Journal of Bacteriology

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.

Type IVa pili (T4aP) are bacterial surface appendages that perform various functions including twitching motility, surface attachment, cell-cell interactions, and DNA uptake for natural transformation. Pivotal to each of these functions is the ability of T4aP to be dynamically extended and retracted from the cell surface. However, the factors that regulate this dynamic activity remain poorly understood. To address this question, we employ the competence T4aP from Vibrio cholerae as a model system. T4aP are composed of major and minor pilin subunits, named based on their relative abundance in the pilus filament. Prior work has established that minor pilins form a complex that initiates T4aP assembly. This allows for the subsequent addition of major pilins to the filament, which promotes T4aP extension. Here, we uncover that the stoichiometry of minor-to-major pilins is a crucial determinant of T4aP dynamic activity. Specifically, we show that either (1) overexpressing minor pilins or (2) underexpressing the major pilin results is a dramatic increase in the frequency of T4aP dynamics. These results indicate that the stoichiometry of major-to-minor pilins, not their absolute abundance, is one mechanism that regulates T4aP dynamic activity. AUTHOR SUMMARYType IVa pili (T4aP) are a broadly conserved family of filamentous bacterial appendages that help bacteria colonize surfaces, move towards or away from stimuli, and gain new traits through a mechanism of horizontal gene transfer called natural transformation. T4aP are primarily composed of protein subunits called major and minor pilins, named based on their relative abundance in the pilus filament. Bacteria can dynamically extend and retract pilus filaments from their surface through polymerization and depolymerization of these pilins. This dynamic activity is critical for the activities that T4aP carry out. However, the factors that regulate this dynamic activity remain incompletely understood. Here, we find that the ratio of minor-to-major pilins is one factor that regulates the frequency of dynamic activity. Minor pilins are a universally conserved feature of T4aP. So, the minor-to-major pilin ratio may be a broadly conserved mechanism for controlling dynamic T4aP activity in diverse bacterial species.

The bacterial peptidoglycan (PG) cell wall, a polymer made of amino-acid-bearing glycan strands, maintains cell shape, provides structural integrity, and protects against osmotic lysis. PG maintenance is an active process that requires regulated PG breakdown to make space for insertion of new PG strands. PG breakdown is accomplished by autolysins, i.e. endogenous enzymes with cell wall cleavage activity. The lytic transglycosylases (LTGs), a class of autolysins, for example, cleave glycan strands during PG remodelling. LTGs are broadly conserved and are highly redundant in bacteria, but their physiological role is poorly-defined. In this study, we interrogated physiological consequences of LTG insufficiency in Vibrio cholerae using TnSeq to gain insights about roles of these enzymes. We identify an uncharacterized transcription factor, Vca0578, which alleviates defects associated with the {Delta}6LTG mutant. We demonstrate that Vca0578 positively regulates the expression of zapC, a typically non-essential Z-ring associated protein. In the absence of zapC, cell division was impaired during perturbations of cell envelope homeostasis caused by absence of LTGs, or by exposure to antibiotics inhibiting cell elongation; either condition rendered zapC conditionally essential. This essentiality could be overcome by increasing FtsZ levels. Lastly, we found that ZapC also contributes to both width and length homeostasis during normal growth. This work thus uncovers a novel transcriptional circuit that contributes to effective cell division in{Delta} 6LTG cells, and suggests an essential role for ZapC in cell division under stress conditions that cause perturbation of cell width homeostasis. AUTHOR SUMMARYBacteria must maintain their outer shell (the cell envelope) in the face of changes in the environment. For this, they use elaborate systems that remodel the cell envelope. How some of these systems work is not well understood. In this study, we describe a new gene circuit that is required to keep cells dividing when the cell envelope is compromised. We found that Vca0578, a putative transcription factor, controls expression of the zapC gene. The protein ZapC then helps bacteria grow and divide when the cell envelope is under stress, for example, in the presence of certain antibiotics. Thus, we have discovered a regulatory circuit that promotes bacterial growth and antibiotic resistance under stress.

