Histidine transport is essential for the growth of Staphylococcus aureus at low pH.

Staphylococcus aureus is an opportunistic pathogen capable of causing many different human diseases. During colonization and infection, S. aureus will encounter a range of hostile environments, including acidic conditions such as those found on the skin and within macrophages. However, little is known about the mechanisms that S. aureus uses to detect and respond to low pH. Here, we employed a transposon sequencing approach to determine on a genome-wide level the genes required or detrimental for growth at low pH. We identified 31 genes that were essential for the growth of S. aureus at pH 4.5 and confirmed the importance of many of them through follow up experiments using mutant strains inactivated for individual genes. Most of the genes identified code for proteins with functions in cell wall assembly and maintenance. These data suggest that the cell wall has a more important role than previously appreciated in promoting bacterial survival when under acid stress. We also identified several novel processes previously not linked to the acid stress response in S. aureus. These include aerobic respiration and histidine transport, the latter by showing that one of the most important genes, SAUSA300_0846, codes for a previously uncharacterized histidine transporter. We further show that under acid stress, the expression of the histidine transporter gene is increased in WT S. aureus. In a S. aureus SAUSA300_0846 mutant strain expression of the histidine biosynthesis genes is induced under acid stress conditions allowing the bacteria to maintain cytosolic histidine levels. This strain is, however, unable to maintain its cytosolic pH to the same extent as a WT strain, revealing an important function specifically for histidine transport in the acid stress response of S. aureus.

Designer broad-spectrum polyimidazolium antibiotics.

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM.

Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes.

Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth.

UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.

Structure-Based Discovery of Lipoteichoic Acid Synthase Inhibitors.

Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely "compound 1771" and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771, namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus. Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate ß-lactams against methicillin-resistant S. aureus. Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.

EslB Is Required for Cell Wall Biosynthesis and Modification in Listeria monocytogenes.

Lysozyme is an important component of the innate immune system. It functions by hydrolyzing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, referred to here as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the MIC to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we showed that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant, indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity.IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB affects not only lysozyme resistance but also endogenous cell lysis, cell wall biosynthesis, cell division, and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is, by means of a yet-unknown mechanism, an important determinant for cell wall integrity in L. monocytogenes.

Phosphoglycerol-type wall and lipoteichoic acids are enantiomeric polymers differentiated by the stereospecific glycerophosphodiesterase GlpQ.

The cell envelope of Gram-positive bacteria generally comprises two types of polyanionic polymers linked to either peptidoglycan (wall teichoic acids; WTA) or to membrane glycolipids (lipoteichoic acids; LTA). In some bacteria, including Bacillus subtilis strain 168, both WTA and LTA are glycerolphosphate polymers yet are synthesized through different pathways and have distinct but incompletely understood morphogenetic functions during cell elongation and division. We show here that the exolytic sn-glycerol-3-phosphodiesterase GlpQ can discriminate between B. subtilis WTA and LTA. GlpQ completely degraded unsubstituted WTA, which lacks substituents at the glycerol residues, by sequentially removing glycerolphosphates from the free end of the polymer up to the peptidoglycan linker. In contrast, GlpQ could not degrade unsubstituted LTA unless it was partially precleaved, allowing access of GlpQ to the other end of the polymer, which, in the intact molecule, is protected by a connection to the lipid anchor. Differences in stereochemistry between WTA and LTA have been suggested previously on the basis of differences in their biosynthetic precursors and chemical degradation products. The differential cleavage of WTA and LTA by GlpQ reported here represents the first direct evidence that they are enantiomeric polymers: WTA is made of sn-glycerol-3-phosphate, and LTA is made of sn-glycerol-1-phosphate. Their distinct stereochemistries reflect the dissimilar physiological and immunogenic properties of WTA and LTA. It also enables differential degradation of the two polymers within the same envelope compartment in vivo, particularly under phosphate-limiting conditions, when B. subtilis specifically degrades WTA and replaces it with phosphate-free teichuronic acids.

