Biology subjects

Cava, F.

Publications and source records attributed to Cava, F..

At least 19 recordsLinked to original sources

bioRxiv2026

An Expanded Lpt Pathway Couples Surface Lipoprotein Export to Outer Membrane Vesicle Biogenesis in Bacteroides

The ecological dominance of Bacteroidota in the human gut microbiota derives from their exceptional capacity to metabolize diverse dietary and host-derived glycans. This ability relies on surface-exposed lipoproteins (SLPs), which are displayed on the bacterial surface and selectively packaged into outer membrane vesicles (OMVs). Although SLP export and OMV biogenesis are tightly coupled, the mechanism linking these processes has remained unknown. Here, we identify LptZ, a previously uncharacterized protein, as a critical link between SLP surface translocation and OMV biogenesis. Cryo-electron tomography of the lptZ mutant revealed chains of vesicles tethered to the outer membrane, offering a rare glimpse into an arrested stage of OMV formation in which vesicle fission is impaired. Proteomic analysis further showed that, in the absence of LptZ, a subset of SLPs fails to reach the cell surface and instead accumulates at the inner membrane. Unexpectedly, LptZ associates with the lipopolysaccharide transport (Lpt) machinery and repurposes it for SLP translocation. Crosslinking experiments revealed selective associations between multiple SLPs and Lpt proteins, providing mechanistic evidence for the direct involvement of the Lpt machinery in SLP trafficking. We further show that the Bacteroides Lpt machinery contains previously unrecognized components, revealing an unanticipated level of specialization relative to canonical Lpt systems. Together, our findings uncover a previously unknown protein export pathway, expand the functional role of the Lpt machinery, and establish a direct mechanistic link between SLP translocation and OMV biogenesis in Bacteroidota.

microbiology↗

bioRxiv2026

Characterisation of the RNA-Binding Properties of the MRSA β-lactam resistance enzyme PBP2a

Analysis of RNA-binding proteome data from different bacterial species revealed many cell wall metabolic enzymes cross-linking to RNA in vivo, hinting that these proteins directly bind RNA. Surprisingly, penicillin-binding proteins (PBPs) were also abundantly identified as putative RNA-binding proteins. The cell surface localisation properties of many of these proteins therefore beg the question at what stage of their cellular life cycle these proteins interact with RNA and what the functional significance is. Here, we characterised the RNA-binding activity of PBP2a, the alternative transpeptidase that confers {beta}-lactam resistance in MRSA. Using in vivo RNA-binding assays, we show that PBP2a interacts with hundreds of transcripts without apparent sequence specificity. Computational analyses identified a possible RNA-binding cleft in PBP2a proximal to its active site. Mutation of only two predicted positively charged residues located in this cleft substantially reduced cross-linking in vivo, implying that RNA recognition is largely dictated by RNA backbone interactions. While PBP2a does not regulate RNA steady-state levels, RNA-binding appears important for proper protein function: an RNA-binding deficient mutant exhibits reduced oxacillin resistance. These findings establish PBP2a as an RNA-binding protein in vivo and provide a framework to investigate how this non-canonical interaction may relate to cell wall biogenesis and {beta}-lactam resistance.

biochemistry↗

bioRxiv2025

A novel mechanism for bacterial sporulation based on programmed peptidoglycan degradation

Many bacteria form spores to endure unfavorable conditions. While Firmicutes generate endospores through cell division, sporulation in non-Firmicutes remains less understood. The Gram-negative bacterium Myxococcus xanthus undergoes sporulation through two distinct mechanisms: rapid sporulation triggered by chemical induction and slow sporulation driven by starvation, both occurring independently of cell division. Instead, these processes depend on the complete degradation of the peptidoglycan (PG) cell wall by two lytic transglycosylases (LTGs), LtgA and LtgB. Remarkably, LtgB programs the pace of PG degradation by LtgA during rapid sporulation, ensuring a controlled process that prevents abrupt PG breakdown and the formation of non-resistant pseudospores. In addition to regulation between LTGs, PG degradation is also influenced by its synthesis; cells exhibiting increased muropeptide production often circumvent sporulation. These findings not only reveal novel mechanisms of bacterial sporulation but also shed light on the regulatory network governing PG dynamics.

