Encoding Dihydropteroate Synthase Research Articles

In vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli in the mammalian gut.

The widespread sulfonamide resistance genes sul1, sul2, and sul3 in food and gut bacteria have attracted considerable attention. In this study, we assessed the in vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli, using a murine model. High fitness costs were incurred for sul1 and sul3 gene-dependent E. coli strains in vivo. A fitness advantage was found in three of the eight mice after intragastric administration of sul2 gene-dependent E. coli strains. We isolated three compensatory mutant strains (CMSs) independently from three mice that outcompeted the parent strain P2 in vivo. Whole-genome sequencing revealed seven identical single nucleotide polymorphism (SNP) mutations in the three CMSs compared with strain P2, an additional SNP mutation in strain S2-2, and two additional SNP mutations in strain S2-3. Furthermore, tandem mass tag-based quantitative proteomic analysis revealed abundant differentially expressed proteins (DEPs) in the CMSs compared with P2. Of these, seven key fitness-related DEPs distributed in two-component systems, galactose and tryptophan metabolism pathways, were verified using parallel reaction monitoring analysis. The DEPs in the CMSs influenced bacterial motility, environmental stress tolerance, colonization ability, carbohydrate utilization, cell morphology maintenance, and chemotaxis to restore fitness costs and adapt to the mammalian gut environment.IMPORTANCESulfonamides are traditional synthetic antimicrobial agents used in clinical and veterinary medical settings. Their long-term excessive overuse has resulted in widespread microbial resistance, limiting their application for medical interventions. Resistance to sulfonamides is primarily conferred by the alternative genes sul1, sul2, and sul3 encoding dihydropteroate synthase in bacteria. Studying the potential fitness cost of these sul genes is crucial for understanding the evolution and transmission of sulfonamide-resistant bacteria. In vitro studies have been conducted on the fitness cost of sul genes in bacteria. In this study, we provide critical insights into bacterial adaptation and transmission using an in vivo approach.

Isolation, identification and whole-genome analysis of an Achromobacter strain with a novel sulfamethazine resistance gene and sulfamethazine degradation gene cluster

A sulfamethazine (SM2) degrading strain, Achromobacter mucicolens JD417, was isolated from sulfonamide-contaminated sludge using gradient acclimation. Optimal SM2 degradation conditions were pH 7, 36 °C, and 5 % inoculum, achieving a theoretical maximum degradation rate of 48 % at 50 ppm SM2. Cell growth followed the Haldane equation across different SM2 concentrations. Whole-genome sequencing of the strain revealed novel functional annotations, including a sulfonamide resistance gene (sul4) encoding dihydropteroate synthase, two flavin-dependent monooxygenase genes (sadA and sadB) crucial for SM2 degradation, and unique genomic islands related to metabolism, pathogenicity, and resistance. Comparative genomics analysis showed good collinearity and homology with other Achromobacter species exhibiting organics resistance or degradation capabilities. This study reveals the novel molecular resistance and degradation mechanisms and genetic evolution of an SM2-degrading strain, providing insights into the bioremediation of sulfonamide-contaminated environments.

Genetic characterization of co-trimoxazole non-susceptible Streptococcus pneumoniae isolates from Indonesia.

We investigated the genetic variation of folA and folP genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes amongst trimethoprim/sulfamethoxazole (co-trimoxazole) non-susceptible Streptococcus pneumoniae isolated from the Indonesian population. Archived S. pneumoniae isolates were screened for the presence and analysis of folA and folP genes using the polymerase chain reaction sequencing method. We found that 80 % of co-trimoxazole non-susceptible isolates (n=30/39) showed a 6 bp insertion in the sulphonamide-binding site of DHPS. The Asp-92-Ala and Ile-100-Leu substitutions were more common on DHFR (42 %; 22/53) followed by the Asp-92–Ala, Glu-94–Asp and Ile-100–Leu substitutions (32 %; 17/53). The combination of the Ile-100–Leu substitution at the DHFR region and the 6 bp insertion was the most dominant combination among isolates having both folA and folP genes.

