Molecular Microbiology

Maintaining proper cation homeostasis is essential for the survival of all living organisms. During infection, Salmonella must adapt to host-imposed magnesium (Mg2+) limitation. To compensate, Salmonella induces high-affinity Mg2+ transporters and synthesis of polyamines, organic cations that partially substitute for Mg2+. Once Mg2+ levels are restored and cells enter stationary phase, excess cations must be exported; failure to do so results in loss of viability. The CorC-domain protein PaeA exports excess polyamines, whereas MgpA and CorC reduce intracellular Mg2+ levels. Here, we identify two new CorC-domain proteins, MgpB (formerly YegH) and CorB, as mediators of Mg2+ homeostasis, showing that they also reduce intracellular Mg2+ accumulation and protect cells from endogenous excess-cation stress. Genetic analyses revealed the functional redundancy among these proteins in conferring resistance to exogenous high Mg2+, endogenous stress induced after Mg2+ starvation, and egg whites. Deletion of mgpB and/or corB conferred little to no phenotype. However, these mutations showed synergistic phenotypes in strains lacking MgpA. This synergistic relationship was also evident in mouse competition assays. Lack of MgpA, CorC, MgpB, and CorB leads to an increase in intracellular Mg2+ levels. Moreover, expression of mgpB and corB are increased in high Mg2+, all consistent with their role as putative Mg2+ exporters. Together, these findings identify MgpB and CorB as new mediators of Mg2+ homeostasis and reveal a multilayered system that enables Salmonella to finely balance intracellular cation levels during environmental and host-imposed stress.

Environmental bacteria have abundant signaling systems wired into complex gene regulatory networks to adapt to fluctuating conditions. In Myxococcus xanthus, starvation triggers a developmental program (specialized biofilm) that produces spore-filled multicellular fruiting bodies surrounded by a distinct quiescent state termed peripheral rods. Fruiting body structure as well as the proportion of cells following each fate can be tuned by a large repertoire of signaling proteins, including numerous orphan histidine kinases. Here, we focus on the histidine kinase TodK which was previously demonstrated to influence developmental progression. We find that loss of TodK produces distinct developmental phenotypes that vary with environmental conditions. To quantify these effects, we developed an image-analysis pipeline that measures aggregation and fruiting body patterning during development on nutrient-limited agar. These analyses revealed the todK mutant precociously aggregates particularly at the peripheries of the colony. Under submerged-culture conditions, initial production of aggregates was not accelerated but aggregates exhibited accelerated progression to mature fruiting bodies. Overexpression of active TodK completely blocked fruiting body formation. Molecular analyses demonstrated that TodK overproduction suppressed expression of core developmental regulators including FruA and CsgA (C-signal). Interestingly, protein accumulation of MrpC, necessary for expression of both FruA and the C-signal was not significantly perturbed suggesting TodK silences MrpC transcriptional activity. Together, these findings establish TodK as a modulator of developmental progression and demonstrate how quantitative phenotyping approaches can reveal biologically meaningful functions for orphan histidine kinases whose mutant phenotypes might otherwise appear subtle. Summary StatementQuantitative analysis of multicellular development reveals previously hidden functions of an orphan histidine kinase, highlighting the importance of robust phenotyping approaches for understanding bacterial signaling networks.