microbiology↗

bioRxiv2025

Disruption of undecaprenyl phosphate recycling suppresses ampC beta-lactamase induction in Pseudomonas aeruginosa

Beta-lactam antibiotics are widely used to treat bacterial infections, but their efficacy is compromised by resistance mechanisms such as the production of beta-lactamases. In Pseudomonas aeruginosa, the chromosomally encoded beta-lactamase AmpC is the primary mediator of beta-lactam resistance. ampC expression is regulated by the transcription factor AmpR, which responds to intracellular peptidoglycan (PG) fragments. Under normal conditions, AmpR binds the PG precursor (UDP-MurNAc-pentapeptide, UDP-M5) and represses ampC expression. However, during beta-lactam treatment or in PG recycling-deficient mutants such as ampD mutants, PG degradation products (anhydromuropeptides, AMP) accumulate and activate AmpR, resulting in elevated ampC expression and beta-lactam resistance. We hypothesized that shifting the balance of PG precursors could modulate AmpR activity and suppress beta-lactamase expression, even in derepressed strains. Undecaprenyl phosphate (UndP) is a lipid carrier essential for translocating PG precursors across the bacterial inner membrane. Recent work has identified members of the DedA superfamily as UndP flippases responsible for recycling this lipid carrier. Disruption of UndP recycling leads to cytoplasmic accumulation of UDP-M5, the known AmpR repressor. Here, we show that deletion of dedA4, which encodes a predicted UndP flippase in P. aeruginosa, causes UDP-M5 accumulation and significantly reduces AmpC production and beta-lactam resistance in an ampD mutant. These findings highlight the influence of PG precursor dynamics on beta-lactamase regulation and identify DedA4 as a promising therapeutic target. Inhibiting UndP recycling offers a novel strategy to counteract beta-lactam resistance in P. aeruginosa and potentially other AmpC-producing pathogens. IMPORTANCEBeta-lactam resistance remains a major clinical challenge, often driven by the overexpression of chromosomally encoded beta-lactamases such as AmpC in Pseudomonas aeruginosa. Strategies to limit ampC activation are urgently needed to preserve the efficacy of these frontline antibiotics. Our study identifies DedA4, a membrane protein involved in undecaprenyl phosphate (UndP) recycling, as a potential target for such intervention. Inactivation of DedA4 disrupts UndP transport, leading to the accumulation of soluble, membrane-unbound PG precursors which act as corepressors of AmpC expression. This disruption in the balance between cell wall synthesis and turnover restores susceptibility to {beta}-lactams, even in strains harboring clinically significant resistance mutations such as those in ampD or dacB. Targeting UndP flippases like DedA4 could therefore represent a novel adjuvant strategy to combat AmpC-mediated beta-lactam resistance.

microbiology↗

bioRxiv2024

Activation of the ChvG-ChvI pathway promotes multiple survival strategies during cell wall stress in Agrobacterium tumefaciens

Agrobacterium tumefaciens shifts from a free-living soil bacterium to a plant-invading state upon encountering the plant root microenvironment. The acid-induced two- component sensor system ChvG-ChvI drives this shift and triggers a complex transcriptional program that promotes host invasion and survival against host immune defenses. Remarkably, ChvG-ChvI is also activated under cell wall stress conditions suggesting that the transcriptional response may have a broader function. Here, we find that blocking cell wall synthesis either genetically or chemically leads to ChvG-ChvI activation. Mutations in key cell wall synthesis or outer membrane proteins, such as PBP1a, FtsW, and AopA1, suppress ChvG-ChvI activation suggesting that providing structural integrity is a primary function of the ChvG-ChvI regulon. Here, we investigated regulon components for this function. First, the exopolysaccharide succinoglycan confers tolerance to multiple {beta}-lactam antibiotics targeting different enzymes by forming a protective barrier around the cells. Next, a Class D {beta}-lactamase is expressed which may contribute to the high level of {beta}-lactam resistance in A. tumefaciens. Finally, outer membrane remodeling compensates for the accumulation of cell wall damage by providing structural integrity. Overall, we expand our understanding of mechanisms driving ChvG-ChvI activation and {beta}-lactam resistance in a bacterial plant pathogen. Significance Statements.O_LIActivation of the ChvG-ChvI two component system promotes survival when the bacterial cell walls are damaged by a variety of genetic or chemical approaches C_LIO_LIThe ChvG-ChvI dependent production of the exopolysaccharide succinoglycan, {beta}-lactamase Cbl activity, and outer membrane proteome remodeling all contribute to survival in the presence of {beta}-lactam antibiotics C_LIO_LIImproved understanding of bacterial stress responses that promote antibiotic tolerance and resistance has the potential to inform development of novel drug targets C_LI

