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Antimicrobial Agents and Chemotherapy, March 2002, p. 769-777, Vol. 46, No. 3
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.3.769-777.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mutations in ponA, the Gene Encoding Penicillin-Binding Protein 1, and a Novel Locus, penC, Are Required for High-Level Chromosomally Mediated Penicillin Resistance in Neisseria gonorrhoeae

Patricia A. Ropp,1 Mei Hu,2 Melanie Olesky,2 and Robert A. Nicholas2*

Departments of Chemistry,1 Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-73652

Received 15 June 2001/ Returned for modification 19 September 2001/ Accepted 6 December 2001


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ABSTRACT
 
Chromosomally mediated penicillin resistance in Neisseria gonorrhoeae occurs in part through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. However, the genetic and molecular mechanisms of transformation of a penicillin-susceptible strain of N. gonorrhoeae to high-level penicillin resistance have not been clearly elucidated. Previous studies suggested that alterations in PBP 1 were involved in high-level penicillin resistance. In this study, we identified a single amino acid mutation in PBP 1 located 40 amino acids N terminal to the active-site serine residue that was present in all chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains for which MICs of penicillin were >=1 µg/ml. PBP 1 harboring this point mutation (PBP 1*) had a three- to fourfold lower rate of acylation (k2/K') than wild-type PBP 1 with a variety of ß-lactam antibiotics. Consistent with its involvement in high-level penicillin resistance, replacement of the altered ponA gene (ponA1) in several CMRNG strains with the wild-type ponA gene resulted in a twofold decrease in the MICs of penicillin. Surprisingly, transformation of an intermediate-level penicillin-resistant strain (PR100; FA19 penA4 mtr penB5) with the ponA1 gene did not increase the MIC of penicillin for this strain. However, we identified an additional resistance locus, termed penC, which was required along with ponA1 to increase penicillin resistance of PR100 to a high level (MIC = 4 µg/ml). The penC locus by itself, when present in PR100, increases the MICs of penicillin and tetracycline twofold each. These data indicate that an additional locus, penC, is required along with ponA1 to achieve high-level penicillin resistance.


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INTRODUCTION
 
Up until 1987, Neisseria gonorrhoeae was treated with a single dose of penicillin, which kills the bacteria by inhibiting the penicillin-binding proteins, or PBPs, that synthesize the cell wall peptidoglycan. N. gonorrhoeae has four PBPs, designated PBP 1, 2, 3, and 4 (1; P. A. Ropp and R. A. Nicholas, unpublished data.). Of these, only PBPs 1 and 2 are essential for cell viability and thus are potential antibiotic killing targets in N. gonorrhoeae. Because penicillin G has an approximately 10-fold higher rate of acylation with PBP 2 than with PBP 1, penicillin G kills N. gonorrhoeae at its MIC by inactivation of PBP 2 (1).

While early isolates of N. gonorrhoeae were extremely sensitive to penicillin (MICs {cong} 0.004 to 0.01 µg/ml), this sensitivity gradually decreased such that by the late 1970s strains emerged that were resistant to penicillin (MICs >= 2 µg/ml). Resistance to other antibiotics, including tetracycline and erythromycin, also increased during this time. Penicillin resistance in the gonococci arose by two independent mechanisms: the plasmid-mediated production of a penicillinase (TEM-1 ß-lactamase) and the chromosomally mediated expression of multiple resistance genes. In 1998, 29.4% of the clinical gonococcal isolates collected throughout the United States by the Gonococcal Isolate Surveillance Project overseen by the Centers for Disease Control and Prevention (CDC) were resistant to either penicillin, tetracycline, or both (6). While the percentage of penicillinase-producing N. gonorrhoeae organisms (PPNG) declined significantly during the years 1991 to 1998, the percentage of chromosomally mediated resistant N. gonorrhoeae organisms (CMRNG) rose.

The genetic mechanisms of chromosomally mediated resistance to penicillin have been investigated in some detail (8, 11). Because N. gonorrhoeae is naturally transformable, penicillin-resistant strains are capable of transferring their resistance genes to susceptible strains in a stepwise manner via transformation and homologous recombination (Fig. 1). The first step in transformation to high-level penicillin resistance (MIC >= 2 µg/ml) is mediated by the penA gene, which encodes altered forms of PBP 2 that display 5- to 10-fold decreases in their rate of acylation by penicillin (28). The second step of transformation is mediated by the mtr locus, which confers nonspecific resistance to erythromycin, rifampin, and detergents through increased expression of the MtrC-MtrD-MtrE efflux pump (17). Transfer of the penB locus leads to further increases in both penicillin and tetracycline resistance (5). Recently, the penB phenotype has been correlated to mutations in the porin P1B allele that presumably decrease the porin-mediated flux of antibiotics across the outer membrane (15).



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  		FIG. 1. Stepwise acquisition of resistance genes in gonococci by DNA uptake and homologous recombination. The stepwise transfer of resistance genes occurs in the order shown, and each step increases resistance to at least one of the three antibiotics shown on the right. The first three resistance genes (penA, mtr, and penB) are well characterized, and transformants with these genes are easy to obtain in the laboratory. In contrast, transformation of an FA19 penA mtr penB strain up to a level of resistance equal to that of the donor strain has been very hard to achieve in the laboratory. The last two steps of transformation to high-level resistance (in blue) are mediated by two resistance loci, penC and ponA1, that are the subjects of this study.