Bacterial cell wall (CW), primarily composed of the biopolymer peptidoglycan, serve as essential protective barriers against external stresses and the internal turgor pressure. The peptidoglycan (PG) biosynthetic pathway encompasses sequential enzymatic reactions in the cytoplasm and in the membrane that involve critical enzymes susceptible to antibiotic targeting. Virtually each step of the pathway is the target of a known antibiotic. Antibiotic-induced inhibition of PG assembly typically weakens the sacculus, often leading to cell lysis. However, the cascade of events that follow inhibition of a specific enzyme of the pathway, and how these culminate in cell death remain largely unknown. Here, we investigated the effects on growing Bacillus subtilis cells of two categories of CW antibiotics: inhibitors of the synthesis of soluble PG precursors in the cytoplasm (fosfomycin and D-cycloserine) and inhibitors of the polymerisation and crosslinking reactions at the outer leaflet of the membrane, which incorporate newly externalised precursors into the existing network (vancomycin and penicillin). In B. subtilis, the latter reactions are catalysed along the sidewalls by the Rod complex, thought to primarily build the sacculus, and by class A penicillin-binding proteins (aPBPs), thought to add to repair it. Our findings reveal that the two antibiotic groups lead to growth arrest, sacculus thinning, and eventual cell lysis. However, while the impact of vancomycin and penicillin G is rapid, lacking morphological deformation, fosfomycin and D-cycloserine induce cell widening and bulging before lysis. During shortage of PG precursors, dysregulated PG hydrolytic activity contributes to elevated cell lysis but is not responsible of bulging. Instead, dispersed PG synthesis by aPBPs persists while the activity of the Rod system is rapidly arrested, resulting in cell rounding. We propose that this facilitates the redirection of the limited PG precursors to sites of CW repair, thereby preserving cell integrity and allowing for prolonged growth during antibiotic challenge.

Inorganic polyphosphate (polyP) is a universally conserved biopolymer central to bacterial stress survival, yet understanding of its roles derives almost entirely from Gram-negative models in which polyP accumulates intracellularly following nutrient downshift. We examined polyP metabolism in the Gram-positive spore-forming bacterium Bacillus cereus using deletions of the polyP kinases PPK1 and PPK2 and the exopolyphosphatase PPX. Intracellular polyP synthesis required PPK1 and was opposed by PPX and PPK2: ppx mutants accumulated polyP in sporulation medium by 24 hours, ppx ppk2 double mutants accumulated more, and no ppk1 mutant accumulated any. A ppk1 ppx double mutant could not be generated, suggesting that unopposed PPK2 activity is lethal. Unlike Escherichia coli and Pseudomonas aeruginosa, B. cereus did not accumulate polyP after shift to minimal medium, increasing only modestly in stationary phase. Fluorescence and transmission electron microscopy localized intracellular polyP to electron-dense granules within ribosome-depleted cytoplasm. Cells bearing these granules remained membrane-intact yet failed to resume growth over 8 hours in rich medium, leading us to propose that polyP drives ribosome sequestration into condensates and a hibernation-like state. Unexpectedly, B. cereus also released close to 100{micro}M polyP extracellularly during late stationary phase, even in a ppk1 ppk2 mutant lacking both known synthetases. Extracellular polyP resisted hydrolysis by purified PPX even after deproteinization, indicating an atypical structure. Bacillus thuringiensis and Bacillus anthracis released similar amounts of extracellular polyP. Together these results identify two distinct polyP pools in the B. cereus group: a PPK1-dependent intracellular pool and an extracellular pool made by an uncharacterized pathway. ImportanceBacillus cereus is a spore-forming bacterium that causes foodborne illness and persists in soil and food-processing environments, where survival depends on managing phosphate and energy reserves during starvation. Inorganic polyphosphate (polyP), an ancient polymer used by nearly all cells to withstand stress, has been studied almost entirely as a molecule stored inside bacteria. We show that Bacillus cereus maintains two separate polyP pools. The internal pool is made by a known enzyme (PPK1) and is associated with dormant cells whose protein-making machinery appears to be packed away. The external pool is made without any known polyP-synthesizing enzyme, pointing to a novel polyP synthesis pathway that is shared with the close relatives Bacillus thuringiensis and Bacillus anthracis.