microbiology↗

bioRxiv2024

Bacteria combine polar- and dispersed-growth to power cell elongation and wall width dynamics

The cell wall is a complex structure. For most bacteria, peptidoglycan is an essential component of their cell wall, with different bacteria having evolved distinct biosynthetic strategies. The mechanisms driving bacterial growth can be divided into three, mutually-exclusive categories: (i) dispersed growth, mediated by MreB and employed by many rod-shaped bacteria; (ii) polar growth, driven by distinct proteins in the actinobacteria and rhizobiales; and (iii) septal growth, fueled by FtsZ in many coccoid bacteria. Here, we show that under conditions of rapid growth, the actinobacterial representative Streptomyces venezuelae transcends these categories, simultaneously employing both canonical polar growth, and MreB-mediated dispersed growth. Our results indicate that MreB is essential for cell wall integrity and culture viability under these growth conditions, promotes dynamic cell wall changes over the course of a growth cycle, and contributes to a wall that is structurally distinct from that of conventionally growing streptomycetes.

microbiology↗

bioRxiv2024

Essentiality of LD-Transpeptidation in Agrobacterium tumefaciens

Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane {beta}-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.

microbiology↗

bioRxiv2024

Phenotypic and Transcriptomic Characterization of ElyC-Defective Escherichia coli Cells Reveal the Importance of ElyC in Cell Envelope Biology at Optimal and Sub-Optimal Temperatures

The bacterial cell envelope acts as the frontline defense against environmental and internal stress, maintaining cellular homeostasis. Understanding envelope biology is crucial for both fundamental research and practical applications. Peptidoglycan (PG) is a key structural element, protecting against mechanical and osmotic stress while maintaining cell shape and integrity. In a previous study, we discovered the importance of ElyC, a highly conserved Escherichia coli protein with an unknown function, in maintaining envelope integrity at low temperatures. ElyC is essential for PG assembly at 21{degrees}C and plays a role in lipid carrier metabolism, a crucial step in PG and other bacterial envelope polysaccharide biosynthesis. At 21{degrees}C, ElyC deficiency leads to complete PG assembly blockage and cell lysis. However, the significance of ElyC in cells grown at 37{degrees}C remained unexplored. In our recent study, we conducted phenotypic and transcriptomic profiling of ElyC-defective E. coli cells grown at 37{degrees}C and 21{degrees}C, compared to wild-type cells. While{Delta} elyC mutant cells grow similarly to wild-type cells at 37{degrees}C, microscopy revealed altered cell morphology due to ElyCs absence. PG quantification confirmed significantly inhibited PG biosynthesis at 37{degrees}C without ElyC, and these mutants showed increased sensitivity to PG-targeting {beta}-lactam antibiotics compared to wild-type cells at the same temperature. RNA-Seq analysis of{Delta} elyC mutant and WT strains at 21{degrees}C and 37{degrees}C revealed that ElyC deletion severely affects the cell envelope at 21{degrees}C and moderately at 37{degrees}C. Several pathways and genes, especially stress response pathways, impact cell envelope functions, including biogenesis, maintenance, repair, metabolism, respiratory chain, peptidoglycan, lipopolysaccharide, membrane, cell wall, oxidative stress, osmotic stress, trehalose, chaperone, oxidoreductase, amino sugar synthesis and metabolism, vancomycin and beta-lactam resistance pathways and are affected. Downregulated transcripts are associated with mobility, arginine metabolism, membrane transport, regulation, outer membrane, transferase, and unknown functions. Our data highlights ElyCs broad role in bacterial cell envelope and peptidoglycan biosynthesis at varying temperatures. IMPORTANCEThe molecular pathways governing bacterial envelope biosynthesis, assembly, regulation, and adaptation remain incompletely understood. Envelope biology is vital for both fundamental microbiological research and the development of novel therapeutic targets. We previously established ElyCs role in sub-optimal temperature envelope biology, showing its essentiality for PG assembly and bacterial survival at 21{degrees}C. In this study, we show that ElyC, a protein containing the highly conserved DUF218 domain of unknown function, is crucial for proper cell morphology, PG biosynthesis, antibiotic tolerance and envelope homeostasis at 37{degrees}C. Our findings emphasize the significance of DUF218-containing ElyC in envelope biology at physiological temperatures and uncover a novel cold-sensitive process in bacterial envelope biology.

