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.

Type I Lipoteichoic Acid (LTA) Purification by Hydrophobic Interaction Chromatography and Structural Analysis by 2D Nuclear Magnetic Resonance (NMR) Spectroscopy.

Type I lipoteichoic acid (LTA) is a glycerol phosphate polymer found in the cell envelope of diverse Gram-positive bacteria. The glycerol phosphate backbone is often further decorated with D-alanine and/or sugar residues. Here, we provide details of a 1-butanol extraction and purification method of type I LTA by hydrophobic interaction chromatography. The protocol has been adapted from methods originally described by Fischer et al. (Eur J Biochem 133:523-530, 1983) and further optimized by Morath et al. (J Exp Med 193:393-397, 2001). We also present information on a 2D nuclear magnetic resonance (NMR) analysis method to gain chemical and structural information of the purified LTA material.

Small-Scale Illumina Library Preparation Using the Illumina Nextera XT DNA Library Preparation Kit.

Here, we describe a protocol for a scaled-down version of a genomic DNA (gDNA)-fragmentation and tagmentation reaction using the Illumina Nextera XT DNA Library Preparation Kit. Using Staphylococcus aureus as an example, which has a genome size of ∼3 Mb, we show how 24 different samples can be pooled for a typical paired-end Illumina high-throughput sequencing run using the MiSeq Reagent V2 300-cycle kit, with which it is possible to sequence 5.1 Gb of DNA. As part of the protocol, a DNA size-selection method using a standard DNA agarose gel-extraction procedure and a final sample quality-control step using a Bioanalyzer are described.

Allelic-Exchange Procedure in Staphylococcus aureus.

This protocol continues a series of methods for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for S. aureus We have previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, then introduced into electrocompetent S. aureus cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker (pheS* gene) and confers chloramphenicol resistance to S. aureus As a specific example, we present the construction of strain RN4220*ΔtagO from strain RN4220 carrying the pIMAY*-ΔtagO plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.

Using CRISPR-Cas9-Based Methods for Genome Editing in Staphylococcus aureus.

Chromosomal mutations and targeted gene deletions and inactivations in Staphylococcus aureus are typically generated using the allelic exchange method. In recent years, however, more rapid methods have been developed, often using CRISPR-Cas9-based systems. Here, we describe recently developed CRISPR-Cas9-based plasmid systems for use in S. aureus, and discuss their use for targeted gene mutation and inactivation. First, we describe how a CRISPR-Cas9 counterselection strategy can be combined with a recombineering strategy to generate gene deletions in S. aureus We then introduce dead Cas9 (dCas9) and Cas9 nickase (nCas9) enzymes, and discuss how the nCas9 enzyme fused to different nucleoside deaminases can be used to introduce specific base changes in target genes. We then discuss how the nCas9-deaminase fusion enzymes can be used for targeted gene inactivation via the introduction of premature stop codons or by mutating the start codon. Together, these tools highlight the power and potential of CRISPR-Cas9-based methods for genome editing in S. aureus.

Introduction of a CRISPR-nCas9 Gene-Targeting Plasmid into Staphylococcus aureus for Gene Disruption.

Methods for gene disruption are essential for functional genomics, and there are multiple approaches for altering gene function in bacteria. One of these methods involves introducing a premature stop codon in a gene of interest, which can be achieved by using the CRISPR-nCas9-cytidine deaminase system. The approach involves the mutation of editable cytidines to thymidines, with the goal of generating a novel stop codon that ultimately results in a nonfunctional gene product. The workflow involves two major sections, one for the identification of editable cytidines, the design of the targeting spacer oligonucleotides for introduction into the CRISPR-nCas9 cytidine deaminase plasmid, and the construction of the gene-targeting CRISPR-nCas9 cytosine deaminase plasmids, and one for the actual introduction of the mutation in the species of interest. Here, we describe the steps for the second part. Specifically, we describe (1) how to introduce the gene-targeting pnCasSA-BEC plasmid into Staphylococcus aureus, (2) how the gene inactivation in S. aureus can be confirmed by PCR and sequencing, and (3) how, following successful gene inactivation, the strain can be cured of the pnCasSA-BEC plasmid. To better illustrate the method, and as specific example, two different geh gene-inactivation mutations are generated here in S. aureus RN4220. The protocol, however, can easily be adapted to generate other gene-inactivating mutations.

