In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer.

Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.

General protein diffusion barriers create compartments within bacterial cells.

In eukaryotes, the differentiation of cellular extensions such as cilia or neuronal axons depends on the partitioning of proteins to distinct plasma membrane domains by specialized diffusion barriers. However, examples of this compartmentalization strategy are still missing for prokaryotes, although complex cellular architectures are also widespread among this group of organisms. This study reveals the existence of a protein-mediated membrane diffusion barrier in the stalked bacterium Caulobacter crescentus. We show that the Caulobacter cell envelope is compartmentalized by macromolecular complexes that prevent the exchange of both membrane and soluble proteins between the polar stalk extension and the cell body. The barrier structures span the cross-sectional area of the stalk and comprise at least four proteins that assemble in a cell-cycle-dependent manner. Their presence is critical for cellular fitness because they minimize the effective cell volume, allowing faster adaptation to environmental changes that require de novo synthesis of envelope proteins.

Evolution-guided discovery of antibiotics that inhibit peptidoglycan remodelling.

Addressing the ongoing antibiotic crisis requires the discovery of compounds with novel mechanisms of action that are capable of treating drug-resistant infections1. Many antibiotics are sourced from specialized metabolites produced by bacteria, particularly those of the Actinomycetes family2. Although actinomycete extracts have traditionally been screened using activity-based platforms, this approach has become unfavourable owing to the frequent rediscovery of known compounds. Genome sequencing of actinomycetes reveals an untapped reservoir of biosynthetic gene clusters, but prioritization is required to predict which gene clusters may yield promising new chemical matter2. Here we make use of the phylogeny of biosynthetic genes along with the lack of known resistance determinants to predict divergent members of the glycopeptide family of antibiotics that are likely to possess new biological activities. Using these predictions, we uncovered two members of a new functional class of glycopeptide antibiotics-the known glycopeptide antibiotic complestatin and a newly discovered compound we call corbomycin-that have a novel mode of action. We show that by binding to peptidoglycan, complestatin and corbomycin block the action of autolysins-essential peptidoglycan hydrolases that are required for remodelling of the cell wall during growth. Corbomycin and complestatin have low levels of resistance development and are effective in reducing bacterial burden in a mouse model of skin MRSA infection.

Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA.

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we use a combination of genetic and cell biological approaches to describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. Specifically, we show that V. cholerae cells retract MSHA pili and detach from a surface in a diffusion-limited, enclosed environment. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP to induce MSHA pilus retraction. CdpA contains a putative nitric oxide (NO)-sensing NosP domain, and we demonstrate that NO is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that the endogenous production of NO (or an NO-like molecule) in V. cholerae stimulates the retraction of MSHA pili. These results extend our understanding of how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

Surface sensing stimulates cellular differentiation in Caulobacter crescentus.

Cellular differentiation is a fundamental strategy used by cells to generate specialized functions at specific stages of development. The bacterium Caulobacter crescentus employs a specialized dimorphic life cycle consisting of two differentiated cell types. How environmental cues, including mechanical inputs such as contact with a surface, regulate this cell cycle remain unclear. Here, we find that surface sensing by the physical perturbation of retracting extracellular pilus filaments accelerates cell-cycle progression and cellular differentiation. We show that physical obstruction of dynamic pilus activity by chemical perturbation or by a mutation in the outer-membrane pilus secretin CpaC stimulates early initiation of chromosome replication. In addition, we find that surface contact stimulates cell-cycle progression by demonstrating that surface-stimulated cells initiate early chromosome replication to the same extent as planktonic cells with obstructed pilus activity. Finally, we show that obstruction of pilus retraction stimulates the synthesis of the cell-cycle regulator cyclic diguanylate monophosphate (c-di-GMP) through changes in the activity and localization of two key regulatory histidine kinases that control cell fate and differentiation. Together, these results demonstrate that surface contact and sensing by alterations in pilus activity stimulate C. crescentus to bypass its developmentally programmed temporal delay in cell differentiation to more quickly adapt to a surface-associated lifestyle.

