Genome-Wide Transposon Mutagenesis Screens Identify Group A Streptococcus Genes Affecting Susceptibility to β-Lactam Antibiotics

ABSTRACT

Group A streptococcus (GAS) is a Gram-positive human bacterial pathogen responsible for more than 700 million infections annually worldwide. Beta-lactam antibiotics are the primary agents used to treat GAS infections. Naturally occurring GAS clinical isolates with decreased susceptibility to beta-lactam antibiotics attributed to mutations in PBP2X have recently been documented. This prompted us to perform a genome-wide screen to identify GAS genes that alter beta-lactam susceptibility in vitro. Using saturated transposon mutagenesis, we screened for GAS gene mutations conferring altered in vitro susceptibility to penicillin G and/or ceftriaxone, two beta-lactam antibiotics commonly used to treat GAS infections. In the aggregate, we found that inactivating mutations in 150 GAS genes are associated with altered susceptibility to penicillin G and/or ceftriaxone. Many of the genes identified were previously not known to alter beta-lactam susceptibility or affect cell wall biosynthesis. Using isogenic mutant strains, we confirmed that inactivation of clpX (Clp protease ATP-binding subunit) or cppA (CppA proteinase) resulted in decreased in vitro susceptibility to penicillin G and ceftriaxone. Deletion of murA1 (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) conferred increased susceptibility to ceftriaxone. Our results provide new information about the GAS genes affecting susceptibility to beta-lactam antibiotics.

IMPORTANCE Beta-lactam antibiotics are the primary drugs prescribed to treat infections caused by group A streptococcus (GAS), an important human pathogen. However, the molecular mechanisms of GAS interactions with beta-lactam antibiotics are not fully understood. In this study, we performed a genome-wide mutagenesis screen to identify GAS mutations conferring altered susceptibility to beta-lactam antibiotics. In the aggregate, we discovered that mutations in 150 GAS genes were associated with altered beta-lactam susceptibility. Many identified genes were previously not known to alter beta-lactam susceptibility or affect cell wall biosynthesis. Our results provide new information about the molecular mechanisms of GAS interaction with beta-lactam antibiotics.

INTRODUCTION

Streptococcus pyogenes (also known as group A streptococcus [GAS]) is a Gram-positive bacterial pathogen causing a variety of human diseases, ranging in severity from relatively mild pharyngitis and impetigo to life-threatening infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. Globally, GAS is responsible for more than 700 million infections and 517,000 deaths annually (1). There is no licensed vaccine to prevent GAS infections (2). Beta-lactam antibiotics such as penicillin and ceftriaxone are commonly used to treat GAS infections. These antibiotics inhibit the transpeptidase activity of the high-molecular-mass penicillin-binding proteins (HMM-PBPs) preventing peptidoglycan synthesis, resulting in bacterial killing by a mechanism not fully known, although promoting a futile cycle of peptidoglycan synthesis and turnover has been proposed as a cause (3).

Although a naturally occurring beta-lactam-resistant GAS isolate has not been documented, multiple studies recently have reported strains causing human infections that have decreased susceptibility to beta-lactam antibiotics in vitro (4–8). Thus far, decreased beta-lactam susceptibility in GAS has only been experimentally proven with amino acid substitutions in the transpeptidase domain of PBP2X, a class B HMM-PBP (4–7). For example, Vannice et al. reported two GAS clinical isolates with a T553K amino acid substitution in PBP2X that had reduced susceptibility to ampicillin, amoxicillin, and cefotaxime (4). Stimulated by this observation, we investigated the genomes of 7,025 GAS clinical isolates and identified 137 strains with amino acid substitutions in PBP2X (5). Many of these strains had decreased susceptibility to multiple beta-lactam antibiotics. Using targeted nucleotide substitution, we experimentally proved that the P601L substitution in PBP2X confers reduced susceptibility to penicillin and ampicillin (5, 8). A subsequent population genomic investigation identified a large clonal population of serotype M12 GAS strains with the PBP2X M593T amino acid substitution that confers reduced beta-lactam susceptibility (6). In an investigation of 26,465 genomes encompassing the current publicly available sequencing data, Beres et al. discovered the first examples of interspecies horizontal acquisition of PBP2X by GAS, likely from a Streptococcus dysgalactiae donor, and showed that GAS strains with a naturally acquired chimeric PBP2X protein have significantly decreased susceptibility to several beta-lactam antibiotics (7).