The phosphodiesterase (PDE) NbdA (NO-induced biofilm dispersion locus A) consists of a membrane-integrated MHYT domain, a degenerated diguanylate cyclase (DGC) AGDEF domain and an EAL domain. The integral membrane domain MHYT is proposed to sense a so far unknown extracellular signal and transfers the information to the cytosolic enzyme domains to modulate cellular c-di-GMP level. Here, we show that full length NbdA from Pseudomonas aeruginosa PAO1 is an active PDE in vivo. In line with its PDE activity, overexpression leads to slightly reduced global c-di-GMP levels, and reduced twitching motility. Surprisingly, overexpression of truncated cytosolic NbdA variants exhibited increased c-diGMP levels, suggesting previously uncharacterized DGC activity despite lacking a canonical GGDEF motif. While full-length NbdA overexpression resulted in only slight c-di-GMP reduction, cytosolic variants induced a significant increase, indicating a potential for nonenzymatic effects like protein-protein interactions. Further investigation revealed a connection between NbdA and type IV pilus (T4P) function. Overexpression of NbdA conferred resistance to the T4P-dependent phage DMS3vir, suggesting interference with T4P assembly or function. Microscopic analysis demonstrated dynamic localization of NbdA, partially co-localizing with T4P components, supporting a role in T4P regulation. However, no clear link was re-established with flagellar motor switching or chemotaxis signaling. These findings position NbdA in the complex signaling network of c-diGMP and T4P-mediated surface behavior in P. aeruginosa. Future work will focus on elucidating the precise mechanisms of NbdAs PDE activity and its interplay with other DGC/PDE networks. ImportanceIn this work, we show the in vivo activity of the membrane-bound phosphodiesterase NbdA of Pseudomonas aeruginosa, its role in c-di-GMP homeostasis, cellular localization and implications in surface behavior. Using strains overexpressing NbdA and truncated protein variants, we detected a strong defect in growth on solid surfaces and an altered phage susceptibility. Co-localization experiments supported further the hypothesis of interaction with the type IV pilus apparatus. We propose for NbdA to be part of the protein network responsible for c-di-GMP level modulation at the cell pole and thereby regulating the function of type IV pilus apparatus.

The envelope of Gram-negative bacteria like Escherichia coli is multilayered with two membranes sandwiching a peptidoglycan cell wall. The inner membrane is a typical phospholipid bilayer whereas the outer membrane is asymmetric with phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. We recently discovered that inactivation of the conserved peptidoglycan synthesis machinery responsible for cell elongation causes defects in both peptidoglycan and LPS synthesis in E. coli. This finding suggests that the isolation of suppressors that rescue the growth phenotype caused by an impaired cell elongation system is an attractive means of identifying factors involved in coordinating the biogenesis of different envelope layers. Here, we report the results of a global, transposon sequencing-based screen for such suppressors. The inactivation of a number of factors including the phospholipid synthesis enzyme PlsX was found to partially suppress the growth defects of a cell elongation mutant. Deletion of plsX also conferred increased resistance to CHIR-090, an inhibitor of the committed step of LPS synthesis catalyzed by LpxC, suggesting that loss of PlsX function stimulates LPS synthesis. Evidence is presented that increased CHIR-090 resistance is not mediated by changes in the activity of the proteolytic system (YejM-LapB-FtsH) controlling LpxC turnover. Rather, our results are consistent with a model in which the phospholipid precursor acyl-phosphate produced by PlsX serves as an inhibitor of LpxC to lower the rate of LPS synthesis when phospholipid synthesis capacity is reduced. IMPORTANCEOver the last several decades, most proteins essential for Gram-negative cell surface assembly have been characterized. However, relatively little is known about how the synthesis of different envelope layers is coordinated to promote uniform surface growth. Here, we report the results of a transposon sequencing-based genetic screen for mutants that suppress defects in the conserved peptidoglycan synthesis machinery responsible for cell elongation. Inactivation of the plsX gene encoding a phospholipid synthesis enzyme was found to both suppress the growth defect of a cell elongation mutant and to confer elevated resistance to an inhibitor of lipopolysaccharide synthesis. Our results suggest the attractive possibility that the product of PlsX, acyl-phosphate, may play a regulatory role in coordinating the phospholipid and lipopolysaccharide synthesis pathways.

