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The rise of antibiotic-resistant bacterial strains has become a critical health concern. According to the World Health Organization, the market introduction of new antibiotics is alarmingly sparse, underscoring the need for novel therapeutic targets. The LytR-CpsA-Psr (LCP) family of proteins, which facilitate the insertion of cell wall glycopolymers (CWGPs) like teichoic acids into peptidoglycan, has emerged as a promising target for antibiotic development. LCP proteins are crucial in bacterial adhesion and biofilm formation, making them attractive for disrupting these processes. This study investigated the structural and functional characteristics of the LCP domain of LytR from Streptococcus dysgalactiae subsp. dysgalactiae. The protein structure was solved by X-ray Crystallography at 2.80 Å resolution. Small-angle X-ray scattering (SAXS) data were collected to examine potential conformational differences between the free and ligand-bound forms of the LytR LCP domain. Additionally, docking and molecular dynamics (MD) simulations were used to predict the interactions and conversion of ATP to ADP and AMP. Experimental validation of these predictions was performed using malachite green activity assays. The determined structure of the LCP domain revealed a fold highly similar to those of homologous proteins while SAXS data indicated potential conformational differences between the ligand-free and ligand-bound forms, suggesting a more compact conformation during catalysis, upon ligand binding. Docking and MD simulations predicted that the LytR LCP domain could interact with ADP and ATP and catalyze their conversion to AMP. These predictions were experimentally validated by malachite green activity assays, confirming the protein's functional versatility. The study provides significant insights into the structural features and functional capabilities of the LCP domain of LytR from S. dysgalactiae subsp. dysgalactiae. These findings pave the way for designing targeted therapies against antibiotic-resistant bacteria and offer strategies to disrupt bacterial biofilm formation.

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Competence development is essential for bacterial transformation since it enables bacteria to take up free DNA from the surrounding environment. The regulation of teichoic acid biosynthesis is tightly controlled during pneumococcal competence; however, the mechanism governing this regulation and its impact on transformation remains poorly understood. We demonstrated that a defect in lipoteichoic acid ligase (TacL)-mediated lipoteichoic acids (LTAs) biosynthesis was associated with impaired pneumococcal transformation. Using a fragment of tacL regulatory probe as bait in a DNA pulldown assay, we successfully identified several regulatory proteins, including ComE. Electrophoretic mobility shift assays revealed that phosphomimetic ComE, but not wild-type ComE, exhibited specific binding to the probe. DNase I footprinting assays revealed the specific binding sequences encompassing around 30 base pairs located 31 base pairs upstream from the start codon of tacL. Expression of tacL was found to be upregulated in the ΔcomE strain, and the addition of exogenous competence-stimulating peptide repressed the tacL transcription in the wild-type strain but not the ΔcomE mutant, indicating that ComE exerted a negative regulatory effect on the transcription of tacL. Mutation in the JH2 region of tacL upstream regulatory sequence led to increased LTAs abundance and displayed higher transformation efficiency. Collectively, our work identified the regulatory mechanisms that control LTAs biosynthesis during competence and thereby unveiled a repression mechanism underlying pneumococcal transformation.

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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.