Peptidoglycan is the major structural component of the bacterial cell wall (9). Beta-lactam antibiotics inhibit transpeptidation, the cross-linking step of peptidoglycan synthesis (10). Inhibition of cross-linking compromises cell wall structural integrity, leading to disruption of bacteria, possibly by means of a futile cycle of peptidoglycan synthesis and turnover (3), albeit additional mechanisms have been proposed (11). The cell wall is a complex and dynamic structure that has to change to accommodate bacterial cell growth and division (12). The complete set of molecular mechanisms and genes/enzymes mediating cell wall synthesis, maintenance, and repair are not known. We hypothesized that molecular mechanisms other than HMM-PBP mutations affect beta-lactam susceptibility. To test this hypothesis and gain a broader understanding of the GAS genes affecting susceptibility to beta-lactam antibiotics, we conducted a genome-wide transposon mutagenesis screen to identify the GAS genes affecting cell fitness and viability in the presence of penicillin G or ceftriaxone. In the aggregate, we discovered 150 GAS genes whose insertional inactivation was associated with altered beta-lactam susceptibility in vitro. Many of these 150 genes have known or inferred functions in cell envelope biogenesis, maintenance, and division, but we also identified genes involved in metabolism, regulation, substrate transportation, protein secretion, and DNA replication. These findings demonstrate that GAS genes/factors other than HMM-PBPs also affect susceptibility to beta-lactam antibiotics.

RESULTS

Genome-wide screen for GAS genes/products affecting susceptibility to penicillin G or ceftriaxone.

We previously generated a highly saturated transposon insertion mutant library from the well-characterized serotype M1 GAS strain MGAS2221 (13). To identify genes whose products affect viability in the presence of beta-lactam antibiotics, we plated our serotype M1 GAS transposon insertion mutant library onto Todd-Hewitt broth supplemented with yeast extract (THY) agar with and without a subinhibitory concentration of penicillin G (6 ng/mL) or ceftriaxone (12 ng/mL) (Fig. 1) (5). After overnight incubation at 37°C, the GAS cells grown under the differing conditions were collected. Transposon insertion site sequencing (TraDIS) was used to characterize and compare the compositions of the mutant pools grown on THY agar with and without added beta-lactam antibiotic (Fig. 1). Specifically, TraDIS analysis was used to identify genes with significantly altered mutant frequencies between the mutant pools, comparing the growth on THY agar supplemented with a beta-lactam antibiotic (i.e., beta-lactam selective conditions) to the growth on THY agar (i.e., beta-lactam nonselective conditions) (Fig. 1). Genes with significantly increased or decreased mutant frequencies (log₂ fold change, less than −2 or >2; q value, <0.001) in the mutant pools recovered from beta-lactam-containing agar were interpreted as affecting beta-lactam susceptibility. Using this strategy, we identified 58 and 81 gene mutations (transposon insertion inactivation) that are associated with increased fitness in the presence of penicillin G and ceftriaxone, respectively (Fig. 2; see Tables S1 and S2 in the supplemental material). Among these 2 sets of genes enhancing fitness in the presence of the beta-lactam antibiotics, 43 genes were identified in common to both conditions. Conversely, we identified mutations in 19 and 44 genes associated with decreased fitness in the presence of penicillin G and ceftriaxone, respectively. A common set of 8 genes contributed to decreased GAS fitness under both conditions (Fig. 2; Tables S3 and S4). In the aggregate, the genome-wide screens found that insertion mutations in 150 GAS genes are associated with altered fitness in the presence of these two beta-lactam antibiotics. Many of these identified genes have known or inferred roles in cell envelope biosynthesis and maintenance (Tables S1 to S4). For example, we found that inactivation of the cardiolipin synthase-encoding gene cls confers significantly increased fitness in the presence of penicillin and ceftriaxone (Tables S1 and S2), in accordance with previous work in Staphylococcus aureus (14). Cardiolipin is an anionic phospholipid located in the inner membrane (15) that plays an important role in bacterial adaptation to osmotic stress (16). In enterococci, a cls mutation is associated with resistance to daptomycin, a membrane-targeting antibiotic (17).

FIG 1.

Strategy for GAS mutant library screening on THY agar without (top) and with (bottom) a subinhibitory concentration of beta-lactam antibiotic selection. TraDIS was used to compare mutant pools grown on THY agar without and with a beta-lactam antibiotic added. Sequencing of the mutant library pools and subsequent read mapping to a reference genome identified the frequency and location of transposon insertions. The relative frequencies of transposon insertions in the input and recovered mutant pools were compared to assess the fitness contribution of individual nonessential genes to the survival of GAS in the presence of the subinhibitory concentrations of the beta-lactam antibiotics.

