repressor of swarming motility. In Vibrio parahaemolyticus, we showed, using genetics and DNA binding assays, that Fis bound directly to the regulatory regions of the qrr genes and was a positive regulator of these genes. In the {Delta}fis mutant, opaR expression was induced and a robust CPS and biofilm was produced, while swarming motility was abolished. Expression analysis and promoter binding assays showed that Fis was a direct activator of both the lateral flagellum laf operon and the surface sensing scrABC operon, both required for swarming motility. In in vitro growth competition assays, {Delta}fis was outcompeted by wild type in minimal media supplemented with intestinal mucus, and we showed that Fis directly modulated catabolism gene expression. In in vivo colonization competition assays, {Delta}fis was outcompeted by wild type, indicating Fis is required for fitness. Overall, these data demonstrate a direct role for Fis in QS, motility, and metabolism in V. parahaemolyticus. IMPORTANCEIn this study, we examined the role of Fis in modulating expression of the five-quorum sensing regulatory sRNAs, qrr1 to qrr5, and showed that Fis is a direct positive regulator of QS, which oppositely controls CPS and swarming motility in V. parahaemolyticus. The {Delta}fis deletion mutant was swarming defective due to a requirement for Fis in lateral flagella and surface sensing gene expression. Thus, Fis links QS and surface sensing to control swarming motility and, indirectly, CPS production. Fis was also required for cell metabolism, acting as a direct regulator of several carbon catabolism loci. Both in vitro and in vivo competition assays showed that the {Delta}fis mutant had a significant defect compared to wild type. Overall, our data demonstrates that Fis plays a critical role in V. parahaemolyticus physiology that was previously unexamined.

YabG is a sporulation-specific protease that is conserved among sporulating bacteria. C. difficile YabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in yabG were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabGC207A (catalytically inactive), C. difficile yabGA46D, C. difficile yabGG37E, and C. difficile yabGP153L strains germinated in response to TA alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the pre sequence from preproSleC. Interestingly, only YabGA46D showed any activity towards purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.

Bacterial surface sensing is often conferred by flagella. The flagellar motor protein FliL plays a key role in this process, but its exact role has been obscured by varying fliL mutant phenotypes. We reanalyzed results from studies on these fliL alleles and found they inadvertently compared mutants with differing length of the retained native N-terminal region, including the transmembrane helix (TM). We find that TM retention in the mutants that lack the native C-terminal domain results in loss of swimming and swarming motility, while alleles that completely lack the TM retain motility. We suggest FliL negatively regulates motor function via its N-terminal region, an observation that may relate to FliL function in mechanosensing.

Pseudomonas aeruginosa causes acute and chronic infections such as those that occur in the lungs of people with cystic fibrosis (CF). In infection environments, oxygen (O2) concentrations are often low. The transcription factor Anr responds to low O2 by upregulating genes necessary for P. aeruginosa fitness in microoxic and anoxic conditions. Anr regulates dnr, a gene encoding a transcriptional regulator that promotes the expression of genes required for using nitrate as an alternative electron acceptor during denitrification. In CF sputum, transcripts involved in denitrification are highly expressed. While Dnr is necessary for the anoxic growth of P. aeruginosa in CF sputum and artificial sputum media (ASMi), the contribution of denitrification to P. aeruginosa fitness in oxic conditions has not been well described. Here we show that P. aeruginosa requires dnr for fitness in ASMi and the requirement for dnr is abolished when nitrate is excluded from the media. Additionally, we show that P. aeruginosa consumes nitrate in lysogeny broth (LB) under microoxic conditions. Furthermore, strains without a functioning quorum sensing regulator LasR, which leads to elevated Anr activity, consume nitrate in LB even in normoxia. There was no growth advantage for P. aeruginosa when nitrate was present at concentrations from 100 {micro}M to 1600 {micro}M. However, P. aeruginosa consumption of nitrate in oxic conditions created a requirement for Dnr and Dnr-regulated NorCB likely due to the need to detoxify nitric oxide. These studies suggest that Anr- and Dnr-regulated processes may impact P. aeruginosa physiology in many common culture conditions. ImportancePseudomonas aeruginosa is an opportunistic pathogen commonly isolated from low-oxygen environments such as the lungs of people with cystic fibrosis. While the importance of P. aeruginosa energy generation by denitrification is clear in anoxic environments, the effects of denitrification in oxic cultures is not clear. Here, we show that nitrate is consumed even in oxic environments and while it does not appear to stimulate growth, it does impact fitness. Further, we report that two regulators that are best known for their roles in anoxic conditions also contribute to P. aeruginosa fitness in commonly- used laboratory media in presence of oxygen.

