Another brick in the wall

The existence of a peptidoglycan cell wall in chlamydiae has been debated for several years. Several studies suggest that these organisms synthesize a cell wall, but some of the components and biosynthetic machinery seem to be missing and a bona fide cell wall has yet to be described. A recent study has revealed that a functional pathway for meso-diaminopimelate, one of the missing bricks for the wall, exists in chlamydiae. Here, I review the chlamydial cell wall paradox and discuss the importance of this new finding.     

In vitro and in vivo functional activity of Chlamydia MurA,a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance

Organisms of Chlamydia spp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli. After transduction of a lethal DeltamurA mutation into the strain, viability of the E. coli strain became dependent upon expression of the C. trachomatis murA. DNA sequence analysis of murA from C. trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA. In E. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth of C. trachomatis was also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to the E. coli strain expressing the chlamydial murA. Conversion of C. trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA. mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C. trachomatis infection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG in Chlamydia spp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.     

A cell wall protein (YqgA) is genetically related to the cell wall-degrading dl-endopeptidases in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis has a thick cell wall. The cell wall contains various proteins, both for secretion and for peptidoglycan (PG) maintenance. Penicillin-binding proteins for PG synthesis, PG hydrolases (autolysins), and regulator proteins for the autolysins are the known components of the PG maintenance system. YqgA was identified as an abundant protein attached to the cell wall of B. subtilis through a proteomics analysis. The YqgA protein was localized at cell division sites during the transition period between the exponential and the stationary phases. YqgA localization was affected by mutations in the dl-endopeptidases (DLEPases), which are the autolysins involved in cell morphogenesis. Furthermore, yqgA mutations on a background of defective DLEPases led to delays in cell growth and cell morphological changes. These results demonstrate that yqgA is genetically related to the genes encoding DLEPases involved in cell morphogenesis.     

Chlamydia species-dependent differences in the growth requirement for lysosomes

Genome reduction is a hallmark of obligate intracellular pathogens such as Chlamydia, where adaptation to intracellular growth has resulted in the elimination of genes encoding biosynthetic enzymes. Accordingly, chlamydiae rely heavily on the host cell for nutrients yet their specific source is unclear. Interestingly, chlamydiae grow within a pathogen-defined vacuole that is in close apposition to lysosomes. Metabolically-labeled uninfected host cell proteins were provided as an exogenous nutrient source to chlamydiae-infected cells, and uptake and subsequent labeling of chlamydiae suggested lysosomal degradation as a source of amino acids for the pathogen. Indeed, Bafilomycin A1 (BafA1), an inhibitor of the vacuolar H(+)/ATPase that blocks lysosomal acidification and functions, impairs the growth of C. trachomatis and C. pneumoniae, and these effects are especially profound in C. pneumoniae. BafA1 induced the marked accumulation of material within the lysosomal lumen, which was due to the inhibition of proteolytic activities, and this response inhibits chlamydiae rather than changes in lysosomal acidification per se, as cathepsin inhibitors also inhibit the growth of chlamydiae. Finally, the addition of cycloheximide, an inhibitor of eukaryotic protein synthesis, compromises the ability of lysosomal inhibitors to block chlamydial growth, suggesting chlamydiae directly access free amino acids in the host cytosol as a preferred source of these nutrients. Thus, chlamydiae co-opt the functions of lysosomes to acquire essential amino acids.     

Bacterial Cell Wall Synthesis: New Insights from Localization Studies

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.     

Bacterial cell wall synthesis: new insights from localization studies.

In order to maintain shape and withstand intracellular pressure, most bacteria are surrounded by a cell wall that consists mainly of the cross-linked polymer peptidoglycan (PG). The importance of PG for the maintenance of bacterial cell shape is underscored by the fact that, for various bacteria, several mutations affecting PG synthesis are associated with cell shape defects. In recent years, the application of fluorescence microscopy to the field of PG synthesis has led to an enormous increase in data on the relationship between cell wall synthesis and bacterial cell shape. First, a novel staining method enabled the visualization of PG precursor incorporation in live cells. Second, penicillin-binding proteins (PBPs), which mediate the final stages of PG synthesis, have been localized in various model organisms by means of immunofluorescence microscopy or green fluorescent protein fusions. In this review, we integrate the knowledge on the last stages of PG synthesis obtained in previous studies with the new data available on localization of PG synthesis and PBPs, in both rod-shaped and coccoid cells. We discuss a model in which, at least for a subset of PBPs, the presence of substrate is a major factor in determining PBP localization.     

Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation

A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination. Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration. Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG. We report analyses of the structure of PG found within immature, developing Bacillus subtilis forespores. The germ cell wall PG is synthesized first, followed by the cortex PG. The germ cell wall is relatively highly cross-linked. The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex. Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration. We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration. Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth.     

Identification of an essential gene responsible for D-Asp incorporation in the Lactococcus lactis peptidoglycan crossbridge

Bacteria such as Lactococcus lactis have D-aspartate (D-Asp) or its amidated derivative D-asparagine (D-Asn), in their peptidoglycan (PG) interpeptide crossbridge. We performed a subtractive genome analysis to identify L. lactis gene yxbA, orthologues of which being present only in bacteria containing D-amino acids in their PG crossbridge, but absent from those that instead insert L-amino acids or glycine. Inactivation of yxbA required a complementing Streptococcus pneumoniae murMN genes, which express enzymes that incorporate L-Ser-L-Ala or L-Ala-L-Ala in the PG crossbridge. Our results show that (i) yxbA encodes D-Asp ligase responsible for incorporation of D-Asp in the PG crossbridge, and we therefore renamed it as aslA, (ii) it is an essential gene, which makes its product a potential target for specific antimicrobials, (iii) the absence of D-Asp may be complemented by L-Ser-L-Ala or L-Ala-L-Ala in the L. lactis PG, indicating that the PG synthesis machinery is not selective for the side-chain residues, and (iv) lactococcal strains having L-amino acids in their PG crossbridge display defects in cell wall integrity, but are able to efficiently anchor cell wall proteins, indicating relative flexibility of lactococcal transpeptidation reactions with respect to changes in PG sidechain composition.     

Lateral Transfers of Serine Hydroxymethyltransferase (glyA) and UDP-N-Acetylglucosamine Enolpyruvyl Transferase (murA) Genes from Free-living Actinobacteria to the Parasitic Chlamydiae

The chlamydiae are important human and animal pathogens which form a phylogentically distinct lineage within the Bacteria. There is evidence that some genes in these obligate intracellular parasites have undergone lateral exchange with other free-living organisms. In the present work, we describe two interesting cases of lateral gene transfer between chlamydiae and actinobacteria, which have been identified based on the shared presence of conserved inserts in two important proteins. In the enzyme serine hydroxymethyltransferase (SHMT or GlyA protein), which links amino acid and nucleotide metabolisms by generating the key intermediate for one-carbon transfer reactions, two conserved inserts of 3 and 31 amino acids (aa) are uniquely present in various chlamydiae species as well as in a subset of Actinobacteria and in the Treponema species. Similarly, in the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which is involved in the synthesis of cell wall peptidoglycan, a 16-aa conserved insert is specifically present in various sequenced chlamydiae and a subset of actinobacteria (i.e., Streptomyces, Actinomyces, Tropheryma, Bifidobacterium, Leifsonia, Arthrobacter, and Brevibacterium). To determine the phylogenetic depths of the GlyA and MurA inserts, the fragments of these genes from two chlamydiae-like species, Simkania negevensis and Waddlia chondrophila, were PCR amplified and sequenced. The presence of the corresponding inserts in both these species strongly indicates that these inserts are distinctive characteristics of the Chlamydiales order. In phylogenetic trees based on GlyA and MurA protein sequences, the chlamydiae species (and also the Treponema species in the case of GlyA) branched with a high affinity with various insert-containing actinobacteria within a clade of other actinobacteria. These results provide strong evidence that the shared presence of these indels in these bacteria is very likely a consequence of ancient lateral gene transfers from actinobacteria to chlamydiae. Pairwise sequence identity and the branching pattern of the GlyA homologues in the phylogenetic tree indicates that the glyA gene was initially transferred from an actinobacteria to an ancestor of the Treponema genus and from there it was acquired by the common ancestor of the Chlamydiales. [Reviewing Editor: Dr. Siv Andersson]     

Cell wall growth during elongation and division: one ring to bind them?

The role of the cell division protein FtsZ in bacterial cell wall (CW) synthesis is believed to be restricted to localizing proteins involved in the synthesis of the septal wall. In this issue of Molecular Microbiology, the groups of Christine Jacobs-Wagner and Waldemar Vollmer provide compelling evidence that in Caulobacter crescentus, FtsZ plays an additional role in CW synthesis in non-dividing cells. During elongation (cell growth) FtsZ is responsible for the incorporation of CW material in a zone at the midcell by recruiting MurG, a protein involved in peptidoglycan (PG) precursor synthesis. This resembles earlier findings of FtsZ mediated PG synthesis activity in Escherichia coli. A role of FtsZ in PG synthesis during elongation forces a rethink of the current model of CW synthesis in rod-shaped bacteria.     