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The threat of infection during implant placement surgery remains a considerable burden for millions of patients worldwide. To combat this threat, clinicians employ a range of anti-infective strategies and practices. One of the most common interventions is the use of prophylactic antibiotic treatment during implant placement surgery. However, these practices can be detrimental by promoting the resilience of biofilm-forming bacteria and enabling them to persist throughout treatment and re-emerge later, causing a life-threatening infection. Thus, it is of the utmost importance to elucidate the events occurring during the initial stages of bacterial surface attachment and determine whether any biological processes may be targeted to improve surgical outcomes. Using gene expression analysis, we identified a cellular mechanism of S. aureus which modifies its cell surface charge following attachment to a medical grade titanium surface. We determined the upregulation of two systems involved in the d-alanylation of teichoic acids and the lysylation of phosphatidylglycerol. We supported these molecular findings by utilizing synchrotron-sourced attenuated total reflection Fourier-transform infrared microspectroscopy to analyze the biomolecular properties of the S. aureus cell surface following attachment. As a direct consequence, S. aureus quickly becomes substantially more tolerant to the positively charged vancomycin, but not the negatively charged cefazolin. The present study can assist clinicians in rationally selecting the most potent antibiotic in prophylaxis treatments. Furthermore, it highlights a cellular process that could potentially be targeted by novel technologies and strategies to improve the outcome of antibiotic prophylaxis during implant placement surgery. STATEMENT OF SIGNIFICANCE: The antibiotic tolerance of bacteria in biofilm is a well-established phenomenon. However, the physiological adaptations employed by Staphylococcus aureus to increase its antibiotic tolerance during the early stages of surface attachment are poorly understood. Using multiple techniques, including gene expression analysis and synchrotron-sourced Fourier-transform infrared microspectroscopy, we generated insights into the physiological response of S. aureus following attachment to a medical grade titanium surface. We showed that this phenotypic transition enables S. aureus to better tolerate the positively charged vancomycin, but not the negatively charged cefazolin. These findings shed light on the antibiotic tolerance mechanisms employed by S. aureus to survive prophylactically administered antibiotics and can help clinicians to protect patients from infections.

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Methicillin-resistant Staphylococcus aureus (MRSA) is a high priority pathogen due to its life-threating infections to human health. Development of prophylactic or therapeutic anti-MRSA vaccine is a potential approach to treat S. aureus infections and overcome the resistance crisis. ß-1,4-GlcNAc glycosylated wall teichoic acids (WTAs) derived from S. aureus are a new type of antigen that is closely associated with ß-lactam resistance. In this study, structure-defined ß-1,4-GlcNAc-modified WTAs varied in chain length and numbers of GlcNAc modification were synthesized by an ionic liquid-supported oligosaccharide synthesis (ILSOS) strategy in high efficiency and chromatography-free approach. Then the obtained WTAs were conjugated with tetanus toxin (TT) as vaccine candidates and were further evaluated in a mouse model to determine the structure-immunogenicity relationship. In vivo immunological studies revealed that the WTAs-TT conjugates provoked robust T cell-dependent responses and elicited high levels of specific anti-WTAs IgG antibodies production associated with the WTAs structure including chain length as well as the ß-1,4-GlcNAc modification pattern. Heptamer WTAs conjugate T6, carrying three copy of ß-1,4-GlcNAc modified RboP, was identified to elicit the highest titers of specific antibody production. The T6 antisera exhibited the highest recognition and binding affinity and the most potent OP-killing activities to MSSA and MRSA cells. This study demonstrated that ß-1,4-GlcNAc glycosylated WTAs are promising antigens for further development against MRSA.

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Teichoic acids are important for the maintenance of cell shape and growth in Gram-positive bacteria. Bacillus subtilis produces major and minor forms of wall teichoic acid (WTA) and lipoteichoic acid during vegetative growth. We found that newly synthesized WTA attachment to peptidoglycan occurs in a patch-like manner on the sidewall with the fluorescent labeling compound of the concanavalin A lectin. Similarly, WTA biosynthesis enzymes fused to the epitope tags were localized in similar patch-like patterns on the cylindrical part of the cell, and WTA transporter TagH was frequently colocalized with WTA polymerase TagF, WTA ligase TagT, and actin homolog MreB, respectively. Moreover, we found that the nascent cell wall patches, decorated with the newly glucosylated WTA, were colocalized with TagH and WTA ligase TagV. In the cylindrical part, the newly glucosylated WTA patchily inserted into the bottom of the cell wall layer and finally reached the outermost layer of the cell wall after approximately half an hour. Incorporation of newly glucosylated WTA was arrested with the addition of vancomycin but restored with the removal of the antibiotic. These results are consistent with the prevailing model that WTA precursors are attached to newly synthesized peptidoglycan. IMPORTANCE In Gram-positive bacteria, the cell wall is composed of mesh-like peptidoglycan and covalently linked wall teichoic acid (WTA). It is unclear where WTA decorates peptidoglycan to create a cell wall architecture. Here, we demonstrate that nascent WTA decoration occurred in a patch-like manner at the peptidoglycan synthesis sites on the cytoplasmic membrane. The incorporated cell wall with newly glucosylated WTA in the cell wall layer then reached the outermost layer of the cell wall after approximately half an hour. Incorporation of newly glucosylated WTA was arrested with the addition of vancomycin but restored with the removal of the antibiotic. These results are consistent with the prevailing model that WTA precursors are attached to newly synthesized peptidoglycan.