Preparation of Electrocompetent Staphylococcus aureus Cells and Plasmid Transformation.

This protocol is part of a series of methodologies for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. Having previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-ΔtagO) can be constructed and isolated from Escherichia coli, we now present details of the next steps in this method-the preparation of electrocompetent S. aureus cells and introduction of the tagO mutant plasmid DNA into the S. aureus cells by electroporation. Colonies containing the plasmid can then be selected on chloramphenicol plates at a low temperature permissive for plasmid replication.

Bacterial Whole-Genome-Resequencing Analysis: Basic Steps Using the CLC Genomics Workbench Software.

In this protocol, we describe the basic steps for bacterial genome resequencing analysis using the QIAGEN CLC Genomics Workbench software. More specifically, we present how a reference genome sequence can be generated from Illumina reads of a wild-type reference bacterial strain and how this reference genome sequence can then be used to identify genomic alterations in mutant strains. As specific examples, Illumina reads from the Staphylococcus aureus RN4220 strain will be used to generate a consensus reference genome based on the publicly available S. aureus NCTC8325 genome sequence. The generated RN4220 consensus reference genome will subsequently be used to identify genomic mutations in an RN4220 mutant strain with increased oxacillin resistance (OxaR strain).

Selection of Antibiotic-Resistant Bacterial Strains and Identification of Genomic Alterations by Whole-Genome Sequencing: Using Staphylococcus aureus and Oxacillin Resistance as an Example.

Here, we discuss methods for the selection of antibiotic-resistant bacteria and the use of high-throughput whole-genome sequencing for the identification of the underlying mutations. We comment on sample requirements and the choice of specific DNA preparation methods depending on the strain used and briefly introduce a workflow we use for the selection of Staphylococcus aureus strains with increased oxacillin resistance and identification of genomic alterations.

Staphylococcus aureus Colony Polymerase Chain Reaction.

Here, we describe a protocol for a colony polymerase chain reaction (PCR) method for Staphylococcus aureus The methodology involves the preparation of small S. aureus lysates by using the enzyme lysostaphin to degrade the peptidoglycan layer. These lysates are prepared using a small patch of bacteria grown on LB agar plates, and the lysates can subsequently be used for PCR analyses.

Identification of Editable Sites, Spacer Oligonucleotide Design, and Generation of the Gene-Targeting CRISPR-nCas9 Plasmid for Gene Disruption in Staphylococcus aureus Using the CRISPR-nCas9 and Cytidine Deaminase System.

Methods for gene disruption are essential for functional genomics, and there are multiple approaches for altering gene function in bacteria. One of these methods involves introducing a premature stop codon in a gene of interest, which can be achieved by using the CRISPR-nCas9-cytidine deaminase system. The approach involves the mutation of editable cytidines to thymidines, with the goal of generating a novel stop codon that ultimately results in a nonfunctional gene product. The workflow involves two major sections, one for the identification of editable cytidines, the design of the targeting spacer oligonucleotides for introduction into the CRISPR-nCas9 cytidine deaminase plasmid, and the construction of the gene-targeting CRISPR-nCas9 cytosine deaminase plasmids, and one for the actual introduction of the mutation in the species of interest. Here, we describe the steps for the first part. To better illustrate the method and oligonucleotide design, we describe the construction of Staphylococcus aureus RN4220 geh mutants with C to T base changes at two different positions, leading to the construction of strains RN4220-geh(160stop) and RN4220-geh(712stop). We outline the steps for (1) the identification of editable cytidines within genes using the CRISPR-CBEI toolkit website, and (2) the design of the targeting spacer oligonucleotides for introduction into the CRISPR-nCas9 cytidine deaminase plasmid pnCasSA-BEC, followed by (3) the construction of the gene-targeting (in this example, geh gene-targeting) CRISPR-nCas9 cytosine deaminase plasmids pnCasSA-BEC-gehC160T and pnCasSA-BEC-gehC712T using the Golden Gate assembly method, plasmid recovery in Escherichia coli, and confirmation by colony PCR and sequencing. The method can be easily adapted to construct gene-inactivation mutants in other S. aureus genes.