FIG 2.

Genome-wide screen for insertion mutations in GAS genes associated with altered susceptibility to penicillin and ceftriaxone. (A and B) Illustrated positionally along the GAS strain MGAS2221 genome are the fold changes in mutant abundance (y axis) for each of the genes (x axis) in the mutant pools recovered from THY agar supplemented with 6 ng/mL of penicillin G (A) or 12 ng/mL of ceftriaxone (B). Genes not significantly altering fitness in the subinhibitory beta-lactam environment are in gray, genes contributing to increased fitness are shown in red, and those contributing to decreased fitness are in blue. (C and D) Venn diagrams summarizing the number of GAS genes contributing to significantly increased (C) or decreased (D) fitness in the presence of penicillin G and/or ceftriaxone.

clpX mutants and cppA mutants are highly abundant in the mutant pools exposed to beta-lactam antibiotics.

Our TraDIS results show that beta-lactam exposure caused an extensive change in the transposon mutant library pool composition (Fig. 3). Of note, the relative abundance of reads mapping to the Clp proteolytic complex ATPase subunit-encoding gene clpX increased from 0.01% in the nonselective environment to 46.7% and 68.7% after the mutant pool was exposed to penicillin G and ceftriaxone, respectively (Fig. 3). Similar to clpX, the relative abundance of reads mapping to the putative protease-encoding gene cppA increased from 0.5% to 16.6% and 7.7% after the mutant pool was exposed to penicillin G and ceftriaxone, respectively. Collectively, the data suggest that abrogating the activities of the clpX and cppA gene products increases the fitness of GAS in the presence of the beta-lactams penicillin G and ceftriaxone.

FIG 3.

Change in the mutant library compositions, comparing gene mutant abundance on unsupplemented THY agar and THY agar supplemented with penicillin G or ceftriaxone. (A and C) Percent distribution by gene of the total number of transposon insertion mutants. (B and D) Mutant library compositional change illustrated again as a stacked bar plot. The environments, either without or with a beta-lactam antibiotic, are indicated. The relative abundance of transposon insertional inactivation mutants is inferred by the TraDIS read counts mapping to each of the genes in the GAS strain MGAS2221 genome. clpX mutants and cppA mutants were the two most abundant in the mutant pools exposed to 6 ng/mL of penicillin G (A and B) or 12 ng/mL of ceftriaxone (C and D).

Insertional inactivation of clpX or cppA confers increased fitness in the presence of penicillin G or ceftriaxone.

We observed that clpX and cppA mutants were enriched in the mutant pools exposed to beta-lactam antibiotics, suggesting that these mutants are less susceptible to these two beta-lactams. To test this hypothesis, we plated the mutant libraries recovered from the penicillin agar and isolated individual mutant strains with transposon insertions in clpX (n = 4 mutants, each at a distinct insertion site) or cppA (n = 2 mutants, each at a distinct insertion site) (Fig. 4). Out of caution, we did not generate targeted gene deletion mutant strains for clpX and cppA because the TraDIS results suggested that deletion of clpX or cppA might result in GAS strains with decreased susceptibility to beta-lactams, the primary antibiotics used to treat GAS infections. Whole-genome sequencing confirmed that the isolated clpX and cppA transposon insertion mutant strains were isogenic derivatives of the parental strain, MGAS2221. That is, no spurious mutations that could confound the genotype-phenotype associations were present in the genomes of these six isolated mutant strains.

FIG 4.

Growth phenotypes in liquid media of mutant strains with transposon insertions in clpX or cppA. (A) Transposon insertion sites of the four isolated clpX mutants. (B to D) Growth of clpX mutants in THY supplemented with the indicated amount of penicillin or ceftriaxone. (E) Transposon insertion sites of the two isolated cppA mutants. (F to H) Growth of cppA mutants in THY supplemented with the indicated amount of penicillin or ceftriaxone. Because all the growth assays shown in this figure were conducted in the same batch experiment, the WT strain growth curves in panels B and F, C and G, and D and H are identical. OD, optical density.