Clostridioides difficile is an important nosocomial pathogen that has been classified as an "urgent threat" by the CDC. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts healthy protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. Nutrient availability and other environmental factors greatly influence toxin production in C. difficile. CodY is a global transcriptional regulator that coordinates metabolism and virulence in Gram-positive pathogens in response to nutrient availability, primarily through sensing branched-chain amino acids (isoleucine, leucine, valine; ILV) and GTP. In C. difficile, CodY indirectly represses toxin production by inhibiting transcription of the positive regulator tcdR and influences sporulation through unknown mechanisms. Here, we characterized two naturally occurring CodY variants, CodY(Y146N) and CodY(V58A). The Y146N substitution is located near the GTP-binding pocket, while V58A is near the ILV-binding site. Ligand binding assays revealed GTP binding ability of CodY(Y146N) is severely compromised. Electrophoretic mobility shift assays (EMSAs) demonstrated that ligand binding differentially influenced promoter binding; in the presence of GTP, CodY(Y146N) bound to the tcdR promoter less efficiently than CodY(WT). In C. difficile, production of either variant resulted in reduced repression of toxin production compared to CodY(WT). Subsequent in vivo experiments in a hamster infection model showed that strains producing CodY(Y146N) or CodY(V58A) were significantly more virulent than the CodY(WT) producing strain. These findings demonstrate that a single amino acid change in this global regulator can alter its ligand affinity and promoter-binding properties to potentially rewire the gene regulatory networks to enhance the pathogenic potential in C. difficile. ImportanceC. difficile has been recognized as an important nosocomial pathogen that causes diarrheal disease as a consequence of antibiotic exposure. CodY controls the expression of numerous metabolic and virulence genes. In this study, we have characterized two CodY variants and have demonstrated that even a single residue change in important domains can affect its function and have implications on bacterial virulence.

Lytic transglycosylases function to degrade peptidoglycan strands that comprise the bacterial cell wall. Degradation of peptidoglycan at the septum following cell division is necessary for cell separation, and a deletion of ltgC in Neisseria gonorrhoeae results in growth in clusters of around 6-20 cells rather than as normal diplococci or monococci. N. gonorrhoeae LtgC is a homolog of Escherichia coli MltA, and comparison of the two proteins shows that LtgC has an extra domain not found in MltA, referred to as domain 3. To better understand the function of LtgC, we characterized N. gonorrhoeae mutants with substitutions in amino acids predicted to be necessary for enzymatic activity or amino acids predicted to be on the surface of domain 3, and we characterized a mutant lacking domain 3. All the mutants showed defects in cell separation, and the bacteria failed to release peptidoglycan-derived disaccharides into the medium. Purified LtgC proteins with the amino acid substitutions had reduced peptidoglycan degradation activity. LtgC was found to bind AmiC in bacterial 2-hybrid assays, and domain 3 mutations reduced binding. In human blood, an ltgC mutant showed decreased survival, suggesting the cell wall defects in the mutant make the bacteria more sensitive to innate immune system components. ImportanceNeisseria gonorrhoeae uses a smaller set of proteins for peptidoglycan breakdown compared to Escherichia coli or other model systems. The peptidoglycan breakdown that occurs at the septum following cell division in N. gonorrhoeae requires three proteins, amidase AmiC, amidase activator NlpD, and lytic transglycosylase LtgC. LtgC has an unusual structure that includes a third domain not found in related proteins. Using mutants that lacked LtgC activity or had amino acid changes in the third domain, we found that the extra domain is involved in interaction of LtgC with AmiC and that it is required for LtgC function for cell separation. All of the ltgC mutants examined showed reduced survival in blood, indicating the importance of LtgC activity for infection.