Preparation of Staphylococcus aureus Genomic DNA Using Promega Nuclei Lysis and Protein Precipitation Solutions, Followed by Additional Cleanup and Quantification Steps.

In this protocol, we describe the isolation of genomic DNA (gDNA) from Staphylococcus aureus using the Promega Nuclei Lysis and Protein Precipitation solutions. Gram-positive bacteria such as S. aureus are harder to lyse than Gram-negative bacteria. Hence, the first step in the procedure for isolating gDNA from Gram-positive bacteria consists of a mechanical lysis step (e.g., using a bead beating grinder or homogenizer) or an enzymatic lysis step. For the method described here, the peptidoglycan layer of S. aureus is digested with an enzyme called lysostaphin. This enzyme cleaves the pentaglycine cross-bridges within the peptidoglycan of S. aureus. After this lysis step, the gDNA can be purified using procedures similar to those used for Gram-negative bacteria. We include additional cleanup and quantification procedures in the final steps of this protocol, in case the gDNA is subsequently used for genome-sequencing projects. By modifying the bacterial lysis step, the procedure can be easily adapted to isolate gDNA from other bacteria.

Preparation of Staphylococcus aureus Genomic DNA Using a Chloroform Extraction and Ethanol Precipitation Method, Followed by Additional Cleanup and Quantification Steps.

In this protocol, we describe the isolation of genomic DNA (gDNA) from Staphylococcus aureus strains using a chloroform extraction and ethanol precipitation method. This gDNA-isolation method is well-suited for downstream whole-genome sequencing applications when working with S. aureus strains that contain plasmids, as only a small amount of plasmid DNA is isolated along with the gDNA. Similar to other gDNA isolation methods for Gram-positive bacteria, the first step in the procedure is a mechanical lysis (e.g., using a bead beating grinder) or an enzymatic lysis step. In this protocol, the peptidoglycan layer of S. aureus is digested with an enzyme called lysostaphin. This enzyme cleaves pentaglycine cross-bridges within the peptidoglycan of S. aureus. After this lysis step, gDNA can be purified using similar procedures as those used for Gram-negative bacteria. We include additional cleanup and quantification procedures in the final steps of this protocol, in case the aim is to use the gDNA for genome-sequencing projects. By modifying the bacterial lysis step, the procedure can be easily adapted to isolate gDNA from other bacteria.

Agar Plate-Based Method for the Selection of Antibiotic-Resistant Bacterial Strains.

Identifying the molecular mechanisms underlying antibiotic resistance is important, as it can reveal key information on the mode of action of a drug and provide insights for the development of novel or improved antimicrobials. Here, we describe an agar-based method for the selection of bacterial strains with increased antibiotic resistance, and how the increase in resistance can be confirmed by a spot-plating assay. As a specific example, we describe the selection of Staphylococcus aureus strains with increased resistance to oxacillin; however, the protocol can be easily adapted and used with other bacteria and antibiotics.

Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli.

We present a protocol for the generation of a gene-deletion allelic-exchange plasmid and its recovery in Escherichia coli for the purpose of constructing an in-frame gene deletion in Staphylococcus aureus Here, we present detailed methodologies for (i) the primer design (using the S. aureus tagO gene as our specific example); (ii) PCR amplification of the required gene fragments; (iii) preparation of the cloning vector (using the S. aureus allelic-exchange vector pIMAY* as an example); (iv) the Gibson assembly cloning method; (v) introduction of the plasmid into E. coli; (vi) confirmation of the plasmid insert in E. coli by colony PCR; and, finally, (vii) confirmation of the insert by sequencing. We also consider the long-term storage of the E. coli strains containing the desired plasmid.