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Bacterial cells are covered with various glycopolymers such as peptidoglycan (PG), lipopolysaccharides (LPS), teichoic acids, and capsules. Among these glycopolymers, PG assembly is the target of some of our most effective antibiotics, consistent with its essentiality and uniqueness to bacterial cells. Biosynthesis of other surface glycopolymers have also been acknowledged as potential targets for developing therapies to control bacterial infections, because of their importance for bacterial survival in the host environment. Moreover, biosynthesis of most surface glycopolymers are closely related to PG assembly because the same lipid carrier is shared for glycopolymer syntheses. In this review, I provide an overview of PG assembly and antibiotics that target this pathway. Then, I discuss the implications of a common lipid carrier being used for assembly of PG and other surface glycopolymers in antibiotic development.

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Bacteriophage endolysins are bacteriolytic enzymes that have been explored as potential weapons to fight antibiotic-resistant bacteria. Despite several studies support the application of endolysins as enzybiotics, detailed knowledge on cellular and enzymatic factors affecting their lytic activity is still missing. The bacterial membrane proton motive force (PMF) and certain cell wall glycopolymers of Gram-positive bacteria have been implicated in some tolerance to endolysins. Here, we studied how the anti-staphylococcal endolysin Lys11, a modular enzyme with two catalytic domains (peptidase and amidase) and a cell binding domain (CBD11), responded to changes in the chemical and/or electric gradients of the PMF (ΔpH and Δψ, respectively). We show that simultaneous dissipation of both gradients enhances endolysin binding to cells and lytic activity. The collapse of ΔpH is preponderant in the stimulation of Lys11 lytic action, while the dissipation of Δψ is mainly associated with higher endolysin binding. Interestingly, this binding depends on the amidase domain. The peptidase domain is responsible for most of the Lys11 bacteriolytic activity. Wall teichoic acids (WTAs) are confirmed as major determinants of endolysin tolerance, in part by severely hindering CBD11 binding activity. In conclusion, the PMF and WTA interfere differently with the endolysin functional domains, affecting both the binding and catalytic efficiencies.

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Methicillin-resistant Staphylococcus aureus (MRSA), a major human pathogen, uses the prophage-encoded tarP gene as an important immune evasion factor. TarP glycosylates wall teichoic acid (WTA) polymers, major S. aureus surface antigens, to impair WTA immunogenicity and impede host defence. However, tarP phages appear to be restricted to only a few MRSA clonal lineages, including clonal complexes (CC) 5 and 398, for unknown reasons. We demonstrate here that tarP-encoding prophages can be mobilized to lysogenize other S. aureus strains. However, transfer is largely restricted to closely related clones. Most of the non-transducible clones encode tarM, which generates a WTA glycosylation pattern distinct from that mediated by TarP. However, tarM does not interfere with infection by tarP phages. Clonal complex-specific Type I restriction-modification systems were the major reasons for resistance to tarP phage infection. Nevertheless, tarP phages were found also in unrelated S. aureus clones indicating that tarP has the potential to spread to distant clonal lineages and contribute to the evolution of new MRSA clones.