The TonB system of Escherichia coli couples the protonmotive force of the cytoplasmic membrane to active transport of nutrients across the outer membrane. In the cytoplasmic membrane, this system consists of three known proteins, TonB, ExbB, and ExbD. ExbB and ExbD appear to harvest the protonmotive force and transmit it to TonB, which then makes direct physical contact with TonB-dependent active transport proteins in the outer membrane. Using two-dimensional gel electrophoresis, we found that ExbD exists as two different species with the same apparent molecular mass but with different pIs. The more basic ExbD species was consistently present, while the more acidic species arose when cells were starved for iron by the addition of iron chelators. The cause of the modification was, however, more complex than simple iron starvation. Absence of either TonB or ExbB protein also gave rise to modified ExbD under iron-replete conditions where the wild-type strain exhibited no ExbD modification. The effect of the tonB or exbB mutations were not entirely due to iron limitation since an equally iron-limited aroB mutation did not replicate the ExbD modification. This constitutes the first report of in vivo modification for any of the TonB system proteins.

In Pseudomonas aeruginosa, alginate biosynthesis gene expression is inhibited by the transmembrane anti-sigma factor MucA, which sequesters the AlgU sigma factor. Cell envelope stress initiates cleavage of the MucA periplasmic domain by site-1 protease AlgW, followed by further MucA degradation to release AlgU. However, after colonizing the lungs of people with cystic fibrosis, P. aeruginosa converts to a mucoid form that produces alginate constitutively. Mucoid isolates often have mucA mutations, with the most common being mucA22, which truncates the periplasmic domain. MucA22 is degraded constitutively, and genetic studies suggested that the Prc protease is responsible. Some studies also suggested that Prc contributes to induction in strains with wild type MucA, whereas others suggested the opposite. However, missing from all previous studies is a demonstration that Prc cleaves any protein directly, which leaves open the possibility that the effect of a prc null mutation is indirect. To address the ambiguities and shortfalls, we reevaluated the roles of AlgW and Prc as MucA and MucA22 site-1 proteases. In vivo analyses using three different assays, and two different inducing conditions, all suggested that AlgW is the only site-1 protease for wild type MucA in any condition. In contrast, genetics suggested that AlgW or Prc act as MucA22 site-1 proteases in inducing conditions, whereas Prc is the only MucA22 site-1 protease in non-inducing conditions. For the first time, we also show that Prc is unable to degrade the periplasmic domain of wild type MucA, but does degrade the mutated periplasmic domain of MucA22 directly. IMPORTANCEAfter colonizing the lungs of individuals with cystic fibrosis, P. aeruginosa undergoes mutagenic conversion to a mucoid form, worsening the prognosis. Most mucoid isolates have a truncated negative regulatory protein MucA, which leads to constitutive production of the extracellular polysaccharide alginate. The protease Prc has been implicated, but not shown, to degrade the most common MucA variant, MucA22, to trigger alginate production. This work provides the first demonstration that the molecular mechanism of Prc involvement is direct degradation of the MucA22 periplasmic domain, and perhaps other truncated MucA variants as well. MucA truncation and degradation by Prc might be the predominant mechanism of mucoid conversion in cystic fibrosis infections, suggesting that Prc activity could be a useful therapeutic target.

Penicillin-binding proteins (PBPs) play critical roles in cell wall construction, cell shape, and bacterial replication. Bacteria maintain a diversity of PBPs, indicating that despite their apparent functional redundancy, there is differentiation across the PBP family. Seemingly redundant proteins can be important for enabling an organism to cope with environmental stressors. We sought to evaluate the consequence of environmental pH on PBP enzymatic activity in Bacillus subtilis. Our data show that a subset of B. subtilis PBPs change activity levels during alkaline shock and that one PBP isoform is rapidly modified to generate a smaller protein (i.e., PBP1a to PBP1b). Our results indicate that a subset of the PBPs are preferred for growth under alkaline conditions, while others are readily dispensable. Indeed, we found that this phenomenon could also be observed in Streptococcus pneumoniae, implying that it may be generalizable across additional bacterial species and further emphasizing the evolutionary benefit of maintaining many, seemingly redundant periplasmic enzymes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/533529v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@bf28c7org.highwire.dtl.DTLVardef@e2e88org.highwire.dtl.DTLVardef@1112259org.highwire.dtl.DTLVardef@1e604ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