Unexpectedly, transformation of a penA mtr penB strain (for which the MIC of penicillin is 0.5 to 1.0 µg/ml) to a level of penicillin resistance equal to that of the donor strain (MIC = 4 µg/ml) has been difficult to achieve in the laboratory (8, 11). Because penicillin-resistant strains for which MICs are >=2 µg/ml appear to express an altered form of PBP 1 that displays a lower rate of acylation by penicillin, the PBP 1 gene likely is involved in transformation to high-level penicillin resistance (7). Dougherty was able to obtain high-level penicillin-resistant transformants at low frequency by using a modified transformation procedure and by selecting transformants at penicillin concentrations two- to fourfold less than the MIC for the donor strain (7). Assessment of the binding of [3H]penicillin G to membranes prepared from these transformants revealed an apparent heterogeneity in the affinity of PBP 1 for penicillin. One group expressed a PBP 1 with an affinity for penicillin G equal to that of the donor strain, whereas another group expressed a PBP 1 with an intermediate affinity. These results suggested that PBP 1 plays a role in mediating high-level penicillin resistance in CMRNG.

We recently cloned the ponA gene encoding PBP 1 from a penicillin-susceptible strain of N. gonorrhoeae (24). In the present study, we isolated and sequenced the ponA gene from several clinical isolates of N. gonorrhoeae for which penicillin MICs were >=1 µg/ml in order to determine the changes in the amino acid sequence that presumably lead to a decreased rate of acylation by penicillin. In addition, we investigated the role of the altered ponA gene in mediating transformation of intermediate-level penicillin-resistant transformants to high-level penicillin resistance. Our results indicate that both the ponA1 gene encoding an altered form of PBP 1 and another resistance locus, termed penC, are required to achieve high-level chromosomally mediated penicillin resistance.


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MATERIALS AND METHODS
 
Bacterial strains and plasmids. Clinical isolates of N. gonorrhoeae for which penicillin G MICs were >=1 µg/ml were obtained from several sources. Strains 2227, 0387, 3391, 5611, and 9634 were kindly provided by Marcia Hobbs and Myron S. Cohen, University of North Carolina at Chapel Hill, from a Wilson county (North Carolina) surveillance program (12). FA19 (21) and FA6140 (for which the penicillin MIC was 4 µg/ml) (5) were obtained from Fred Sparling, University of North Carolina at Chapel Hill. Piliated FA6140 was obtained from William Shafer, Emory University. Strains 111, 114, 131, 151, and 154 were obtained from Joan Knapp at the CDC. Chromosomal DNA from CMRNG strains CDC77-124615 (8) and CDC84-060418 (7) was kindly provided by Brian Spratt, Imperial College, London.

The plasmids pPR16 and pPR17 harbor the coding regions of the wild-type and mutant ponA genes, respectively, with an extra 546 bp of downstream sequence to facilitate homologous recombination. To aid in selection, each plasmid also contained the {Omega} fragment encoding spectinomycin and streptomycin resistance (23), inserted 68 bp downstream of the ponA stop codon (see Fig. 2). pMutS-erm, which contained the gonococcal mutS gene inactivated by insertion of an erythromycin resistance cassette, was provided by Janne Cannon, University of North Carolina at Chapel Hill.




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  		FIG. 2. Location of the Leu-421->Pro mutation in PBP 1*. (A) The ponA gene is shown along with the locations of the two functional domains, the transglycosylase domain and the transpeptidase domain. Locations of the three highly conserved sequence motifs found in all penicillin-interacting proteins are shown above the transpeptidase domain. The location of the Leu-421->Pro mutation is shown by an asterisk. Also shown are the DNA sequences of the ponA and ponA1 genes. The mutation fortuitously disrupts a unique PstI restriction site. (B) Schematic showing the construct (pPR17) used to create the strains containing the ponA1 gene. The {Omega} fragment, which encodes resistance to both spectinomycin and streptomycin, was used to identify transformants containing the ponA1 mutation (see text for details). pPR16 is identical to pPR17, except that it contains the wild-type ponA gene.

Media, growth conditions, and MIC determinations. N. gonorrhoeae strains were grown on GC medium base (GCB) agar (Difco Laboratories, Detroit, Mich.) with supplements I and II (20) at 37°C in 5% CO2. Liquid cultures were grown in GCB broth containing supplements I and II, supplement B, and 10 mM NaHCO3 in a shaking incubator at 37°C. MICs were determined according to the method of Sparling et al. (27) or by the spot method. Briefly, cells were resuspended in GCB broth, and 1,000 colonies were spread onto GCB agar plates containing increasing concentrations of the appropriate antibiotic. The MIC was defined as the minimal concentration of antibiotic at which no more than five colonies were observed after 24 h. Alternatively, colonies were suspended in GCB broth and 5 x 104 colonies in 5 µl were spotted onto GCB agar plates containing increasing twofold concentrations of the antibiotic. The MIC was defined as the minimal concentration of antibiotic that inhibited growth after 24 h. The two methods gave very similar results.