Prostaglandin synthesis by the cochlea of the guinea pig. Influence of aspirin, gentamicin, and acoustic stimulation

This study describes the synthesis of prostaglandins (PGs) by the vascular structures of the inner ear (lateral wall = stria vascularis and spiral ligament) in vitro. The main PGs produced were PGI2, PGF2 alpha and PGE2. PGI2 and PGF2 alpha were also found in the perilymph. A 350 mg/kg ip injection of aspirin decreased PG synthesis by the lateral wall and PG levels in perilymph. This effect was reversed after 3 days. Gentamicin (10(-9) to 10(-5) M) decreased significantly and reversibly PG synthesis in vitro, as did 100 mg/kg ip injection. Acoustic stimulation increased ex vivo PGI2 and PGE2 synthesis without modifying PG levels in perilymph. Results suggest that PGs could be one humoral mediator of the cochlear microcirculation homeostasis, and, possibly, of the circulatory disturbances reported after acoustic stimulation. The decreased PG synthesis after gentamicin treatment could account for the angiotoxic component observed in aminoglycoside ototoxicity.     

Revisiting the chlamydial type III protein secretion system: clues to the origin of type III protein secretion

The bacterial type III secretion pathway delivers effector proteins into eukaryotic cells. Analysis of the type III system and flagellar export genes in the obligate parasites of the family Chlamydiales suggests that the type III system arose from the flagellar export system in chlamydiae or related bacteria.     

Altered proteoglycan synthesis via the false acceptor pathway can be dissociated from beta-D-xyloside inhibition of proliferation.

beta-D-Xylosides have been used to perturb proteoglycan (PG) synthesis to elucidate the function of PGs in a number of cellular processes, including proliferation, migration, and differentiation. This study was designed to examine whether specific xylosides affect the proliferation of several different cell types and, if so, whether this effect is dependent on altered PG synthesis via the false acceptor pathway. Both methylumbelliferyl beta-D-xylopyranoside and p-nitrophenyl beta-D-xylopyranoside (PNP beta-xyloside) inhibit cell proliferation and modulate PG synthesis; however, the alpha form of PNP xyloside which does not perturb PG synthesis inhibits the proliferation of cultured cells on a molar basis equally as well as the beta form. Conversely, beta-methyl xylopyranoside stimulates the synthesis of free glycosaminoglycan chains equally as well as PNP beta-xyloside and yet has no measurable effect on cell proliferation at comparable doses, indicating that cells can grow normally while experiencing disruption of their proteoglycan metabolism. At doses ranging from 0.5 to 5 mM, PNP beta-xyloside arrests cells in the G1 phase of the cell cycle at the same time point as serum starvation. It also delays the exist of cycling cells from the S phase. This treatment is not cytotoxic and is rapidly reversed by the replacement of PNP beta-xyloside containing medium with control medium. Dimethyl sulfoxide, the most commonly used solvent for beta-xyloside in proteoglycan studies, potentiates the inhibitory effect of PNP beta-xyloside on cell proliferation. These results indicate that the perturbation of PG synthesis via the false acceptor pathway can be uncoupled from control of cell proliferation.     

Staphylococcus aureus Survives with a Minimal Peptidoglycan Synthesis Machine but Sacrifices Virulence and Antibiotic Resistance

Many important cellular processes are performed by molecular machines, composed of multiple proteins that physically interact to execute biological functions. An example is the bacterial peptidoglycan (PG) synthesis machine, responsible for the synthesis of the main component of the cell wall and the target of many contemporary antibiotics. One approach for the identification of essential components of a cellular machine involves the determination of its minimal protein composition. Staphylococcus aureus is a Gram-positive pathogen, renowned for its resistance to many commonly used antibiotics and prevalence in hospitals. Its genome encodes a low number of proteins with PG synthesis activity (9 proteins), when compared to other model organisms, and is therefore a good model for the study of a minimal PG synthesis machine. We deleted seven of the nine genes encoding PG synthesis enzymes from the S. aureus genome without affecting normal growth or cell morphology, generating a strain capable of PG biosynthesis catalyzed only by two penicillin-binding proteins, PBP1 and the bi-functional PBP2. However, multiple PBPs are important in clinically relevant environments, as bacteria with a minimal PG synthesis machinery became highly susceptible to cell wall-targeting antibiotics, host lytic enzymes and displayed impaired virulence in a Drosophila infection model which is dependent on the presence of specific peptidoglycan receptor proteins, namely PGRP-SA. The fact that S. aureus can grow and divide with only two active PG synthesizing enzymes shows that most of these enzymes are redundant in vitro and identifies the minimal PG synthesis machinery of S. aureus. However a complex molecular machine is important in environments other than in vitro growth as the expendable PG synthesis enzymes play an important role in the pathogenicity and antibiotic resistance of S. aureus.     