The CenIR regulatory system of Clostridioides difficile comprises a predicted transcriptional repressor, CenI, and a predicted membrane metalloprotease, CenR. The physiological role of CenIR and activating signal(s) are not known. CenIR belongs to the BlaIR family of regulators that mediate resistance to {beta}-lactam antibiotics. In canonical BlaIR systems, binding of a {beta}-lactam to the extracellular transpeptidase domain of BlaR triggers proteolysis of BlaI and thus induction of a closely linked {beta}-lactamase gene. However, CenR lacks a {beta}-lactam-binding domain and transposon mutagenesis indicated CenI is essential for viability even when {beta}-lactams are not present. Here we confirmed essentiality of CenIR and determined its regulon contains [~]12 genes, including an exported protein of unknown function (CDR_0474) that is induced about 500-fold and a peptidoglycan hydrolase (Cwp6) that is induced about 7-fold when cells are depleted of CenIR. There are no essential genes or {beta}-lactamases in the regulon. Phenotypic characterization of CenIR-depletion strains revealed slower growth, mild elongation and cell lysis. Deletion of cdr_0474 corrected all three defects, while deletion of cwp6 only rescued the lysis phenotype. It was possible to delete cenIR in either a {Delta}cdr_0474 or {Delta}cwp6 background. We propose that CenIR is essential because its absence leads to lysis due to Cwp6 overproduction. Bioinformatic analyses revealed the predicted extracellular sensing domains in annotated "BlaR" proteins are diverse. Thus, BlaIR systems are not dedicated to defense against {beta}-lactams but probably enable bacteria to adapt to a variety of environmental stimuli. ImportanceMany of the regulatory systems for controlling cell envelope biogenesis and stress responses have yet to be studied. Here we characterize a Clostridioides difficile BlaIR-like regulatory system that we have named CenIR for cell envelope. Unlike canonical BlaIR systems, which bind {beta}-lactams and induce a {beta}-lactamase, CenIR lacks a {beta}-lactam binding domain and is essential for viability even in the absence of antibiotics. We identified the genes in the regulon and found that CenIR is essential because its absence leads to overproduction of the Cwp6 peptidoglycan hydrolase. We also show that most annotated BlaIR-like systems lack a {beta}-lactam-binding domain, from which we infer that these systems have much broader physiological roles than generally appreciated.

Type IV pili and type II secretion systems assemble dynamic fibers used by bacteria and archaea for diverse functions. The pilus fiber is made up of major and minor pilin subunits containing a hydrophobic -helical spine and a globular head. Purifying minor pilins is complicated by the hydrophobic -helical spine, frequently present disulfide bonds, and low abundance within the fiber. These challenges have impeded structural and functional studies of pilin protomers. Here, we describe a method for expression and purification of soluble type IV pilin proteins from Escherichia coli. Signal peptidase I cleavage sites are engineered into the -helix of the pilin proteins. This allows their globular domains to be purified from the periplasmic fraction. We used this method to obtain the Neisseria gonorrhoeae minor pilins PilI and PilK in soluble form. In a third case, where the minor pilin PilJ could not be obtained on its own, coexpression with PilI and purification of a PilI-PilJ heterodimer was possible. We suggest that PilI and PilJ form an obligate heterodimer that is essential for their function. ImportanceType IV pili are essential to many bacteria responsible for disease. They can be found in both Gram-negative and Gram-positive bacteria, as well as archaea, making them likely present in the last common ancestor of all life on Earth. Despite their significance in a variety of species, there are large gaps in our understanding of the structure of these diverse biological machines. One roadblock to this research has been the difficulty of purifying the minor pilin proteins that serve different functions in the fiber. Here, we describe a novel method for the purification of these proteins and demonstrate the ability of this method to identify a protein-protein interaction between two minor pilins of Nesseria gonorrhoeae.

Glutathione (GSH) peroxidases are conserved enzymes found in prokaryotic and eukaryotic organisms that reduce H2O2 to protect cells from oxidative stress damage. In this study, we identified and characterized the GpoA glutathione peroxidase of S. pneumoniae, one of the most important human bacterial pathogens. This bacterium is unable to synthesize endogenous GSH as a cofactor for this enzyme but acquires GSH from host tissues via the GshT transporter. We demonstrated that recombinant GpoA exhibits GSH peroxidase activity and that the Cys36 residue is essential for this function. The gpoA transcripts, as well as tpxD and ahpD, which encode the TpxD thiol peroxidase and the AhpD alkylhydroperoxidase, respectively, were upregulated when pneumococci were exposed to H2O2. The{Delta} gpoA, {Delta}tpxD, {Delta}ahpD, and{Delta} gshT mutants exhibited increased susceptibility to H2O2, and also impaired intracellular survival in pneumocytes, macrophages, and neutrophils compared to the wild-type strain. These findings indicate that GpoA, TpxD, and AhpD constitute a robust H2O2 detoxification system that depends on extracellular GSH uptake. These three peroxidases also contribute to the fluoroquinolone persistence mechanism, which is closely associated with the oxidative stress response in S. pneumoniae. Additionally, we investigated the effect of GpoA on virulence in a murine model. The {Delta}gpoA mutant exhibited diminished survival across multiple organs relative to the wild-type strain, suggesting that GpoA contributes to pneumococcal pathogenesis. The molecular, biochemical, and functional analysis of GpoA elucidates an effective bacterial mechanism that incorporates the TpxD and AhpD peroxidases alongside the GshT transporter, promoting extracellular and intracellular survival under oxidative stress conditions.