Bacteria tightly regulate intracellular zinc homeostasis by coordinating zinc uptake and efflux systems. We previously showed that deletion of the ribosomal protein gene rpmJ confers zinc resistance in Escherichia coli in a manner dependent on the zinc efflux transporter zntA. Here, we analyzed the effect of rpmJ deficiency on zntA expression. Under zinc excess conditions, zntA mRNA levels were markedly higher in the rpmJ mutant than in the wild-type strain. Enhanced zntA expression required the native zntA promoter, the native Shine-Dalgarno sequence, and the N-terminal coding region of zntA, indicating that translation initiated from the native Shine-Dalgarno sequence and extending through the N-terminal coding region is required for enhanced transcription initiation from the native zntA promoter. Furthermore, ectopic expression of ykgO, a paralog of rpmJ that is known to replace RpmJ on the ribosome under zinc-limited conditions, abolished the increased zntA expression and zinc resistance conferred by rpmJ deletion. Collectively, these findings suggest that ribosomes lacking RpmJ or YkgO promote transcription initiation from the native zntA promoter through translation of zntA mRNA. IMPORTANCEBacteria must carefully control the amount of zinc inside their cells. Too little zinc prevents essential cellular processes, whereas too much zinc is toxic. We found that removing a small ribosomal protein called RpmJ allows Escherichia coli to survive high zinc levels by increasing production of the zinc exporter zntA. Surprisingly, this increase depends not only on the zntA promoter but also on translation of the beginning of the zntA coding region. Our findings suggest that changes in ribosome composition can stimulate gene transcription through early translation of the same messenger RNA, revealing a previously unrecognized mechanism linking translation and transcription during bacterial adaptation to zinc stress.

G-protein-coupled receptor (GPCR)-mediated signalling pathways govern critical cellular responses in fungi, including mating, biofilm formation, and virulence. The mating pathway of Saccharomyces cerevisiae, in which peptide-sensing GPCRs (peptide GPCRs) activate a MAP kinase cascade and the transcription factor Ste12, has thereby served as the canonical model for fungal peptide GPCR signalling. Homologues of this pathway are conserved across many fungi, including the high-priority pathogen Nakaseomyces glabratus. Yet the function of this pathway in this mating-incompetent pathogen remains unclear. Here, we investigated whether the central transcription factor Ste12 of N. glabratus (NgSte12(1)) can act as a peptide GPCR-coupled transcription factor using a component-swapping strategy in which pathway elements from N. glabratus and S. cerevisiae were reciprocally exchanged and assessed using fluorescent and growth arrest readouts. We show that NgSte12(1) is in principle capable of supporting peptide-GPCR-dependent activation in S. cerevisiae, however, subtle and dependent on promoter identity. Ste12 chimaeras further demonstrate that all three NgSte12(1) subdomains are individually functional and compatible with their S. cerevisiae counterparts. Heterologous expression of ScSte12 in N. glabratus shows constitutive pathway activation, while NgSte12(1) does not show pathway activation in N. glabratus. Peptide-GPCR expression experiments indicate a block in N. glabratus signalling upstream of Ste12 (1): the S. cerevisiae peptide-GPCR Ste2 (ScSte2) activated the N. glabratus Ste12 GPCR-dependent downstream signalling constitutively, while NgSte2 failed to activate signalling in its native environment. Together, these results indicate that NgSte12(1) is a competent pheromone-responsive transcription factor that can couple to a heterologous GPCR in a heterologous context but is unresponsive to its own cognate upstream peptide/GPCR pair, either because it binds a yet unknown ligand or the signalling is rewired in this pathogen.