microbiology↗

bioRxiv2024

Breaking Barriers: pCF10 Type 4 Secretion System relies on a self-regulating muramidase to modulate the cell wall

Conjugative Type 4 Secretion Systems (T4SS) are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient mating bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has 3 predicted extracellular hydrolase domains, LytM, SLT and CHAP. Here, we report the structure of the LytM domain, and show that its active site is degenerate and lacks the active site metal. Further, we show that only the predicted SLT domain is functional, and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Further, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information towards understanding the function of Gram-positive T4SSs.

biochemistry↗

bioRxiv2024

Staphylococcus aureus counters organic acid anion-mediated inhibition of peptidoglycan cross-linking through robust alanine racemase activity

Weak organic acids are commonly found in host niches colonized by bacteria, and they can inhibit bacterial growth as the environment becomes acidic. This inhibition is often attributed to the toxicity resulting from the accumulation of high concentrations of organic anions in the cytosol, which disrupts cellular homeostasis. However, the precise cellular targets that organic anions poison and the mechanisms used to counter organic anion intoxication in bacteria have not been elucidated. Here, we utilize acetic acid, a weak organic acid abundantly found in the gut to investigate its impact on the growth of Staphylococcus aureus. We demonstrate that acetate anions bind to and inhibit D-alanyl-D-alanine ligase (Ddl) activity in S. aureus. Ddl inhibition reduces intracellular D-alanyl-D-alanine (D-Ala-D-Ala) levels, compromising staphylococcal peptidoglycan cross-linking and cell wall integrity. To overcome the effects of acetate-mediated Ddl inhibition, S. aureus maintains a substantial intracellular D-Ala pool through alanine racemase (Alr1) activity and additionally limits the flux of D-Ala to D-glutamate by controlling D-alanine aminotransferase (Dat) activity. Surprisingly, the modus operandi of acetate intoxication in S. aureus is common to multiple biologically relevant weak organic acids indicating that Ddl is a conserved target of small organic anions. These findings suggest that S. aureus may have evolved to maintain high intracellular D-Ala concentrations, partly to counter organic anion intoxication. SignificanceUnder mildly acidic conditions, weak organic acids like acetic acid accumulate to high concentrations within the cytosol as organic anions. However, the physiological consequence of organic anion accumulation is poorly defined. Here we investigate how the acetate anion impacts S. aureus. We show that acetate anions directly bind Ddl and inhibit its activity. The resulting decrease in intracellular D-Ala-D-Ala pools impacts peptidoglycan integrity. Since acetate is a weak inhibitor of Ddl, mechanisms that maintain a high intracellular D-Ala pools are sufficient to counter the effect of acetate-mediated Ddl inhibition in S. aureus.

