Explore the vast range of titles available.

Peptidoglycan is an essential structural component of the cell wall in the majority of bacteria, but the obligate intracellular human pathogen Chlamydia trachomatis was thought to be one of the few exceptions; here a click chemistry approach is used to label peptidoglycan in replicating C. trachomatis with novel d -amino acid dipeptide probes. The chlamydial anomaly resolved The sugar amino acid polymer peptidoglycan is an essential cell-wall component in most free-living bacteria. The Chlamydiales, Gram-negative parasites including the human pathogen Chlamydia trachomatis , were thought to be a rare exception: they encode genes for peptidoglycan biosynthesis and are susceptible to β -lactam antibiotics, yet attempts to detect chlamydial peptidoglycans had failed. Now this paradox, known as the 'chlamydial anomaly', has been resolved. This study, using a novel click chemistry technique to label peptidoglycans with D -amino acid dipeptide probes, demonstrates the presence of peptidoglycans in replicating C. trachomatis . Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure 1 . Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia harbour genes for PG biosynthesis 2 , 3 , 4 , 5 , 6 , 7 and exhibit susceptibility to ‘anti-PG’ antibiotics 8 , 9 , yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’ 10 ). We used a novel approach to metabolically label chlamydial PG using d -amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis were labelled with these probes throughout their biphasic developmental life cycle, and the results of differential probe incorporation experiments conducted in the presence of ampicillin are consistent with the presence of chlamydial PG-modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence so far that chlamydial species possess functional PG.

Staphylococcus aureus is an aggressive pathogen and a model organism to study cell division in sequential orthogonal planes in spherical bacteria. However, the small size of staphylococcal cells has impaired analysis of changes in morphology during the cell cycle. Here we use super-resolution microscopy and determine that S. aureus cells are not spherical throughout the cell cycle, but elongate during specific time windows, through peptidoglycan synthesis and remodelling. Both peptidoglycan hydrolysis and turgor pressure are required during division for reshaping the flat division septum into a curved surface. In this process, the septum generates less than one hemisphere of each daughter cell, a trait we show is common to other cocci. Therefore, cell surface scars of previous divisions do not divide the cells in quadrants, generating asymmetry in the daughter cells. Our results introduce a need to reassess the models for division plane selection in cocci. Staphylococci are spherical bacteria that divide in sequential orthogonal planes. Here, the authors use super-resolution microscopy to show that staphylococcal cells elongate before dividing, and that the division septum generates less than one hemisphere of each daughter cell, generating asymmetry.

To colonize and survive in the host, bacterial pathogens like Acinetobacter baumannii must acquire zinc (Zn). To maintain Zn homeostasis, A. baumannii synthesizes proteins of the COG0523 family which are predicted to chaperone Zn to metalloproteins. Bioinformatic tools identified A. baumannii A1S_0934 as a COG0523 protein, and yeast two-hybrid screening revealed that MurD, an essential muramyl ligase, interacts with A1S_0934. As such, we have named A1S_0934 MurD interacting GTPase COG0523 (MigC). Here we show that MigC is a GTPase whose activity is stimulated upon Zn coordination to a characteristic CxCC (C = Cys; x = Leu/Ile/Met) motif to form a S 3 (N/O) complex. MigC-deficient strains (Δ migC ) display sensitivity to Zn depletion and exhibit altered cell wall architecture in vitro . Biochemical and functional assays confirm the MigC-MurD interaction, which inhibits the catalytic activity of MurD. CRISPRi knockdowns of murD reduce A. baumannii fitness and increase filamentation during Zn depletion, a phenotype reversed in Δ migC strains, suggesting that MigC also inhibits MurD activity in cells. Δ migC cells are elongated and sensitized to ceftriaxone, a cephalosporin antibiotic, consistent with decreased cell wall integrity. The Δ migC strain has reduced ability to colonize in a murine model of pneumonia highlighting the importance of the MigC-MurD interaction induced by A. baumannii infection. Together these data suggest that MigC impacts cell wall biogenesis, in part through interactions with MurD, emphasizing the importance of MigC and MurD to the survival and pathogenicity of A. baumannii while expanding the potential functions of the COG0523 family of enzymes.

The peptidoglycan (PG) layer stabilizes the bacterial cell envelope to maintain the integrity and shape of the cell. Penicillin-binding proteins (PBPs) synthesize essential 4–3 cross-links in PG and are inhibited by β-lactam antibiotics. Some clinical isolates and laboratory strains of Enterococcus faecium and Escherichia coli achieve high-level β-lactam resistance by utilizing β-lactam–insensitive LD-transpeptidases (LDTs) to produce exclusively 3–3 cross-links in PG, bypassing the PBPs. In E. coli, other LDTs covalently attach the lipoprotein Lpp to PG to stabilize the envelope and maintain the permeability barrier function of the outermembrane. Here we show that subminimal inhibitory concentration of copper chloride sensitizes E. coli cells to sodium dodecyl sulfate and impair survival upon LPS transport stress, indicating reduced cell envelope robustness. Cells grown in the presence of copper chloride lacked 3–3 cross-links in PG and displayed reduced covalent attachment of Braun’s lipoprotein and reduced incorporation of a fluorescent D-amino acid, suggesting inhibition of LDTs. Copper dramatically decreased the minimal inhibitory concentration of ampicillin in E. coli and E. faecium strains with a resistance mechanism relying on LDTs and inhibited purified LDTs at submillimolar concentrations. Hence, our work reveals how copper affects bacterial cell envelope stability and counteracts LDT-mediated β-lactam resistance.

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.

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. Microbial communities provide protection to their hosts by excluding colonizing pathogens. Here the authors study plasmid transfer and plasmid-mediated effects on host colonization and persistence of Enterococcus faecalis in the intestinal tract of mice.

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. 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 .

Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae , the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure–function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division. Placement of the bacterial division site is crucial for the creation of identical daughter cells. Here, the authors solve the structure of the MapZ protein, which helps to position the cell division protein FtsZ at the cell centre, and further analyse the function of the protein in vivo .

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. 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. In , 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 β-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.

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. Rod-shaped bacteria typically elongate and divide by transverse fission, but a few species are known to divide longitudinally. Here, the authors use genomic, phylogenetic and microscopy techniques to shed light on the evolution of cell shape, multicellularity and division mode within the family Neisseriaceae .