To establish infection, uropathogens must overcome several host defenses including the glycosaminoglycan (GAG) layer coating the apical surface of the bladder urothelium. GAGs are thought to protect against urinary tract infection (UTI) by serving as scaffolding sites for commensals, providing barrier function and preventing uropathogen adherence. However, the ability of uropathogens to degrade and utilize GAGs and the contribution of these activities toward UTI progression is largely unknown. We previously discovered that the uropathogen Proteus mirabilis, a common cause of catheter-associated UTI (CAUTI), degrades the GAG chondroitin sulfate (CS). In this study we sought to define the kinetics and regulation of CS degradation by diverse P. mirabilis strains clinically isolated from both recurrent UTI and CAUTI patients. We found variation in CS degradation kinetics between P. mirabilis strains and media types. However, CS degradation depended on conserved putative chondroitin sulfate ABC endo- and exolyases in all strains. Furthermore, we found that CS degradation in Pm123 was repressed by urea and that this repression was dependent on P. mirabilis urease activity. Complementation of the Pm123 endolyase into urea-insensitive HI4320 resulted in a urea-sensitive CS degradation phenotype suggesting functional differences between the Pm123 and HI4320 endolyases. Sequence alignment and structural modeling analysis identified two unique point mutations within the Pm123 endolyase that may contribute to urea sensitivity. Finally, unlike urea-insensitive P. mirabilis strains, Pm123 demonstrated attenuated swarming and loss of chondroitin endolyase activity had no effect on Pm123 virulence in a mouse CAUTI model. Our results suggest that the kinetics and regulation of CS degradation differ between P. mirabilis strains and in urea-sensitive strains, thus reduces the contribution of CS degradation to urovirulence during murine CAUTI. ImportanceThis work demonstrates that the ability to degrade a common component of bladder mucosal surfaces, chondroitin sulfate, is a phenotype that is shared by multiple strains of the common catheter-associated UTI (CAUTI) pathogen P. mirabilis. We find that this activity is dependent on encoded chondroitin ABC endo- and exolyases, first described in Proteus vulgaris. Additionally, we discovered that for P. mirabilis strain Pm123, degradation of CS is negatively regulated by the presence of urea, a major component of urine. The repression of CS degradation by urea is dependent on the activity of the P. mirabilis urease enzyme, which breaks down urea producing ammonia which raises pH. We found expression of the Pm123 CS endolyase was sufficient to confer a urea-sensitive CS-degradation phenotype and identified two unique mutations within the Pm123 enzyme that may contribute to urea sensitivity. Finally, we find that while CS-degradation plays a role in progression and severity of murine CAUTI model in urea-insensitive P. mirabilis, there was not significant difference in CAUTI outcomes between the urea-sensitive Pm123 wild-type and chondroitinase knockout strains. This study represents a major step forward in understanding the diversity of CS degradation activity and regulation among clinical strains of the critically important CAUTI pathogen P. mirabilis as well as its contribution to urovirulence.