Metals are essential for all forms of life and are therefore the subject of tight competition at the host-pathogen interface. Manganese (Mn) is an important cofactor for enzymes involved in various processes in bacteria, including the response to oxidative stress, and the ability to scavenge Mn through the expression of high-affinity Mn transporters has been associated with virulence of many extracellular and vacuolar pathogens. While the cytosol has been established to be restrictive for growth of non-adapted pathogens, the extent to which metal limitation contributes to this is not well understood. Here, we investigate whether high-affinity Mn acquisition is important for intracellular adaptation and virulence of the cytosolic pathogen Listeria monocytogenes (Lmo). To do so, we generated a strain of Lmo in which both of its predicted high-affinity Mn transporters, MntH and MntABC, are disrupted. We found that this strain was severely defective in growth in vitro under Mn-limited conditions and had lower intracellular Mn content. Additionally, we observed Mn-dependent transcriptional regulation of the mntH and mntA genes. Together, these data strongly support that MntH and MntABC are high-affinity Mn transporters and are likely to be the only such dedicated Mn transporters expressed in Lmo. Using the {Delta}mntH {Delta}mntA strain, we provide evidence that Mn limitation sensitizes Lmo to oxidative stress in vitro, consistent with the known role of Mn in mitigating reactive oxygen species. Ultimately, we demonstrate that expression of high-affinity Mn transporters is essential for intracellular replication of Lmo in macrophages, plaquing on fibroblast monolayers, and for virulence in an animal model of systemic infection. Our work, therefore, suggests that Lmo is challenged by Mn limitation in the cytosol and requires Mn scavenging to adapt to its intracellular niche.

Magnesium supplementation permits deletion of otherwise essential genes involved in cell envelope biogenesis in the Gram-positive model bacterium Bacillus subtilis. Yet, the specific underlying mechanism has remained elusive. To address this key knowledge gap, we made use of a mutant lacking ezrA and gpsB. Deletion of both of these genes involved in cell wall synthesis leads to severe growth inhibition which is ameliorated by magnesium addition. Our results indicate that, in the absence of magnesium, this mutant contains elevated levels of labile iron, is impaired in activating the oxidative stress response, and displays extreme sensitivity to iron and manganese intoxication. Intriguingly, we find that an ezrA single deletion, but not gpsB, exhibits heightened susceptibility to excess iron and manganese. This observation allowed us to investigate the source of toxicity and how EzrA may support metal homeostasis. Our data suggests that the major contributor of ROS is the electron transport system involved in cellular respiration. Both genetic and chemical means to reprogram the cells in favor of fermentation alleviate the metal toxicity in cells lacking ezrA. Collectively, our data shows that magnesium limits iron availability and redirects metabolism towards pathways that are preferred during iron scarcity. Consequently, these mechanisms result in reduced ROS production and oxidative stress mitigation. This explains why magnesium supplementation may render essential genes dispensable. In support of this model, we find that addition of magnesium helps cells to circumvent lysis typically caused by the treatment of an antibiotic that disrupts cell wall synthesis. Taken together, our results suggest that unmitigated oxidative stress fueled by labile iron is likely responsible for the detrimental effects of specific gene disruptions and certain antibiotic treatments. By reducing the pool of free iron and reprogramming cellular metabolism, magnesium mitigates oxidative damage and protects cells from ROS-mediated death.

Inorganic polyphosphate (polyP) is a ubiquitous, multifunctional biomolecule that supports stress survival in bacteria. During nitrogen (N) starvation, Escherichia coli rapidly accumulates polyP, which drives the formation of RNA protein granules that sustain long term survival. What triggers this upregulation has remained unknown. Here we show that polyamines, a group of highly conserved, amino acid-derived polycations, regulate polyP accumulation during N starvation. We found that deleting all nine polyamine biosynthesis genes elevates polyP during exponential growth and by more than 5fold under N starvation, with shorter chains preferentially accumulating. To dissect which polyamine matters, we further deleted the three catabolic enzymes that consume polyamines as a nitrogen source, which enabled complementation assays. Supplying any one of the three physiological polyamines, putrescine, spermidine, or cadaverine, restored wild type polyP levels during N starvation. We found that the absence of polyamines did not affect the steady state levels of either the polyP synthesizing kinase PPK or the exopolyphosphatase PPX, pointing instead to posttranslational control of enzyme activity or to as yet unidentified polyamine dependent regulators. These findings establish a direct metabolic link between two universal, functionally intertwined biomolecules and suggest that the polyamine decline upon N starvation contributes to the observed polyP accumulation.

