To identify the genetic basis of reduced susceptibility in GCGS1095, we transformed a ceftriaxone susceptible (CRO^S) recipient strain with genomic DNA from GCGS1095. We reasoned that minimizing the genetic distance between the resistant donor and susceptible recipient would improve the likelihood of identifying the causative locus for CRO^RS and would reduce potentially confounding effects of divergent genomic backgrounds. Consequently, we selected GCGS0457, a close phylogenetic neighbor of GCGS1095 (Figure 1), as the CRO^S recipient for these experiments. Transformants were selected on CRO plates, and those that acquired the CRO^RS phenotype were characterized by whole genome sequencing to determine which variants they had inherited from GCGS1095.

We recovered multiple CRO^RS transformants. GCGS0457 did not develop spontaneous CRO^RS during culture or when transformed with autologous GCGS0457 genomic DNA. Whole genome sequencing of the CRO^RS transformants showed that each transformant inherited the same single nucleotide polymorphism (SNP) in rpoB (nucleotide change G602A), which encodes the RNA polymerase beta subunit RpoB (amino acid substitution R201H) (Figure 2). We named this allele rpoB1. These CRO^RS transformants did not acquire variants in genes known to contribute to reduced cephalosporin susceptibility, such as penA, ponA, pilQ, or mtr (Eyre et al., 2017).

As RNA polymerase mutations have not been associated with cephalosporin resistance in Neisseria, we tested whether the rpoB1 mutation is sufficient to confer CRO^RS. We performed directed transformation of GCGS0457 with a ~ 1.5 kilobase PCR product surrounding the variant position of rpoB1. With the exception of the single rpoB G602A nucleotide change, the sequence of this PCR product was identical to the parental GCGS0457 sequence. Transformation with the rpoB1 allele amplified from GCGS1095 yielded multiple CRO^RS transformants, whereas transformation with control PCR products amplified from a strain containing wild-type rpoB yielded none. Sanger sequencing confirmed the presence of the expected SNP in each resistant transformant. Representative CRO^RS transformants were further examined by whole genome sequencing, which ruled out spontaneous mutations. The CRO MIC of the rpoB1 transformant strain GCGS0457 RpoB^R201H was 0.19 μg/mL, a more than ten-fold increase from recipient strain GCGS0457 (CRO MIC 0.012 μg/mL) (Table 1). This transformant MIC is similar to the MIC of the parental CRO^RS isolate GCGS1095 (Table 1), indicating that introduction of this SNP is sufficient to fully recapitulate the CRO^RS phenotype in the GCGS0457 background.

GCGS1014 is a phylogenetic neighbor of GCGS1095 that also has unexplained CRO^RS (Figure 1; Table 1). The close phylogenetic relationship between these isolates led us to hypothesize that they would share the same mechanism of CRO^RS. However, the GCGS1014 isolate lacks the rpoB1 allele identified in GCGS1095. In fact, among the 1102 sequenced isolates in the GISP collection, the rpoB1 allele is unique to GCGS1095 (Supplementary file 1).

To define the genetic basis of CRO^RS in GCGS1014, we used the same unbiased transformation approach as described above, again using the CRO^S neighbor GCGS0457 as the recipient strain. The CRO^RS transformants from GCGS1014 gDNA did not have mutations in rpoB or in other characterized resistance-associated genes. Instead, all CRO^RS transformants inherited a single nucleotide substitution in rpoD (nucleotide G292A), which encodes the major housekeeping sigma factor RpoD, or σ⁷⁰ (amino acid substitution E98K) (Figure 2). This allele, rpoD1, is present in the CRO^RS donor GCGS1014 but not in GCGS1095 (Table 1) or in any other isolate from the GISP collection (Supplementary file 1). To test the ability of the rpoD1 allele to confer CRO^RS to a susceptible strain, we transformed GCGS0457 with a ~ 1.5 kilobase PCR product that includes the single variant position in rpoD1. As with the directed transformation of rpoB1, the rpoD1 allele amplified from GCGS1014 transformed GCGS0457 to high-level CRO^RS, but control PCR products amplified from strains with wild-type rpoD did not. The presence of the rpoD1 allele in resistant transformants was confirmed by Sanger sequencing and by whole genome sequencing, which also ruled out the possibility of spontaneous mutations causing the CRO^RS phenotype. The rpoD1 transformant strain GCGS0457 RpoD^E98K had a CRO MIC of 0.125 μg/mL, the same as that of the parental CRO^RS isolate GCGS1014 (Table 1).