Whole genome global insight of antibiotic resistance gene repertoire and virulome of high - risk multidrug-resistant Uropathogenic Escherichiacoli

Elucidation of genetic determinants via whole genome sequence (WGS) analyses can help understand the high risk multidrug-resistant (MDR) Uropathogenic Escherichia coli (UPEC) associated with urinary tract infections (UTI) and its evasion strategies from treatment. We investigated the WGS of 30 UPEC strains from UTI samples across the world (2016–2019) and found 25 UPEC strains carrying 2–23 antibiotic resistance genes (ARGs) scattered across 1–3 plasmids per strain. Different ARGs (blaTEM, blaCTXM, blaNDM, blaOXA, blaCMY) encoding extended-spectrum beta-lactamases (TEM, CTXM, CMY) and carbapenemases (NDM, OXA) were found in 24/30, ARGs encoding aminoglycoside modifying enzymes (AAC, APH, AAD) variants in 23/30, trimethoprim ARGs (dfrA17, dfrA12, dfrA5, dfrB4 variants) encoding dihydrofolate reductase in 19/30 and sulfonamide ARGs (sul1, sul2, sul3) encoding dihydropteroate synthase and macrolide ARGs (mph1) encoding macrolide 2’ phosphotransferase in 15/30 UPEC strains. Collectively the ARGs were distributed in different combinations in 40 plasmids across UPEC strains with 20 plasmids displaying co-occurrence of multiple ARGs conferring resistance to beta lactam, aminoglycoside, sulfonamide, trimethoprim and macrolide antibiotics. These resistance plasmids belonged to seven incompatibility groups (IncF, IncI, IncC, IncH, IncN, IncB and Col), with IncFI and IncFII being the predominant resistance plasmids. Additionally, we observed co-occurrence of specific mutation pattern in quinolone resistance determining region (QRDR) viz., DNA gyrase (gyrA: S83L, D87N), and topoisomerase IV (parC: S80I, E84V; parE: I529L) in 18/30 strains. The strains also harbored diverse virulence genes, such as fimH, gad, iss, iha, ireA, iroN, cnf1 and san. Multilocus sequence typing (MLST) reconfirmed ST131(n = 10) as the predominant global high-risk clonal strain causing UTI. In summary, our findings contribute to better understand the plasmid mediated ARGs and its encoded enzymes that may contribute in antibiotic inactivation/modification or alteration in the antibiotic target site in high risk MDR hypervirulent UPEC strains causing UTI. The study reinforces the need to characterize and design appropriate inhibitors to counterattack different enzymes and devise strategies to curtail resistance plasmid.

High Frequency Mutations in pfdhfr and pfdhps of Plasmodium falciparum in Response to Sulfadoxine-Pyrimethamine: A Cross-Sectional Survey in Returning Chinese Migrants From Africa.

BackgroundSulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment in Africa against Plasmodium falciparum infection. However, increasing SP resistance (SPR) of P. falciparum affects the therapeutic efficacy of SP, and pfdhfr (encoding dihydrofolate reductase) and pfdhps (encoding dihydropteroate synthase) genes are widely used as molecular markers for SPR surveillance. In the present study, we analyzed single nucleotide polymorphisms (SNPs) of pfdhfr and pfdhps in P. falciparum isolated from infected Chinese migrant workers returning from Africa.MethodsIn total, 159 blood samples from P. falciparum-infected workers who had returned from Africa to Anhui, Shangdong, and Guangxi provinces were successfully detected and analyzed from 2017 to 2019. The SNPs in pfdhfr and pfdhps were analyzed using nested PCR. The genotypes and linkage disequilibrium (LD) were analyzed using Haploview.ResultsHigh frequencies of the Asn51Ile (N51I), Cys59Arg(C59R), and Ser108Asn(S108N) mutant alleles were observed, with mutation frequencies of 97.60, 87.43, and 97.01% in pfdhfr, respectively. A triple mutation (IRN) in pfdhfr was the most prevalent haplotype (86.83%). Six point mutations were detected in pfdhps DNA fragment, Ile431Val (I431V), Ser436Ala (S436A), Ala437Gly (A437G), Lys540Glu(K540E), Ala581Gly(A581G), Ala613Ser(A613S). The pfdhps K540E (27.67%) was the most predominant allele, followed by S436A (27.04%), and a single mutant haplotype (SGKAA; 62.66%) was predominant in pfdhps. In total, 5 haplotypes of the pfdhfr gene and 13 haplotypes of the pfdhps gene were identified. A total of 130 isolates with 12 unique haplotypes were found in the pfdhfr-pfdhps combined haplotypes, most of them (n = 85, 65.38%) carried quadruple allele combinations (CIRNI-SGKAA).ConclusionA high prevalence of point mutations in the pfdhfr and pfdhps genes of P. falciparum isolates was detected among Chinese migrant workers returning from Africa. Therefore, continuous in vitro molecular monitoring of Sulfadoxine-Pyrimethemine combined in vivo therapeutic monitoring of artemisinin combination therapy (ACT) efficacy and additional control efforts among migrant workers are urgently needed.