Many bacteria use flagella to swim individually through bulk liquid, or swarm collectively over a semi-solid surface. In E. coli, c-di-GMP inhibits swimming via the effector protein YcgR. We show in this study that contrary to its effect on swimming, a certain threshold level of c-di-GMP is required for swarming. Gene expression profiles first indicated that several c-di-GMP synthases - dgcJ, dgcM, dgcO - were upregulated during swarming. Of these, we found DgcO to play a critical role. DgcO is reported to affect PGA (poly-{beta}-1,6-N-acetylglucosamine) synthesis in E. coli. We show that DgcO also promotes production of colanic acid - one of the three major exopolysaccharides in E. coli, and that colanic acid has hitherto-unknown surfactant properties that are expected to aid swarming. ImportanceIt is well-established that in bacteria, c-di-GMP inhibits flagella-driven motility at various points in the pathway. Concomitantly, elevated c-di-GMP levels induce the expression and synthesis of a variety of exopolysaccharides that enmesh the bacteria in a biofilm, thereby also interfering with flagella function. This study reports the surprising finding that in E. coli, the exopolysaccharide colanic acid is required to enable surface navigation and that the diguanylate cyclase DgcO is employed for this purpose. For surface navigation, there appears to be a sweet spot where c-di-GMP levels are just right to produce polysaccharides that can serve as surfactants and wetting agents rather than promote formation of biofilms.

Colanic acid and enterobacterial common antigen (ECA) are cell surface polysaccharides that are produced by many E. coli isolates. Colanic acid is induced under low pH, low temperature, and hyperosmotic conditions and is important in E. coli resistance to these stresses; however, the role of the ECA is unclear. In this study, we observed that knockout of the flippase wzxE, which converts ECA intermediates from the cytoplasmic side of the inner membrane to the periplasmic side, resulted in low pH sensitivity in E. coli. The wzxE-knockout mutant showed reduced growth potential and viable counts in the extracts of several vegetables (cherry tomatoes, carrots, celery, lettuce, and spinach), which are known to be low pH environments. A double knockout strain of wzxE and wecF, which encodes an enzyme that synthesizes an ECA intermediate, did not show sensitivity to low pH, nor did a double knockout mutant of wzxE and wcaJ, which encodes a colanic acid synthase. The wzxE-knockout mutant was sensitive to low temperature or hyperosmotic conditions, which induced colanic acid synthesis, and these sensitivities were abolished by the additional knockout of wcaJ. These results suggest that ECA intermediates cause E. coli susceptibility to low pH, low temperature, and high osmotic pressure in a colanic acid-dependent manner, and that wzxE suppresses this negative effect. ImportancePolysaccharides covering bacterial cell surfaces, such as colanic acid, confer resistance to various stresses, such as low pH. However, the role of enterobacterial common antigens, carbohydrate antigens that are conserved throughout enterobacteria, in stress resistance is unclear. Our results suggest that lipid III enterobacterial common antigen, a substrate of flippase, causes sensitivity of Escherichia coli to low pH, low temperature, and high osmolarity in dependence on colanic acid synthesis, while wzxE inhibits this negative effect. The wzxE-knockout mutant was sensitive to crude vegetable extracts, suggesting that the creation of WzxE inhibitors could lead to new food poisoning prevention agents.

Clostridioides difficile is a major gastrointestinal pathogen that is transmitted as a dormant spore. As an intestinal pathogen, C. difficile must contend with variable environmental conditions, including fluctuations in pH and nutrient availability. Nutrition and pH both influence growth and spore formation, but how pH and nutrition jointly influence sporulation are not known. In this study, we investigated the dual impact of pH and pH-dependent metabolism on C. difficile sporulation. Specifically, we examined the impacts of pH and the metabolite acetoin on C. difficile growth and sporulation. We found that expression of the predicted acetoin dehydrogenase operon, acoRABCL, was pH-dependent and regulated by acetoin. Regulation of the C. difficile aco locus is distinct from other characterized systems and appears to involve a co-transcribed DeoR-family regulator rather than the sigma54-dependent activator. In addition, an acoA null mutant produced significantly more spores and initiated sporulation earlier than the parent strain. However, unlike other Firmicutes, growth and culture density of C. difficile was not increased by acetoin availability or disruption of the aco pathway. Together, these results indicate that acetoin, pH, and the aco pathway play important roles in nutritional repression of sporulation in C. difficile, but acetoin metabolism does not support cell growth as a stationary phase energy source. IMPORTANCEClostridioides difficile, or C. diff, is an anaerobic bacterium that lives within the gut of many mammals and causes infectious diarrhea. C. difficile is able to survive outside of the gut and transmit to new hosts by forming dormant spores. It is known that the pH of the intestine and the nutrients available both affect the growth and sporulation of C. diffiicile, but the specific conditions that result in sporulation in the host are not clear. In this study, we investigated how pH and the metabolite acetoin affect the ability of C. difficile to grow, proliferate, and form spores. We found that a mutant lacking the predicted acetoin metabolism pathway form more spores, but their growth is not impacted. These results show that C. difficile uses acetoin differently than many other species and that acetoin has an important role as an environmental metabolite that influences spore formation.