High-throughput transposon sequencing highlights the cell wall as an important barrier for osmotic stress in methicillin resistant Staphylococcus aureus and underlines a tailored response to different osmotic stressors.

Staphylococcus aureus is an opportunistic pathogen that can cause soft tissue infections but is also a frequent cause of foodborne illnesses. One contributing factor for this food association is its high salt tolerance allowing this organism to survive commonly used food preservation methods. How this resistance is mediated is poorly understood, particularly during long-term exposure. In this study, we used transposon sequencing (TN-seq) to understand how the responses to osmotic stressors differ. Our results revealed distinctly different long-term responses to NaCl, KCl and sucrose stresses. In addition, we identified the DUF2538 domain containing gene SAUSA300_0957 (gene 957) as essential under salt stress. Interestingly, a 957 mutant was less susceptible to oxacillin and showed increased peptidoglycan crosslinking. The salt sensitivity phenotype could be suppressed by amino acid substitutions in the transglycosylase domain of the penicillin-binding protein Pbp2, and these changes restored the peptidoglycan crosslinking to WT levels. These results indicate that increased crosslinking of the peptidoglycan polymer can be detrimental and highlight a critical role of the bacterial cell wall for osmotic stress resistance. This study will serve as a starting point for future research on osmotic stress response and help develop better strategies to tackle foodborne staphylococcal infections.

Identification of the main glutamine and glutamate transporters in Staphylococcus aureus and their impact on c-di-AMP production.

A Staphylococcus aureus strain deleted for the c-di-AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in opuD, encoding the main glycine-betaine transporter, and alsT, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a dacA mutant to near wild-type (WT) size, while inactivation of AlsT does not. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the S. aureus dacA mutant. In addition, GltS was identified as a glutamate transporter. By performing growth curves with WT, alsT and gltS mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of S. aureus. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT and hence likely a higher intracellular glutamine concentration inhibited c-di-AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c-di-AMP signalling in S. aureus.

Galactosylated wall teichoic acid, but not lipoteichoic acid, retains InlB on the surface of serovar 4b Listeria monocytogenes.

Listeria monocytogenes is a Gram-positive, intracellular pathogen harboring the surface-associated virulence factor InlB, which enables entry into certain host cells. Structurally diverse wall teichoic acids (WTAs), which can also be differentially glycosylated, determine the antigenic basis of the various Listeria serovars. WTAs have many physiological functions; they can serve as receptors for bacteriophages, and provide a substrate for binding of surface proteins such as InlB. In contrast, the membrane-anchored lipoteichoic acids (LTAs) do not show significant variation and do not contribute to serovar determination. It was previously demonstrated that surface-associated InlB non-covalently adheres to both WTA and LTA, mediating its retention on the cell wall. Here, we demonstrate that in a highly virulent serovar 4b strain, two genes gtlB and gttB are responsible for galactosylation of LTA and WTA respectively. We evaluated the InlB surface retention in mutants lacking each of these two genes, and found that only galactosylated WTA is required for InlB surface presentation and function, cellular invasiveness and phage adsorption, while galactosylated LTA plays no role thereof. Our findings demonstrate that a simple pathogen-defining serovar antigen, that mediates bacteriophage susceptibility, is necessary and sufficient to sustain the function of an important virulence factor.

Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM.

c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion.

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

Inactivation of the Monofunctional Peptidoglycan Glycosyltransferase SgtB Allows Staphylococcus aureus To Survive in the Absence of Lipoteichoic Acid.