The unique chlamydial developmental cycle comprises three stages: primary differentiation of infectious elementary bodies (EBs) into reticulate bodies (RBs), RB replication, and secondary differentiation into progeny EBs. Extensive chromosome remodeling during RB-to-EB differentiation is thought to be mediated by the histones HctA and HctB. Here, we used an inducible CRISPR interference system to repress hctA, hctB, or both genes during development in Chlamydia trachomatis. Surprisingly, repression of either histone gene alone or in combination caused only modest reductions in EB yield and did not prevent nucleoid condensation during the parental developmental cycle. In contrast, when progeny EBs generated under histone-repressing conditions were used to initiate secondary infections in the absence of inducer, histone deficiency during EB maturation profoundly impaired fitness in the next infection cycle. Secondary cultures initiated with HctA-deficient EBs exhibited a delayed onset of genome replication, consistent with inefficient primary EB-to-RB differentiation, whereas combined repression of hctA and hctB caused both delayed genome replication and persistently reduced genome accumulation, indicative of defects in RB formation and subsequent growth. Repression of hctB alone did not measurably affect genome replication in secondary cultures. Together, these findings reveal a transgenerational role for chlamydial histones and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles. IMPORTANCEChlamydial histones HctA and HctB are unusual among bacterial chromatin-binding proteins in that they share sequence homology with mammalian histones and are developmentally regulated during the formation of infectious particles. Here, we show that reduced expression of HctA and HctB has only modest effects on genome condensation and EB production, consistent with partial functional redundancy between the two histones and suggesting that additional chromatin factors contribute to EB chromosome compaction. In contrast, deficiency of HctA and HctB during EB maturation has profound consequences in the next infection cycle, impairing primary EB-to-RB differentiation and subsequent RB growth. These findings reveal a previously unrecognized transgenerational role for chlamydial histones and establish chromosome organization during EB maturation as a key determinant of developmental fitness across infection cycles.

Pseudomonas aeruginosa, an opportunistic pathogen, uses a trio of quorum sensing (QS) systems to regulate the production of some virulence factors. Two of these, the las and rhl systems, involve acyl-homoserine lactone signals; the third, called pqs, primarily uses the signal 2-heptyl-3-hydroxy-4(1H)-quinolone ("PQS"). We aimed to identify how interbacterial interactions are regulated between P. aeruginosa and Stenotrophomonas maltophilia, which co-occur in the airways of people with cystic fibrosis. We explored P. aeruginosa and S. maltophilia interactions using a co-culture model. S. maltophilia in co-culture with P. aeruginosa grows for 12 hours and thereafter exhibits a large decline in CFU, demonstrating that P. aeruginosa is killing S. maltophilia. Co-culture of S. maltophilia with P. aeruginosa deficient in las, rhl, or pqs QS resulted in greater S. maltophilia viability than co-culture with the wildtype. This inhibition was not attributable to las and rhl-regulated toxins. Therefore, we interrogated the role of PQS and found that co-culture of S. maltophilia with P. aeruginosa deficient in PQS biosynthesis showed similar CFUs to monoculture. Exogenous PQS did not complement this phenotype, suggesting that another quinolone is the effector. We found that S. maltophilia killing is reduced in competition with a mutant that cannot make the quinolone HQNO. We show that full killing of S. maltophilia by P. aeruginosa requires three components: HQNO, the chaperone PqsE, and intact PQS biosynthesis. Our work identifies quinolone biosynthesis as a driver for interactions between P. aeruginosa and S. maltophilia and, more generally, in modulating interbacterial interactions.

Bacterial cell division is a tightly regulated process that is carried out by a complex of proteins called the divisome, which is assembled in a defined sequential order. Upon assembly the complex is allosterically activated, which stimulates cell wall synthesis at the division site. Bacteria inhibit division during DNA damage by blocking either divisome assembly or activation. While the regulation of cell division is known to be important during M. tuberculosis infection, little is known about mycobacterial mechanisms of divisome function and regulation in DNA damage. By using M. smegmatis as a model organism, we find here that divisome factor SepIVA is involved in septum initiation, and that it is recruited to the mid-cell by FtsQ but it is not a recruitment factor itself. We find that a sepIVA loss-of-function defect can be suppressed by overexpression of ftsW, supporting a role for SepIVA in activation of the divisome complex. When cell division is inhibited during DNA damage, we find that SepIVA is delocalized from the division site, while the septal localization of FtsZ, FtsQ and FtsW are not impacted. We also find that the interaction between FtsQ and SepIVA is inhibited during DNA damage. Our results suggest that SepIVA is a trigger factor for activation of cell division during normal growth, and show that the signaling to inhibit cell division during DNA damage involves inhibition of its interaction with FtsQ. IMPORTANCECell division is critical for bacterial cells to propagate and cause infection. Despite its importance, division is a dangerous process as it requires building and subsequently hydrolyzing new cell wall material, in a place where the chromosome typically resides. If cell division is done before chromosome segregation is completed, or if cell wall metabolism is improperly regulated, then cell death results. The divisome is a protein complex that regulates cell division - coordinating it with the status of the chromosome and ensuring that the cell wall metabolic enzymes are carefully controlled. Many of the proteins in the divisome complex are highly conserved across bacterial Phyla; however, the factor that stimulates the complex to activate and initiate cell wall synthesis is not widely conserved. Here, we study the activation of the divisome in Mycobacterium smegmatis, a model for cell physiology of Mycobacterium tuberculosis. We find evidence that SepIVA, a protein found only in Actinobacteria, is likely the factor that stimulates activation of the divisome in mycobacteria. We also show that the association of SepIVA with the divisome is blocked under DNA damage, when cell division is inhibited. These results provide a model for the regulation of cell division in mycobacteria in growth and stress, and also provide insights into how bacteria with different types of septa may regulate division differently.

Mycobacteria form rough and smooth colonies. The Mycobacterium marinum strain 1218S is a smooth colony forming variant isolated from the 1218R strain, which forms rough colonies and is more virulent than 1218S in infecting fish. Genes for the type VII secretion ESX-1 system, which includes mycobacterial virulence genes, have been partially duplicated in M. marinum and is refered to as ESX-6. We recently reported that several ESX-1 genes are missing in the 1218S strain. On the basis of the complete genomes of these two and three other M. marinum strains we provide insight into strain differences and similarities focusing on 1218R and 1218S, and ESX genes, selected virulence genes, and LOS genes, which are involved in lipooligosaccharide synthesis and smooth colony formation. We provide RNA-Seq data for 1218R and 1218S and two other well-characterized M. marinum strains suggesting that loss of ESX-1 genes in 1218S results in increased transcript levels of ESX-6 genes. Furthermore, while there is no difference in gene synteny and sequence of LOS genes comparing 1218R and 1218S, with the exception of duplication of lsr2, a regulator of LOS genes, in 1218S. Our RNA-Seq data show increased transcript levels of LOS genes in stationary 1218S cells relative to 1218R indicating that transcription and/or RNA degradation of LOS genes influence smooth and rough colony formation. We finally provide data suggesting that Ms1 RNA affect the transcription of LOS genes (and ESX-1 genes), and that loss of ESX-1 genes influence biofilm formation.

Glycosaminoglycans (GAGs) are negatively charged polysaccharides composed of repeating disaccharide units and are essential components of the extracellular matrix throughout numerous tissues. The bladder urothelium has a thick protective GAG layer that primarily consists of chondroitin sulfate (CS), heparan sulfate (HS), and hyaluronic acid (HA), and urinary tract pathogens must either degrade or otherwise circumvent this layer to infect the urothelium. In this study, we investigated GAG degradation by Proteus mirabilis, a common and persistent colonizer of the catheterized urinary tract. Almost all P. mirabilis urinary tract isolates harbor a putative chondroitin endolyase (PMI2127), exolyase (PMI2128), and sulfatase (PMI2124). By generating mutant and complemented strains of these genes, we determined that P. mirabilis strain HI4320 degrades multiple forms of CS under numerous culture conditions, including during growth in human urine, and can use CS degradation products as a carbon source. Sulfatase and endolyase activity were required for efficient degradation of all CS types, while the exolyase only contributed to using CS-B or CS-C as carbon source. Interestingly, only endolyase activity contributed to colonization in a murine model of CAUTI, although the colonization defect was even more pronounced when the endolyase and exolyase were both disrupted. The colonization defect was specific to the CAUTI model, likely due to the impact of catheterization on the GAG landscape of the bladder. Limiting CS degradation by P. mirabilis may therefore represent a strategy for reducing risk of ascending infection in catheterized patients. ImportanceGlycosaminoglycans (GAGs) are a family of negatively charged heteropolysaccharides that are ubiquitously expressed throughout the body, forming a significant component of the extracellular matrix and a luminal GAG layer in the bladder. This GAG layer functions as a physical barrier for the bladder surface, protecting it from bacterial infection. Disruption of this barrier through physical forces, such as catheter insertion, or enzymatic degradation by bacteria may contribute to infection outcomes. In this study, we defined the contribution of three putative chondroitin sulfate degrading enzymes (PMI2124, PMI2127, PMI2128) to the pathogenesis of a common pathogen in the catharized urinary tract, Proteus mirabilis. We found that P. mirabilis can utilize chondroitin sulfate as a carbon source, and that chondroitin sulfate degradation contributes to infection in a model of catheterized urinary tract infection. This work contributes to a growing understanding of how uropathogens subvert host defenses and acquire nutrients within the bladder.

Autoinducer-2 (AI-2) is a LuxS-dependent product of the activated methyl cycle (AMC) that functions as a quorum-sensing signal in diverse bacteria. Fusobacterium nucleatum is a genetically heterogeneous oral anaerobe comprising four subspecies: nucleatum (FNN), vincentii (FNV), polymorphum (FNP), and animalis (FNA). Previous studies have reported that FNN and FNP strains produce AI-2 and have proposed that AI-2-mediated quorum sensing contributes to biofilm formation and virulence. However, the distribution and functional relevance of AI-2 across all subspecies have not been systematically examined. Here, we show that AI-2 production is restricted to FNA strains. Genomic analysis revealed that FNN and FNV lack luxS, whereas FNP carries a disrupted luxS homolog. Consistent with these findings, AI-2 bioassays using the Vibrio harveyi BB170 reporter detected AI-2 exclusively in FNA strains. Deletion of luxS in FNA abolished AI-2 production but resulted in minimal transcriptional changes, and exogenous AI-2 failed to elicit global gene expression responses in non-producing subspecies. These results demonstrate that AI-2 production in F. nucleatum is subspecies-specific and uncoupled from quorum sensing. Our findings revise current assumptions regarding AI-2-mediated communication in F. nucleatum and reveal previously unrecognized metabolic divergence within the species complex. IMPORTANCEPeriodontitis affects nearly half of adults in the United States and remains a leading cause of tooth loss worldwide. Fusobacterium nucleatum is a central member of oral biofilms and has also been linked to adverse pregnancy outcomes and colorectal cancer. Although AI-2-mediated quorum sensing has been proposed to contribute to its biofilm formation and virulence, our study demonstrates that AI-2 production is confined to subsp. animalis and is absent in other subspecies. Moreover, AI-2 does not function as a conserved quorum-sensing regulator in this species. These findings fundamentally revise prevailing assumptions about AI-2 signaling in F. nucleatum and suggest that subspecies-specific metabolic traits, rather than universal quorum sensing, may underlie ecological adaptation and host association.

The release of extracellular vesicles (EV) is a universally conserved process. Bacterial EVs package diverse cargo, including proteins and nucleic acids, and influence bacterial adaptation and survival as well as host-pathogen interactions. Currently, our understanding of the mechanisms underlying global principles in Gram-positive EV biogenesis and release is limited, partly due to labor-intensive vesicle isolation and assessment methods. Here, we describe a moderately high-throughput approach to analyze the Nebraska Transposon Mutant Library to identify genetic determinants of EV production in S. aureus. We show that the agr quorum sensing system dictates EV production in response to nutrient availability, likely through communication with the adaptive stress response. This study demonstrates the contribution of nutritional stress to vesiculogenesis and supports a conserved communication strategy that allows metabolic state to influence EV production.

Lig E is a periplasm-targeted ATP-dependent DNA ligase found in many Gram-negative bacteria including Neisseria gonorrhoeae. Although Lig E has been shown to have a role in biofilm formation, many Lig E-possessing bacteria are also naturally competent, suggesting a possible function in transformation with extracellular DNA. Here, we demonstrate that Lig E participates in bacterial competence by increasing transformation with nuclease-damaged extracellular DNA that contains single-stranded or cohesive breaks. We show that increased transformation with this restricted DNA is ATP-dependent, and that the ATP concentration increases in the extracellular milieu during maintenance of N. gonorrhoeae in liquid culture. Impact StatementNatural transformation is an important route of horizontal gene transfer that enables competent bacteria to acquire novel phenotypic traits such as antibiotic resistance or virulence factors. By demonstrating that Lig E increases transformation of N. gonorrhoeae with damaged resistance-encoding DNA, we provide a mechanism which competent bacteria can use to overcome nucleolytic damage sustained by environmental DNA, making this more readily available as a source of novel and potentially pathogen-enhancing genes.