As the CRO^RS phenotypes in GCGS1095 and GCGS1014 are caused by distinct RNAP mutations, we examined alleles of RNAP components in each of the 1102 genomes in the GISP collection (Grad et al., 2016) to identify additional RNAP variants that might explain other cases of uncharacterized CRO^RS (Supplementary file 1). We found that the isolate GCGS1013 has a 12-basepair in-frame internal deletion in rpoD, resulting in the deletion of amino acids 92–95 (Figure 2); GCGS1013 has a CRO^RS phenotype but does not have a CRO^RS-associated penA allele (CRO MIC 0.19 μg/mL; Table 1). Transformation of the GCGS1013 rpoD allele, rpoD2, into the susceptible recipient GCGS0457 increased CRO resistance to 0.19 μg/mL (Table 1). The rpoD2 allele is also present in a clinical gonococcal isolate from a recent United Kingdom strain collection (De Silva et al., 2016). This isolate, GCPH44 (genome accession number SRR3360905), has a CRO^RS phenotype (CRO MIC 0.125 μg/mL) and belongs to distinct phylogenetic lineage from that of GCGS1013 (Figure 1). Like GCGS1013, the CRO^RS phenotype of GCPH44 is not attributable to variation in penA or other known resistance determinants (Table 1).

The amino acids deleted in the rpoD2 allele (Δ92–95) are in a similar region of the RpoD protein as the single amino acid change in the rpoD1 allele (E98K) (Figure 2A). This flexible portion of the σ⁷⁰ 1.1 domain is not included in published RNA polymerase holoenzyme crystal structures. Modeling of E. coli RNAP predicts that σ⁷⁰ 1.1 interacts with the DNA channel formed by the β subunit encoded by rpoB prior to open complex formation (Murakami, 2013). In E. coli, the RpoB residue homologous to the substituted amino acid in rpoB1 (E. coli RpoB R197) is located on the surface of the β lobe structure that makes up this same channel (Figure 2B). Taken together, these results demonstrate that RNAP-mediated CRO^RS has arisen multiple times in clinical isolates of N. gonorrhoeae, and that these different mutations affect a similar region of the RNAP holoenzyme. They may therefore act via a similar mechanism to effect phenotypic CRO^RS.

The rpoB1 and rpoD1 alleles each differ from wild-type by a single nucleotide. The ability of these mutations to cause high-level CRO^RS when introduced into the susceptible strain GCGS0457 indicates that some gonococcal isolates are a single-step mutation from CRO^RS. Because three of the four clinical isolates we identified with RNAP-mediated CRO^RS are clustered in one part of the gonococcal phylogeny (Figure 1), we tested whether acquisition of CRO^RS via this pathway is limited to strains within this particular clade – which would suggest that one or more genetic background factors are required – or whether RNAP mutation represents a broadly accessible evolutionary trajectory to CRO^RS.

To address this question, we sought to transform genetically diverse recipient strains to CRO^RS with the rpoB1 allele. In a panel of 17 clinical isolates from the GISP collection, we found five additional recipients dispersed throughout the phylogeny that acquired phenotypic CRO^RS following directed transformation with rpoB1 (Figure 1; Supplementary file 1). The remaining 12 isolates, as well as the laboratory strain 28BL, did not readily produce CRO^RS rpoB1 transformants after 1–2 transformation attempts. These results indicate that genetic background compatibility with this resistance mechanism is not limited to a single gonococcal lineage, to recipients with particular alleles of any RNA polymerase component (Supplementary file 1), or to recipients with variants in ponA or mtrR associated with β-lactam resistance (Eyre et al., 2017) (Supplementary file 2). As transformation efficiency in these diverse isolates is variable, the failure to recover CRO^RS transformants from any particular strain following a single transformation attempt may reflect low efficiency rather than an absolute genetic incompatibility with an RNAP-mediated resistance mechanism. The successful transformation of rpoB1 into these five additional isolates therefore represents a lower bound on, rather than exact count of, the number of isolates in this panel that can develop resistance via this pathway.