Cell lysis by beta-lactam antibiotics correlates with the bacterial rate of growth (18). Broadly, a reduced bacterial growth rate results in reduced beta-lactam susceptibility. Therefore, we examined the clpX and cppA mutants for altered growth relative to the wild-type (WT) parental strain. The clpX mutants had a lag in growth when grown in THY liquid medium without antibiotics (Fig. 4B). In contrast, the cppA mutants grew without an initial lag, similarly to the WT parental strain (Fig. 4F). In THY supplemented with penicillin G or ceftriaxone, mutant strains with transposon insertions in clpX or cppA grew to significantly higher optical densities than the WT parental strain (Fig. 4). These results demonstrate that inactivation of clpX or cppA caused significantly increased fitness in vitro in the presence of the subinhibitory concentrations of penicillin G or ceftriaxone and did not confer a substantial change in growth.

Deletion of murA1 confers decreased fitness in the presence of ceftriaxone.

Many Gram-positive bacteria have two homologs of murA, murA1 and murA2, that encode functionally active and redundant enzymes with UDP-N-acetylglucosamine 1-carboxyvinyl transferase activity (19). MurA catalyzes the synthesis of UDP-N-acetylglucosamine enolpyruvate, the first committed step of peptidoglycan synthesis (19). The TraDIS results suggested that the insertional inactivation of either murA1 or murA2 confers significantly decreased survival in the presence of ceftriaxone (Table S4). To test this hypothesis, we constructed an isogenic markerless murA1 deletion mutant strain and examined its growth phenotype with and without 12 ng/mL of ceftriaxone in the medium. In unsupplemented THY, the growth curves of the WT parental strain and the murA1 deletion mutant strain are superimposable (Fig. 5). In contrast, in THY supplemented with 12 ng/mL of ceftriaxone, the murA1 deletion mutant strain grew to significantly lower optical densities than the WT parental strain (Fig. 5), as expected. This result indicates that inactivation of murA1 confers significantly decreased fitness in vitro in the presence of ceftriaxone.

FIG 5.

Growth phenotype of murA1 deletion mutant in liquid media. (A) Whole-genome sequencing result of the murA1 deletion mutant. Illumina sequencing reads mapping to the MGAS2221 genome confirmed deletion of the murA1 gene. (B and C) Growth of the isogenic murA1 mutant strain in plain THY (B) and THY supplemented with ceftriaxone (C).

DISCUSSION

Beta-lactams are the primary antibiotics used to treat GAS infections. Recently, GAS clinical isolates with decreased in vitro beta-lactam susceptibility have been documented (4–6). The molecular mechanisms underlying the interactions between GAS and beta-lactam antibiotics are not fully understood. To identify genes whose products affect GAS susceptibility to beta-lactam antibiotics, we conducted a genome-wide transposon mutagenesis screen and identified 150 genes associated with altered susceptibility to penicillin G and/or ceftriaxone.

HMM-PBPs are the primary targets of beta-lactams, and it is well established that mutations in the genes encoding HMM-PBPs can alter susceptibility to this class of antibiotics. However, among the 150 genes we identified by TraDIS as affecting beta-lactam susceptibility, only one, pbp2a, encodes an HMM-PBP (see Table S1 in the supplemental material). We surmise that there are likely two reasons why other GAS HMM-PBP genes were not identified by the genetic screens. First, in GAS, mutations that have been documented to confer altered beta-lactam susceptibility are all function-modulating point mutations or homologous recombinations that result in amino acid substitutions in the HMM-PBPs (4–7, 20) and are not loss-of-function mutations. The transposon insertional mutagenesis screens that we used detect bacterial fitness changes primarily resulting from gene disruption function-inactivating mutations. Second, certain GAS HMM-PBP genes are individually essential for viability, such as pbp2x and pbp1a (21). Thus, transposon insertions in these essential PBP genes are lethal to GAS. Therefore, the impact of these and all other essential genes are not assayable by the TraDIS genetic screening approach. Importantly, our genetic screens identified many genes other than HMM-PBP genes that also affect GAS susceptibility to beta-lactam antibiotics.

Although most identified GAS fitness genes do not encode HMM-PBPs, many have known or inferred roles in cell membrane and cell wall biosynthesis and maintenance (Tables S1 to S4). Of note, we found that inactivation of cls (encoding cardiolipin synthase) confers significantly increased fitness in the presence of penicillin and ceftriaxone (Tables S1 and S2), as found for daptomycin in S. aureus (14). Cardiolipin is an anionic phospholipid located in the inner membrane (15) that plays a crucial role in adaptation to osmotic stress (16). In enterococci, cls mutation is associated with resistance to daptomycin, a membrane-targeting antibiotic (17). Also, cardiolipin affects lipid II binding to MurJ, an essential step in cell wall peptidoglycan synthesis (22, 23). Due to the impact of cardiolipin on osmotic stress response and cell wall synthesis, it is not surprising that cls inactivation affects bacterial susceptibility to cell wall-targeting antibiotics. Future research should investigate the presence of naturally occurring cls mutations in clinical GAS isolates and their contribution to the altered susceptibility of GAS strains to beta-lactam antibiotics.

