Molecular Microbiology

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 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.

Like other cells, parasitic and other trypanosomatids sense and regulate Zn2+ transport, but the mechanisms involved remained unknown. Here we identify a trypanosome RNA-binding protein which specifically eliminates ZIP3 Zn2+-transporter mRNA in Zn2+-replete conditions. We first demonstrated that Trypanosoma brucei ZIP3 mRNA abundance is subject to 3-untranslated region (3-UTR) and Zn2+-dependent negative control. A genome-wide RNA interference library screen, using a reporter associated with the ZIP3 3-UTR, identified Tb927.11.9510 as a candidate Zn2+-sensor. We name this protein Zinc Nuclear Knuckles 1 (ZNK1) since it localises to the nucleus and contains several Zn2+-knuckle motifs. ZNK1 is conserved among trypanosomatids, and a PIN-domain suggests a ribonuclease-based mechanism. We validate ZNK1 as a ZIP3 3-UTR dependent negative regulator and identify a GU-repeat motif in the ZIP3 3-UTR that is predictive of negative control by ZNK1. We use Cas9-editing to knockout ZNK1, and RNA-seq to assess the consequences, revealing highly specific accumulation of ZIP3 transcripts in znk1-null cells. We conclude that ZNK1 senses Zn2+-abundance and eliminates ZIP3 mRNA in a Zn2+-dependent manner. We suggest that trypanosomatid ZNK1 is an RNA-specific zinc finger nuclease that binds ZIP3 3-UTRs and degrades ZIP3 mRNA only when the tandem sensor modules are coordinated with Zn2+. Key pointsO_LITrypanosome Zinc Nuclear Knuckles 1 (ZNK1) is a zinc-sensor that eliminates zinc transporter mRNA. C_LIO_LIZNK1 negative control operates via the transporter mRNA 3-untranslated region. C_LIO_LIThe findings indicate that trypanosomatid ZNK1 is a conserved RNA-specific zinc finger nuclease. C_LI

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.

During infection, Mycobacterium tuberculosis (Mtb) encounters multiple environmental stressors, including nitric oxide (NO) and iron limitation, and an ability to mount an integrated response is essential for the bacteriums adaptation and continued survival. Iron-containing prosthetic groups in key enzymes are critical for Mtb sensing and detoxification of NO, and there is significant overlap between NO- and low iron-responsive genes. However, how Mtb adapts to these two stressors concurrently is largely unknown. Here, we find that exposure to NO globally augments expression of low iron-responsive genes and vice versa, with a two gene operon, rv3839-rv3840, among the most highly upregulated. Deletion of rv3839-rv3840 resulted in increased growth under prolonged iron limitation and early exit of Mtb from an adaptive state of growth arrest induced upon exposure to NO/low iron. {Delta}rv3839-rv3840 Mtb exhibited an elongated cell morphology compared to wild type Mtb in NO/low iron conditions, indicating effects of this operon on cell growth and division under stress conditions, with Rv3839 as the key driver of this phenotype. Coproporphyrin III tetramethyl ester (TMC), a modified precursor molecule in the endogenous Mtb heme biosynthesis pathway, was found to accumulate in {Delta}rv3839-rv3840 Mtb under iron limiting conditions. Further, intrabacterial heme levels were increased in {Delta}rv3839-rv3840 Mtb under NO stress and iron limitation. Together, these findings reveal Rv3839-Rv3840 as proteins involved in the downregulation of heme biosynthesis under NO stress and iron limitation, and highlight the link between Mtb growth control in response to NO/low iron and endogenous heme biosynthesis. IMPORTANCESlowed growth is a physiologic adaptation to key environmental cues important for survival of Mycobacterium tuberculosis (Mtb) in the host. Nitric oxide (NO) is one such signal, but while regulation of NO response by the DosRS(T) two-component system is well-studied, NO stress also provokes a broad transcriptional response outside of DosRS(T) regulation that overlaps with the transcriptional response to iron limitation. Here, we show that Rv3839-Rv3840 contribute to Mtb maintenance of NO and low iron-induced growth arrest and find that this inability to maintain growth arrest is connected to dysregulation of the endogenous heme biosynthetic pathway. Little is known about regulation of endogenous heme biosynthesis in Mtb and its role in Mtb survival under stress conditions, and our results reveal a previously unknown interplay between NO and iron limitation response regulation and heme homeostasis.

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.

The multi-copy phage resistance gene (mpr) of M. smegmatis is a major factor in resistance to the lytic mycobacteriophage D29. Mpr is a membrane-bound exonuclease that cleaves phage DNA post injection, hence blocking downstream stages in the phage infection cycle. The mechanism of resistance allows for adsorption, is non-abortive and independent of any mutation in the gene. Rather, it depends on overexpression of a wild type copy of the gene. However, the underlying factor behind mpr overexpression in spontaneous D29-resistant mutants of M. smegmatis remained elusive. Here, we report that D29 infection triggers insertion sequence (IS) rearrangements, including the transposition and integration of IS6120 directly upstream of mpr. Mutants with IS6120 integration upstream of mpr show highly elevated Mpr expression. Whole genome sequence analysis reveals that IS6120 introduced a putative transcription factor-binding site and a canonical -35 promoter element at the integration site, hence reconstituting a fuller promoter (rcp) than the original promoter (wtp) at the site. Promoter reporter assays suggest that rcp is a far stronger promoter than wtp, implying that elevated mpr expression in D29-resistant mutants with this transposition event could be due to promoter reconstitution. While strains with this transposition event appear to grow normally, rcp-driven, vector-borne Mpr overexpression appears to be toxic as it barely allows for colony formation on agar plates. This study reports a previously unknown factor likely behind mpr regulation in M. smegmatis, adding to the existing knowledge of mycobacterial anti-phage defense mechanisms and guiding rational phage engineering efforts for therapeutic applications.

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.

Envelope biogenesis in Staphylococcus aureus is concentrated at the septum and includes peptidoglycan synthesis, lipo- and wall-teichoic acid production, and the targeted secretion of YSIRK/GXXS signal peptide-bearing proteins. How S. aureus confines these processes to the dividing crosswall remains unclear. EzrA, a scaffolding protein structurally related to eukaryotic spectrins, has been implicated in linking cell division to envelope synthesis, yet its precise role is poorly understood. Here, we re-examine the function of EzrA for its contribution to envelope biogenesis and homeostasis. We observe that ezrA null mutants synthesize excess peptidoglycan that is incorporated in a dispersed pattern, no longer strictly confined to the septum. A similar loss of spatial restriction was observed for protein A, a surface protein whose YSIRK/GXXS signal peptide directs septal secretion and anchoring. In wild-type cells, newly synthesized peptidoglycan co-localized with nascent protein A anchoring sites at the septum, whereas this spatial coupling was disrupted in the absence of EzrA. In addition, loss of EzrA resulted in impaired nucleoid occlusion with septal guillotining of the chromosome. Together, these findings support a model in which EzrA acts as a molecular organizer of cell division, coordinating septal biosynthesis and envelope assembly while ensuring proper nucleoid occlusion.

Bone infections, which are predominantly caused by Staphylococcus (S.) aureus, can be difficult to treat and have high rates of chronicity and reoccurrence. We previously identified that osteoclasts, the cells that break down bone matrix, may contribute to disease progression by allowing S. aureus to replicate intracellularly. There we identified that this bacteriums ability to grow intracellularly is tied to the maturation of osteoclasts. In this study we addressed whether osteoclast differentiation supports intracellular growth by changing the host cells response to infection or by altering the host cell environment to better support S. aureus. Using dual species RNA-sequencing we analyzed host and bacterial transcripts of infected osteoclast and precursor bone marrow macrophage (BMM) cultures. Host transcript analysis suggests that infected osteoclasts are slow to upregulate bacterial response genes compared to BMMs. We also identify that the S. aureus transcriptional response is primarily determined by the host cell type, and that bacteria in osteoclasts upregulate carbon metabolism genes compared to those inside BMMs. By utilizing intracellular survival assays on S. aureus mutants deficient in carbon metabolism and related pathways we determine that S. aureus require glycolysis, acetyl-CoA synthesis, and aspartate biosynthesis for proliferation inside osteoclasts, although bacteria can survive without them. With differentiation, osteoclasts increase glutamine uptake, and this metabolite is required for S. aureus intracellular growth. Taken together, these findings suggest that osteoclasts support S. aureus intracellular growth by providing nutrients required to replicate in the context of a blunted antimicrobial response. IMPORTANCEInfectious osteomyelitis, bone infection, is frequently caused by the bacterium Staphylococcus aureus. Intracellular infection of cells that build bone, osteoblasts, and cells that resorb bone, osteoclasts, have been implicated in disease progression by providing a niche for immune evasion. While S. aureus in osteoblasts are largely quiescent, bacteria in osteoclasts proliferate and therefore may be a source of reemergent infection. Factors that promote this growth in osteoclasts are poorly characterized. In this study we find that osteoclasts have a diminished transcriptional response to infection and show that S. aureus acquire glucose and glutamine, which have high flux in osteoclasts, to support intracellular growth. We further observe that S. aureus in osteoclasts require aspartate synthesis to grow intracellularly. This work highlights the importance of host cellular metabolism for the intracellular fate of S. aureus as an added factor beyond the direct antimicrobial response.

