The function of CozE proteins is linked to lipoteichoic acid biosynthesis in Staphylococcus aureus.

Coordinated membrane and cell wall synthesis is vital for maintaining cell integrity and facilitating cell division in bacteria. However, the molecular mechanisms that underpin such coordination are poorly understood. Here we uncover the pivotal roles of the staphylococcal proteins CozEa and CozEb, members of a conserved family of membrane proteins previously implicated in bacterial cell division, in the biosynthesis of lipoteichoic acids (LTA) and maintenance of membrane homeostasis in Staphylococcus aureus. We establish that there is a synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc2DAG. By contrast, in cells lacking LtaA, the flippase of Glc2DAG, the essentiality of CozE proteins was alleviated, suggesting that the function of CozE proteins is linked to the synthesis and flipping of the glycolipid anchor. CozE proteins were indeed found to modulate the flipping activity of LtaA in vitro. Furthermore, CozEb was shown to control LTA polymer length and stability. Together, these findings establish CozE proteins as novel players in membrane homeostasis and LTA biosynthesis in S. aureus.IMPORTANCELipoteichoic acids are major constituents of the cell wall of Gram-positive bacteria. These anionic polymers are important virulence factors and modulators of antibiotic susceptibility in the important pathogen Staphylococcus aureus. They are also critical for maintaining cell integrity and facilitating proper cell division. In this work, we discover that a family of membrane proteins named CozE is involved in the biosynthesis of lipoteichoic acids (LTAs) in S. aureus. CozE proteins have previously been shown to affect bacterial cell division, but we here show that these proteins affect LTA length and stability, as well as the flipping of glycolipids between membrane leaflets. This new mechanism of LTA control may thus have implications for the virulence and antibiotic susceptibility of S. aureus.

The F pilus serves as a conduit for the DNA during conjugation between physically distant bacteria.

Horizontal transfer of F-like plasmids by bacterial conjugation is responsible for disseminating antibiotic resistance and virulence determinants among pathogenic Enterobacteriaceae species, a growing health concern worldwide. Central to this process is the conjugative F pilus, a long extracellular filamentous polymer that extends from the surface of plasmid donor cells, allowing it to probe the environment and make contact with the recipient cell. It is well established that the F pilus can retract to bring mating pair cells in tight contact before DNA transfer. However, whether DNA transfer can occur through the extended pilus has been a subject of active debate. In this study, we use live-cell microscopy to show that while most transfer events occur between cells in direct contact, the F pilus can indeed serve as a conduit for the DNA during transfer between physically distant cells. Our findings enable us to propose a unique model for conjugation that revises our understanding of the DNA transfer mechanism and the dissemination of drug resistance and virulence genes within complex bacterial communities.

New insights into the Undecaprenol monophosphate recycling pathway of Streptococcus pneumoniae.

Recycling of undecaprenol pyrophosphate is critical to regenerate the pool of undecaprenol monophosphate required for cell wall biosynthesis. Undecaprenol pyrophosphate is dephosphorylated by membrane-associated undecaprenyl pyrophosphate phosphatases such as UppP or type 2 Phosphatidic Acid Phosphatases (PAP2) and then transferred across the cytoplasmic membrane by Und-P flippases such as PopT (DUF368-containing protein) or UptA (a DedA family protein). While the deletion of uppP in S. pneumoniae has been reported to increase susceptibility to bacitracin and reduce infectivity in a murine infection model, the presence of PAP2 family proteins or Und-P flippases and their potential interplay with UppP in S. pneumoniae remained unknown. In this report, we identified two PAP2 family proteins and a DUF368-containing protein and investigated their roles together with that of UppP in cell growth, cell morphology and susceptibility to bacitracin in S. pneumoniae. Our results suggest that the undecaprenol monophosphate recycling pathway in S. pneumoniae could result from a functional redundancy between UppP, the PAP2-family protein Spr0434 and the DUF368-containing protein Spr0889.

The morphogenic protein CopD controls the spatio-temporal dynamics of PBP1a and PBP2b in Streptococcus pneumoniae.

IMPORTANCE: Penicillin-binding proteins (PBPs) are essential for proper bacterial cell division and morphogenesis. The genome of Streptococcus pneumoniae encodes for two class B PBPs (PBP2x and 2b), which are required for the assembly of the peptidoglycan framework and three class A PBPs (PBP1a, 1b and 2a), which remodel the peptidoglycan mesh during cell division. Therefore, their activities should be finely regulated in space and time to generate the pneumococcal ovoid cell shape. To date, two proteins, CozE and MacP, are known to regulate the function of PBP1a and PBP2a, respectively. In this study, we describe a novel regulator (CopD) that acts on both PBP1a and PBP2b. These findings provide valuable information for understanding bacterial cell division. Furthermore, knowing that ß-lactam antibiotic resistance often arises from PBP mutations, the characterization of such a regulator represents a promising opportunity to develop new strategies to resensitize resistant strains.

Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic Actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis. These bacteria have multi-layered cell walls composed of the mycolyl-arabinogalactan-peptidoglycan complex and a polar growth mode, thus requiring tight coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the coiled-coil protein Wag31. Here, using C. glutamicum, we report the discovery of two divisome members: a gephyrin-like repurposed molybdotransferase (Glp) and its membrane receptor (GlpR). Our results show how cell cycle progression requires interplay between Glp/GlpR, FtsZ and Wag31, showcasing a crucial crosstalk between the divisome and elongasome machineries that might be targeted for anti-mycobacterial drug discovery. Further, our work reveals that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis, similar to the gephyrin/GlyR system that mediates synaptic signalling in higher eukaryotes through network organization of membrane receptors and the microtubule cytoskeleton.

Recent progress in our understanding of peptidoglycan assembly in Firmicutes.

Years of intense research have shown that the assembly of peptidoglycan, the extracellular mesh-like polymer surrounding the bacterial cell, is incredibly complex. It requires a suite of reactions catalyzed by dynamic macromolecular protein complexes whose localization and activity should be finely regulated in space and time. In this review, we focus on the main developments reported over the last five years for the assembly of peptidoglycan in Firmicutes, a bacterial phylum that comprises monoderm bacteria and that encompasses well studied bacterial models with different cell shapes and lifestyles.

A CozE Homolog Contributes to Cell Size Homeostasis of Streptococcus pneumoniae.

Control of peptidoglycan assembly is critical to maintain bacterial cell size and morphology. Penicillin-binding proteins (PBPs) are crucial enzymes for the polymerization of the glycan strand and/or their cross-linking via peptide branches. Over the last few years, it has become clear that PBP activity and localization can be regulated by specific cognate regulators. The first regulator of PBP activity in Gram-positive bacteria was discovered in the human pathogen Streptococcus pneumoniae This regulator, named CozE, controls the activity of the bifunctional PBP1a to promote cell elongation and achieve a proper cell morphology. In this work, we studied a previously undescribed CozE homolog in the pneumococcus, which we named CozEb. This protein displays the same membrane organization as CozE but is much more widely conserved among Streptococcaceae genomes. Interestingly, cozEb deletion results in cells that are smaller than their wild-type counterparts, which is the opposite effect of cozE deletion. Furthermore, double deletion of cozE and cozEb results in poor viability and exacerbated cell shape defects. Coimmunoprecipitation further showed that CozEb is part of the same complex as CozE and PBP1a. However, although we confirmed that CozE is required for septal localization of PBP1a, the absence of CozEb has no effect on PBP1a localization. Nevertheless, we found that the overexpression of CozEb can compensate for the absence of CozE in all our assays. Altogether, our results show that the interplay between PBP1a and the cell size regulators CozE and CozEb is required for the maintenance of pneumococcal cell size and shape.IMPORTANCE Penicillin-binding proteins (PBPs), the proteins catalyzing the last steps of peptidoglycan assembly, are critical for bacteria to maintain cell size, shape, and integrity. PBPs are consequently attractive targets for antibiotics. Resistance to antibiotics in Streptococcus pneumoniae (the pneumococcus) are often associated with mutations in the PBPs. In this work, we describe a new protein, CozEb, controlling the cell size of pneumococcus. CozEb is a highly conserved integral membrane protein that works together with other proteins to regulate PBPs and peptidoglycan synthesis. Deciphering the intricate mechanisms by which the pneumococcus controls peptidoglycan assembly might allow the design of innovative anti-infective strategies, for example, by resensitizing resistant strains to PBP-targeting antibiotics.

RocS drives chromosome segregation and nucleoid protection in Streptococcus pneumoniae.