microbiology↗

bioRxiv2023

The regulation of polar flagella assembly in Pseudomonas putida

The Gram-negative bacterium Pseudomonas putida bears a tuft of flagella at a single cell pole. New flagella must be assembled de novo every cell cycle to secure motility of both daughter cells. Here we show that the coordinated action of FimV, FlhF and FleN sets the location, timing and number of flagella assembled. The polar landmark proteins FimV and FlhF are independently targeted to the nascent new pole during or shortly after cell division, but FimV stabilizes FlhF association with the cell poles. FlhF determines the polar position of the flagella by targeting early flagellar components to the cell pole and preventing their nucleation at non-polar sites. FlhF also promotes efficient flagellar assembly and indirectly stimulates Class III flagellar promoter activation by promoting secretion of the anti-FliA anti-{sigma} factor FlgM. The MinD-like ATPase FleN partitions between the cell poles and the cytoplasm. Cytoplasmic FleN regulates flagellar number by preventing excessive accumulation of FlhF at the cell poles that may otherwise lead to hyperflagellation, likely by antagonizing FleQ-dependent transcriptional activation. FimV is essential to FleN polar location. FimV and FleN temporally regulate the onset of flagellar assembly by preventing premature polar targeting of FlhF and the ensuing premature targeting of additional flagellar components. Our results shed new light on the mechanisms that ensure the timely assembly of the appropriate number of flagella at the correct polar location in polarly flagellated bacteria. HIGHLIGHTSO_LIFimV, FlhF and FleN determine the position, number and timing of flagellar assembly C_LIO_LIFimV is essential to the normal intracellular distribution of FlhF and FleN C_LIO_LIFlhF restricts flagellar location to the cell poles and promotes efficient assembly C_LIO_LISoluble, cytoplasmic FleN prevents polar FlhF accumulation and hyperflagellation C_LIO_LIPole-bound FimV and FleN prevent premature FlhF recruitment and flagellar assembly C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/571843v5_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f57b6dorg.highwire.dtl.DTLVardef@7a38c7org.highwire.dtl.DTLVardef@259c58org.highwire.dtl.DTLVardef@13b6f20_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Maintenance of cell wall remodeling and vesicle production are connected in Mycobacterium tuberculosis

Pathogenic and nonpathogenic mycobacteria secrete extracellular vesicles (EVs) under various conditions. EVs produced by Mycobacterium tuberculosis (Mtb) have raised significant interest for their potential in cell communication, nutrient acquisition, and immune evasion. However, the relevance of vesicle secretion during tuberculosis infection remains unknown due to the limited understanding of mycobacterial vesicle biogenesis. We have previously shown that a transposon mutant in the LCP-related gene virR (virRmut) manifested a strong attenuated phenotype during experimental macrophage and murine infections, concomitant to enhanced vesicle release. In this study, we aimed to understand the role of VirR in the vesicle production process in Mtb. We employ genetic, transcriptional, proteomics, ultrastructural and biochemical methods to investigate the underlying processes explaining the enhanced vesiculogenesis phenomenon observed in the virRmut. Our results establish that VirR is critical to sustain proper cell permeability via regulation of cell envelope remodeling possibly through the interaction with similar cell envelope proteins, which control the link between peptidoglycan and arabinogalactan. These findings advance our understanding of mycobacterial extracellular vesicle biogenesis and suggest that these set of proteins could be attractive targets for therapeutic intervention.

microbiology↗

bioRxiv2023

A distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha and Betaproteobacteria

Most bacteria are surrounded by an essential protective mesh-like structure called peptidoglycan, made of glycan chains crosslinked through short peptides by enzymes known as transpeptidases. Of these, penicillin-binding DD-transpeptidases connect adjacent peptide stems between their 4th and 3rd amino acids (4,3-type), D-alanine and a meso-diaminopimelic acid (mDAP) in Gram negatives, whereas LD-transpeptidases make the 3,3-type between mDAP3 residues. While these two processes explain the formation of crosslinks in most bacteria, recent investigations involving non-model species have brought to light novel crosslinking mechanisms that point to the existence of less-explored groups of peptidoglycan crosslinking enzymes. Here, we present the identification and characterization of a novel LD-transpeptidase found in the acetic acid bacterial Gluconobacter oxydans, named LDTGo, which performs 1,3-type crosslinks between L-Ala1 and mDAP3. LDTGo-like proteins are conserved among Alpha and Betaproteobacteria species that do not encode LD3,3-transpeptidases. Using a highly active ortholog, we demonstrated in vitro that this enzyme can work with non-terminal peptide bonds in the crosslinking process. This property is different from the strict specificity of typical LD- and DD-transpeptidases, which only deal with terminal peptide bonds. The high-resolution crystal structure of LDTGo revealed significant distinctions when compared to 3,3-type LD-transpeptidases. These include a proline-rich region near the N-terminus that restricts substrate access to the active site, and an unprecedented cavity designed to accommodate both the glycan chain and the peptide stem from donor muropeptides, a feature that exhibits broad conservation among LD1,3-transpeptidases. Finally, we demonstrated the involvement of DD-crosslinking turnover in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between structurally distinct crosslinking mechanisms in maintaining cell wall integrity in G. oxydans.