Helicobacter pylori is a prevalent bacterial pathogen that chronically colonizes the human gastric epithelium, but the bacteriums physiological mechanisms that promote this are understudied. Dormancy and low growth are known to facilitate other microbial chronic infections. A critical feature of low growth states is the down regulation of ribosome translational activity via regulation factors. The H. pylori genome is predicted to encode only one ribosome regulation factor, called RsfS (Ribosomal Silencing Factor S). In other bacterial species, RsfS prevents ribosome assembly by binding to a protein called L14 on the 50S large ribosomal subunit. Although H. pylori RsfS has not been experimentally investigated prior to this work, it conserves key residues, suggesting it is a bona fide RsfS homolog. To investigate phenotypes associated with rsfS, the gene was deleted and mutant phenotypes characterized. H. pylori rsfS null mutants had no defects during exponential phase but had viability defects in stationary phase and low growth factor conditions. Additionally, rsfS null mutants could not form biofilms, and instead were only able to form monolayers of multicellular aggregates. These defects were corrected by the re-introduction of rsfS in a second site on the chromosome. To explore whether rsfS is required in vivo, a mouse model was employed. rsfS mutants initially colonized in low numbers in both the glands and total stomach but were unable to develop robust long-term colonization. This work supports that H. pylori requires RsfS for survival in low growth states and to maintain chronic infections in the host. ImportanceH. pylori chronic infections are difficult to cure in part because H. pylori is proposed to adopt low-growth states known to render bacteria tolerant to antibiotics. One key signature of a low growth state includes low translation via ribosome regulation factors. Unlike other bacterial species, H. pylori contain only one known ribosome regulation factor called Ribosomal Silencing Factor S (RsfS). This gene was previously found to be transcriptionally upregulated in at least one low growth state, biofilms. In this work, we found that H. pylori rsfS is required for this microbe to thrive in low growth states and during infection. This study is one of only two studies that investigates the phenotypes of rsfS knockout mutants in any bacterial species and the first to address knowledge gaps in ribosomal regulation by H. pylori in vivo.

2.Streptomyces bacteria have complex life cycles involving hyphal growth, sporulation, and the production of diverse specialised metabolites, including antibiotics. In this study, we investigated the role of the highly conserved orphan response regulator OrrA in Streptomyces venezuelae NRRL B-65442. We show that S. venezuelae {Delta}orrA mutants are defective in sporulation and, using ChIP-seq, identify five OrrA binding sites in vivo. Tandem-mass-tag proteomics revealed that OrrA directly activates two of these putative target genes, wblA and vnz_04640, a finding consistent with previous work on OrrA in the distantly related S. coelicolor. We also demonstrate that deleting wblA blocks sporulation and that overexpressing wblA restores sporulation in the {Delta}orrA mutant. Additionally, chloramphenicol biosynthesis is upregulated in both the {Delta}orrA and {Delta}wblA mutants compared with the wild type. Taken together, these results indicate that the primary function of OrrA is to regulate WblA production, and that reduced intracellular WblA levels underlie the phenotypes observed in the {Delta}orrA mutant. 3. Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article, through supplementary data files and via public databases.

Vibrio cholerae secretes a variety of effector proteins that are freely released into the extracellular space via its Type II Secretion System (T2SS) including cholera toxin, the causative agent of the disease cholera. In contrast to cholera toxin, a growing number of T2SS effectors is increasingly understood to remain associated with the cell surface. The serine protease VesB from V. cholerae is a surface protein that is produced with a short C-terminal motif, called GlyGly-CTERM. This motif is linked to the rest of VesB via a predicted unstructured linker. In addition to VesB, V. cholerae encodes five additional GlyGly-CTERM proteins including the serine proteases VesA and VesC, a putative metalloprotease VCA0065, the DNase Xds, and VC1485, a protein of unknown function. Proteins with a GlyGly-CTERM are co-distributed in bacteria with a specific rhomboid protease called rhombosortase (RssP), and it has been demonstrated that VesB requires processing by RssP for surface localization and activation. Here, we investigate the intrinsic function of the GlyGly-CTERM by proteomics, enzyme assays and heterologous expression of alternative motifs on model protein VesB as well as on unrelated periplasmic and extracellular proteins. We show that the GlyGly-CTERM and processing by RssP is sufficient for membrane association, but a secondary secretion signal is required for outer membrane translocation. Unexpectedly, VesC is released from the cells through autoproteolytic processing at a site within the unstructured linker. We propose that the GlyGly-CTERM facilitates efficient secretion of proteins via its intrinsic ability to target them to RssP resulting in membrane association. ImportanceVibrio cholerae is responsible for the disease cholera. Without treatment, V. cholerae causes massive dehydration with high mortality rates (1). It utilizes the Type II Secretion System to export the causative agent of disease, cholera toxin, as well as a suite of additional effector proteins that are involved in pathogenesis. Here, we investigate the unique transport mechanism of a subset of effectors secreted by this pathogen that are targeted to the cell surface.