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.

The Mycobacterium genus includes more than 190 species that occupies diverse ecological niches. Some are nonpathogenic and environmental, whereas others cause severe diseases both in humans and animals, e.g. tuberculosis (TB) and leprosy. SelenoCysteine (SeC) is present in all three domains of life. Here we report the presence of the SeC-machinery (selA, selB, selC and selD) genes and selenoprotein (fdhA) genes in roughly 40% of 244 mycobacterial genomes. Their presence is distributed evenly among slow and rapid growing mycobacteria and our data indicate that they were acquired through horizontal gene transfer. Some mycobacteria however lost these genes during the evolution of the genus. We provide RNA-Seq data showing transcript levels of the SeC-machinery genes and fdhA in different mycobacteria grown under different conditions. Finally, we suggest that selC (the tRNASeC gene), positioned immediately upstream of selA-selB, is involved in the regulation of the expression of the SeC-machinery genes selA-selB. Together our data expand our understanding of selenocysteine metabolism and its evolution within the Mycobacterium genus.

We used transposon sequencing (Tn-seq) to define genetic requirements for intracellular survival of Brucella neotomae, a rodent-associated species. A near-saturating mutant library was subjected to selection during infection of J774A.1 macrophages, identifying 54 genes required for intracellular fitness. These included core components of the VirB type IV secretion system, multiple regulatory factors, an aquaporin gene with a strong fitness defect, and a set of metabolic genes involved in amino acid biosynthesis. Targeted mutagenesis revealed that methionine and histidine biosynthesis are indispensable for intracellular growth, whereas tryptophan biosynthesis was required for full intracellular fitness, with mutants exhibiting significant but incomplete attenuation. Notably, these auxotrophs grew normally in minimal medium under axenic conditions, indicating that their requirement is specific to the intracellular environment. Amino acid supplementation rescued intracellular growth in a concentration- and time-dependent manner, consistent with increased metabolic demand during intracellular replication. Disruption of the aquaporin gene similarly impaired intracellular survival, suggesting a role for water homeostasis during adaptation to the macrophage vacuolar environment. Beyond metabolic and osmotic adaptation, we identify OmpR1 as an upstream regulator of B. neotomae virulence. Biochemical, genetic, and transcriptional analyses establish a hierarchical regulatory cascade in which OmpR1 activates the BvrR/BvrS system, which in turn controls VjbR and downstream VirB expression. Under the conditions examined, OmpR1 is required for activation of this cascade. Consistent with this, OmpR1 loss is not rescued by VjbR and requires BvrR activity for restoration of intracellular growth. Phylogenetic analysis places OmpR1 in a distinct lineage relative to other well-characterized Brucella transcriptional regulators, suggesting that this regulatory pathway has been underappreciated across the genus. Together, these findings reveal that intracellular fitness in Brucella depends on metabolic capacity, osmotic homeostasis, and a hierarchical regulatory cascade centered on OmpR1. Author SummaryBrucella species are bacteria that survive and replicate inside immune cells called macrophages, where they cause persistent infection. To live within these cells, the bacteria must carefully balance their metabolism with the expression of genes required for virulence. We used a genome-wide genetic approach to determine which genes are specifically required for intracellular survival of Brucella neotomae, a rodent-associated species. We found that several amino acid biosynthesis pathways, including those required to produce methionine and histidine, are essential for replication inside macrophages but are not required during growth in laboratory media. This indicates that the intracellular environment imposes nutrient limitations not apparent in culture. We also discovered that a gene encoding an aquaporin, which regulates water movement across the bacterial membrane, is critical for intracellular survival, highlighting the importance of maintaining water balance within the host cell vacuole. In addition, we identify OmpR1 as an upstream regulator that controls a hierarchical virulence cascade required for intracellular growth. Our findings show that successful infection depends on metabolic capacity, virulence regulation and water homeostasis, and provide new insight into how Brucella adapts to its host environment.