microbiology↗

bioRxiv2023

A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions

Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve (Bb). We generated a high density, randomly barcoded transposon insertion pool in Bb, and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.

microbiology↗

bioRxiv2023

Metabolic reprogramming and flux to cell envelope precursors in a pentose phosphate pathway mutant increases MRSA resistance to β-lactam antibiotics

Central metabolic pathways controls virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to {beta}-lactam antibiotics, particularly in chemically defined media with glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased {beta}-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. Further evidence of the pleiotropic effect of the pgl mutation was reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Reduced binding of wheat germ agglutinin (WGA) to pgl was indicative of lower wall teichoic acid/lipoteichoic acid levels or altered teichoic acid structures. Mutations in the vraFG or graRS loci reversed the increased OX resistance phenotype and restored WGA binding to wild-type levels. VraFG/GraRS was previously implicated in susceptibility to cationic antimicrobial peptides and vancomycin, and these data reveal a broader role for this multienzyme membrane complex in the export of cell envelope precursors or modifying subunits required for resistance to diverse antimicrobial agents. Altogether our study highlights important roles for the PPP and VraFG/GraRS in {beta}-lactam resistance, which will support efforts to identify new drug targets and reintroduce {beta}-lactams in combination with adjuvants or other antibiotics for infections caused by MRSA and other {beta}-lactam resistant pathogens. Author summaryHigh-level resistance to penicillin-type ({beta}-lactam) antibiotics significantly limits the therapeutic options for patients with MRSA infections necessitating the use of newer agents, for which reduced susceptibility has already been described. Here we report for the first time that the central metabolism pentose phosphate pathway controls MRSA resistance to penicillin-type antibiotics. We comprehensively demonstrated that mutation of the PPP gene pgl perturbed metabolism in MRSA leading to increased flux to cell envelope precursors to drive increased antibiotic resistance. Moreover, increased resistance was dependent on the VraRG/GraRS multienzyme membrane complex previously implicated in resistance to antimicrobial peptides and vancomycin. Our data thus provide new insights on MRSA mechanisms of {beta}-lactam resistance, which will support efforts to expand the treatment options for infections caused by this and other antimicrobial resistant pathogens.

microbiology↗

bioRxiv2023

Peptidoglycan remodeling in response to cell wall acting antibiotics in Bacillus subtilis

Most bacteria are encased into a load-bearing rigid framework, the cell wall (CW). The peptidoglycan (PG) layer, a network composed of glycan strands cross-linked by stem peptides, is the main component of the CW. During PG synthesis, precursors are first synthetized intracellularly, before being incorporated into the existing PG meshwork by transglycosylation (TG) and transpeptidation (TP) reactions. Covalent modifications of the PG meshwork such as amidation and acetylation participate in PG homeostasis by regulating PG-associated enzymes like PG hydrolases. Because of its essential role, PG synthesis represents a primary target for antibiotic action. Here, we investigated the effect on PG composition of antibiotics targeting intracellular and extracellular steps of PG synthesis: inhibitors of PG precursors synthesis (fosfomycin, D-cycloserine, bacitracin and tunicamycin) and TG/TP inhibitors (vancomycin and penicillin G), respectively. Our study revealed interesting correlations between crosslinking and both de-N-acetylation and amidation of the sacculus. A thorough analysis of muropeptides composition put into light an unexpected anti-correlation between the degree of PG crosslinking and accumulation of de-amidated disaccharide-tripeptide monomer subunit (M3) in the presence of TP inhibitors. We confirmed these observations by analyzing mutants of the PG synthesis pathway.

microbiology↗

bioRxiv2022

Quantitative analysis of morphogenesis and growth dynamics in an obligate intracellular bacterium