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A major feature of the pathogenicity of Staphylococcus aureus is its ability to secrete cytolytic toxins. This process involves the translocation of the toxins from the cytoplasm through the bacterial membrane and the cell wall to the external environment. The process of their movement through the membrane is relatively well defined, involving both general and toxin-specific secretory systems. Movement of the toxins through the cell wall was considered to involve the passive diffusion of the proteins through the porous cell wall structures; however, recent work suggests that this is more complex, and here we demonstrate a role for the wall teichoic acids (WTA) in this process. Utilizing a genome-wide association approach, we identified a polymorphism in the locus encoding the WTA biosynthetic machinery as associated with the cytolytic activity of the bacteria. We verified this association using an isogenic mutant set and found that WTA are required for the release of several cytolytic toxins from the bacterial cells. We show that this effect is mediated by a change in the electrostatic charge across the cell envelope that results from the loss of WTA. As a major target for the development of novel therapeutics, it is important that we fully understand the entire process of cytolytic toxin production and release. These findings open up a new aspect to the process of toxin release by a major human pathogen while also demonstrating that clinical isolates can utilize WTA production to vary their cytotoxicity, thereby altering their pathogenic capabilities. IMPORTANCE The production and release of cytolytic toxins is a critical aspect for the pathogenicity of many bacterial pathogens. In this study, we demonstrate a role for wall teichoic acids, molecules that are anchored to the peptidoglycan of the bacterial cell wall, in the release of toxins from S. aureus cells into the extracellular environment. Our findings suggest that this effect is mediated by a gradient of electrostatic charge which the presence of the negatively charged WTA molecules create across the cell envelope. This work brings an entirely new aspect to our understanding of the cytotoxicity of S. aureus and demonstrates a further means by which this major human pathogen can adapt its pathogenic capabilities.

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Penicillin and related antibiotics disrupt cell wall synthesis in bacteria causing the downstream misactivation of cell wall hydrolases called autolysins to induce cell lysis. Despite the clinical importance of this phenomenon, little is known about the factors that control autolysins and how penicillins subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae (Sp), LytA is the major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked teichoic acids (LTAs). Because LytA binds to both species of teichoic acids, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation and lysis during exponential growth . We show that WhyD is a WTA hydrolase that restricts the WTA content of the wall to areas adjacent to active peptidoglycan (PG) synthesis. Our results support a model in which the WTA tailoring activity of WhyD during exponential growth directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA.

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Listeria innocua is genetically closely related to the foodborne human pathogen Listeria monocytogenes. However, as most L. innocua strains are non-pathogenic, it has been proposed as a surrogate organism for determining the efficacy of antimicrobial strategies against L. monocytogenes. Teichoic acids are one of the three major cell wall components of Listeria, along with the peptidoglycan backbone and cell wall-associated proteins. The polymeric teichoic acids make up the majority of cell wall carbohydrates; the type of teichoic acids directly attached to the peptidoglycan are termed wall teichoic acids (WTAs). WTAs play vital physiological roles, are important virulence factors, antigenic determinants, and phage-binding ligands. The structures of the various WTAs of L. monocytogenes are well known, whereas those of L. innocua are not. In the present study, the WTA structure of L. innocua ZM39 was determined mainly by 1D and 2D NMR spectroscopy and it was found to be the following: [→4)-[α-D-GlcpNAc-(1→3)]-ß-D-GlcpNAc-(1→4)-D-Rbo-(1P→]n This structure is new with respect to all currently known Listeria WTAs and it shares structural similarities with type II WTA serovar 6a. In addition, the genome of strain L. innocua ZM39 was sequenced and the majority of putative WTA synthesis genes were identified.