Survival of bacteria in their natural habitat requires dynamic responses and adaptation to environmental cues. In Bacillus subtilis, one adaptive strategy is cannibalism, a form of programmed cell death during post-exponential development. Cannibalism enhances multicellular differentiation by prolonging or preventing commitment to endospore formation under starvation conditions. B. subtilis produces three cannibalism toxins: the sporulation delay protein, the sporulation killing factor, and the epipeptide EPE. Production of the latter is encoded in the epeXEPAB operon. Expression of this operon is transcriptionally controlled by the stationary phase regulators Spo0A and AbrB. Here, we demonstrate that EPE production is also post-transcriptionally regulated by two RNA binding proteins, Kre and SpoVG. Deletion of comK, the master regulator of competence development, abolished EPE production. This defect was reversed by additionally deleting kre. The RNA-binding protein, Kre, binds the epeX transcript and acts as a bidirectional ComK repressor, indicating that ComK indirectly regulates EPE biosynthesis via Kre. A second RNA-binding protein, SpoVG, also binds to the epeX mRNA. While Kre acts as a negative regulator, SpoVG was essential for EPE production. These findings reveal a novel regulatory connection between competence and cannibalism, expanding our understanding of how programmed cell death is coordinated in B. subtilis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/716078v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@57e20dorg.highwire.dtl.DTLVardef@1b9f4e5org.highwire.dtl.DTLVardef@17cfbc9org.highwire.dtl.DTLVardef@76824d_HPS_FORMAT_FIGEXP M_FIG C_FIG

DNA-binding transcriptional regulators control gene expression in response to environmental cues. A subset of these proteins, called transmembrane transcriptional regulators (TTRs), directly bind DNA to regulate transcription while remaining anchored in the cytoplasmic membrane. Prior work has shown that in the presence of the polysaccharide chitin, two TTRs, TfoS and ChiS, coordinate to induce the expression of TfoR, a small RNA that is critical for natural transformation in Vibrio cholerae. Specifically, it was shown that ChiS recruits the PtfoR locus to the membrane, which allows for the subsequent activation of this promoter by TfoS. However, it was also shown that increasing TfoS protein levels bypasses this coordination, allowing TfoS to activate the promoter independently. It therefore remains unclear what molecular mechanisms drive the requirement for ChiS in native conditions. Here, we show that ChiS binds PtfoR with a higher affinity than TfoS. We hypothesized that the low affinity of TfoS for PtfoR helps reinforce its dependence on ChiS for activation. To test this, we isolated a mutant allele of the TfoS DNA-binding domain that has a higher affinity for PtfoR. We show that this high-affinity TfoS allele promotes ChiS-independent activation of PtfoR. These results demonstrate that the relative DNA-binding affinity of TTRs is a critical feature that drives their coordination. IMPORTANCEDNA-binding transmembrane transcriptional regulators (TTRs) are critical for some bacterial species to properly sense and respond to their environments. Recent work highlights that pairs of TTRs can coordinate their activities to regulate gene expression, allowing them to sensitively control behaviors like virulence and horizontal gene transfer. However, the mechanisms that enable this coordination remain poorly understood. Here, we show that the relative DNA-binding affinity of paired TTRs is a critical feature that can drive their coordination.

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.