While screening isolates for genetic compatibility with rpoB-mediated CRO^RS in the directed transformation experiments described above, we tested isolates in parallel for spontaneous development of CRO^RS. We found that the CRO^S isolate GCGS0364, located in a similar part of the phylogeny as the resistant isolates GCGS1095, GCGS1014, and GCGS1013 (Figure 1), produced stably CRO^RS derivatives when exposed to CRO in vitro. Sanger sequencing of the rpoB locus in two of these derivatives identified two different spontaneous rpoB mutations: rpoB2 (nucleotide change C470T, amino acid change P157L) and rpoB3 (nucleotide change G473T, amino acid change G158V) (Figure 2A). These de novo RpoB mutations are predicted to occur in the same β-sheet as the rpoB1 variant position (R201H) identified in GCGS1095 (Figure 2B). Directed transformation of rpoB2 or rpoB3 into the parental (CRO^S) GCGS0364 isolate conferred phenotypic CRO^RS, increasing the CRO MIC more than 20-fold from 0.023 μg/mL to 0.5–0.75 μg/mL (Table 1).

Becasue optimal RNA polymerase functionality is required for bacterial fitness, we tested the hypothesis that these RNAP mutations are not specific cephalosporin resistance mutations, but instead cause a slow-growth or general stress response phenotype that nonspecifically increases resistance to diverse classes of antimicrobial compounds.

As expected, the rpoB1, rpoD1, and rpoD2 alleles conferred reduced susceptibility to ceftriaxone as well as cefixime (another third-generation cephalosporin) when transformed into the CRO^S recipient GCGS0457. However, these alleles did not affect susceptibility to antimicrobial drugs that do not target the cell wall, including ciprofloxacin, tetracycline, azithromycin, and rifampicin. Surprisingly, these mutations also failed to confer resistance to the non-cephalosporin β-lactams penicillin and ertapenem. The apparent cephalosporin specificity of these RNAP mutations in the GCGS0457 background is consistent with the antibiotic susceptibility profiles of the three CRO^RS clinical isolates in which they were first identified (GCGS1095 for rpoB1, GCGS1014 for rpoD1, and GCGS1013 for rpoD2) (Table 2).

GCGS0457 rpoB1 and GCGS0457 rpoD1 transformants were further examined for evidence of a slow growth phenotype that might increase antibiotic tolerance. When grown on GCB agar (Difco), neither of these transformants had a reduced growth rate compared to the parental GCGS0457 strain (Figure 3A). Furthermore, the rpoB1 and rpoD1 transformants had no gross defects in cell morphology by transmission electron microscopy (TEM) (Figure 3B), although transformants were slightly smaller than the parental GCGS0457 strain (Figure 3—figure supplement 1). Taken together, these results do not support the hypothesis that RNAP mutations reduce CRO susceptibility via a slow growth or nonspecific tolerance phenotype.

To measure the functional effect of the rpoB1 and rpoD1 alleles on RNA polymerase, we characterized the transcriptional profiles of the CRO^RS isolates GCGS1095 (rpoB1) and GCGS1014 (rpoD1) and the susceptible isolate GCGS0457 by RNA-seq. The transcriptional profiles of the CRO^RS isolates were similar to one another but distinct from GCGS0457 (Figure 4—figure supplement 1), with 1337 and 1410 annotated ORFs significantly altered in abundance, respectively. The transcriptomic differences we observe may partly reflect the genetic distance between these isolates (Wadsworth et al., 2019), as the resistant strains GCGS1095 and GCGS1014 are more closely related to one another than they are to GCGS0457. We therefore compared GCGS0457 to its isogenic CRO^RS transformants GCGS0457 RpoB^R201H and GCGS0457 RpoD^E98K. The rpoB1 and rpoD1 alleles each profoundly altered the transcriptional profile of GCGS0457: 1278 annotated ORFs in GCGS0457 RpoD^E98K and 1218 annotated ORFs in the GCGS0457 RpoB^R201H were differentially expressed relative to the parental GCGS0457 strain. The transcriptional profiles of the two transformants were very similar to one another (Figure 4—figure supplement 1), indicating that these different mutations have similar functional consequences. Overall, a total of 890 annotated ORFs were differentially expressed in all four CRO^RS strains (the two CRO^RS isolates and two transformants in the GCGS0457 background) compared to GCGS0457. The vast majority of the transcriptional changes are small in magnitude: 805/890 (90%) are less than 2-fold differentially expressed in one or more of the CRO^RS strains.