General methods. Chromosomal DNA was isolated by ethidium bromide-CsCl equilibrium centrifugation by a modification of the method of Dowson et al. (10). In some instances, chromosomal DNA was isolated with the DNAzol reagent (Gibco/BRL, Gaithersburg, Md.) following the manufacturer's instructions. p-Trimethylstannyl-penicillin V (a gift from Larry Blaszczak; Eli Lilly and Co.) was converted to [125I]iodopenicillin ([125I]IPV) as described previously (2). Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, Calif.).

Isolation and sequencing of the ponA gene from penicillin-resistant strains. The ponA1 gene was cloned initially from FA6140. The complete coding region of the ponA gene was amplified by PCR from FA6140 DNA with primers GC23 (5'-GTGAGTAACCGTTTCGGTATCC-3'), which hybridizes 124 bp upstream of the ATG start codon, and GC44 (5'-TTAAAACAGGGAATCCAACTGC-3'), an antisense oligonucleotide that hybridizes to the last 22 bp of the ponA gene. The amplified product was subcloned into pUC19-K (pUC19 containing the kanamycin resistance gene). Both strands of the plasmid harboring the ponA gene as well as the ponA PCR product were sequenced with the Amplicycle CS sequencing kit (Perkin-Elmer, Foster City, Calif.). The same sequence was obtained from multiple independent amplifications, verifying that the mutation was not a PCR artifact. Sequences of the ponA genes from the other penicillin-resistant strains examined in this study were amplified by PCR and sequenced directly as described above.

Genetic transformation. N. gonorrhoeae was transformed as described previously (25). Briefly, cells were passaged on GCB agar, and a single piliated colony was streaked onto a fresh GCB plate and allowed to grow overnight. Cells were scraped from the plate and gently resuspended in prewarmed GCB broth containing supplements I and II and 10 mM MgCl2. Resuspended cells were diluted in prewarmed GCB broth to a cell density of 108/ml (optical density at 560 nm, 0.18), followed by the addition of NaHCO3 to a final concentration of 10 mM. Aliquots (900 µl) of diluted cells were mixed gently with 100 µl of 20-µg/ml donor DNA (either plasmid or chromosomal DNA) and allowed to incubate for 5 h at 37°C in a humidified 5% CO2 atmosphere. Various amounts of cells were then plated onto GCB agar containing the appropriate antibiotic concentrations (described below) and grown 24 to 48 h at 37°C in 5% CO2.

PR100 (FA19 penA4 mtrR penB5; see Table 3) was constructed by transforming FA19 with FA6140 genomic DNA and selecting for each resistance gene at the following antibiotic concentrations: (i) penA4, 0.16 µg of penicillin G per ml; (ii) mtr, 0.5 µg of erythromycin per ml; and (iii) penB5, 0.37 µg of tetracycline per ml. Each of these genes was amplified by PCR from PR100 genomic DNA and sequenced; all three genes corresponded exactly to the sequences obtained from FA6140. Other transformants were selected on GCB agar containing antibiotic concentrations as indicated. Gonococci transformed with pPR16 or pPR17 plasmid DNA harboring the {Omega} fragment were selected on GCB agar containing 100 µg of spectinomycin per ml, whereas cells transformed with plasmid constructs containing the erythromycin resistance gene (i.e., for disruption of mutS) were selected on 10 µg of erythromycin per ml. Since pUC plasmids are not replicative in N. gonorrhoeae, transformation occurred via homologous recombination into the genome. To test for the presence of the ponA1 mutation in transformation experiments, individual colonies were resuspended in 100 µl of 10 mM Tris-HCl-1 mM EDTA, pH 8.0, placed in a boiling water bath for 10 min, and then centrifuged. The ponA gene was then amplified from 1 µl of the boiled lysates with the following primers: 5'-CGCGGTGCGGAAAACTGATATCGAT-3' (bp 955 to 978 of ponA open reading frame) and 5'-AGCCCGGATCGGTTACCATACGTT-3' (bp 2218 to 2195 of ponA open reading frame). Aliquots (5 µl) of the amplification reaction mixture were used for subsequent PstI digestion without further purification.


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  		TABLE 3. Derivation of and MICs for strains of N. gonorrhoeae used in this study

To examine if the increased resistance in penC strains was due to an additional mutation(s) in one of the genes comprising the mtr operon, we amplified the mtrR + mtrC, mtrD, mtrE, and mtrF genes from the mtr operon by PCR from 1 ng of PR102 DNA and tested the ability of each fragment to transform PR100 to PR100 penC. The primers were designed such that each amplified fragment contained at least one uptake sequence. Following amplification, the fragments were purified from an agarose gel and mixed with piliated PR100 cells, and resistant transformants were selected on GCB plates containing 0.95 µg of penicillin per ml. As a positive control, the mtrR + mtrC fragment (which contains the original mtr mutation) was used to transform FA19 penA4 to FA19 penA4 mtr (transformation frequency, {approx}3 x 10-5).