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Staphylococcus aureus, which lacks pili and flagella, is nonmotile. However, it hitchhikes motile bacteria, such as Pseudomonas aeruginosa, to migrate in the environment. This study demonstrated that the hitchhiking motility of S. aureus SA113 was reduced after the tagO, which encodes an enzyme for wall teichoic acids (WTA) synthesis, was deleted. The hitchhiking motility was restored after the mutation was complemented by transforming a plasmid expressing TagO into the mutant. We also showed that adding purified lipopolysaccharide (LPS) to a culture that contains S. aureus SA113 and P. aeruginosa PAO1, reduced the movement of S. aureus, showing that WTA and LPS are involved in the hitchhiking motility of S. aureus. This study also found that P. aeruginosa promoted the movement of S. aureus in the digestive tract of Caenorhabditis elegans and in mice. In conclusion, this study reveals how S. aureus hitchhikes P. aeruginosa for translocation in an ecosystem. The results from this study improve our understanding on how a nonmotile pathogen moves in the environment and spreads in animals.

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Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections. Due to the rapid evolution of antibiotic resistance that leads to treatment failure, it is important to understand the underlying mechanisms. Here, the cell wall structures of several laboratory vancomycin-intermediate S. aureus (VISA) strains were analyzed. Among the VISA strains were S. aureus VC40, which accumulated 79 mutations, including most importantly 2 exchanges in the histidine-kinase VraS, and developed full resistance against vancomycin (MIC, 64 µg/ml); a revertant S. aureus VC40R, which has an additional mutation in vraR (MIC, 4 µg/ml); and S. aureus VraS(VC40), in which the 2 vraS mutations were reconstituted into a susceptible background (MIC, 4 µg/ml). A ultraperformance liquid chromatography (UPLC) analysis showed that S. aureus VC40 had a significantly decreased cross-linking of the peptidoglycan. Both S. aureus VC40 and S. aureus VraS(VC40) displayed reduced autolysis and an altered autolysin profile in a zymogram. Most striking was the significant increase in d-alanine and N-acetyl-d-glucosamine (GlcNAc) substitution of the wall teichoic acids (WTAs) in S. aureus VC40. Nuclear magnetic resonance (NMR) analysis revealed that this strain had mostly ß-glycosylated WTAs in contrast to the other strains, which showed only the α-glycosylation peak. Salt stress induced the incorporation of ß-GlcNAc anomers and drastically increased the vancomycin MIC for S. aureus VC40R. In addition, ß-glycosylated WTAs decreased the binding affinity of AtlA, the major autolysin of S. aureus, to the cell wall, compared with α-glycosylated WTAs. In conclusion, there is a novel connection between wall teichoic acids, autolysis, and vancomycin susceptibility in S. aureus. IMPORTANCE Infections with methicillin-resistant Staphylococcus aureus are commonly treated with vancomycin. This antibiotic inhibits cell wall biosynthesis by binding to the cell wall building block lipid II. We set out to characterize the mechanisms leading to decreased vancomycin susceptibility in a laboratory-generated strain, S. aureus VC40. This strain has an altered cell wall architecture with a thick cell wall with low cross-linking, which provides decoy binding sites for vancomycin. The low cross-linking, necessary for this resistance mechanism, decreases the stability of the cell wall against lytic enzymes, which separate the daughter cells. Protection against these enzymes is provided by another cell wall polymer, the teichoic acids, which contain an unusually high substitution with sugars in the ß-conformation. By experimentally increasing the proportion of ß-N-acetyl-d-glucosamine in a closely related isolate through the induction of salt stress, we could show that the ß-conformation of the sugars plays a vital role in the resistance of S. aureus VC40.