Sulfonamides removal under different redox conditions and microbial response to sulfonamides stress during riverbank filtration: A laboratory column study

Riverbank filtration (RBF) as a barrier of pathogenic microorganisms and organic micropollutants recently has been proven capable of removing sulfonamides. However, the study about the effect of redox conditions on biodegradation of common and persistent sulfonamides in RBF is limited and the response of microbial communities to sulfonamides stress during RBF is unknown. In this study, two column set-ups (with residence time 5 days and 11 days respectively), simulating different redox conditions of riverbank filtration systems, were operated for seven months to investigate 1) the long-term effect of redox conditions on ng∙L−1 level sulfonamides (sulfapyridine, sulfadiazine, sulfamethoxazole, sulfamethazine, sulfaquinoxaline) removal, and 2) the microbial community evolution represented by the phylogenetic and metabolic function shift under non-lethal selective pressures of sulfonamides. The results showed that sulfonamides were more degradable under anoxic conditions than oxic and suboxic conditions. In the sulfonamides stressed community, the phylogenetic diversity increased slightly. Relative abundance of an intrinsic sulfonamides resistant bacteria Bacillus spp. increased, suggesting that sulfonamide resistance developed in specific bacteria under sulfonamides contamination pressure in RBF systems. At the same time, an activated transport function in the stressed microbial community was noticed. The predicted relative abundance of gene folP, which encodes dihydropteroate synthase, also increased significantly, indicating a detoxification mechanism and sulfonamides resistance potential under non-lethal selective pressures of sulfonamides in RBF systems.

High Prevalence of Dihydropteroate Synthase Mutations in Pneumocystis jirovecii Isolated from Patients with Pneumocystis Pneumonia in South Africa

Pneumocystis jirovecii pneumonia (PCP) is an important cause of morbidity and mortality in immunocompromised patients. Sulfa-containing drugs are used for the treatment and prophylaxis of PCP. Mutations in the P. jirovecii fas gene, which encodes dihydropteroate synthase (DHPS), are associated with prior exposure to sulfa drugs, and their appearance suggests the emergence of variants with reduced sulfa susceptibility. The present study examined the prevalence of DHPS mutations in P. jirovecii strains isolated from South African patients with PCP. P. jirovecii infection was investigated by immunofluorescence microscopy and quantitative real-time PCR with respiratory specimens from 712 patients (93% of whom were >15 years of age) with suspected PCP consecutively received for the detection of P. jirovecii over 1 year. PCR amplification and sequencing of the DHPS fas gene was attempted with DNA from the P. jirovecii-positive samples. P. jirovecii infection was confirmed by immunofluorescence microscopy in 168/712 (24%) of the patients. Carriage of the fungus was revealed by real-time PCR in 17% of the patients with negative microscopy results. The P. jirovecii fas gene was successfully amplified from specimens from 151 patients and sequenced. Mutations resulting in the Thr55Ala and/or Pro57Ser amino acid substitution were detected in P. jirovecii strains from 85/151 (56%) patients. The high frequency of PCP episodes with P. jirovecii harboring DHPS mutations in South Africa indicates that populations of this fungus are evolving under the considerable selective pressure exerted by sulfa-containing antibiotics. These results, similar to previous observations of sulfa drug resistance in bacterial populations, underscore the importance of the rational use of sulfa medications either prophylactically against PCP or for the treatment of other infections.