HfaE Is a Component of the Holdfast Anchor Complex That Tethers the Holdfast Adhesin to the Cell Envelope.

Bacteria use adhesins to colonize different surfaces and form biofilms. The species of the Caulobacterales order use a polar adhesin called holdfast, composed of polysaccharides, proteins, and DNA, to irreversibly adhere to surfaces. In Caulobacter crescentus, a freshwater Caulobacterales species, the holdfast is anchored at the cell pole via the holdfast anchor (Hfa) proteins HfaA, HfaB, and HfaD. HfaA and HfaD colocalize with holdfast and are thought to form amyloid-like fibers that anchor holdfast to the cell envelope. HfaB, a lipoprotein, is required for the translocation of HfaA and HfaD to the cell surface. Deletion of the anchor proteins leads to a severe defect in adherence resulting from holdfast not being properly attached to the cell and shed into the medium. This phenotype is greater in a ΔhfaB mutant than in a ΔhfaA ΔhfaD double mutant, suggesting that HfaB has other functions besides the translocation of HfaA and HfaD. Here, we identify an additional HfaB-dependent holdfast anchoring protein, HfaE, which is predicted to be a secreted protein. HfaE is highly conserved among Caulobacterales species, with no predicted function. In planktonic culture, hfaE mutants produce holdfasts and rosettes similar to those produced by the wild type. However, holdfasts from hfaE mutants bind to the surface but are unable to anchor cells, similarly to other anchor mutants. We showed that fluorescently tagged HfaE colocalizes with holdfast and that HfaE forms an SDS-resistant high-molecular-weight species consistent with amyloid fiber formation. We propose that HfaE is a novel holdfast anchor protein and that HfaE functions to link holdfast material to the cell envelope. IMPORTANCE For surface attachment and biofilm formation, bacteria produce adhesins that are composed of polysaccharides, proteins, and DNA. Species of the Caulobacterales produce a specialized polar adhesin, holdfast, which is required for permanent attachment to surfaces. In this study, we evaluate the role of a newly identified holdfast anchor protein, HfaE, in holdfast anchoring to the cell surface in two different members of the Caulobacterales with drastically different environments. We show that HfaE plays an important role in adhesion and biofilm formation in the Caulobacterales. Our results provide insights into bacterial adhesins and how they interact with the cell envelope and surfaces.

Competence pili in Streptococcus pneumoniae are highly dynamic structures that retract to promote DNA uptake.

The competence pili of transformable Gram-positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram-negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram-positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) leave transient cavities in the cell wall that facilitate DNA passage, (2) form static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane-embedded transporters, or (3) play an active role in translocating bound DNA via dynamic activity. Here, we use a recently described pilus labeling approach to demonstrate that competence pili in Streptococcus pneumoniae are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling the principal pilus monomer, ComGC, with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results suggest that Gram-positive competence pili in other species may also be dynamic and retractile structures that play an active role in DNA uptake.

The mechanism of force transmission at bacterial focal adhesion complexes.

Various rod-shaped bacteria mysteriously glide on surfaces in the absence of appendages such as flagella or pili. In the deltaproteobacterium Myxococcus xanthus, a putative gliding motility machinery (the Agl-Glt complex) localizes to so-called focal adhesion sites (FASs) that form stationary contact points with the underlying surface. Here we show that the Agl-Glt machinery contains an inner-membrane motor complex that moves intracellularly along a right-handed helical path; when the machinery becomes stationary at FASs, the motor complex powers a left-handed rotation of the cell around its long axis. At FASs, force transmission requires cyclic interactions between the molecular motor and the adhesion proteins of the outer membrane via a periplasmic interaction platform, which presumably involves contractile activity of motor components and possible interactions with peptidoglycan. Our results provide a molecular model of bacterial gliding motility.

Sequential evolution of bacterial morphology by co-option of a developmental regulator.