Purification of recombinant PBP 1 and PBP 1*. The coding sequences of the ponA genes from both FA19 and FA6140 were amplified by PCR with Taq DNA polymerase (Gibco/BRL), cloned into pET15b-K (pET15b containing the kanamycin resistance gene in place of the ß-lactamase gene), and transformed into Escherichia coli BL21(DE3) as previously described (24). These constructs result in the fusion of 20 additional amino acids, including a hexahistidine tag, to the amino termini of PBP 1 and PBP 1* (PBP 1 containing the Leu-421->Pro mutation).

E. coli BL21(DE3) cells containing PBP 1 expression plasmids were grown in Luria-Bertani medium supplemented with kanamycin (50 µg/ml). Overnight growth resulted in significant expression of PBP 1 in the absence of induction. Cells were pelleted, resuspended in cold lysis buffer (50 mM sodium phosphate buffer [pH 8.0], 10% glycerol, 0.5 mM phenylmethylsulfonyl chloride), and lysed by three passes through an Aminco French pressure cell (Champaign, Ill.) at 16,000 lb/in2. Membranes were isolated from the cell lysate by centrifugation at 225,000 x g, washed, resuspended, and stored at -20°C. Crude membranes were solubilized with an equal volume of 50 mM sodium phosphate buffer (pH 8.0)-2 M NaCl-2% Triton X-100-40 mM imidazole for 1 h with stirring at room temperature.

Following centrifugation at 225,000 x g, the supernatants containing solubilized PBP 1 or PBP 1* were loaded directly onto Ni2+-NTA columns (Qiagen, Chatsworth, Calif.) and washed with 50 mM sodium phosphate (pH 8.0)-1 M NaCl-10% glycerol-0.1% Triton X-100-15 mM imidazole. PBP 1 and PBP 1* were eluted with an increasing linear gradient of imidazole (15 to 500 mM) in the above buffer. Fractions containing PBP 1 or PBP 1* were pooled, concentrated by ultrafiltration, and then dialyzed extensively against 50 mM sodium phosphate (pH 8.0)-0.5 M NaCl-10% glycerol-0.1% Triton X-100 to remove imidazole. The purified proteins were stored at -80°C at a concentration of 2 to 4 mg/ml.

Determination of the kinetic constants for the interaction of ß-lactam antibiotics with PBP 1 and PBP 1*. k2/K' constants of recombinant PBP 1 and PBP 1* for [125I]IPV were determined from time courses of acyl-enzyme formation as described by Frere et al. (14). Briefly, purified proteins were incubated at 30°C with [125I]IPV concentrations ranging from 0.625 to 5 mM in 50 mM sodium phosphate-1 mM EDTA-0.1% Triton X-100 (pH 7.4), and the reaction was stopped by the addition of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer at 0.5- to 1-min time intervals. The samples were run on a sodium dodecyl sulfate-8% polyacrylamide gel, and the dried gel was exposed to a phosphorimaging screen for 12 to 24 h. Levels of acyl-enzyme formed were quantitated with ImageQuant software following imaging on a Storm 840 Phosphorimager (Molecular Dynamics, Sunnyvale, Calif.). The k2/K' constants was derived from the slope of a plot of the apparent first-order rate constant ka versus the concentration of [125I]IPV. The k2/K' values for penicillin, ceftriaxone, and cephaloridine were determined by the competition method as described previously (14).

k3 values were determined by monitoring the formation of free PBP from samples of PBP-antibiotic complexes. PBPs were incubated with unlabeled antibiotics for 15 min at 30°C, and excess antibiotics were removed by dialysis at 4°C against the same buffer as above with the addition of 10% glycerol. The addition of glycerol was necessary to ensure the stability and activity of PBP 1 proteins during prolonged incubations. Following dialysis, the protein complex was incubated at 30°C, aliquots were removed at various times, and the amount of free PBP was assessed by incubation with a saturating concentration of [125I]IPV. The relative amounts of labeled PBPs were derived by phosphorimager analysis as described above. No significant hydrolysis was observed over 3 days of incubation.


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RESULTS
 
Identification of an altered form of the ponA gene from CMRNG strains. Previous studies have shown that high-level penicillin resistance in N. gonorrhoeae is correlated with expression of PBP 1 displaying an apparent decrease in its affinity for penicillin (7, 8). These data suggested that one or more mutations were present in the primary sequence of PBP 1 that decreased its rate of acylation with penicillin. To test this hypothesis, we isolated the ponA gene from the high-level penicillin-resistant strain FA6140 (5) (penicillin MIC = 4 µg/ml) by PCR amplification of genomic DNA and compared its nucleotide sequence to that of the ponA gene from the penicillin-susceptible strain FA19 (24). Sequence analyses of several independent clones isolated from separate amplification reactions identified a single point mutation, a T-to-C transition, at nucleotide 1261 of the ponA coding region (hereafter designated ponA1) (Fig. 2A). This mutation, which results in the change of Leu-421->Pro, was the only difference observed in the entire 2,400-bp coding region.

To determine if this mutation occurs in the ponA gene from other penicillin-resistant strains, the ponA genes from 10 geographically and temporally distinct CMRNG isolates for which penicillin MICs were >=1 µg/ml were amplified and sequenced. Serovars of these strains suggest that these strains were descended from different lineages (Table 1). Nine of these strains harbored the ponA1 gene containing a point mutation identical to that observed in the ponA gene from FA6140 (Table 1). Two of these strains, CDC120177 and FA6140, were isolated in the late 1970s; strains 111, 114, and 131 were isolated in Cincinnati, Ohio, in 1994; and strains 0387, 3391, 5611, and 9634 were isolated in rural North Carolina in 1993 from a gonococcal surveillance program. As we observed with the ponA1 gene from FA6140, there were no other differences in the ponA genes from these other strains. CDC84-060418, the only strain that did not contain the mutation, is known to be different from the majority of CMRNG strains in that it expresses an altered PBP 2 with an extremely low rate of acylation by penicillin, but with little to no change in the rate of acylation of PBP 1 (7). Thus, with the exception of CDC84-060418, all CMRNG strains for which the MIC of penicillin was >=1 µg/ml showed the presence of the altered codon in the ponA gene.


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  		TABLE 1. Characteristics of the N. gonorrhoeae strains used in this study

Interaction of PBP 1 and PBP 1* with ß-lactam antibiotics. The T-to-C transition in the ponA1 gene results in a change of Leu-421->Pro in the PBP 1 amino acid sequence (hereafter referred to as PBP 1*; Fig. 2A). Leu-421 is 40 amino acids to the amino-terminal side of the active-site serine residue (Ser-461) and thus is near (but not necessarily within) the active-site cavity. To assess the functional consequences of the Leu-421->Pro amino acid mutation in PBP 1*, both the wild-type and mutant forms of PBP 1 were expressed in E. coli and purified, and the kinetic constants for their interaction with ß-lactam antibiotics were determined. ß-Lactam antibiotics interact with PBPs according to the following scheme:

where E is the enzyme, S is a ß-lactam antibiotic, and P is the inactive degradation product (13, 22). The effectiveness of an antibiotic is defined by two parameters: (i) the second-order specificity constant, k2/K', where K' = k-1/k+1; and (ii) the rate constant for hydrolysis of the acyl-enzyme complex, k3. The Leu-421->Pro mutation results in a fourfold decrease in the k2/K' constant for [125I]IPV and approximately three- to fourfold decreases in the k2/K' constants for penicillin G, ceftriaxone, and cephaloridine (Table 2). No changes in the rates of hydrolysis of the acyl-enzyme complex were observed, indicating that resistance arises from a lower rate of acylation by ß-lactam antibiotics and not by an increase in the rate of deacylation.


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  		TABLE 2. Kinetic constantsa of PBP 1 and PBP 1* for interaction with ß-lactam antibiotics

Role of the ponA1 gene in transformation of an intermediate-level resistant strain to high-level penicillin resistance. The identification of a mutation in PBP 1 that decreases its rate of acylation with ß-lactam antibiotics suggests that the ponA1 gene is involved in conferring high-level penicillin resistance. To determine the role of PBP 1* in resistance, we first produced an intermediate-level penicillin-resistant strain (PR100; FA19 penA4 mtr penB5) as described in Materials and Methods. PCR amplification and sequencing of the penA4, mtr, and penB5 genes from PR100 verified that these genes were identical to those in FA6140 (data not shown). For PR100, both penicillin and tetracycline have a MIC of 1.0 µg/ml, and this strain also shows increased resistance (relative to FA19) to both ceftriaxone and cephaloridine (Table 3).

PR100 was transformed with the plasmid pPR17, which contains the entire ponA1 gene and 546 bp of the 3' flanking sequence with the {Omega} fragment (encoding spectinomycin resistance) (23) inserted 68 bp downstream of the ponA1 stop codon (Fig. 2B). The transformed cells were plated on GCB plates containing either 2 µg of penicillin per ml or 100 µg of spectinomycin per ml. No colonies grew on the GCB-penicillin plates, even though spectinomycin-resistant colonies were isolated at a frequency of {approx}10-5. We screened the spectinomycin-resistant transformants for the successful recombination of the linked ponA1 mutation by PCR amplification of the ponA gene and subsequent digestion of the PCR products with PstI. Fortuitously, the ponA1 mutation destroys a unique PstI site within the ponA gene, and thus loss of the PstI site in the amplified fragments is diagnostic for the presence of ponA1 (Fig. 2A). Approximately half of the spectinomycin-resistant colonies had incorporated the ponA1 mutation (data not shown). Surprisingly, there was no increase in the MIC of penicillin for spectinomycin-resistant transformants containing the ponA1 mutation (PR101; Table 3). Thus, acquisition of the ponA1 gene by an intermediate-level penicillin-resistant strain does not increase the MIC of penicillin to the level seen with FA6140.

To determine whether PBP 1* is involved in high-level penicillin resistance at all, we replaced the ponA1 gene in FA6140 with the wild-type ponA gene and determined the MIC of penicillin for the resulting isogenic strain. FA6140 was transformed with pPR16, a plasmid containing the wild-type ponA gene and the linked {Omega} fragment, and spectinomycin-resistant colonies were screened for the presence of the wild-type gene by PstI digestion of the amplified ponA gene. For several such transformants, designated FA6140 ponA-wt, consistent twofold decreases in the MIC of penicillin (2 µg/ml; Table 3) were observed, indicating that the ponA1 gene is involved in high-level penicillin resistance in FA6140. The MIC of tetracycline was unchanged. Identical results were obtained with the CMRNG strains 111, 114, and 131.

Identification of penC, a previously uncharacterized resistance gene. In our attempts to obtain penicillin-resistant colonies following transformation of PR100 with either pPR17 plasmid DNA or FA6140 chromosomal DNA, we observed the growth of a few (5 to 25) colonies following plating on GCB plates containing 0.95 µg of penicillin per ml. For all of these colonies, twofold increases in the MICs of both penicillin (2.0 µg/ml) and tetracycline (2.0 µg/ml) and similar increases in the MICs of ceftriaxone and cephaloridine (Table 3) were observed. PCR amplification of the ponA gene and digestion with PstI showed that none of these colonies had recombined the ponA1 gene (data not shown), which was consistent with our previous data showing that the ponA1 gene does not increase the MIC of penicillin when transformed into PR100. Moreover, we were able to isolate penicillin- and tetracycline-resistant colonies for which the MICs showed twofold increases simply by plating PR100 (FA19 penA mtr penB) on GCB-penicillin or GCB-tetracycline plates (i.e., in the absence of DNA transformation). The increase in MICs observed with these colonies (designated PR102; Table 3) was stable to repeated passages on GCB agar over several days with no antibiotics present, strongly suggesting that resistance was due to a genetic event and not to an induction of an enzyme in response to antibiotic challenge.

These data suggest that the twofold increase in penicillin and tetracycline resistance is due to a low-frequency, spontaneous mutation within the population of PR100. If true, then plating PR100 containing a disrupted mutS gene (which increases mutation frequency) on selective media should result in a large increase in the numbers of penicillin- and tetracycline-resistant colonies. Consistent with this hypothesis, plating PR103 (PR100 mutS) on selective media produced colonies at a frequency 17-fold higher on average than for PR100 (n = 3; data not shown). Moreover, the MICs of both penicillin and tetracycline for all of the resistant colonies derived from PR103 increased twofold. This experiment provides additional evidence that the increased resistance is due to a spontaneously arising genetic mutation in an as yet unidentified locus. This locus has been designated penC.

Repeated attempts to transform PR100 to PR100 penC with DNA from the CMRNG strains FA6140, 114, and 131 were unsuccessful. However, we were able to transform penC into PR100 with chromosomal DNA isolated from PR102 (PR100 penC; Table 3) at a frequency of {approx}10-5. (While this transformation frequency is lower than that obtained when penA4 is transformed into FA19 [{approx}3 x 10-4], similar transformation frequencies were obtained when PR100 was transformed with either plasmid or chromosomal DNA containing different resistance markers. These results suggest that the lower transformation frequency of PR100 is a characteristic of the strain and is not related to the penC locus.) The inability to transform the penC locus into PR100 from FA6140, 114, and 131 indicates that these CMRNG strains do not contain the penC locus and suggests that a novel mechanism or another as yet unidentified mutation is involved in high-level penicillin resistance in these strains. However, given that penC can arise spontaneously, we suspect that the penC locus will be found in other CMRNG strains when a more extensive search is conducted.

Since both ponA1 and penC are required to mediate high-level penicillin resistance in FA19, we attempted to identify a similar resistance locus in FA6140 by transforming PR101 (PR100 ponA1) with FA6140 DNA to high-level penicillin resistance. However, these attempts were unsuccessful, suggesting that in FA6140 an additional resistance locus that is not easily transformable or a novel genetic mechanism is involved in high-level resistance.

The penC locus does not encode additional mutations in previously characterized resistance genes. We also considered the possibility that additional mutations in the three known resistance loci (penA, mtr, and penB) might underlie the increased resistance observed in penC strains. To test this possibility, we sequenced the penA and penB genes from both PR100 and PR102 (PR100 penC). No differences were noted in either gene obtained from the two strains. To determine if mutation(s) in one or more of the genes encoding the Mtr efflux pump might be responsible for the increased resistance observed in penC strains, we amplified the mtrR + mtrC, mtrD, mtrE, and mtrF genes from PR102 by PCR and used these fragments in an attempt to transform PR100 into PR100 penC. None of the fragments gave rise to penicillin- or tetracycline-resistant colonies at a frequency higher than that of a no-DNA control, while DNA from PR102 transformed PR100 into PR102 with a frequency of {approx}10-5. These data are consistent with the idea that penC is a novel resistance locus and not an already-characterized resistance gene that has acquired an additional mutation.

Transformation of a penC strain to high-level penicillin resistance with ponA1. Even though acquisition of the ponA1 gene by PR100 by itself did not increase penicillin resistance, acquisition of penC may be required before the ponA1 gene can increase penicillin resistance. To test this hypothesis, we transformed the penC strain PR102 with pPR17 and screened spectinomycin-resistant transformants for the presence of the linked ponA1 gene as described above. For the resulting transformants, designated PR105, the MIC of penicillin was 4 µg/ml, the same as for FA6140 (Table 3). The MIC of cephaloridine increased following acquisition of ponA1, whereas no increases were observed for ceftriaxone and tetracycline. We were unable to transform PR102 to higher penicillin resistance with FA6140 chromosomal DNA or pPR17 plasmid DNA by selecting on GCB-penicillin plates, but this may simply reflect the difficulty in selecting transformants having such a small increase in penicillin resistance (7). These data demonstrate that both ponA1 and penC are necessary to transform an intermediate-level resistant recipient strain to high-level penicillin resistance. Moreover, the penC locus must already be present in order for ponA1 to increase resistance. Thus, increasing the penicillin resistance of FA19 to a high level (MIC = 4 µg/ml) requires mutations in five genes or loci (penA, mtr, penB, penC, and ponA1), including both essential PBPs (Fig. 1).


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DISCUSSION
 
The mechanism by which chromosomally mediated penicillin-resistant strains arise in N. gonorrhoeae is different from that utilized by most other bacteria. Susceptible strains of N. gonorrhoeae become resistant to penicillin by acquiring multiple resistance genes in a stepwise fashion. As each gene is acquired, penicillin resistance increases until treatment failure occurs. While the genes involved in transforming N. gonorrhoeae to an intermediate level of penicillin resistance are well established, those that mediate high-level resistance have not been identified. In this study, we show that two loci, the ponA1 gene encoding an altered form of PBP 1 and a newly identified locus, penC, are required to transform an intermediate-level penicillin-resistant strain to high-level resistance.

The ponA1 gene encodes PBP 1 containing a single amino acid mutation, Leu-421->Pro, which decreases the rate of acylation with ß-lactam antibiotics three- to fourfold compared to the wild type. An identical mutation was observed in the ponA genes from penicillin-resistant strains isolated 17 years apart, from geographically distinct regions, and with different serovars. Transformation of an intermediate-level penicillin-resistant strain with the ponA1 gene increased penicillin resistance only when an additional locus, penC, was already present. These data demonstrate that a penicillin-susceptible strain must acquire five resistance genes, penA, mtr, penB, penC, and ponA1, to reach the level of resistance of a high-level, chromosomally mediated penicillin-resistant strain such as FA6140.

The mechanism of the emergence and propagation of the ponA1 mutation in resistant strains of N. gonorrhoeae is in marked contrast to that described for the penA gene encoding PBP 2. PenA is the first resistance gene in the stepwise transfer of resistance genes from a resistant donor strain to a susceptible recipient strain (11). Analysis of the penA genes from penicillin-resistant strains of N. gonorrhoeae has shown that the coding sequences contain blocks of DNA with high sequence divergence from penA genes of susceptible strains (28). Further studies showed that in the closely related Neisseria species N. meningitidis, the divergent blocks of DNA in altered penA genes arose by horizontal transfer of resistant penA genes from commensal strains such as N. flavescens or N. cinerea (3, 29, 30). These alterations lead to multiple amino acid changes in PBP 2, with the most important change being an amino acid insertion (Asp-345a) that lowers the rate of acylation of PBP 2 with penicillin four- to fivefold (4, 26).

In contrast to the high sequence divergence observed in altered forms of PBP 2, alteration of PBP 1 is due to a single base change in the ponA gene that results in mutation of Leu-421 to proline. We have sequenced the ponA genes from at least 10 different CMRNG isolates, and the only difference within the entire 2,400 bp of coding sequence is at codon 421. These isolates include one of the original CMRNG isolates from 1977, CDC77-124615, and resistant strains isolated nearly 20 years later in both Cincinnati and North Carolina. This mutation evidently arose in N. gonorrhoeae, since the ponA genes from several Neisseria commensal species show significant sequence divergence from the ponA gene of N. gonorrhoeae, with overall DNA sequence identities of 88% (N. lactamica; accession no. AF085689), 85% (N. cinerea; AF085340), and 73% (N. flavescens; AF087677) (Ropp and Nicholas, unpublished). Although it is possible that the mutation arose in N. meningitidis, whose ponA gene is 100% identical to its gonococcal homologue in the region of the mutation (24), it seems unlikely since high-level penicillin resistance in this species has not yet been noted.

Substitution of Leu-421 with proline causes a three- to fourfold decrease in the acylation rate of the protein when assessed with a variety of antibiotics. One of the most unusual aspects of this mutation is that the alteration is located on the amino-terminal side of the active-site serine residue. In other PBPs whose acylation rates have been altered by amino acid mutations, these mutations occur on the C-terminal side of the active-site serine residue. For example, mutations in altered forms of PBP 2 are located near two conserved active-site sequence motifs, the SXN triad and the KTG triad (28), and a similar location of alterations is observed in the PBPs from penicillin-resistant strains of Streptococcus pneumoniae (9, 16, 18). Because no high-resolution crystal structure of a class A PBP is available, it is not possible at this time to understand at a molecular level how this mutation causes a decrease in the rate of acylation. Although it is possible that Leu-421 is near the active site, mutation to proline may simply impart a structural perturbation (due to the structural effects of proline) that is propagated from a distance and disturbs the active-site architecture. However, in the absence of structural information for PBP 1 or one of its homologues, the mechanism by which the mutation decreases acylation remains highly speculative. Because the fold decrease in k2/K' for each of the antibiotics tested were very similar, i.e., approximately three- to fourfold, it appears that the structural alteration is general in nature and not specific to penicillin. However, a more extensive analysis with additional antibiotics is necessary to confirm this hypothesis.

The original reports detailing the stepwise transformation of susceptible strains of N. gonorrhoeae by donor DNA from high-level penicillin-resistant strains noted that transformation of recipient strains to a level of penicillin resistance equivalent to that of the donor strain could not be achieved (8, 11). However, Dougherty was able to isolate transformants at low frequency for which the MICs of penicillin were equal to those observed with the donor strain by using high concentrations of DNA and a modified transformation protocol and by selecting at penicillin concentrations fourfold below the MIC (7). Given the low frequency of transformation, Dougherty concluded that possibly two genes were involved in high-level penicillin resistance. Our study confirms and extends this hypothesis. We have shown that acquisition of ponA1 increases the MIC only when the penC mutation is present. It is probable that Dougherty also isolated colonies containing spontaneous mutations in penC in his experiments, since well over half of the transformants isolated were cross resistant to tetracycline, even though no selection for tetracycline was carried out (7).

Despite multiple attempts, we could not obtain colonies containing the penC locus (with frequencies greater than those of no-DNA controls) by transforming PR100 with donor DNA from several high-level penicillin-resistant strains. Our inability to obtain penC transformants was not due to penC being a nontransformable locus, since we were able to transform PR100 to PR100 penC with PR102 DNA at reasonably high frequencies (i.e., {approx}10-5). This result indicates that the penC mutation is not present in the CMRNG isolates that we tested and suggests that a novel mechanism or perhaps a different genetic background is responsible for resistance in these strains. It is interesting that the penicillin MIC for FA6140 ponA-wt is 2 µg/ml, twofold higher than that of FA19 containing the first three resistance genes from FA6140 (PR100; FA19 penA4 mtr penB5). The fact that the MIC for FA6140 ponA-wt is higher than for PR100 implies that the novel mechanism or genetic background involved in high-level penicillin resistance in FA6140 increases the MIC of penicillin above that which can be accounted for by the penA4, mtr, and penB5 genes. This scenario is reminiscent of the effects of the penC locus.

Introduction of the ponA1 gene into FA19, FA19 penA4, and FA19 penA4 mtr penB5 (PR100) had no effect on the MIC of penicillin (Table 3), indicating that penicillin kills these strains by inhibition of PBP 2. These data also show that PBP 2 remains the killing target even after its rate of acylation is markedly reduced by alterations in its coding sequence (i.e., penA2). However, PBP 1* clearly has a role in clinically relevant high-level penicillin resistance, since replacing the ponA1 gene with the wild-type gene in FA6140 (Table 3) and three other CMRNG strains resulted in twofold decreases in the penicillin MICs for these strains. The fact that introduction of the ponA1 gene into PR102 (PR100 penC) increases the MIC of penicillin indicates that the penC gene must in some way decrease the rate of acylation of PBP 2 such that PBP 1 becomes the killing target. A similar argument can be made for CMRNG strains such as FA6140, although the modifying gene(s) in these strains is not penC but some other as yet unknown gene or genetic mechanism. It is tempting to speculate that in both cases the membrane surface is altered in such a way that the rate of acylation of PBP 2 within the cell division complex is decreased below that of PBP 1, allowing PBP 1* to increase the MIC of penicillin.

In conclusion, this study is the first to show unequivocally that PBP 1 is involved in penicillin resistance. However, transformation experiments demonstrate that a decrease in the rate of acylation of PBP 1 is still not sufficient to impart high-level penicillin resistance (MIC = 2 to 4 µg/ml) to an intermediate-level resistant strain, implicating the involvement of an additional resistance locus in the transformation of gonococci to high-level resistance. In our studies, that additional locus is penC; however, in several clinical isolates, the identity of this locus is not penC but an as yet unidentified locus. Studies are in progress to identify the penC locus and to define the mechanism by which it increases both penicillin and tetracycline resistance.


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ACKNOWLEDGMENTS
 
This work was supported by grant AI-36901 from the National Institutes of Health.

We gratefully acknowledge the help and advice of Janne Cannon and Joanne Demsey, Janne Cannon for comments on the manuscript, and Marcia Hobbs for helping with serotyping the resistant strains.


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FOOTNOTES
 
* Corresponding author. Mailing address: University of North Carolina at Chapel Hill, Department of Pharmacology, CB# 7365 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7365. Phone: (919) 966-6547. Fax: (919) 966-5640. E-mail: nicholas{at}med.unc.edu. Back


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Antimicrobial Agents and Chemotherapy, March 2002, p. 769-777, Vol. 46, No. 3
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.3.769-777.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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