Onion tissues produce antimicrobial thiosulfinates after tissue damage and cellular decompartmentalization. Burkholderia gladioli pv. alliicola (Bga), a common onion pathogen, encodes a thiosulfinate tolerance gene (TTG) cluster that protects the bacterium during thiosulfinate exposure. Previous work showed that the TTG cluster contributes to foliar infection but has little effect on infection of onion bulb tissue. To further examine Bga-thiosulfinate interactions in foliar and bulb tissues, we used a thiosulfinate-responsive PaltR-Lux reporter strain to determine when and where Bga encounters thiosulfinates. In leaves, Bga-induced necrosis was associated with de-repression of the PaltR-Lux reporter and coincided with a contribution of the TTG cluster to bacterial population size, indicating thiosulfinate exposure during foliar infection. In contrast, TTG mutants and wild-type (WT) strains showed similar growth in scales, and PaltR-Lux signal declined as scale necrosis progressed, suggesting limited thiosulfinate exposure during bulb colonization. However, when necrosis was induced by the non-native toxin pantaphos, PaltR-Lux was de-repressed and recovery of the TTG mutant was reduced. These results indicate that Bga encounters thiosulfinates during foliar infection but largely avoids exposure during bulb infection. Preconditioning the TTG mutant in onion scale tissue did not alter its thiosulfinate sensitivity in vitro, arguing against an infection-associated thiosulfinate exclusion mechanism. In contrast, partial rescue of the TTG mutant by the WT strain in zone-of-inhibition co-plating assays suggests extracellular thiosulfinate detoxification. Together, these findings indicate that Bga detoxifies thiosulfinates released during bulb necrosis, limiting thiosulfinate exposure during onion bulb infection. The molecular basis for detoxification and tissue specificity remain unresolved.

Mycobacterium tuberculosis has a flavin-intensive metabolism, with more than 180 proteins that require flavin or deazaflavin cofactors. M. tuberculosis requires riboflavin biosynthesis for growth in standard culture conditions in vitro, which can be overcome by providing exogenous riboflavin. It is unknown whether M. tuberculosis also requires riboflavin synthesis to grow or persist in infected host cells or tissues. Here we used a M. tuberculosis strain that conditionally expresses ribA2, which encodes a bifunctional enzyme that catalyzes the first steps in both branches of riboflavin biosynthesis, to determine whether M. tuberculosis requires riboflavin biosynthesis during infection. Transcriptional repression of ribA2 caused death of M. tuberculosis in vitro without riboflavin supplementation. RNAseq revealed that riboflavin starved M. tuberculosis induce a transcriptional program indicating decreased electron transport chain function and broad changes to central metabolism. Transcriptional repression of ribA2 prevented M. tuberculosis growth in cultured macrophages and caused clearance of M. tuberculosis from infected tissues at all stages of infection in both C57BL/6 mice that form cellular lung lesions and C3HeB/FeJ mice that develop necrotic lung granulomas. Our results demonstrate that M. tuberculosis requires RibA2 for viability at all stages of infection and support further characterization of the riboflavin biosynthesis pathway as a potential target for development of new anti-tubercular drugs.