Allelic Exchange: Construction of an Unmarked In-Frame Deletion in Staphylococcus aureus.

Here we describe an allelic-exchange procedure for the construction of an unmarked gene deletion in the bacterium Staphylococcus aureus As a practical example, we outline the construction of a tagO gene deletion in S. aureus using the allelic-exchange plasmid pIMAY*. We first present the general principles of the allelic-exchange method, along with information on counterselectable markers. Furthermore, we summarize relevant cloning procedures, such as the splicing by overhang extension (SOE) polymerase chain reaction (PCR) and Gibson assembly methods, and we conclude by giving some general consideration to performing genetic modifications in S. aureus.

Introduction of a Recombineering Oligonucleotide and a CRISPR-Cas9 Gene-Targeting Plasmid into Staphylococcus aureus for Generating a Gene-Deletion Strain.

Gene deletions can be generated in Staphylococcus aureus using recombineering in combination with a CRISPR-Cas9 counterselection approach. The method involves first designing the recombineering oligonucleotides and generating the relevant plasmids, and then introducing these elements into S. aureus to generate the desired gene deletion. Here, we describe the second part of this workflow; the introduction of the gene-targeting plasmid and the recombineering oligonucleotide(s) into S. aureus to generate the gene-deletion strain. Specifically, we outline the steps to (1) generate the S. aureus recipient strain for the recombineering CRISPR-Cas9 counterselection method by introducing plasmid pCN-EF2132tet, (2) introduce the recombineering oligonucleotide(s) and gene-targeting plasmid into the pCN-EF2132tet plasmid-containing S. aureus strain, (3) confirm the gene deletion in S. aureus by colony PCR and sequencing, and (4) curate the plasmids following successful gene deletion. To illustrate the method, we give a specific example of how to generate a 55-bp deletion in the geh gene of S. aureus strain RN4220. The protocol, however, can be easily adapted to other strain backgrounds and to generate deletions in other genes.

Oligonucleotide Design and Construction of a Gene-Targeting CRISPR-Cas9 Plasmid in Escherichia coli for Generating a Gene-Deletion Strain in Staphylococcus aureus.

Gene deletions can be constructed in Staphylococcus aureus using recombineering in combination with a CRISPR-Cas9 counterselection approach. The method involves first designing the recombineering oligonucleotides and generating the relevant plasmids, and then introducing these elements into S. aureus to generate the desired gene deletion. Here, we describe the first part of this workflow, oligonucleotide design and plasmid generation. To better illustrate the method and oligonucleotide design, the construction of a 55-bp out-of-frame deletion in the S. aureus geh gene will be presented as a specific example. To this end, we describe the use of geh gene-specific recombineering oligonucleotides and the construction of a geh gene-targeting CRISPR-Cas9 plasmid. The protocol is divided into three parts: (1) design of the gene-specific targeting spacer oligonucleotides for introduction into the CRISPR-Cas9 plasmid pCas9-counter, (2) design of 90-mer recombineering oligonucleotides to generate a 55-bp out-of-frame gene deletion, and (3) construction of the gene-targeting CRISPR-Cas9 plasmid pCas9-geh, plasmid recovery in Escherichia coli, and confirmation by colony PCR and sequencing. The method can easily be adapted to design deletions for other S. aureus genes.

Purine Nucleosides Interfere with c-di-AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To ß-Lactam Antibiotics.

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes.

The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen Listeria monocytogenes is intrinsically resistant towards lysozyme, mainly due to deacetylation and O-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An eslB mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less O-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of eslB phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the eslB mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-N-acetylglucosamine (UDP-GlcNAc) production or distribution.