We found that certain mutant strains were enriched and highly abundant in the mutant pools exposed to penicillin G or ceftriaxone (Fig. 3). Notably, the relative abundance of clpX mutants increased from 0.01% in the environment without antibiotics to 47.4% and 68.5% in the penicillin G- or ceftriaxone-containing environments, respectively. clpX mutants were the most abundant mutant population in the mutant pools exposed to beta-lactam antibiotics (Fig. 3). Also, we found that isogenic clpX mutants grew significantly better than their WT parental strain in the presence of a subinhibitory concentration of penicillin G or ceftriaxone (Fig. 4). The clpX gene encodes the ATP-dependent specificity component of the ClpXP proteolytic complex (24). Unlike in Streptococcus pneumoniae, ClpX is not essential for viability in GAS (25, 26). Our finding that GAS clpX mutants had reduced susceptibility to penicillin G and ceftriaxone is analogous to the findings that Staphylococcus aureus mutant strains lacking ClpX had decreased susceptibility to several types of antibiotics targeting the cell wall (24, 27–29). Bæk et al. also showed that S. aureus cells lacking ClpX had thicker cell walls and increased peptidoglycan cross-linking (30). It would be interesting to investigate whether the inactivation of clpX in GAS also increases cell wall thickness and peptidoglycan cross-linking.

The second most abundant transposon insertional inactivated gene in the mutant pools exposed to penicillin G or ceftriaxone was cppA (Fig. 3). Using isogenic mutants, we confirmed that inactivation of the cppA gene resulted in decreased penicillin susceptibility in vitro (Fig. 4). cppA encodes a proteinase that is present in multiple streptococcal pathogens (31). In S. pneumoniae, CppA is a virulence factor that contributes to bacterial survival in blood and assists nasopharyngeal transmission in animal models (32). Although CppA lacks a typical secretion signal sequence, it has been used as a vaccine candidate in S. pneumoniae (33). Aside from its role in virulence, it is noteworthy that cppA is located immediately upstream of RS07915, a gene encoding a peptidoglycan transpeptidase family protein (Fig. 4E). Based on the effect of cppA inactivation on penicillin susceptibility, and the close proximity of cppA and RS07915, we speculate that cppA and RS07915 encode functionally related enzymes that play a role in maintaining cell wall homeostasis. Detailed functional studies are needed to test this hypothesis.

Aside from the enriched mutants, we also identified mutants that were preferentially eliminated in the presence of beta-lactams. For example, inactivation of murA1 or murA2 confers significantly decreased fitness in the presence of ceftriaxone (Table S4). In Gram-positive bacteria, murA1 and murA2 each encode a homolog of MurA, a UDP-N-acetylglucosamine 1-carboxyvinyl transferase that catalyzes the first committed step of cell wall peptidoglycan synthesis (19). Similar to our observation, Vesic et al. reported that deletion of murAA (homolog of GAS murA1) in Enterococcus faecalis increases its susceptibility to cephalosporins (34). We hypothesize that MurA1 and MurA2 are functionally redundant in GAS. Although inactivation of either murA1 or murA2 individually is not lethal to GAS, we speculate that inactivation of either individually decreases the overall UDP-N-acetylglucosamine 1-carboxyvinyl transferase activity, reducing peptidoglycan synthesis, resulting in GAS that are more vulnerable to peptidoglycan synthesis-inhibiting beta-lactam antibiotics. MurA is the drug target of fosfomycin (35). Many studies have documented the synergistic activity of fosfomycin and beta-lactam antibiotics against bacterial infections (36–38). Since the transposon inactivation of murA1 or murA2 renders GAS more vulnerable to ceftriaxone, it may be useful in future studies to examine the synergistic activity of fosfomycin and beta-lactam antibiotics against GAS in vitro.