The cell wall of Staphylococcus aureus is composed of peptidoglycan and the anionic polymers lipoteichoic acid (LTA) and wall teichoic acid. LTA is required for growth and normal cell morphology in S. aureus Strains lacking LTA are usually viable only when grown under osmotically stabilizing conditions or after the acquisition of compensatory mutations. LTA-negative suppressor strains with inactivating mutations in gdpP, which resulted in increased intracellular c-di-AMP levels, were described previously. Here, we sought to identify factors other than c-di-AMP that allow S. aureus to survive without LTA. LTA-negative strains able to grow in unsupplemented medium were obtained and found to contain mutations in sgtB, mazE, clpX, or vraT The growth improvement through mutations in mazE and sgtB was confirmed by complementation analysis. We also showed that an S. aureussgtB transposon mutant, with the monofunctional peptidoglycan glycosyltransferase SgtB inactivated, displayed a 4-fold increase in the MIC of oxacillin, suggesting that alterations in the peptidoglycan structure could help bacteria compensate for the lack of LTA. Muropeptide analysis of peptidoglycans isolated from a wild-type strain and sgtB mutant strain did not reveal any sizable alterations in the peptidoglycan structure. In contrast, the peptidoglycan isolated from an LTA-negative ltaS mutant strain showed a significant reduction in the fraction of highly cross-linked peptidoglycan, which was partially rescued in the sgtB ltaS double mutant suppressor strain. Taken together, these data point toward an important function of LTA in cell wall integrity through its necessity for proper peptidoglycan assembly.IMPORTANCE The bacterial cell wall acts as a primary defense against environmental insults such as changes in osmolarity. It is also a vulnerable structure, as defects in its synthesis can lead to growth arrest or cell death. The important human pathogen Staphylococcus aureus has a typical Gram-positive cell wall, which consists of peptidoglycan and the anionic polymers LTA and wall teichoic acid. Several clinically relevant antibiotics inhibit the synthesis of peptidoglycan; therefore, it and teichoic acids are considered attractive targets for the development of new antimicrobials. We show that LTA is required for efficient peptidoglycan cross-linking in S. aureus and inactivation of a peptidoglycan glycosyltransferase can partially rescue this defect, together revealing an intimate link between peptidoglycan and LTA synthesis.

Discovery of genes required for lipoteichoic acid glycosylation predicts two distinct mechanisms for wall teichoic acid glycosylation.

The bacterial cell wall is an important and highly complex structure that is essential for bacterial growth because it protects bacteria from cell lysis and environmental insults. A typical Gram-positive bacterial cell wall is composed of peptidoglycan and the secondary cell wall polymers, wall teichoic acid (WTA) and lipoteichoic acid (LTA). In many Gram-positive bacteria, LTA is a polyglycerol-phosphate chain that is decorated with d-alanine and sugar residues. However, the function of and proteins responsible for the glycosylation of LTA are either unknown or not well-characterized. Here, using bioinformatics, genetic, and NMR spectroscopy approaches, we found that the Bacillus subtilis csbB and yfhO genes are essential for LTA glycosylation. Interestingly, the Listeria monocytogenes gene lmo1079, which encodes a YfhO homolog, was not required for LTA glycosylation, but instead was essential for WTA glycosylation. LTA is polymerized on the outside of the cell and hence can only be glycosylated extracellularly. Based on the similarity of the genes coding for YfhO homologs that are required in B. subtilis for LTA glycosylation or in L. monocytogenes for WTA glycosylation, we hypothesize that WTA glycosylation might also occur extracellularly in Listeria species. Finally, we discovered that in L. monocytogenes, lmo0626 (gtlB) was required for LTA glycosylation, indicating that the encoded protein has a function similar to that of YfhO, although the proteins are not homologous. Together, our results enable us to propose an updated model for LTA glycosylation and also indicate that glycosylation of WTA might occur through two different mechanisms in Gram-positive bacteria.

Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions.

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus However, the exact role of this molecule has not been identified. dacA, the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA, and qoxB, coding proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these findings reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus.

Use of the counter selectable marker PheS* for genome engineering in Staphylococcus aureus.