Dihydropteroate synthase gene mutations in Pneumocystis jiroveci strains isolated from immunocompromised patients

The emergence of Pneumocystis jiroveci drug resistance has been suggested recently by the mutations in the gene encoding dihydropteroate synthase ( DHPS). The aim of the present study was to determine the prevalence of DHPS mutations in P. jiroveci strains isolates from bronchoalveolar lavages (BAL) and sputum samples of 21 immunocompromised patients. We used the touchdown-PCR for amplification of DHPS gene and the restriction fragment length polymorphism (RFLP) technique for discrimination of wild and mutant DHPS genotypes. The DHPS amplification was positive in 17 patients (81%). The association of wild genotype and mutant genotype was detected in two patients after the enzymatic digestion of the PCR products by AccI and HaeIII. No mutations in the DHPS gene were seen in 15 patients. In addition, no variation was observed in DHPS genotypes detected in the repeated specimens (BAL and sputum) from some patients. The touchdown PCR-RFLP technique is a simple and rapid method for revelation of DHPS gene mutations in P. jiroveci strains. It could be advantageously used in clinical laboratory to control the prevalence of mutations associated with sulfa resistance.

Resistance to trimethoprim/sulfamethoxazole and Tropheryma whipplei

Whipple's disease (WD) is a chronic infection caused by Tropheryma whipplei. A 1-year treatment of oral trimethoprim/sulfamethoxazole (SXT) is commonly used. Advances in the culture of T. whipplei have allowed for full genome sequencing and antibiotic susceptibility testing, which has demonstrated resistance of T. whipplei to trimethoprim. Several mutations in the folP gene that encodes dihydropteroate synthase, the target of sulphonamides, has been reported for one patient with clinically acquired resistance to SXT. Here we report three new patients who experienced clinically acquired resistance to SXT during treatment and one patient with biological failure. Sixty-two folP sequences from DNA samples of 59 WD patients were also obtained. Among the detected amino acid changes, two positions (N4S and S234F) significantly predicted secondary sulfamethoxazole failure (four of five). We suggest that these mutations should be detected at the time of WD diagnosis by sequencing folP in order to avoid sulfamethoxazole monotherapy.

Genome Comparison Analysis of Molecular Mechanisms of Resistance to Antibiotics in the Rickettsia Genus

In this study we describe molecular mechanisms of resistance to several classes of antibiotics within drug targets by in silico genome comparisons for bacteria of the genus Rickettsia. Apart from the mutations in the rpoB gene in naturally rifampin-resistant Rickettsia species previously reported by our team, we found that typhus group (TG) rickettsiae had a triple amino acid difference in the highly conserved region of the L22 ribosomal protein as compared to the spotted fever group rickettsiae (SFG), which could explain the natural resistance of SFG rickettsia to erythromycin. We found also that the genome of R. conorii contains an aminoglycoside 3'-phosphotransferase. Finally, either folA gene (encoding dihydrofolate reductase) and/or folP gene (encoding dihydropteroate synthase) was missing in the genome of rickettsial strains explaining the natural resistance to cotrimoxazole. Finally, multiple genes encoding for pump efflux were found especially in the genome of R. conorii that could be involved in resistance to antibiotics. Five specific ORFs related to antibiotic resistance have been identified in the genome of R. felis including a streptomycin resistance protein homologue, a class C beta-lactamase, a class D beta-lactamase, a penicillin acylase homologue, and an ABC-type multidrug transporter system. For the first time, using this approach, an experimental beta-lactamase activity has been shown for this bacterium. We believe that whole genome sequence analysis may help to predict several phenotypic characters, in particular resistance to antibiotics for obligate intracellular bacteria.

Mutations in folP Associated with Elevated Sulfonamide MICs for Neisseria meningitidis Clinical Isolates from Five Continents

Sulfonamide resistance in meningococci is associated with mutations in the chromosomal gene folP, which encodes dihydropteroate synthase. Several mutations associated with resistance have been previously described, including amino acid substitutions at codons 31 and 194, a glycine-serine insertion at codons 195 and 196, and, recently, an additional mutation at nucleotide 682 (C682A). In this study, sulfisoxazole MICs were determined for 424 geographically diverse clinical isolates of Neisseria meningitidis, including all major subtypes. A subset of 134 isolates with MICs ranging from 0.5 to >64 microg/ml were assayed for the C682A mutation by real-time PCR, and 25 isolates were selected for folP gene sequencing. All isolates for which the sulfisoxazole MIC was >/=8 possessed the C682A mutation by real-time PCR or folP sequencing, and 34 of 35 isolates with a MIC of </=2 lacked this mutation. Of 16 sequenced isolates for which the sulfisoxazole MIC was >/=4, 15 possessed previously described mutations, including 10 at codon 31, 1 at codon 194, and 4 with the 2-amino-acid insertion codons 195 and 196; all 16 possessed the C682A mutation. The C682A mutation predicted elevated sulfonamides MICs for a large number of geographically diverse clinical isolates of meningococci. Detection of this mutation by real-time PCR or other methods may allow more wide-scale detection of meningococcal isolates with for which the sulfonamide MICs are elevated without resorting to multiple assays or folP gene sequencing, providing a simple, high-throughput screening method for use in public health and epidemiologic settings.