The second messenger c-di-GMP plays a critical role in regulating cell size and growth in the cyanobacterium Anabaena sp. PCC 7120. While all 16 c-di-GMP metabolic enzymes and the primary receptor CdgR have been identified, how metabolic enzyme activity is controlled to maintain c-di-GMP homeostasis remains largely unexplored. Here, using a suppressor-screening approach based on c-di-GMP-deficient mutants, we identify All0729 (CdgKA) as a regulator that orchestrates c-di-GMP homeostasis and cell size regulation. Genetic and quantitative analyses demonstrate that CdgKA acts upstream of All2306 (CdgSHA), the reported major c-di-GMP hydrolase, to modulate the activity of the latter. While c-di-GMP synthesis mutants displayed reduced c-di-GMP levels and decreased cell size, inactivation of cdgKA or cdgSHA in these mutant backgrounds rescued both the c-di-GMP levels and cell size. We propose that CdgKA and CdgSHA form a complex two-component system, and the phosphorylation of CdgSHA enhances its c-di-GMP-degrading activity. While the previously identified two-component system composed of CdgK-CdgS regulates c-di-GMP synthesis, the CdgKA-CdgSHA system controls its degradation. Together, they constitute a dual TCS network that precisely tunes c-di-GMP levels and cell size, establishing a regulatory link between two-component signaling and second messenger homeostasis in cyanobacteria.

Integrative and conjugative elements (ICEs) are widespread in bacteria and typically confer important phenotypes to their hosts. Following excision from the host chromosome, an ICE can transfer to a new cell through the element-encoded conjugation machinery (a type IV secretion system) to create a transconjugant. Tn916, a well-studied ICE that confers tetracycline resistance, is found in several Gram-positive pathogens and has been widely studied in Bacillus subtilis. Unlike most ICEs, Tn916 causes host-cell death following its activation (excision and gene expression). We found that orf7 and orf8 (renamed sig7 and sig8), two regulatory genes in Tn916, encode a two-subunit (split) sigma factor of RNA polymerase, Sig7-8, that activates transcription from Porf7 (now Psig7) and is required for excision of the element. Importantly, production of Sig7-8, either from Psig7 or another strong promoter, caused cell death. Using a Cre recombinase reporter to detect gene expression, we detected little or no transcription from Psig7 in transconjugants. This lack of transcription from Psig7 in transconjugants was essential for fitness of both the element and its host: forcing transcription from Psig7 in transconjugants caused either loss of Tn916 or death of nascent transconjugants. Our findings indicate that, unlike other well-studied ICEs (e.g., ICEBs1) that use repressors to control gene expression and are expressed in nascent transconjugants, most Tn916 genes remain off following transfer to a new host. This regulation prevents Tn916 from killing transconjugants and allows successful horizontal transfer and maintains the potential for future activation in the new host.

Bacteria routinely respond to changing environments, with vastly different with nutrient availability. When nutritional changes are predictable, bacteria can deploy mechanisms to anticipate them. In these cases, an organism's past environment can shape its response to its current environment, which is often referred to as a history-dependent behaviour (HDB). Using nutritional shift assays, we investigated how prior growth phase influences subsequent growth across different carbon sources. Here, we show evidence of a novel HDB regulating galactose metabolism in Escherichia coli, where previous growth to exponential phase leads to a growth advantage in a galactose environment, with no comparable effect observed in all other carbon environments tested. We demonstrate a role of the galactose repressors, GalR and GalS, in regulating this behaviour, as it is lost in strains deficient in either repressor. Further, we show how this HDB is generalizable across a collection of E. coli isolates, but not Salmonella - a closely related species with a similar natural history. Together, these findings build on the existing knowledge of HDBs governing carbohydrate metabolism by demonstrating how growth phase history can influence an organism's response to fluctuating nutritional environments.

Ribosomal RNAs contain numerous conserved nucleotide modifications, yet the functional importance of most of these modifications remains unclear. In Escherichia coli, deletion of individual 16S rRNA modification enzymes generally produces only minor phenotypes, raising questions about their biological significance. Here, we generated a comprehensive collection of deletion mutants lacking individual and combined 16S rRNA modifications, culminating in a strain lacking all known 30S ribosomal subunit modifications. Despite the absence of all known 16S rRNA modifications, cells remained viable, exhibiting a fitness defect of ~30% at 37 {degrees}C that increased to ~50% at 20 {degrees}C, consistent with impaired ribosome biogenesis. We identified strong epistatic interactions between modifications in the 3' major and 3' minor domains of 16S rRNA, resulting in disproportionately large effects on both fitness and antibiotic susceptibility. Live-cell single-molecule tracking revealed a marked increase in the fraction of non-translating ribosomes and a prolonged time required to enter productive translation, whereas translational elongation by actively engaged 70S ribosomes remained largely unaffected. In addition, fluorescence-based measurements showed that unmodified ribosomes exhibited increased stringency during translation initiation, reducing utilization of near-cognate start codons. Together, these findings demonstrate that 16S rRNA modifications are not essential for viability but collectively enhance the efficiency, robustness, and fidelity of ribosome assembly and translational initiation.

Despite growing interest in the MreBCD morphogenetic complex as a potential antimicrobial target, its function in Pseudomonas aeruginosa remains poorly understood. While previous studies using the MreB inhibitor A22 have established its role in cell shape maintenance and pilus regulation, the impact of mreB deletion has not been comprehensively investigated. Using genetic and microscopy-based approaches, we show that deletion of mreB is viable in P. aeruginosa, but results in spherical cells that lose all forms of motility despite retaining flagella. Importantly, we uncover a previously overlooked polar effect of the in-frame mreB deletion on the downstream mreCD genes and show, using CRISPRi-mediated silencing, that mreCD expression is essential for viability.{Delta} mreB mutants also display increased sensitivity to {beta}-lactam antibiotics and enhanced initial surface attachment, yet form more compact biofilms with reduced dispersal. In mixed-culture biofilms, spherical{Delta} mreB cells are outcompeted by rod-shaped wild-type cells and remain confined to the biofilm base. The identification of natural P. aeruginosa isolates carrying truncated mreB alleles further indicates that loss of MreB function can be tolerated in natural populations. Together, our findings reveal important contributions of the MreBCD system to viability, morphogenesis, motility and biofilm development in P. aeruginosa, providing new insights into bacterial adaptation and informing the development of targeted antimicrobial strategies.

Bacterial operons enable co-transcription of multiple genes, yet post-transcriptional mechanisms permitting differential regulation of individual genes within a polycistronic transcript remains incompletely understood. We demonstrate that a cis-encoded RNA-RNA interaction within the bicistronic yabJ-spoVG transcript forms an RNase III substrate that represses the transcription factor SpoVG in Staphylococcus aureus. Disruption of the cis-encoded regulation elevated monocistronic spoVG expression, reduced biofilm formation and cell wall peptidoglycan thickness, and increased sensitivity to cell wall-targeting antimicrobials in clinical vancomycin-intermediate S. aureus, consistent with phenotypes observed in an independent SpoVG overexpression strain. These findings uncover a previously undescribed mode of cis-regulatory control within a polycistronic operon and establish regulatory mRNA-mRNA interactions as a mechanism for sub-operonic gene regulation.

Resistance to {beta}-lactam antibiotics in the human pathogen Streptococcus pneumoniae is mainly attributed to the acquisition of mutated versions of pbp2x, pbp2b and pbp1a, encoding functional penicillin binding proteins with reduced affinity to {beta}-lactams. This enables the bacterium to synthesise peptidoglycan required for cell division in the presence of higher {beta}-lactam concentrations. In addition, it has recently been shown that increased levels of the second messenger molecule cyclic di-AMP, resulting from mutations that compromise the cyclic di-AMP degrading phosphodiesterase Pde1, contribute to decreased {beta}-lactam susceptibility in this species. A link between cyclic di-AMP levels and beta-lactam resistance has also been reported in several other Gram-positive bacteria. Here, we further investigated this link in S. pneumoniae and present evidence supporting that cyclic di-AMP could be involved in regulation of cell division. We confirm that elevated cyclic di-AMP levels decreased penicillin susceptibility and cell size in pde1 mutants. Next, we show that low cyclic di-AMP levels had the opposite effects in which cells became more susceptible to penicillin and displayed an elongated morphology with multiple incomplete septa. Using fluorescence microscopy and bacterial two-hybrid assays, we found that proteins involved in cyclic di-AMP synthesis (CdaA, CdaR) and degradation (Pde1) were enriched around the division site and that they most probably interact with each other. Finally, we identified changes to the cell wall stem peptide composition of {Delta}pde1 cells. Together our findings indicate that cyclic di-AMP could function as a regulator of the division machinery.