Type III secretion genes identify a putative virulence locus of Chlamydia

Four genes of Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), whose predicted products are highly homologous to structural and regulatory components of a contact-dependent or type III secretion apparatus, were isolated. Related to genes present in several animal and plant bacterial pathogens, these genes may represent a section of a previously undetected chromosomal virulence locus analogous to several recently described virulence-associated type III secretion loci. The existence of contact-dependent secretion in Chlamydia strongly suggests that these bacteria use pathogenic mechanisms that are similar to those of other intracellular bacterial pathogens. Unlike other intracellular bacteria, however, chlamydiae are metabolically inactive extracellularly and only become capable of global protein synthesis several hours after infection. This implies that chlamydial contact-dependent secretion is only active from within, uniquely after the bacteria have been internalized by eukaryotic cells. The possible role(s) of this pathway in chlamydial pathogenesis are discussed.     

A high throughput screen for inhibitors of fungal cell wall synthesis

Fungal cell wall synthesis is essential for viability, requiring the activity of genes involved in environmental sensing, precursor synthesis, transport, secretion, and assembly. This multitude of potential targets, the availability of known agents targeting this pathway, and the unique nature of fungal cell wall synthesis make this pathway an appealing target for drug discovery. Here we describe the adaptation of an assay monitoring cell wall synthesis for high-throughput screening. The assay requires fungal cell growth, in the presence of the test compound, for 3 h before the cells are subjected to osmotic shock in the presence of a dye that stains DNA. Miniaturization of the assay to a 384-well plate format and removing a mechanical transfer led to subtle changes in the assay characteristics. Validation of the assay with a library of known pharmacologically active agents has identified a number of different classes of compounds that are active in this assay, causing aberrant cell wall morphology and in many cases the inhibition of fungal cell growth.     

Hydrolysis of peptidoglycan is modulated by amidation of meso‐diaminopimelic acid and Mg2+ in Bacillus subtilis

The ability of excess Mg²⁺ to compensate the absence of cell wall related genes in Bacillus subtilis has been known for a long time, but the mechanism has remained obscure. Here, we show that the rigidity of wild‐type cells remains unaffected with excess Mg²⁺, but the proportion of amidated meso‐diaminopimelic (mDAP) acid in their peptidoglycan (PG) is significantly reduced. We identify the amidotransferase AsnB as responsible for mDAP amidation and show that the gene encoding it is essential without added Mg²⁺. Growth without excess Mg²⁺ causes ΔasnB mutant cells to deform and ultimately lyse. In cell regions with deformations, PG insertion is orderly and indistinguishable from the wild‐type. However, PG degradation is unevenly distributed along the sidewalls. Furthermore, ΔasnB mutant cells exhibit increased sensitivity to antibiotics targeting the cell wall. These results suggest that absence of amidated mDAP causes a lethal deregulation of PG hydrolysis that can be inhibited by increased levels of Mg²⁺. Consistently, we find that Mg²⁺ inhibits autolysis of wild‐type cells. We suggest that Mg²⁺ helps to maintain the balance between PG synthesis and hydrolysis in cell wall mutants where this balance is perturbed in favor of increased degradation.     

The GpsB files: the truth is out there

Peptidoglycan (PG), an essential stress‐bearing component of the bacterial cell wall, is synthesised by penicillin binding proteins (PBPs). PG synthesis at the cell division septum is necessary for constructing new poles of progeny cells, and cells cannot elongate without inserting new PG in the side‐wall. The cell division regulator GpsB appears to co‐ordinate PG synthesis at the septum during division and at the side‐wall during elongation in rod‐shaped and ovococcoid Gram‐positive bacteria. How the control over PG synthesis is exerted is unknown. In this issue of Molecular Microbiology, Rued et al. show that in pneumococci GpsB forms complexes with PBP2a and PBP2b, and that deletion or depletion of GpsB prevents closure of the septal ring that in itself is PBP2x‐dependent. Loss of GpsB can be suppressed by spontaneous mutations, including within the gene encoding the only PP2C Ser/Thr phosphatase in Streptococcus pneumoniae, indicating that GpsB plays a key – but unknown – role in protein phosphorylation in pneumococci. Rued et al. combine phenotypic and genotypic analyses of mutant strains that suggest discrepancies in the literature concerning GpsB might have arisen from accumulation of unidentified suppressors, highlighting the importance and power of strain validation and whole genome sequencing in this context.