The bacterial cell wall is a covalently linked meshwork of peptidoglycan (PG) that establishes cell shape and prevents osmotic lysis. This structure must be flexible enough to accommodate transenvelope protein complexes, but strong enough to withstand high intracellular pressure. In order to elongate and divide, cells must remodel the cell wall through the concerted action of PG synthesis and degradation. Endopeptidases, a class of PG degrading enzymes, facilitate cell growth by hydrolyzing PG crosslinks. Vibrio cholerae encodes several functionally redundant endopeptidases, two of which are nearly identical: ShyA and ShyC. To investigate differential roles of these enzymes, we assessed growth and morphology of ShyA and ShyC mutants. We found that ShyA, but not ShyC, is required for normal adaptation to low osmolarity medium. Cells lacking ShyA exhibited longer lag phase and aberrant morphology during adaptation, and reduced survival in the presence of a beta-lactam antibiotic. Lastly, our experiments revealed that cells lacking ShyAs LysM domain exhibited more severe defects than cells lacking ShyA altogether, implicating the LysM domain in proper regulation of ShyA activity.

Pseudomonas aeruginosa have a versatile metabolism; they can adapt to many stressors, including limited oxygen and nutrient availability. This versatility is especially important within a biofilm where multiple microenvironments are present. As a facultative anaerobe, P. aeruginosa can survive under anaerobic conditions utilizing denitrification. This process produces nitric oxide (NO) which has been shown to result in cell elongation. However, the molecular mechanism underlying this phenotype is poorly understood. Our laboratory has previously shown that NosP is a NO-sensitive hemoprotein that works with the histidine kinase NahK to regulate biofilm in P. aeruginosa. In this study, we identify NahK as a novel regulator of denitrification under anaerobic conditions. Under anaerobic conditions, deletion of nahK leads to a reduction of growth coupled with reduced transcriptional expression and activity of the denitrification reductases. Further, during stationary phase under anaerobic conditions, {Delta}nahK does not exhibit cell elongation, which is characteristic of P. aeruginosa. We determine the loss of cell elongation is due to changes in NO accumulation in{Delta} nahK. We further provide evidence that NahK may regulate denitrification through modification of RsmA activity. ImportanceP. aeruginosa is an opportunistic multi-drug resistance pathogen that is associated with hospital acquired infections. P. aeruginosa is highly virulent, in part due to its versatile metabolism and ability to form biofilms. Therefore, better understanding of the molecular mechanisms that regulate these processes should lead to new therapeutics to treat P. aeruginosa infections. The histidine kinase NahK has been previously shown to be involved in both NO signaling and quorum sensing through RsmA. The data presented here demonstrate that NahK is responsive to NO produced during denitrification to regulate cell morphology. Understanding NahKs role in metabolism under anaerobic conditions has larger implications in determining Nahks role in a heterogeneous metabolic environment such as a biofilm.

Bacteria use chemotaxis to move to favorable ecological niches. For many pathogenic bacteria, chemotaxis is required for full virulence, particularly for the initiation of host colonization. There do not appear to be limits to the type of compounds that attract bacteria, and we are just beginning to understand how chemotaxis adapts them to their lifestyles. Quantitative capillary assays for chemotaxis show that P. aeruginosa is strongly attracted to serotonin, dopamine, epinephrine, and norepinephrine. Chemotaxis to these compounds is greatly decreased in a mutant lacking the TlpQ chemoreceptor, and complementation of this mutant with a plasmid harboring the tlpQ gene restores wild-type-like chemotaxis. Microcalorimetric titrations of the TlpQ sensor domain with these four compounds indicate that they bind directly to TlpQ. All four compounds are hormones and neurotransmitters that control a variety of processes and are also important signal molecules involved in the virulence of P. aeruginosa. They modulate motility, biofilm formation, the production of virulence factors and serve as siderophores that chelate iron. Therefore, chemotaxis to these four compounds is likely to alter P. aeruginosa virulence. Additionally, we believe that this is the first report of bacterial chemotaxis to serotonin and dopamine. This study provides an incentive for research to define the contribution of chemotaxis to these host signaling molecules to the virulence of P. aeruginosa.