Chromosome segregation in bacteria is poorly understood outside some prominent model strains1-5 and even less is known about how it is coordinated with other cellular processes. This is the case for the opportunistic human pathogen Streptococcus pneumoniae (the pneumococcus)6, which lacks the Min and the nucleoid occlusion systems7, and possesses only an incomplete chromosome partitioning Par(A)BS system, in which ParA is absent8. The bacterial tyrosine kinase9 CpsD, which is required for capsule production, was previously found to interfere with chromosome segregation10. Here, we identify a protein of unknown function that interacts with CpsD and drives chromosome segregation. RocS (Regulator of Chromosome Segregation) is a membrane-bound protein that interacts with both DNA and the chromosome partitioning protein ParB to properly segregate the origin of replication region to new daughter cells. In addition, we show that RocS interacts with the cell division protein FtsZ and hinders cell division. Altogether, this work reveals that RocS is the cornerstone of a nucleoid protection system ensuring proper chromosome segregation and cell division in coordination with the biogenesis of the protective capsular layer.

Bacterial adhesion at the single-cell level.

The formation of multicellular microbial communities, called biofilms, starts from the adhesion of a few planktonic cells to the surface. The transition from a free-living planktonic lifestyle to a sessile, attached state is a multifactorial process that is determined by biological, chemical and physical properties of the environment, the surface and the bacterial cell. The initial weak, reversible interactions between a bacterium and a surface strengthen to yield irreversible adhesion. In this Review, we summarize our understanding of the mechanisms governing bacterial adhesion at the single-cell level, including the physical forces experienced by a cell before reaching the surface, the first contact with a surface and the transition from reversible to permanent adhesion.

Restricted Localization of Photosynthetic Intracytoplasmic Membranes (ICMs) in Multiple Genera of Purple Nonsulfur Bacteria.

In bacteria and eukaryotes alike, proper cellular physiology relies on robust subcellular organization. For the phototrophic purple nonsulfur bacteria (PNSB), this organization entails the use of a light-harvesting, membrane-bound compartment known as the intracytoplasmic membrane (ICM). Here we show that ICMs are spatially and temporally localized in diverse patterns among PNSB. We visualized ICMs in live cells of 14 PNSB species across nine genera by exploiting the natural autofluorescence of the photosynthetic pigment bacteriochlorophyll (BChl). We then quantitatively characterized ICM localization using automated computational analysis of BChl fluorescence patterns within single cells across the population. We revealed that while many PNSB elaborate ICMs along the entirety of the cell, species across as least two genera restrict ICMs to discrete, nonrandom sites near cell poles in a manner coordinated with cell growth and division. Phylogenetic and phenotypic comparisons established that ICM localization and ICM architecture are not strictly interdependent and that neither trait fully correlates with the evolutionary relatedness of the species. The natural diversity of ICM localization revealed herein has implications for both the evolution of phototrophic organisms and their light-harvesting compartments and the mechanisms underpinning spatial organization of bacterial compartments.IMPORTANCE Many bacteria organize their cellular space by constructing subcellular compartments that are arranged in specific, physiologically relevant patterns. The purple nonsulfur bacteria (PNSB) utilize a membrane-bound compartment known as the intracytoplasmic membrane (ICM) to harvest light for photosynthesis. It was previously unknown whether ICM localization within cells is systematic or irregular and if ICM localization is conserved among PNSB. Here we surveyed ICM localization in diverse PNSB and show that ICMs are spatially organized in species-specific patterns. Most strikingly, several PNSB resolutely restrict ICMs to regions near the cell poles, leaving much of the cell devoid of light-harvesting machinery. Our results demonstrate that bacteria of a common lifestyle utilize unequal portions of their intracellular space to harvest light, despite light harvesting being a process that is intuitively influenced by surface area. Our findings therefore raise fundamental questions about ICM biology and evolution.

Author Correction: Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation.

In the original version of this Article, a grant number and acknowledgement were omitted. The Acknowledgements section should have stated that one of the 3D SIM microscopes used for this research was supported by Medical Research Council UK grant (MR/K015753/1) to S. Foster, University of Sheffield, UK, and that the authors thank C. Walther and S. Foster for the access and their kind help with this. This has now been corrected in all versions of the Article.

Obstruction of pilus retraction stimulates bacterial surface sensing.

It is critical for bacteria to recognize surface contact and initiate physiological changes required for surface-associated lifestyles. Ubiquitous microbial appendages called pili are involved in sensing surfaces and facilitating downstream behaviors, but the mechanism by which pili mediate surface sensing has been unclear. We visualized Caulobacter crescentus pili undergoing dynamic cycles of extension and retraction. Within seconds of surface contact, these cycles ceased, which coincided with synthesis of the adhesive holdfast required for attachment. Physically blocking pili imposed resistance to pilus retraction, which was sufficient to stimulate holdfast synthesis without surface contact. Thus, to sense surfaces, bacteria use the resistance on retracting, surface-bound pili that occurs upon surface contact.

Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation.

Modification of essential bacterial peptidoglycan (PG)-containing cell walls can lead to antibiotic resistance; for example, ß-lactam resistance by L,D-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG-labelling approach utilizing timed pulses of multiple fluorescent D-amino acids, we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall, L,D-transpeptidaseBd-mediated D-amino acid modifications strengthening prey PG during Bdellovibrio invasion, and a zonal mode of predator elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division.

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.

MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis.

Single-cell analysis of bacteria and subcellular protein localization dynamics has shown that bacteria have elaborate life cycles, cytoskeletal protein networks and complex signal transduction pathways driven by localized proteins. The volume of multidimensional images generated in such experiments and the computation time required to detect, associate and track cells and subcellular features pose considerable challenges, especially for high-throughput experiments. There is therefore a need for a versatile, computationally efficient image analysis tool capable of extracting the desired relationships from images in a meaningful and unbiased way. Here, we present MicrobeJ, a plug-in for the open-source platform ImageJ(1). MicrobeJ provides a comprehensive framework to process images derived from a wide variety of microscopy experiments with special emphasis on large image sets. It performs the most common intensity and morphology measurements as well as customized detection of poles, septa, fluorescent foci and organelles, determines their subcellular localization with subpixel resolution, and tracks them over time. Because a dynamic link is maintained between the images, measurements and all data representations derived from them, the editor and suite of advanced data presentation tools facilitates the image analysis process and provides a robust way to verify the accuracy and veracity of the data.

Single-Cell Analysis of Growth and Cell Division of the Anaerobe Desulfovibrio vulgaris Hildenborough.

Recent years have seen significant progress in understanding basic bacterial cell cycle properties such as cell growth and cell division. While characterization and regulation of bacterial cell cycle is quite well-documented in the case of fast growing aerobic model organisms, no data has been so far reported for anaerobic bacteria. This lack of information in anaerobic microorganisms can mainly be explained by the absence of molecular and cellular tools such as single cell microscopy and fluorescent probes usable for anaerobes and essential to study cellular events and/or subcellular localization of the actors involved in cell cycle. In this study, single-cell microscopy has been adapted to study for the first time, in real time, the cell cycle of a bacterial anaerobe, Desulfovibrio vulgaris Hildenborough (DvH). This single-cell analysis provides mechanistic insights into the cell division cycle of DvH, which seems to be governed by the recently discussed so-called incremental model that generates remarkably homogeneous cell sizes. Furthermore, cell division was reversibly blocked during oxygen exposure. This may constitute a strategy for anaerobic cells to cope with transient exposure to oxygen that they may encounter in their natural environment, thereby contributing to their aerotolerance. This study lays the foundation for the first molecular, single-cell assay that will address factors that cannot otherwise be resolved in bulk assays and that will allow visualization of a wide range of molecular mechanisms within living anaerobic cells.

Adhesins Involved in Attachment to Abiotic Surfaces by Gram-Negative Bacteria.

During the first step of biofilm formation, initial attachment is dictated by physicochemical and electrostatic interactions between the surface and the bacterial envelope. Depending on the nature of these interactions, attachment can be transient or permanent. To achieve irreversible attachment, bacterial cells have developed a series of surface adhesins promoting specific or nonspecific adhesion under various environmental conditions. This article reviews the recent advances in our understanding of the secretion, assembly, and regulation of the bacterial adhesins during biofilm formation, with a particular emphasis on the fimbrial, nonfimbrial, and discrete polysaccharide adhesins in Gram-negative bacteria.

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature.

Nutritional stress induces exchange of cell material and energetic coupling between bacterial species.

Knowledge of the behaviour of bacterial communities is crucial for understanding biogeochemical cycles and developing environmental biotechnology. Here we demonstrate the formation of an artificial consortium between two anaerobic bacteria, Clostridium acetobutylicum (Gram-positive) and Desulfovibrio vulgaris Hildenborough (Gram-negative, sulfate-reducing) in which physical interactions between the two partners induce emergent properties. Molecular and cellular approaches show that tight cell-cell interactions are associated with an exchange of molecules, including proteins, which allows the growth of one partner (D. vulgaris) in spite of the shortage of nutrients. This physical interaction induces changes in expression of two genes encoding enzymes at the pyruvate crossroads, with concomitant changes in the distribution of metabolic fluxes, and allows a substantial increase in hydrogen production without requiring genetic engineering. The stress induced by the shortage of nutrients of D. vulgaris appears to trigger the interaction.