Altered expression of resistance-associated genes such as efflux pumps and β-lactamases is a common mechanism of transcriptionally-mediated drug resistance. Although these strains lack a TEM-1 β-lactamase (Grad et al., 2016), increased expression of the gonococcal MtrCDE efflux pump is known to increase resistance to β-lactams, including cephalosporins (Lindberg et al., 2007). However, transcription of the operon encoding this pump is not elevated in the CRO^RS RNAP mutants (Figure 4—figure supplement 2), nor is it differentially upregulated in response to drug exposure in these strains (Figure 4—figure supplement 3).

In vitro evolution studies that evolved cephalosporin resistance in N. gonorrhoeae identified inactivating mutations in the type IV pilus pore subunit PilQ (Johnson et al., 2014; Zhao et al., 2005), although similar pilQ mutations have not been identified in clinical isolates, presumably because colonization requires pilus-mediated adhesion to epithelial cells (Kellogg et al., 1963; Rudel et al., 1992). Notably, pilQ transcription is reduced in the GCGS0457 rpoB1 and GCGS0457 rpoD1 transformants, as well as in the CRO^RS isolates GCGS1095 and GCGS1014 (Figure 4—figure supplements 2 and 3). The effect of hypomorphic pilQ expression on cephalosporin susceptibility has not been described, but it is possible that reducing pilQ transcript levels may confer the benefits of inactivating pilQ mutations without altogether eliminating pilus-mediated attachment and colonization.

Since ESCs, like other β-lactams, block cell wall biosynthesis by covalently inhibiting penicillin-binding proteins (PBPs), we examined how CRO^RS-associated RNAP mutations affect expression of enzymes in this target pathway. N. gonorrhoeae encodes four known PBPs (Obergfell et al., 2018; Sauvage et al., 2008): the essential high-molecular weight bifunctional transpeptidase/transglycosylase PBP1 (encoded by ponA); the essential high-molecular weight transpeptidase PBP2 (encoded by penA) (Zhao et al., 2009), which is the primary target of ESCs; and the nonessential carboxypeptidases PBP3 (encoded by dacB) and PBP4 (encoded by pbpG). In addition to these canonical PBPs, N. gonorrhoeae genomes include DacC (encoded by dacC), a third putative low-molecular weight carboxypeptidase that lacks conserved active site motifs (Obergfell et al., 2018). A homologue of the L,D-transpeptidase YnhG is also present in the genome, although it has not been reported to be functional in N. gonorrhoeae.

Increased expression of drug targets can lead to decreased drug susceptibility, but CRO^RS-associated RNAP mutations did not increase mRNA or protein abundance of the CRO target PBP2 (Figure 4). Transcription of the putative L,D-transpeptidase gene ynhG is also unchanged in the CRO^RS strains (Figure 4—figure supplement 2). By contrast, PBP1 (ponA) is upregulated in each of the CRO^RS strains compared to GCGS0457, while expression of PBP3, pbpG, and dacC is decreased in these strains (Figure 4A–B). Similar expression patterns were observed in the presence of sub-inhibitory CRO (Figure 4—figure supplement 3).

These altered PBP expression patterns are reflected in the cell wall structure of the CRO^RS strains: reduced expression of the carboxypeptidases results in more peptidoglycan with pentapeptide stems (Figure 5 and Supplementary file 3), in agreement with reported structural changes of cell walls from strains lacking the DacB carboxypeptidase (Obergfell et al., 2018). The increased expression of PBP1 in these RNAP mutants does not appear to increase the abundance of crosslinked peptidoglycan (Figure 5 and Supplementary file 3).

Strains are presented in Supplementary file 4. Metadata for the 1102 GISP isolates included in Figure 1 was previously published (Grad et al., 2016). Except where otherwise specified, N. gonorrhoeae was cultured on GCB agar (Difco) with Kellogg’s supplements (GCB-K) (Kellogg et al., 1963) at 37°C in a 5% CO₂ atmosphere. Antibiotic susceptibility testing of N. gonorrhoeae strains was performed on GCB media supplemented with 1% IsoVitaleX (Becton Dickinson) via agar dilution (for cefixime, azithromycin, tetracycline, ciprofloxacin, and rifampicin) or Etest (bioMérieux) (for ceftriaxone, penicillin G, and ertapenem). A CRO^RS breakpoint of ≥0.125 μg/mL was used, in accordance with the GISP breakpoint (Kirkcaldy et al., 2016).

The CRO^S recipient strain GCGS0457 was transformed with genomic DNA from GCGS1014 and GCGS1095. Transformations were conducted in liquid culture as described (Morse et al., 1986; Wadsworth et al., 2018). Briefly, piliated N. gonorrhoeae was suspended in GCP medium (15 g/L protease peptone 3, 1 g/L soluble starch, 4 g/L dibasic K₂HPO₄, 1 g/L monobasic KH₂PO₄, 5 g/L NaCl) with Kellogg’s supplement, 10 mM MgCl₂, and approximately 100 ng genomic DNA. Suspensions were incubated for 10 min at 37°C with 5% CO₂. Transformants were allowed to recover on nonselective agar for 4–5 hr. After recovery, transformants were plated on GCB-K ceftriaxone gradient agar plates, which were prepared by allowing a 40 μL droplet of 5 μg/mL ceftriaxone to dry onto a GCB-K agar plate. Transformations performed with GCGS0457 genomic DNA served as controls to monitor for spontaneous CRO^RS mutation. After outgrowth at 37°C in 5% CO₂, colonies growing within the ceftriaxone zone of inhibition were subcultured for further analysis.

~1.5 kb PCR fragments surrounding the rpoB and rpoD loci of interest were amplified using the primer pairs RpoB-US (5’-ATGCCGTCTGAATATCAGATTGATGCGTACCGTT-3’) and RpoB-DS (5’-CGTACTCGACGGTTGCCCAAG-3’) or RpoD-US (5’-AACTGCTCGGACAGGAAGCG-3’) and RpoD-DS (5’-CGCGTTCGAGTTTGCGGATGTT-3’). The 12 bp DNA uptake sequence (DUS) for N. gonorrhoeae (Ambur et al., 2007) was added to the 5’ end of the RpoB-US primer (underlined) to enhance transformation efficiency with the PCR product; a DUS was not added to the RpoD-US or RpoD-DS primers, as the PCR product amplified by these primers includes two endogenous DUS regions. CRO^S recipient strains were transformed with 200–300 ng purified PCR products and transformants were selected for CRO^RS as above. Transformation reactions performed with wildtype alleles (rpoB⁺ from GCGS1014; rpoD⁺ from GCGS1095) served as controls to monitor for spontaneous CRO^RS mutation.

The following isolates were used for experiments examining the compatibility of rpoB1-mediated CRO^RS with diverse genetic backgrounds: GCGS0092, GCGS0275, GCGS0336, GCGS0465, GCGS0524 (all successfully); and GCGS0126, GCGS0161, GCGS0249, GCGS0353, GCGS0374, GCGS0489, GCGS0497, GCGS0501, GCGS0560, GCGS0576, GCGS0698, and GCGS0769. CRO susceptibility and RNA polymerase allele information for each of these isolates is available in Supplementary file 1.

Following undirected transformation of GCGS0457 with GCGS1014 or GCGS1095 genomic DNA, or directed transformation of GCGS0457 with PCR products, CRO^RS transformants were analyzed by whole genome sequencing. Genomic DNA of transformants was purified with the PureLink Genomic DNA Mini kit (Life Technologies) and sequencing libraries were prepared as described Kim et al. (2017). Paired-end sequencing of these libraries was performed on an Illumina MiniSeq (Illumina). Reads were aligned to the de novo assembly of the GCGS0457 genome (European Nucleotide Archive, accession number ERR855051) using bwa v0.7.8 (Li, 2013), and variant calling was performed with pilon v1.22 (Walker et al., 2014).