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The cytoplasm of bacteria is maintained at a higher osmolality than the growth medium, which generates a turgor pressure. The cell membrane (CM) cannot support a large turgor, so there are two possibilities for transferring the pressure to the peptidoglycan cell wall (PGW): (1) the CM could be pressed directly against the PGW, or (2) the CM could be separated from the PGW by a periplasmic space that is isoosmotic with the cytoplasm. There is strong evidence for gram-negative bacteria that a periplasm exists and is isoosmotic with the cytoplasm. No comparable studies have been done for gram-positive bacteria. Here I suggest that a periplasmic space is probably essential in order for the periplasmic proteins to function, including especially the PBPs that remodel the peptidoglycan wall. I then present a semi-quantitative analysis of how teichoic acids could support a periplasm that is isoosmotic with the cytoplasm. The fixed anionic charge density of teichoic acids in the periplasm is ∼0.5 M, which would bring in ∼0.5 M Na+ neutralizing ions. This approximately balances the excess osmolality of the cytoplasm that would produce a turgor pressure of 19 atm. The 0.5 M fixed charge density is similar to that of proteoglycans in articular cartilage, suggesting a comparability ability to support pressure. An isoosmotic periplasm would be especially important for cell division, since it would allow CM constriction and PGW synthesis to avoid turgor pressure.

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Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (α-GlcNAc at the RboP C4; ß-GlcNAc at the RboP C4; ß-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains.

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Background: Wall teichoic acid (WTA) genes are essential for production of cell walls in gram-positive bacteria and necessary for survival and variability in the cassette has led to recent antibiotic resistance acquisition in pathogenic bacteria.  Methods: Using a pan-genome approach, we examined the evolutionary history of WTA genes in  Bacillus subtilis ssp.  subtilis.  Results: Our analysis reveals an interesting pattern of evolution from the type-strain WTA gene cassette possibly resulting from horizontal acquisition from organisms with similar gene sequences. The WTA cassettes have a high level of variation which may be due to one or more independent horizontal transfer events during the evolution of  Bacillus subtilis ssp.  subtilis. This swapping of entire WTA cassettes and smaller regions within the WTA cassettes is an unusual feature in the evolution of the  Bacillus subtilis genome and highlights the importance of horizontal transfer of gene cassettes through homologous recombination within  B. subtilis or other bacterial species.  Conclusions: Reduced sequence conservation of these WTA cassettes may indicate a modified function like the previously documented WTA ribitol/glycerol variation. An improved understanding of high-frequency recombination of gene cassettes has ramifications for synthetic biology and the use of  B. subtilis in industry.

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Bacterial wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising the transmembrane and ATPase subunits TagG and TagH. Here the dimeric structure of the N-terminal domain of TagH (TagH-N) was solved by single-wavelength anomalous diffraction using a selenomethionine-containing crystal, which shows an ATP-binding cassette (ABC) architecture with RecA-like and helical subdomains. Besides significant structural differences from other ABC transporters, a prominent patch of positively charged surface is seen in the center of the TagH-N dimer, suggesting a potential binding site for the glycerol phosphate chain of WTA. The ATPase activity of TagH-N was inhibited by clodronate, a bisphosphonate, in a non-competitive manner, consistent with the proposed WTA-binding site for drug targeting.

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Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth.IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

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The exopolymer (EPSp) produced by the strain B. licheniformis IDN-EC was isolated and characterized using different techniques (MALDI-TOF, NMR, ATR-FTIR, TGA, DSC, SEM). The results showed that the low molecular weight EPSp contained a long polyglutamic acid and an extracellular teichoic acid polysaccharide. The latter was composed of poly(glycerol phosphate) and was substituted at the 2-position of the glycerol residues with a αGal and αGlcNH2. The αGal O-6 position was also found to be substituted by a phosphate group. The antiviral capability of this EPSp was also tested on both enveloped (herpesviruses HSV, PRV and vesicular stomatitis VSV) and non-enveloped (MVM) viruses. The EPSp was efficient at inhibiting viral entry for the herpesviruses and VSV but was not effective against non-enveloped viruses. The in vivo assay of the EPSp in mice showed no signs of toxicity which could allow for its application in the healthcare sector.

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