What mechanisms underlie the transitions responsible for the diverse shapes observed in the living world? Although bacteria exhibit a myriad of morphologies, the mechanisms responsible for the evolution of bacterial cell shape are not understood. We investigated morphological diversity in a group of bacteria that synthesize an appendage-like extension of the cell envelope called the stalk. The location and number of stalks varies among species, as exemplified by three distinct subcellular positions of stalks within a rod-shaped cell body: polar in the genus Caulobacter and subpolar or bilateral in the genus Asticcacaulis. Here we show that a developmental regulator of Caulobacter crescentus, SpmX, is co-opted in the genus Asticcacaulis to specify stalk synthesis either at the subpolar or bilateral positions. We also show that stepwise evolution of a specific region of SpmX led to the gain of a new function and localization of this protein, which drove the sequential transition in stalk positioning. Our results indicate that changes in protein function, co-option and modularity are key elements in the evolution of bacterial morphology. Therefore, similar evolutionary principles of morphological transitions apply to both single-celled prokaryotes and multicellular eukaryotes.

MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumoniae.

In every living organism, cell division requires accurate identification of the division site and placement of the division machinery. In bacteria, this process is traditionally considered to begin with the polymerization of the highly conserved tubulin-like protein FtsZ into a ring that locates precisely at mid-cell. Over the past decades, several systems have been reported to regulate the spatiotemporal assembly and placement of the FtsZ ring. However, the human pathogen Streptococcus pneumoniae, in common with many other organisms, is devoid of these canonical systems and the mechanisms of positioning the division machinery remain unknown. Here we characterize a novel factor that locates at the division site before FtsZ and guides septum positioning in pneumococcus. Mid-cell-anchored protein Z (MapZ) forms ring structures at the cell equator and moves apart as the cell elongates, therefore behaving as a permanent beacon of division sites. MapZ then positions the FtsZ ring through direct protein-protein interactions. MapZ-mediated control differs from previously described systems mostly on the basis of negative regulation of FtsZ assembly. Furthermore, MapZ is an endogenous target of the Ser/Thr kinase StkP, which was recently shown to have a central role in cytokinesis and morphogenesis of S. pneumoniae. We show that both phosphorylated and non-phosphorylated forms of MapZ are required for proper Z-ring formation and dynamics. Altogether, this work uncovers a new mechanism for bacterial cell division that is regulated by phosphorylation and illustrates that nature has evolved a diversity of cell division mechanisms adapted to the different bacterial clades.

The Two Chemotaxis Clusters in Caulobacter crescentus Play Different Roles in Chemotaxis and Biofilm Regulation.

The holdfast polysaccharide adhesin is crucial for irreversible cell adhesion and biofilm formation in Caulobacter crescentus Holdfast production is tightly controlled via developmental regulators, as well as via environmental and physical signals. Here, we identify a novel mode of regulation of holdfast synthesis that involves chemotaxis proteins. We characterized the two identified chemotaxis clusters of C. crescentus and showed that only the previously characterized major cluster is involved in the chemotactic response toward different carbon sources. However, both chemotaxis clusters encoded in the C. crescentus genome play a role in biofilm formation and holdfast production by regulating the expression of hfiA, the gene encoding the holdfast inhibitor HfiA. We show that CheA and CheB proteins act in an antagonistic manner, as follows: while the two CheA proteins negatively regulate hfiA expression, the CheB proteins are positive regulators, thus providing a modulation of holdfast synthesis and surface attachment.IMPORTANCE Chemosensory systems constitute major signal transduction pathways in bacteria. These systems are involved in chemotaxis and other cell responses to environment conditions, such as the production of adhesins to enable irreversible adhesion to a surface and surface colonization. The C. crescentus genome encodes two complete chemotaxis clusters. Here, we characterized the second novel chemotaxis-like cluster. While only the major chemotaxis cluster is involved in chemotaxis, both chemotaxis systems modulate C. crescentus adhesion by controlling expression of the holdfast synthesis inhibitor HfiA. Here, we identify a new level in holdfast regulation, providing new insights into the control of adhesin production that leads to the formation of biofilms in response to the environment.

Comparative Analysis of Ionic Strength Tolerance between Freshwater and Marine Caulobacterales Adhesins.