The gold standard method for the creation of gene deletions in Staphylococcus aureus is homologous recombination using allelic exchange plasmids with a temperature-sensitive origin of replication. A knockout vector that contains regions of homology is first integrated into the chromosome of S. aureus by a single crossover event selected for at high temperatures (non-permissive for plasmid replication) and antibiotic selection. Next, the second crossover event is encouraged by growth without antibiotic selection at low temperature, leading at a certain frequency to the excision of the plasmid and the deletion of the gene of interest. To detect or encourage plasmid loss, either a beta-galactosidase screening method or, more typically, a counterselection step is used. We present here the adaptation of the counter-selectable marker pheS*, coding for a mutated subunit of the phenylalanine tRNA synthetase, for use in S. aureus. The PheS* protein variant allows for the incorporation of the toxic phenylalanine amino acid analogue para-chlorophenylalanine (PCPA) into proteins and the addition of 20-40 mM PCPA to rich media leads to drastic growth reduction for S. aureus and supplementing chemically defined medium with 2.5-5 mM PCPA leads to complete growth inhibition. Using the new allelic exchange plasmid pIMAY*, we delete the magnesium transporter gene mgtE in S. aureus USA300 LAC* (SAUSA300_0910/SAUSA300_RS04895) and RN4220 (SAOUHSC_00945) and demonstrate that cobalt toxicity in S. aureus is mainly mediated by the presence of MgtE. This new plasmid will aid the efficient and easy creation of gene knockouts in S. aureus.

Lipoteichoic acid synthesis and function in gram-positive bacteria.

Lipoteichoic acid (LTA) is an important cell wall polymer found in gram-positive bacteria. Although the exact role of LTA is unknown, mutants display significant growth and physiological defects. Additionally, modification of the LTA backbone structure can provide protection against cationic antimicrobial peptides. This review provides an overview of the different LTA types and their chemical structures and synthesis pathways. The occurrence and mechanisms of LTA modifications with D-alanyl, glycosyl, and phosphocholine residues will be discussed along with their functions. Similarities between the production of type I LTA and osmoregulated periplasmic glucans in gram-negative bacteria are highlighted, indicating that LTA should perhaps be compared to these polymers rather than lipopolysaccharide, as is presently the case. Lastly, current efforts to use LTAs as vaccine candidates, synthesis proteins as novel antimicrobial targets, and LTA mutant strains as improved probiotics are highlighted.

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria.

The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases--RsgA, RbgA, Era, HflX, and ObgE--as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.

Evolutionary Adaptation of the Essential tRNA Methyltransferase TrmD to the Signaling Molecule 3',5'-cAMP in Bacteria.

The nucleotide signaling molecule 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) plays important physiological roles, ranging from carbon catabolite repression in bacteria to mediating the action of hormones in higher eukaryotes, including human. However, it remains unclear whether 3',5'-cAMP is universally present in the Firmicutes group of bacteria. We hypothesized that searching for proteins that bind 3',5'-cAMP might provide new insight into this question. Accordingly, we performed a genome-wide screen and identified the essential Staphylococcus aureus tRNA m1G37 methyltransferase enzyme TrmD, which is conserved in all three domains of life as a tight 3',5'-cAMP-binding protein. TrmD enzymes are known to use S-adenosyl-l-methionine (AdoMet) as substrate; we have shown that 3',5'-cAMP binds competitively with AdoMet to the S. aureus TrmD protein, indicating an overlapping binding site. However, the physiological relevance of this discovery remained unclear, as we were unable to identify a functional adenylate cyclase in S. aureus and only detected 2',3'-cAMP but not 3',5'-cAMP in cellular extracts. Interestingly, TrmD proteins from Escherichia coli and Mycobacterium tuberculosis, organisms known to synthesize 3',5'-cAMP, did not bind this signaling nucleotide. Comparative bioinformatics, mutagenesis, and biochemical analyses revealed that the highly conserved Tyr-86 residue in E. coli TrmD is essential to discriminate between 3',5'-cAMP and the native substrate AdoMet. Combined with a phylogenetic analysis, these results suggest that amino acids in the substrate binding pocket of TrmD underwent an adaptive evolution to accommodate the emergence of adenylate cyclases and thus the signaling molecule 3',5'-cAMP. Altogether this further indicates that S. aureus does not produce 3',5'-cAMP, which would otherwise competitively inhibit an essential enzyme.

New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus.

Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5'-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism.