Clinical Strains of Mycobacterium tuberculosis Representing Different Genotype Families Exhibit Distinct Propensities to Adopt the Differentially Culturable State.

Growing evidence points to the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens from individuals with active tuberculosis (TB) disease. These bacteria are unable to grow on solid media but can resuscitate in liquid media. Given the epidemiological success of certain clinical genotype families of Mycobacterium tuberculosis, we hypothesize that different strains may have distinct mechanisms of adaptation and tolerance. We used an in vitro carbon starvation model to determine the propensity of strains from lineages 2 and 4 that included the Beijing and LAM families respectively, to generate DCTB. Beijing strains were associated with a greater propensity to produce DCTB compared to LAM strains. Furthermore, LAM strains required culture filtrate (CF) for resuscitation whilst starved Beijing strains were not dependent on CF. Moreover, Beijing strains showed improved resuscitation with cognate CF, suggesting the presence of unique growth stimulatory molecules in this family. Analysis of starved Beijing and LAM strains showed longer cells, which with resuscitation were restored to a shorter length. Cell wall staining with fluorescent D-amino acids identified strain-specific incorporation patterns, indicating that cell surface remodeling during resuscitation was distinct between clinical strains. Collectively, our data demonstrate that M. tuberculosis clinical strains from different genotype lineages have differential propensities to generate DCTB, which may have implications for TB treatment success.

Bacterial growth dynamics in a rhythmic symbiosis.

The symbiotic relationship between the bioluminescent bacterium Vibrio fischeri and the bobtail squid Euprymna scolopes serves as a valuable system to investigate bacterial growth and peptidoglycan (PG) synthesis within animal tissues. To better understand the growth dynamics of V. fischeri in the crypts of the light-emitting organ of its juvenile host, we showed that, after the daily dawn-triggered expulsion of most of the population, the remaining symbionts rapidly proliferate for about 6 h. At that point the population enters a period of extremely slow growth that continues throughout the night until the next dawn. Further, we found that PG synthesis by the symbionts decreases as they enter the slow-growing stage. Surprisingly, in contrast to the most mature crypts (i.e., Crypt 1) of juvenile animals, most of the symbiont cells in the least mature crypts (i.e., Crypt 3) were not expelled and, instead, remained in the slow-growing state throughout the day, with almost no cell division. Consistent with this observation, the expression of the gene encoding the PG-remodeling enzyme, L,D-transpeptidase (LdtA), was greatest during the slowly growing stage of Crypt 1 but, in contrast, remained continuously high in Crypt 3. Finally, deletion of the ldtA gene resulted in a symbiont that grew and survived normally in culture, but was increasingly defective in competing against its parent strain in the crypts. This result suggests that remodeling of the PG to generate additional 3-3 linkages contributes to the bacterium's fitness in the symbiosis, possibly in response to stresses encountered during the very slow-growing stage. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

Amidation of glutamate residues in mycobacterial peptidoglycan is essential for cell wall cross-linking.

Introduction: Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors. Methods: Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation. Results and discussion: In Mycobacterium smegmatis, we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and ß-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.

Structure-Activity Relationship Study of Cannabidiol-Based Analogs as Negative Allosteric Modulators of the µ-Opioid Receptor.

The US faces an unprecedented surge in fatal drug overdoses. Naloxone, the only antidote for opiate overdose, competes at the mu opioid receptor (µOR) orthosteric site. Naloxone struggles against fentanyl-class synthetic opioids that now cause ∼80% of deaths. Negative allosteric modulators (NAMs) targeting secondary sites may noncompetitively downregulate µOR activation. (-)-Cannabidiol ((-)-CBD) is a candidate µOR NAM. To explore its therapeutic potential, we evaluated the structure-activity relationships among CBD analogs to identify NAMs with increased potency. Using a cyclic AMP assay, we characterize reversal of µOR activation by 15 CBD analogs, several of which proved more potent than (-)-CBD. Comparative docking investigations suggest that potent compounds interact with a putative allosteric pocket to stabilize the inactive µOR conformation. Finally, these compounds enhance naloxone displacement of fentanyl from the orthosteric site. Our results suggest that CBD analogs offer considerable potential for the development of next-generation antidotes for opioid overdose.

Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis.

Enterococcus faecalis virulence requires cell wall-associated proteins, including the sortase-assembled endocarditis and biofilm associated pilus (Ebp), important for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that sortases attach substrates to lipid II peptidoglycan (PG) precursors, prior to their incorporation into the growing cell wall. Contrary to prevailing dogma, by following the distribution of Ebp and PG throughout the E. faecalis cell cycle, we found that cell surface Ebp do not co-localize with newly synthesized PG. Instead, surface-exposed Ebp are localized to the older cell hemisphere and excluded from sites of new PG synthesis at the septum. Moreover, Ebp deposition on the younger hemisphere of the E. faecalis diplococcus appear as foci adjacent to the nascent septum. We propose a new model whereby sortase substrate deposition can occur on older PG rather than at sites of new cell wall synthesis. Consistent with this model, we demonstrate that sequestering lipid II to block PG synthesis via ramoplanin, does not impact new Ebp deposition at the cell surface. These data support an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto uncrosslinked cell wall, independent of new PG synthesis.

Evolution of longitudinal division in multicellular bacteria of the Neisseriaceae family.

Rod-shaped bacteria typically elongate and divide by transverse fission. However, several bacterial species can form rod-shaped cells that divide longitudinally. Here, we study the evolution of cell shape and division mode within the family Neisseriaceae, which includes Gram-negative coccoid and rod-shaped species. In particular, bacteria of the genera Alysiella, Simonsiella and Conchiformibius, which can be found in the oral cavity of mammals, are multicellular and divide longitudinally. We use comparative genomics and ultrastructural microscopy to infer that longitudinal division within Neisseriaceae evolved from a rod-shaped ancestor. In multicellular longitudinally-dividing species, neighbouring cells within multicellular filaments are attached by their lateral peptidoglycan. In these bacteria, peptidoglycan insertion does not appear concentric, i.e. from the cell periphery to its centre, but as a medial sheet guillotining each cell. Finally, we identify genes and alleles associated with multicellularity and longitudinal division, including the acquisition of amidase-encoding gene amiC2, and amino acid changes in proteins including MreB and FtsA. Introduction of amiC2 and allelic substitution of mreB in a rod-shaped species that divides by transverse fission results in shorter cells with longer septa. Our work sheds light on the evolution of multicellularity and longitudinal division in bacteria, and suggests that members of the Neisseriaceae family may be good models to study these processes due to their morphological plasticity and genetic tractability.

Dynamics of plasmid-mediated niche invasion, immunity to invasion, and pheromone-inducible conjugation in the murine gastrointestinal tract.

Microbial communities provide protection to their hosts by resisting pathogenic invasion. Microbial residents of a host often exclude subsequent colonizers, but this protection is not well understood. The Enterococcus faecalis plasmid pCF10, whose conjugative transfer functions are induced by a peptide pheromone, efficiently transfers in the intestinal tract of mice. Here we show that an invading donor strain established in the gastrointestinal tract of mice harboring resident recipients, resulting in a stable, mixed population comprised of approximately 10% donors and 90% recipients. We also show that the plasmid-encoded surface protein PrgB (Aggregation Substance), enhanced donor invasion of resident recipients, and resistance of resident donors to invasion by recipients. Imaging of the gastrointestinal mucosa of mice infected with differentially labeled recipients and donors revealed pheromone induction within microcolonies harboring both strains in close proximity, suggesting that adherent microcolonies on the mucosal surface of the intestine comprise an important niche for cell-cell signaling and plasmid transfer.

FtsZ-mediated fission of a cuboid bacterial symbiont.

Less than a handful of cuboid and squared cells have been described in nature, which makes them a rarity. Here, we show how Candidatus Thiosymbion cuboideus, a cube-like gammaproteobacterium, reproduces on the surface of marine free-living nematodes. Immunostaining of symbiont cells with an anti-fimbriae antibody revealed that they are host-polarized, as these appendages exclusively localized at the host-proximal (animal-attached) pole. Moreover, by applying a fluorescently labeled metabolic probe to track new cell wall insertion in vivo, we observed that the host-attached pole started septation before the distal one. Similarly, Ca. T. cuboideus cells immunostained with an anti-FtsZ antibody revealed a proximal-to-distal localization pattern of this tubulin homolog. Although FtsZ has been shown to arrange into squares in synthetically remodeled cuboid cells, here we show that FtsZ may also mediate the division of naturally occurring ones. This implies that, even in natural settings, membrane roundness is not required for FtsZ function.

The bacterial tyrosine kinase system CpsBCD governs the length of capsule polymers.

Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse-chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.

Characterisation of a putative M23-domain containing protein in Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis remains a global health concern, further compounded by the high rates of HIV-TB co-infection and emergence of multi- and extensive drug resistant TB, all of which have hampered efforts to eradicate this disease. As a result, novel anti-tubercular interventions are urgently required, with the peptidoglycan component of the M. tuberculosis cell wall emerging as an attractive drug target. Peptidoglycan M23 endopeptidases can function as active cell wall hydrolases or degenerate activators of hydrolases in a variety of bacteria, contributing to important processes such as bacterial growth, division and virulence. Herein, we investigate the function of the Rv0950-encoded putative M23 endopeptidase in M. tuberculosis. In silico analysis revealed that this protein is conserved in mycobacteria, with a zinc-binding catalytic site predictive of hydrolytic activity. Transcript analysis indicated that expression of Rv0950c was elevated during lag and log phases of growth and reduced in stationary phase. Deletion of Rv0950c yielded no defects in growth, colony morphology, antibiotic susceptibility or intracellular survival but caused a reduction in cell length. Staining with a monopeptide-derived fluorescent D-amino acid, which spatially reports on sites of active PG biosynthesis or repair, revealed an overall reduction in uptake of the probe in ΔRv0950c. When stained with a dipeptide probe in the presence of cell wall damaging agents, the ΔRv0950c mutant displayed reduced sidewall labelling. As bacterial peptidoglycan metabolism is important for survival and pathogenesis, the role of Rv0950c and other putative M23 endopeptidases in M. tuberculosis should be explored further.

Unipolar Peptidoglycan Synthesis in the Rhizobiales Requires an Essential Class A Penicillin-Binding Protein.

Members of the Rhizobiales are polarly growing bacteria that lack homologs of the canonical Rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant pathogen Agrobacterium tumefaciens. The development of fluorescent d-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an additional mode of cell wall synthesis that requires ld-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation. IMPORTANCE While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation. Yet the vast majority of our understanding regarding bacterial elongation is derived from model organisms exhibiting lateral elongation. Here, we explore the role of penicillin-binding proteins in unipolar elongation of Agrobacterium tumefaciens and related bacteria within the Rhizobiales. Our findings suggest that penicillin-binding protein 1a, along with a subset of ld-transpeptidases, drives unipolar growth. Thus, these enzymes may serve as attractive targets for biocontrol of pathogenic Rhizobiales.

Class A Penicillin-Binding Protein-Mediated Cell Wall Synthesis Promotes Structural Integrity during Peptidoglycan Endopeptidase Insufficiency in Vibrio cholerae.

The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth, and structural integrity. PG is synthesized by class A/B penicillin-binding proteins (a/bPBPs) and shape, elongation, division, and sporulation (SEDS) proteins like RodA (as part of the Rod system cell elongation machinery) and degraded by "autolytic" enzymes to accommodate growth processes. It is thought that autolysins (particularly endopeptidases [EPs]) are required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and PG synthesis remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae We found that EP depletion resulted in severe morphological and division defects, but these cells continued to increase in mass and aberrantly incorporated new cell wall material. Mass increase proceeded in the presence of Rod system inhibitors, but cells lysed upon inhibition of aPBPs, suggesting that aPBPs are required for structural integrity under these conditions. The Rod system, although not essential for the observed mass increase, remained functional even after prolonged EP depletion. Last, heterologous expression of an EP from Neisseria gonorrhoeae fully complemented growth and morphology of an EP-insufficient V. cholerae, highlighting the possibility that the PG synthases may not necessarily function via direct interaction with EPs. Overall, our findings suggest that during EP insufficiency in V. cholerae, aPBPs become essential for structural integrity while the Rod system is unable to promote proper cell expansion.IMPORTANCE Synthesis and turnover of the bacterial cell wall must be tightly coordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines, the aPBPs and the Rod system. Our results suggest that in Vibrio cholerae, one class of turnover enzymes, the endopeptidases, are necessary for proper cell elongation and division. aPBPs become essential for maintaining structural integrity during EP insufficiency, while the Rod system remains active but contributes little to cell expansion under these conditions. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the coordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes.

Organization of peptidoglycan synthesis in nodes and separate rings at different stages of cell division of Streptococcus pneumoniae.

Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid-shaped Streptococcus pneumoniae (Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Labeling with fluorescent D-amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin-binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading-edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA-marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced "nodes" of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.

Synthesis of 9-Dechlorochrysophaentin A Enables Studies Revealing Bacterial Cell Wall Biosynthesis Inhibition Phenotype in B. subtilis.

Chrysophaentin A is an antimicrobial natural product isolated from the marine alga C. taylori in milligram quantity. Structurally, chrysophaentin A features a macrocyclic biaryl ether core incorporating two trisubstituted chloroalkenes at its periphery. A concise synthesis of iso- and 9-dechlorochrysophaentin A enabled by a Z-selective ring-closing metathesis (RCM) cyclization followed by an oxygen to carbon ring contraction is described. Fluorescent microscopy studies revealed 9-dechlorochrysophaentins leads to inhibition of bacterial cell wall biosynthesis by disassembly of key divisome proteins, the cornerstone to bacterial cell wall biosynthesis and division.

A Division of Labor in the Recruitment and Topological Organization of a Bacterial Morphogenic Complex.

Bacteria come in an array of shapes and sizes, but the mechanisms underlying diverse morphologies are poorly understood. The peptidoglycan (PG) cell wall is the primary determinant of cell shape. At the molecular level, morphological variation often results from the regulation of enzymes involved in cell elongation and division. These enzymes are spatially controlled by cytoskeletal scaffolding proteins, which both recruit and organize the PG synthesis complex. How then do cells define alternative morphogenic processes that are distinct from cell elongation and division? To address this, we have turned to the specific morphotype of Alphaproteobacterial stalks. Stalk synthesis is a specialized form of zonal growth, which requires PG synthesis in a spatially constrained zone to extend a thin cylindrical projection of the cell envelope. The morphogen SpmX defines the site of stalk PG synthesis, but SpmX is a PG hydrolase. How then does a non-cytoskeletal protein, SpmX, define and constrain PG synthesis to form stalks? Here, we report that SpmX and the bactofilin BacA act in concert to regulate stalk synthesis in Asticcacaulis biprosthecum. We show that SpmX recruits BacA to the site of stalk synthesis. BacA then serves as a stalk-specific topological organizer for PG synthesis activity, including its recruiter SpmX, at the base of the stalk. In the absence of BacA, cells produce "pseudostalks" that are the result of unconstrained PG synthesis. Therefore, the protein responsible for recruitment of a morphogenic PG remodeling complex, SpmX, is distinct from the protein that topologically organizes the complex, BacA.

The Pneumococcal Iron Uptake Protein A (PiuA) Specifically Recognizes Tetradentate FeIIIbis- and Mono-Catechol Complexes.

Streptococcus pneumoniae (Spn) is an important Gram-positive human pathogen that causes millions of infections worldwide with an increasing occurrence of antibiotic resistance. Fe acquisition is a crucial virulence determinant in Spn; further, Spn relies on exogenous FeIII-siderophore scavenging to meet nutritional Fe needs. Recent studies suggest that the human catecholamine stress hormone, norepinephrine (NE), facilitates Fe acquisition in Spn under conditions of transferrin-mediated Fe starvation. Here we show that the solute binding lipoprotein PiuA from the piu Fe acquisition ABC transporter PiuBCDA, previously described as an Fe-hemin binding protein, binds tetradentate catechol FeIII complexes, including NE and the hydrolysis products of enterobactin. Two protein-derived ligands (H238, Y300) create a coordinately saturated FeIII complex, which parallel recent studies in the Gram-negative intestinal pathogen Campylobacter jejuni. Our in vitro studies using NMR spectroscopy and 54Fe LC-ICP-MS confirm the FeIII can move from transferrin to apo-PiuA in an NE-dependent manner. Structural analysis of PiuA FeIII-bis-catechol and GaIII-bis-catechol and GaIII-(NE)2 complexes by NMR spectroscopy reveals only localized structural perturbations in PiuA upon ligand binding, largely consistent with recent descriptions of other solute binding proteins of type II ABC transporters. We speculate that tetradentate FeIII complexes formed by mono- and bis-catechol species are important Fe sources in Gram-positive human pathogens, since PiuA functions in the same way as SstD from Staphylococcus aureus.

Non-walled spherical Acinetobacter baumannii is an important type of persister upon ß-lactam antibiotic treatment.

Bacterial persistence is one of the major causes of antibiotic treatment failure and the step stone for antibiotic resistance. However, the mechanism by which persisters arise has not been well understood. Maintaining a dormant state to prevent antibiotics from taking effect is believed to be the fundamental mechanistic basis, and persisters normally maintain an intact cellular structure. Here we examined the morphologies of persisters in Acinetobacter baumannii survived from the treatment by three major classes of antibiotics (i.e. ß-lactam, aminoglycoside, and fluoroquinolone) with microcopy and found that a fraction of enlarged spherical bacteria constitutes a major sub-population of bacterial survivors from ß-lactam antibiotic treatment, whereas survivors from the treatment of aminoglycoside and fluoroquinolone were less changed morphologically. Further studies showed that these spherical bacteria had completely lost their cell wall structures but could survive without any osmoprotective reagent. The spherical bacteria were not the viable-but-non-culturable cells and they could revive upon the removal of ß-lactam antibiotics. Importantly, these non-walled spherical bacteria also persisted during antibiotic therapy in vivo using Galleria mellonella as the infection model. Additionally, the combinational treatment on A. baumannii by ß-lactam and membrane-targeting antibiotic significantly enhanced the killing efficacy. Our results indicate that in addition to the dormant, structure intact persisters, the non-wall spherical bacterium is another important type of persister in A. baumannii. The finding suggests that targeting the bacterial cell membrane during ß-lactam chemotherapy could enhance therapeutic efficacy on A. baumannii infection, which might also help to reduce the resistance development of A. baumannii.

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum.

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.

Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori.

Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. The helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod H. pylori. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape.

Round spheres, straight rods, and twisting corkscrews, bacteria come in many different shapes. The shape of bacteria is dictated by their cell wall, the strong outer barrier of the cell. As bacteria grow and multiply, they must add to their cell wall while keeping the same basic shape. The cells walls are made from long chain-like molecules via processes that are guided by protein scaffolds within the cell. Many common antibiotics, including penicillin, stop bacterial infections by interrupting the growth of cell walls. Helicobacter pylori is a common bacterium that lives in the gut and, after many years, can cause stomach ulcers and stomach cancer. H. pylori are shaped in a twisting helix, much like a corkscrew. This shape helps H. pylori to take hold and colonize the stomach. It remains unclear how H. pylori creates and maintains its helical shape. The helix is much more curved than other bacteria, and H. pylori does not have the same helpful proteins that other curved bacteria do. If H. pylori grows asymmetrically, adding more material to the cell wall on its long outer side to create a twisting helix, what controls the process? To find out, Taylor et al. grew H. pylori cells and watched how the cell walls took shape. First, a fluorescent dye was attached to the building blocks of the cell wall or to underlying proteins that were thought to help direct its growth. The cells were then imaged in 3D, and images from hundreds of cells were reconstructed to analyze the growth patterns of the bacteria's cell wall. A protein called CcmA was found most often on the long side of the twisting H. pylori. When the CcmA protein was isolated in a dish, it spontaneously formed sheets and helical bundles, confirming its role as a structural scaffold for the cell wall. When CcmA was absent from the cell of H. pylori, Taylor et al. observed that the pattern of cell growth changed substantially. This work identifies a key component directing the growth of the cell wall of H. pylori and therefore, a new target for antibiotics. Its helical shape is essential for H. pylori to infect the gut, so blocking the action of the CcmA protein may interrupt cell wall growth and prevent stomach infections.

Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis.

Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.