Bacillus subtilis grows and divides by binary fission, directed by medial localization of cell division protein FtsZ. Disruption of either the Min system or EzrA results in aberrant FtsZ positioning. Here we compare FtsZ dynamics in cells disrupted for either MinD or EzrA when grown in microfluidic channels. Here we show that cells lacking MinD or EzrA appear to be similarly defective in Z-ring disassembly after septation, but play different roles as simultaneous disruption results in a synergistic defect in division. Moreover, we account for a low frequency of minicell formation in the absence of EzrA, as MinD but not EzrA is necessary for removal of ZapA from polar Z-rings. Finally, overexpression of MinCD results inhibits division through pervasive Z-ring disassembly but appears to concentrate ZapA through localized sequestration. Combined, our results indicate a closer relationship between MinCD and ZapA than previously recognized and show that Z-ring disassembly can be genetically separated into discrete steps. We propose a two-step model for Z-ring disassembly that mirrors the assembly process, such that after and/or during septation, the Z-ring separately decondenses and protofilaments are disassembled to monomers for recycling.

Bacteria in the Mycobacteriales order like mycobacteria and corynebacteria surround themselves with a multilayered cell envelope. Their cytoplasmic membrane is fortified by a peptidoglycan cell wall that is decorated with branched arabinogalactan (AG) polymers. The AG glycans are further modified with mycolic acids to form an outer membrane. Biogenesis of this mycomembrane requires the transport of mycolates from their site of synthesis in the cytoplasmic membrane to the cell surface. How mycolate transport is controlled and coordinated with AG synthesis has remained unclear. Mycolate transport is mediated by the essential RND-family transporter MmpL3 in mycobacteria and a pair of related, partially redundant transporters called CmpL1 and CmpL4 in corynebacteria. The acetyltransferase TmaT has also been implicated in mycolate transport in both types of bacteria. In corynebacteria, it is required for the production of acetylated mycolates, and this modification has been proposed to promote mycolate transport via both CmpL transporters in the model organism Corynebacterium glutamicum (Cglu). Here, we reinvestigated the function of TmaT in Cglu and found that it and several factors encoded in the tmaT locus are specifically required for mycolate transport via the CmpL4 transporter pathway. Notably, one of these additional genes encodes the arabinosyltransferase AftD involved in AG biogenesis. TmaT and AftD were found to interact, and our results indicate that this interaction is required for acetylated mycolate production, mycolate transport via the CmpL4 pathway, and normal arabinan synthesis. Thus, the TmaT-AftD interaction may serve as a regulatory link connecting mycolate transport with AG biogenesis. SIGNIFICANCEMycobacteriales bacteria, including pathogens like Mycobacterium tuberculosis (Mtb), have a complex cell surface comprising an inner membrane, a cell wall modified with arabinogalactan (AG), and an outer mycomembrane made of mycolic acids linked to AG polymers. Because these surface biogenesis pathways are targeted by frontline anti-Mtb drugs, there is great interest in elucidating their underlying mechanisms. Here, we identify an interaction between factors involved in mycomembrane (TmaT) and AG biosynthesis (AftD) in the model organism Corynebacterium glutamicum. We show that this interaction is important for proper surface biogenesis and may therefore function to coordinate mycomembrane assembly with AG synthesis. This and other potential regulatory connections controlling envelope biogenesis represent attractive targets for future antibiotic development.