To summarize, we screened a highly saturated transposon insertion mutant library made in a clinically relevant serotype M1 GAS strain for gene mutations conferring altered susceptibility to penicillin G and ceftriaxone. Our genetic screens identified many GAS genes that affect bacterial susceptibility to beta-lactams. These findings may ultimately lead to a better understanding of GAS interaction with beta-lactam antibiotics and bacterial cell wall homeostasis in general.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Strain MGAS2221 is genetically representative of the pandemic clone of serotype M1 GAS that originated in the 1980s and rapidly spread worldwide (39). The transposon insertion mutant library (13) and the isolated transposon insertion mutant strains used in this study were derived from the parental strain MGAS2221. GAS strains were cultured in THY (Todd-Hewitt broth supplemented with 0.5% yeast extract) with the indicated dose of penicillin G or ceftriaxone. Isolated transposon insertion mutant strains were whole-genome sequenced to determine the site of transposon insertion and to confirm that the mutant strains were isogenic derivatives of the parental strain MGAS2221. Briefly, the genomes of GAS transposon insertion mutant strains were sequenced using an Illumina NextSeq 550 instrument. The sequence reads were quality filtered, adapters and artifacts were trimmed, and base call errors were corrected using Trimmomatic and Musket (40); then, the reads were mapped to the genome of strain MGAS2221 using SMALT (https://www.sanger.ac.uk/tool/smalt-0/). Single-nucleotide polymorphisms (SNPs) and insertions (including transposon insertion sites) and deletions were identified with FreeBayes and Pilon (41).

Screen for GAS gene mutations conferring altered susceptibility to penicillin G or ceftriaxone.

To screen for GAS gene mutations (transposon insertional inactivation mutants) associated with altered beta-lactam susceptibility, we plated 50 μL of the M1 GAS MGAS2221 transposon insertion mutant library stock (~10⁷ CFU) (13) onto THY agar plates with and without 6 ng/mL of penicillin G or 12 ng/mL of ceftriaxone. For each genetic screen (penicillin G or ceftriaxone), TraDIS was performed by analyzing three technical replicates. Specifically, GAS transposon insertion mutant library stock was plated onto three THY agar plates supplemented with an indicated beta-lactam antibiotic. After 12 h incubation at 37°C, mutant pools grown on each of the three agar plates were collected by washing the colonies off the agar surface with THY broth containing 25% glycerol and stored at −80°C. The three collected mutant pools constituted the three technical replicates for the subsequent analysis. TraDIS was used to characterize and compare the compositions of the mutant pools grown on THY agar with and without penicillin G or ceftriaxone (Fig. 1A) according to a previously described method (13).

Sequencing the transposon insertion mutant libraries.

Preparation of genomic DNA and sequencing of transposon mutant libraries was performed as previously described (13, 21, 42–46). Briefly, genomic DNA of each of the collected mutant pools was isolated using the DNeasy blood and tissue kit (Qiagen). PCR was used to amplify the insertion site transposon-chromosome junction. The PCR-amplified libraries were sequenced with a NextSeq 550 instrument (Illumina) using a single-end 75-cycle protocol.

Processing of DNA sequencing reads and data analysis.

The sequencing reads of the mutant libraries were analyzed with the TraDIS toolkit (47) according to previously described methods (13). Briefly, bacteria_tradis was used to trim transposon tag sequences and map the reads to the reference genome of strain MGAS2221. The plot files generated by bacteria_tradis were analyzed using tradis_gene_insert_sites to generate spreadsheets listing the read count, insertion count, and insertion index for each gene. The output files from the tradis_gene_insert_sites analysis were transferred to tradis_comparison.R to compare the compositions of the mutant libraries grown on THY agar plates with and without the indicated beta-lactam antibiotics. Gene mutations with significantly altered frequency (log₂ fold change, >2 or less than −2; q value, <0.001) with respect to the input mutant pools grown without antibiotic were regarded as altering GAS fitness.

Isolation of clpX and cppA mutants from the transposon insertion mutant pool.

Because clpX and cppA mutants were highly abundant in mutant pools exposed to penicillin G or ceftriaxone, these mutants were readily recovered from the transposon insertion mutant pools (Fig. 3). To isolate individual mutants, we diluted and plated the mutant pool exposed to penicillin G and isolated four clpX mutants and two cppA mutants. Each isolated mutant had a unique transposon insertion site in either clpX or cppA (Fig. 4). Whole-genome sequencing confirmed that the isolated clpX and cppA mutants were isogenic relative to their parental strain, MGAS2221. That is, no spurious mutations were present elsewhere in the genome in any of the six mutants.

Construction of the isogenic murA1 deletion mutant strain.

The isogenic gene deletion mutant strain ΔmurA1 was constructed using the parental strain MGAS2221. Gene deletion was accomplished by allelic exchange as previously described (48). Primers used for mutant construction are listed in Table S5 in the supplemental material. The genome of the mutant strain was sequenced to rule out the introduction of spurious mutations.