Bacterial adhesion is affected by environmental factors, such as ionic strength, pH, temperature, and shear forces. Therefore, marine bacteria must have developed adhesins with different compositions and structures than those of their freshwater counterparts to adapt to their natural environment. The dimorphic alphaproteobacterium Hirschia baltica is a marine budding bacterium in the clade CaulobacteralesH. baltica uses a polar adhesin, the holdfast, located at the cell pole opposite the reproductive stalk, for surface attachment and cell-cell adhesion. The holdfast adhesin has been best characterized in Caulobacter crescentus, a freshwater member of the Caulobacterales, and little is known about holdfast compositions and properties in marine Caulobacterales Here, we use H. baltica as a model to characterize holdfast properties in marine Caulobacterales We show that freshwater and marine Caulobacterales use similar genes in holdfast biogenesis and that these genes are highly conserved among the species in the two genera. We determine that H. baltica produces a larger holdfast than C. crescentus and that the holdfasts have different chemical compositions, as they contain N-acetylglucosamine and galactose monosaccharide residues and proteins but lack DNA. Finally, we show that H. baltica holdfasts tolerate higher ionic strength than those of C. crescentus We conclude that marine Caulobacterales holdfasts have physicochemical properties that maximize binding in high-ionic-strength environments.IMPORTANCE Most bacteria spend a large part of their life spans attached to surfaces, forming complex multicellular communities called biofilms. Bacteria can colonize virtually any surface, and therefore, they have adapted to bind efficiently in very different environments. In this study, we compare the adhesive holdfasts produced by the freshwater bacterium C. crescentus and a relative, the marine bacterium H. baltica We show that H. baltica holdfasts have a different morphology and chemical composition and tolerate high ionic strength. Our results show that the H. baltica holdfast is an excellent model to study the effect of ionic strength on adhesion and provides insights into the physicochemical properties required for adhesion in the marine environment.

Flagellar Mutants Have Reduced Pilus Synthesis in Caulobacter crescentus.

Surface appendages, such as flagella and type IV pili, mediate a broad range of bacterial behaviors, including motility, attachment, and surface sensing. While many species harbor both flagella and type IV pili, little is known about how or if their syntheses are coupled. Here, we show that deletions of genes encoding different flagellum machinery components result in a reduction of pilus synthesis in Caulobacter crescentus First, we show that different flagellar mutants exhibit different levels of sensitivity to a pilus-dependent phage and that fewer cells within populations of flagellar mutants make pili. Furthermore, we find that single cells within flagellar mutant populations produce fewer pili per cell. We demonstrate that these gene deletions result in reduced transcription of pilus-associated genes and have a slight but significant effect on general transcription profiles. Finally, we show that the decrease in pilus production is due to a reduction in the pool of pilin subunits that are polymerized into pilus fibers. These data demonstrate that mutations in flagellar gene components not only affect motility but also can have considerable and unexpected consequences for other aspects of cell biology.IMPORTANCE Most bacterial species synthesize surface-exposed appendages that are important for environmental interactions and survival under diverse conditions. It is often assumed that these appendages act independently of each other and that mutations in either system can be used to assess functionality in specific processes. However, we show that mutations in flagellar genes can impact the production of type IV pili, as well as alter general RNA transcriptional profiles compared to a wild-type strain. These data demonstrate that seemingly simple mutations can broadly affect cell-regulatory networks.

A Multiprotein Complex Anchors Adhesive Holdfast at the Outer Membrane of Caulobacter crescentus.

Adhesion allows microbes to colonize surfaces and is the first stage in biofilm formation. Stable attachment of the freshwater alphaproteobacterium Caulobacter crescentus to surfaces requires an adhesive polysaccharide called holdfast, which is synthesized at a specific cell pole and ultimately found at the tip of cylindrical extensions of the cell envelope called stalks. Secretion and anchoring of holdfast to the cell surface are governed by proteins HfsDAB and HfaABD, respectively. The arrangement and organization of these proteins with respect to each other and the cell envelope, and the mechanism by which the holdfast is anchored on cells, are unknown. In this study, we have imaged a series of C. crescentus mutants using electron cryotomography, revealing the architecture and arrangement of the molecular machinery involved in holdfast anchoring in cells. We found that the holdfast is anchored to cells by a defined complex made up of the HfaABD proteins and that the HfsDAB secretion proteins are essential for proper assembly and localization of the HfaABD anchor. Subtomogram averaging of cell stalk tips showed that the HfaABD complex spans the outer membrane. The anchor protein HfaB is the major component of the anchor complex located on the periplasmic side of the outer membrane, while HfaA and HfaD are located on the cell surface. HfaB is the critical component of the complex, without which no HfaABD complex was observed in cells. These results allow us to propose a working model of holdfast anchoring, laying the groundwork for further structural and cell biological investigations.IMPORTANCE Adhesion and biofilm formation are fundamental processes that accompany bacterial colonization of surfaces, which are of critical importance in many infections. Caulobacter crescentus biofilm formation proceeds via irreversible adhesion mediated by a polar polysaccharide called holdfast. Mechanistic and structural details of how the holdfast is secreted and anchored on cells are still lacking. Here, we have assigned the location and described the arrangement of the holdfast anchor complex. This work increases our knowledge of the relatively underexplored field of polysaccharide-mediated adhesion by identifying structural elements that anchor polysaccharides to the cell envelope, which is important in a variety of bacterial species.

Feedback regulation of Caulobacter crescentus holdfast synthesis by flagellum assembly via the holdfast inhibitor HfiA.

To permanently attach to surfaces, Caulobacter crescentusproduces a strong adhesive, the holdfast. The timing of holdfast synthesis is developmentally regulated by cell cycle cues. When C. crescentusis grown in a complex medium, holdfast synthesis can also be stimulated by surface sensing, in which swarmer cells rapidly synthesize holdfast in direct response to surface contact. In contrast to growth in complex medium, here we show that when cells are grown in a defined medium, surface contact does not trigger holdfast synthesis. Moreover, we show that in a defined medium, flagellum synthesis and regulation of holdfast production are linked. In these conditions, mutants lacking a flagellum attach to surfaces over time more efficiently than either wild-type strains or strains harboring a paralyzed flagellum. Enhanced adhesion in mutants lacking flagellar components is due to premature holdfast synthesis during the cell cycle and is regulated by the holdfast synthesis inhibitor HfiA. hfiA transcription is reduced in flagellar mutants and this reduction is modulated by the diguanylate cyclase developmental regulator PleD. We also show that, in contrast to previous predictions, flagella are not necessarily required for C. crescentus surface sensing in the absence of flow, and that arrest of flagellar rotation does not stimulate holdfast synthesis. Rather, our data support a model in which flagellum assembly feeds back to control holdfast synthesis via HfiA expression in a c-di-GMP-dependent manner under defined nutrient conditions.

Diversity Takes Shape: Understanding the Mechanistic and Adaptive Basis of Bacterial Morphology.

The modern age of metagenomics has delivered unprecedented volumes of data describing the genetic and metabolic diversity of bacterial communities, but it has failed to provide information about coincident cellular morphologies. Much like metabolic and biosynthetic capabilities, morphology comprises a critical component of bacterial fitness, molded by natural selection into the many elaborate shapes observed across the bacterial domain. In this essay, we discuss the diversity of bacterial morphology and its implications for understanding both the mechanistic and the adaptive basis of morphogenesis. We consider how best to leverage genomic data and recent experimental developments in order to advance our understanding of bacterial shape and its functional importance.

Mutations in Sugar-Nucleotide Synthesis Genes Restore Holdfast Polysaccharide Anchoring to Caulobacter crescentus Holdfast Anchor Mutants.

Attachment is essential for microorganisms to establish interactions with both biotic and abiotic surfaces. Stable attachment of Caulobacter crescentus to surfaces requires an adhesive polysaccharide holdfast, but the exact composition of the holdfast is unknown. The holdfast is anchored to the cell envelope by outer membrane proteins HfaA, HfaB, and HfaD. Holdfast anchor gene mutations result in holdfast shedding and reduced cell adherence. Translocation of HfaA and HfaD to the cell surface requires HfaB. The Wzx homolog HfsF is predicted to be a bacterial polysaccharide flippase. An hfsF deletion significantly reduced the amount of holdfast produced per cell and slightly reduced adherence. A ΔhfsF ΔhfaD double mutant was completely deficient in adherence. A suppressor screen that restored adhesion in the ΔhfsF ΔhfaD mutant identified mutations in three genes: wbqV, rfbB, and rmlA Both WbqV and RfbB belong to a family of nucleoside-diphosphate epimerases, and RmlA has similarity to nucleotidyltransferases. The loss of wbqV or rfbB in the ΔhfsF ΔhfaD mutant reduced holdfast shedding but did not restore holdfast synthesis to parental levels. Loss of wbqV or rfbB did not restore adherence to a ΔhfsF mutant but did restore adherence and holdfast anchoring to a ΔhfaD mutant, confirming that suppression occurs through restoration of holdfast anchoring. The adherence and holdfast anchoring of a ΔhfaA ΔhfaD mutant could be restored by wbqV or rfbB mutation, but such mutations could not suppress these phenotypes in the ΔhfaB mutant. We hypothesize that HfaB plays an additional role in holdfast anchoring or helps to translocate an unknown factor that is important for holdfast anchoring.IMPORTANCE Biofilm formation results in increased resistance to both environmental stresses and antibiotics. Caulobacter crescentus requires an adhesive holdfast for permanent attachment and biofilm formation, but the exact mechanism of polysaccharide anchoring to the cell and the holdfast composition are unknown. Here we identify novel polysaccharide genes that affect holdfast anchoring to the cell. We identify a new role for the holdfast anchor protein HfaB. This work increases our specific knowledge of the polysaccharide adhesin involved in Caulobacter attachment and the general knowledge regarding production and anchoring of polysaccharide adhesins by bacteria. This work also explores the interactions between different polysaccharide biosynthesis and secretion systems in bacteria.

Peptidoglycan O-acetylation is functionally related to cell wall biosynthesis and cell division in Streptococcus pneumoniae.

The peptidoglycan is a rigid matrix required to resist turgor pressure and to maintain the cellular shape. It is formed by linear glycan chains composed of N-acetylmuramic acid-(ß-1,4)-N-acetylglucosamine (MurNAc-GlcNAc) disaccharides associated through cross-linked peptide stems. The peptidoglycan is continually remodelled by synthetic and hydrolytic enzymes and by chemical modifications, including O-acetylation of MurNAc residues that occurs in most Gram-positive and Gram-negative bacteria. This modification is a powerful strategy developed by pathogens to resist to lysozyme degradation and thus to escape from the host innate immune system but little is known about its physiological function. In this study, we have investigated to what extend peptidoglycan O-acetylation is involved in cell wall biosynthesis and cell division of Streptococcus pneumoniae. We show that O-acetylation driven by Adr protects the peptidoglycan of dividing cells from cleavage by the major autolysin LytA and occurs at the septal site. Our results support a function for Adr in the formation of robust and mature MurNAc O-acetylated peptidoglycan and infer its role in the division of the pneumococcus.

Pathogenic Chlamydia Lack a Classical Sacculus but Synthesize a Narrow, Mid-cell Peptidoglycan Ring, Regulated by MreB, for Cell Division.

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe's developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.

Biological consequences and advantages of asymmetric bacterial growth.

Asymmetries in cell growth and division occur in eukaryotes and prokaryotes alike. Even seemingly simple and morphologically symmetric cell division processes belie inherent underlying asymmetries in the composition of the resulting daughter cells. We consider the types of asymmetry that arise in various bacterial cell growth and division processes, which include both conditionally activated mechanisms and constitutive, hardwired aspects of bacterial life histories. Although asymmetry disposes some cells to the deleterious effects of aging, it may also benefit populations by efficiently purging accumulated damage and rejuvenating newborn cells. Asymmetries may also generate phenotypic variation required for successful exploitation of variable environments, even when extrinsic changes outpace the capacity of cells to sense and respond to challenges. We propose specific experimental approaches to further develop our understanding of the prevalence and the ultimate importance of asymmetric bacterial growth.