Whole genome sequencing reads from the GISP collection were assembled using Velvet (Zerbino and Birney, 2008) as previously described Grad et al. (2016). Spades v 3.12 (Bankevich et al., 2012) was used for de novo assembly of GCPH44. Contigs were corrected by mapping reads back to the assembly (--careful), and contigs with less than 10X coverage or fewer than 500 nucleotides were removed. Reads were also mapped to the reference genome NCCP11945 (NC_011035.1) with bwa mem v0.7.15 (Li, 2013). Variants were identified with Pilon v 1.16 (Walker et al., 2014) using a minimum depth of 10 and minimum mapping quality of 20. SNPs, small deletions, and uncertain positions were incorporated into the reference genome to create pseudogenomes. For each isolate in the GISP collection and in the United Kingdom collection (De Silva et al., 2016), the sequence of RNA polymerase components was identified using BLASTn (ncbi-blast v2.2.30) (Supplementary file 1).

Recombinant regions of the pseudogenome alignment were detected by Gubbins (Croucher et al., 2015), and the maximum likelihood phylogenetic tree was estimated from this masked alignment using RAxML (Stamatakis, 2014). The phylogeny was visualized using ITOL (Letunic and Bork, 2019).

Bacterial cells grown overnight on GCB-K plates were suspended in tryptic soy broth to OD₆₀₀ 0.01. Three replicate GCB-K plates per time point were inoculated with 0.1 mL of this suspension and incubated at 37°C with 5% CO₂. At each time point, the lawn of bacterial growth from each of three replicate plates was suspended in tryptic soy broth. Serial dilutions were plated on GCB-K to determine total CFUs per plate. CFU counts were normalized to the initial inoculum density (CFUs measured at time 0) for each replicate.

Bacterial cells grown overnight on GCB-K plates were suspended in liquid GCP medium with 1% Kellogg’s supplement and 0.042% NaHCO₃ to OD₆₀₀ 0.1. Suspensions were incubated at 37°C with aeration for 2 hr to allow cultures to return to log phase. Cell pellets were collected by centrifugation and fixed for at least two hours at room temperature in 0.2M cacodylate buffer with 2.5% paraformaldehyde, 5% glutaraldehyde, and 0.06% picric acid. Fixed pellets were washed in 0.1M cacodylate buffer and postfixed with 1% Osmiumtetroxide (OsO₄)/1.5% Potassiumferrocyanide (KFeCN6) for 1 hr, washed 2x in water, 1x Maleate buffer (MB) 1x and incubated in 1% uranyl acetate in MB for 1 hr followed by two washes in water and subsequent dehydration in grades of alcohol (10 min each; 50%, 70%, 90%, 2 × 10 min 100%). The samples were then put in propyleneoxide for 1 hr and infiltrated ON in a 1:1 mixture of propyleneoxide and TAAB Epon (Marivac Canada Inc St. Laurent, Canada). The following day the samples were embedded in TAAB Epon and polymerized at 60 degrees C for 48 hr. Ultrathin sections (~60 nm) were cut on a Reichert Ultracut-S microtome, picked up on to copper grids stained with lead citrate and examined in a JEOL 1200EX Transmission electron microscope or a TecnaiG² Spirit BioTWIN and images were recorded with an AMT 2 k CCD camera.

RNA isolation and sequencing was performed from N. gonorrhoeae strains as described Wadsworth et al. (2018). Bacterial cells grown on GCB-K plates for 17 hr were suspended in liquid GCP medium with 1% IsoVitaleX and 0.042% NaHCO₃ to OD₆₀₀ 0.1. Suspensions were incubated at 37°C with aeration for 2–3 hr to allow cultures to return to log phase. Cells were collected to measure baseline transcriptional profiles. For samples measuring the effect of drug exposure, 0.008 μg/mL CRO was added and cultures were incubated at 37°C for an additional 90 min before cell collection. RNA was purified with the Direct-Zol kit (Zymo). Transcriptome libraries were prepared at the Broad Institute at the Microbial ‘Omics Core using a modified version of the RNAtag-seq protocol (Shishkin et al., 2015). Five hundred nanograms of total RNA was fragmented, depleted of genomic DNA, dephosphorylated, and ligated to DNA adapters carrying 5’-AN8-3’ barcodes of known sequence with a 5’ phosphate and a 3’ blocking group. Barcoded RNAs were pooled and depleted of rRNA using the RiboZero rRNA depletion kit (Epicentre). Pools of barcoded RNAs were converted to Illumina cDNA libraries in two steps: (i) reverse transcription of the RNA using a primer designed to the constant region of the barcoded adapter with addition of an adapter to the 3’ end of the cDNA by template switching using SMARTScribe (Clontech) as described previously (Zhu et al., 2001); (ii) PCR amplification using primers whose 5’ ends target the constant regions of the 3’ or 5’ adapter and whose 3’ ends contain the full Illumina P5 or P7 sequences. cDNA libraries were sequenced on the Illumina Nextseq 500 platform to generate 50 bp paired-end reads. Barcode sequences were removed, and reads were aligned to the FA1090 reference genome. Differential expression analysis was conducted in DESeq2 v.1.16.1 (Love et al., 2014).