MmpL proteins play an important role in the various mechanisms associated with mycobacterial virulence. Identification of interacting protein partners required for a detailed understanding of their role remains hampered because of their large size (> 100 kDa) and the presence of twelve transmembrane domains by classical methods. In this study, we used two independent biotin proximity labelling assays (APEX2 and BioID) to define the proxisome of MmpL11 in M. smegmatis. Indeed, these techniques are performed directly in the organism of interest, allowing the detection of potentially transient or weak interactions in multiprotein complexes and preserving the subcellular structures and the presence of cofactors or post-translational modifications that can also impact protein-protein interactions. BioID leads to the biotinylation of lysine residues, whereas APEX2 leads to the biotinylation of mainly tyrosine residues; they have also been shown to have different effective labelling radii. On one hand, an interaction was detected between the cytoplasmic C-terminal domain of MmpL11 and MSMEG_0240, a protein of unknown function, using BioID. This interaction was confirmed using both MmpL11 and MSMEG_0240 as fusions with BirA and was corroborated by AlphaFold3 prediction. On the other hand, APEX2 failed to detect an interaction between MmpL11 and MSMEG_0240, probably due to the absence of accessible tyrosines. However, both approaches identified MSMEG_0940 as an additional interactant with MmpL11 that also depends on the C-terminal domain. Overall, this study demonstrates that APEX2 and BioID as complementary tools for defining the proxisome of mycobacterial proteins.

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.

Mycobacterium abscessus are non-tuberculous mycobacteria that are widespread in the environment and of increasing global clinical significance. Accumulating evidence shows that M. abscessus has emerged as an important pathogen, driven by highly drug-resistant lineages, enhanced transmissibility and the acquisition of specific virulence factors. In this study, we describe a previously uncharacterised ESX secretion system encoded on a 123-kbp plasmid identified in a clinical isolate of M. abscessus. This ESX system, termed ESX-pMA07, is distinct from ESX systems previously reported in M. abscessus in both sequence composition and locus organisation, characterised by a unique arrangement of core ESX components and low sequence identity to ESX-3, ESX-4 and plasmid-borne ESX-P systems. ESX-pMA07 was detected in geographically diverse clinical isolates but was restricted to particular genotypes within the global M. abscessus phylogeny. Transcriptional profiling revealed expression of ESX-pMA07 components in artificial cystic fibrosis media and during intracellular growth in macrophage cell lines. Using CRISPR interference, we show that inducible silencing of eccC, encoding the ATPase component of ESX-pMA07, significantly reduced intracellular survival of M. abscessus within macrophages. To our knowledge, this is the first characterisation of a functional, plasmid-borne ESX secretion system in M. abscessus, demonstrating that mobile genetic elements contribute to the pathogens intracellular persistence and may influence its evolving virulence. Author SummaryMycobacterium abscessus is a rapidly emerging, highly drug-resistant bacterium that causes chronic infections, particularly in people with underlying lung disease such as cystic fibrosis. The factors that enable certain M. abscessus strains to persist inside host cells are not fully understood. In this study, we identified a previously unrecognised type VII secretion system (ESX) encoded on a large plasmid in a clinical isolate of M. abscessus. This plasmid-borne ESX system, which we termed ESX-pMA07, is genetically distinct from the ESX systems normally found on the chromosome and was detected in geographically diverse clinical isolates, but restricted to specific lineages within the global M. abscessus population. We show that ESX-pMA07 genes are expressed under conditions relevant to lung infection and during intracellular growth in macrophages. Using inducible CRISPR interference to silence the ESX ATPase gene eccC, we demonstrate that ESX-pMA07 contributes to intracellular survival of M. abscessus in macrophages. These findings reveal that mobile genetic elements can encode functional secretion systems that enhance intracellular persistence, providing a mechanism for the emergence and spread of virulence traits in this important pathogen.

In Gram-negative bacteria, co-translational insertion of membrane proteins into the plasma membrane may be coupled to ongoing transcription, a phenomenon known as transertion. Transertion results in a physical shift of the coding gene from the nucleoid towards the membrane and is one of the determinants of the shape of the nucleoid and placement of cell division in Gram-negative bacteria. In contrast, the existence of functional coupling of transcription and translation in Bacillus subtilis and potentially other Gram-positive bacteria has been questioned, suggesting that transertion may not happen. Here, by imaging vertically oriented B. subtilis cells, we show that the gene of a transmembrane protein changes its localization from inside the nucleoid to the plasma membrane upon induction of its transcription. Localization is restored to the nucleoid when induction of transcription has ceased. The shift of the gene towards the membrane is strictly dependent on transcription, its induction, translation and transmembrane nature of the coded protein. These results suggest that, at least, some principles of cellular regulation based on functional coupling of transcription and translation may be conserved between Gram-positive and Gram-negative bacteria.