Obligate intracellular bacteria of the order Rickettsiales include numerous arthropod-borne human pathogens. However, our understanding of the basic biology of Rickettsia species is limited by technical challenges imposed by their obligate intracellular lifestyle. To overcome this roadblock, we developed quantitative methods to assess the cell wall composition, intracellular growth, and morphology of Rickettsia parkeri, a human pathogen in the Spotted Fever Group of the Rickettsia genus. Analysis of the cell wall composition of R. parkeri revealed unique features including a high M3 monomer fraction and absence of LD-crosslinks. Using a novel fluorescence microscopy approach, we quantified the cell morphology of R. parkeri in live host cells and found that bacterial morphology is maintained stably during exponential growth in two different epithelial cell lines. To assess population growth kinetics in a high-throughput and high-resolution manner, we developed an imaging-based growth assay and applied this to determine the growth rate of up to 24 infected cultures at a time. We also sought to gain insight into the cell cycle regulation of R. parkeri. To this end, we developed methods to quantify the fraction of the population preparing to divide as well as those undergoing active constriction. These approaches permitted a quantitative analysis of cell cycle status across a population of R. parkeri. Finally, as a proof of concept, we applied the above tools to quantitatively determine how MreB, a bacterial actin homolog, contributes to the growth and morphogenesis of R. parkeri. Inhibition of MreB with the small molecule MP265 led to cell rounding and slowed growth, suggesting that MreB is required for the growth and shape maintenance of R. parkeri. Collectively, we developed a toolkit of high-throughput, quantitative tools to understand intracellular growth and morphogenesis of R. parkeri that is translatable to other obligate intracellular bacteria. AUTHOR SUMMARYThe obligate intracellular lifestyle of members of the bacterial order Rickettsiales, which includes important human pathogens, has hindered our progress in understanding their biology. Here we developed and applied high-throughput, quantitative tools to analyze essential features of rickettsial cell biology such as morphology and growth in living host cells. By applying these tools in a proof of concept, we showed that the bacterial actin homolog, MreB is required for the regulation of rod shape and intracytoplasmic growth.

microbiology↗

bioRxiv2022

Identification of genes required for long term survival of L. pneumophila in water.

Long-term survival of Legionella pneumophila in aquatic environments is thought to be important for establishing an ecological niche necessary for epidemic outbreaks in humans. Eliminating bacterial colonization in plumbing systems is the primary strategy that depletes this reservoir and prevents disease. To uncover L. pneumophila determinants facilitating survival in water, a Tn-seq strategy was used to identify survival-defective mutants during 50-day starvation in tap water at 42{degrees}C. The mutants with most drastic survival defects carried insertions in electron transport chain genes, indicating that membrane energy charge and/or ATP synthesis requires the generation of a proton gradient by the respiratory chain to maintain survival in the presence of water stress. In addition, periplasmically-localized proteins that are known (EnhC) or hypothesized (lpg1697) to stabilize the cell wall against turnover were essential for water survival. To test that the identified mutations disrupted water survival, candidate genes were knocked down by CRISPRi. The vast majority of knockdown strains with verified transcript depletion showed remarkably low viability after 50-day incubations. To demonstrate that maintenance of cell wall integrity was an important survival determinant, a deletion mutation in lpg1697, in a gene encoding a predicted L,D-transpeptidase domain, was analyzed. The loss of this gene resulted in increased osmolar sensitivity and carbenicillin hypersensitivity relative to the WT, as predicted for loss of an L,D-transpeptidase. These results indicate that the L. pneumophila envelope has been evolutionarily selected to allow survival under conditions in which the bacteria are subjected to long-term exposure to starvation and low osmolar conditions. ImportanceWater is the primary vector for transmission of L. pneumophila to humans and the pathogen is adapted to persist in this environment for extended periods of time. Preventing survival of L. pneumophila in water is therefore critical for prevention of Legionnaires disease. We analyzed dense transposon mutation pools for strains with severe survival defects during a 50-day water incubation at 42{degrees}C. By tracking the associated transposon insertion sites in the genome, we defined a distinct essential gene set for water survival and demonstrate that a predicted peptidoglycan crosslinking enzyme, lpg1697, and components of the electron transport chain are required to ensure survival of the pathogen. Our results indicate that select characteristics of the cell wall and components of the respiratory chain of L. pneumophila are primary evolutionary targets being shaped to promote its survival in water.

microbiology↗