Genomic and transcriptomic read libraries are available from the NCBI SRA database (accession number PRJNA540288).

Protein abundance of PBP1, PBP2, and PBP3 was calculated using the fluorescent penicillin derivative bocillin-FL (Thermo Fisher). N. gonorrhoeae strains from overnight cultures were suspended in liquid GCP medium supplemented with 1% IsoVitalex and 0.042% NaHCO₃ to a density of OD₆₀₀ 0.1. Suspensions were incubated with aeration at 37°C for 2.5–3 hr. Bacterial cells were collected by centrifugation, washed once with 1 mL of sterile phosphate-buffered saline (PBS), and resuspended in PBS 5 μg/mL bocillin-FL and 0.1% dimethyl sulfoxide (DMSO) to a final concentration of 1 mL of OD₆₀₀ 0.5 per 50 μL suspension. Bocillin-FL suspensions were incubated for 5 min. An equal volume 2x SDS-PAGE sample buffer (Novex) was added and samples were boiled for 5 min. Proteins in 30 μL of each suspension were separated by SDS-PAGE on 4–12% Tris-Glycine protein gels (Novex), which were visualized on a Typhoon imager (Amersham) (excitation 488 nm/emission 526 nm) to detect bocillin-FL fluorescence. Densitometry was performed with ImageJ (Schneider et al., 2012). Total bocillin-FL fluorescent signal was calculated for each sample, and the proportional contribution of individual PBPs to this signal was reported.

N. gonorrhoeae strains were cultured for approximately 18 hr at 37°C with agitation in GCP medium supplemented with Kellogg’s reagent and 0.042% NaHCO₃. Peptidoglycan was isolated and digested as described Kühner et al. (2015) with minor modifications. Briefly, bacterial cells were pelleted by centrifugation, suspended in 1 mL 1M NaCl, and incubated at 100°C for 20 min. Samples were centrifuged for one minute at 18,000 × g. Pellets were washed three times with water, suspended in 1 mL water, and placed in a bath sonicator for 30 min. 0.5 mL 0.1M Tris pH 6.8, 40 μg/mL RNase, and 16 μg/mL DNase were added to each sample; samples were incubated with shaking at 37°C for two hours, with the addition of 16 μg/mL trypsin after the first hour of incubation. Samples were heated to 100°C for 5 min to inactivate enzymes, then centrifuged for 3 min at 18,000 × g to pellet peptidoglycan. Pellets were washed with 1 mL aliquots of water until the suspension pH measured between 5 and 5.5. Peptidoglycan was then resuspended in 0.2 mL 12.5 mM NaH₂PO₄ pH 5.5 with 500 U/mL mutanolysin and incubated with shaking at 37°C for 16 hr. Samples were heated to 100°C for 5 min to inactivate mutanolysin and centrifuged for 5 min at 18,000 × g to pellet debris. The supernatant, containing solubilized muropeptides, was removed to new tubes, and 50 μL 10 mg/mL NaBH₄ was added to each. Samples were incubated at room temperature for 30 min and then the pH was adjusted to 2–3 with 85% H₃PO₄.

LC-MS was conducted using an Agilent Technologies 1200 series HPLC in line with an Agilent 6520 Q-TOF mass spectrometer operating with electrospray ionization (ESI) and in positive ion mode. The muropeptide fragments were separated on a Waters Symmetry Shield RP18 column (5 μm, 4.6 × 250 mm) using the following method: 0.5 mL/minute solvent A (water, 0.1% formic acid) for 10 min followed by a linear gradient of 0% solvent B (acetonitrile, 0.1% formic acid) to 20% B over 90 min.