Prevalence of dihydropteroate synthase mutants in HIV-infected South African children with Pneumocystis jiroveci pneumonia.

Pneumocystis jiroveci (formerly Pneumocystis carinii) pneumonia (PCP) is a major cause of mortality in human immunodeficiency virus (HIV)-infected infants in Africa, but the prevalence of mutations in the gene encoding dihydropteroate synthase (DHPS) in isolates from Africa has not been reported. This study investigated the prevalence of DHPS mutations in P. jiroveci isolates from South African HIV-infected children with PCP by amplifying DNA using 2 different polymerase chain reactions. P. jiroveci DNA from 30 respiratory specimens was amplified; 26 specimens (86.7%) contained wild-type DHPS alleles. Of the 4 samples (13.3%) with DHPS mutations, 2 contained a homogenous population with single DHPS mutations, 1 contained a homogenous population with 2 DHPS mutations, and the fourth contained a heterogenous population of organisms with both wild-type and single-mutant DHPS genotypes. Only 1 child was receiving trimethoprim-sulphamethoxazole (TMP-SMZ) prophylaxis; this patient was infected with wild-type P. jiroveci. The mortality rate (overall, 20 [66.7%] of 30 children) was not significantly different between children infected with wild-type P. jiroveci (17 [65.4%] of 26) and those infected with mutant strains (3 [75%] of 4; P=.8). DHPS mutations are uncommon in P. jiroveci isolates from South Africa. However, increasing use of TMP-SMZ prophylaxis may result in widespread development of mutations.

Rapid real-time PCR genotyping of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum.

The resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) is an emerging public health threat. Resistance to these drugs is associated with point mutations in the genes encoding dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). We describe here an assay using real-time PCR and sequence-specific probes that detects these mutations. Using DNA from plasmids, cultured strains, and clinical samples, real-time PCR could distinguish four DHPS polymorphisms (codons 437, 540, 581, and 613) and three DHFR polymorphisms (codons 51, 59, and 108). This assay is rapid and sensitive, with a detection limit of 10 copies in most cases. This assay is amenable to large-scale studies of drug resistance.

Sulfonamide Resistance in Haemophilus influenzae Mediated by Acquisition of sul2 or a Short Insertion in Chromosomal folP

Determinants of sulfonamide resistance were investigated in clinical isolates of Haemophilus influenzae from the United Kingdom and Kenya. The mechanism of sulfonamide resistance in H. influenzae has not previously been reported. Eight isolates requiring at least 1,024 microg of sulfamethoxazole per ml for inhibition carried the sul2 gene, a common mediator of acquired sulfonamide resistance in enteric bacteria. In other isolates with similarly high levels of resistance, the chromosomal gene encoding dihydropteroate synthase, folP, was found to carry an insertion of 15 bp together with other missense mutations relative to folP of H. influenzae strain Rd RM118 (MIC, 8 microg/ml); the folP sequence was identical in all seven such isolates investigated, although they represented three different strains by restriction pattern analysis. The 15-bp insertion was absent in isolates inhibited by sulfamethoxazole at 2 to 64 microg/ml (although these exhibited considerable divergence in folP sequence) and in highly resistant isolates carrying sul2. Transformation with a 599-bp fragment of folP containing the insertion but no other differences conferred high-level resistance on a recipient strain, confirming the role of the insertion. Other amino acid substitutions in dihydropteroate synthase may modulate the level of sulfonamide inhibition in susceptible isolates and those with more moderate levels of resistance. The two mechanisms of resistance, mediated by sul2 and modified folP, were detected in isolates from both the United Kingdom and Kenya.

Sequence variations in the genes encoding dihydropteroate synthase and dihydrofolate reductase and clinical response to sulfadoxine-pyrimethamine in patients with acute uncomplicated falciparum malaria.

Mutations in dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) are associated with in vitro resistance to sulfadoxine and pyrimethamine, respectively. The response of 75 patients to sulfadoxine-pyrimethamine was determined, and the genes of the corresponding Plasmodium falciparum isolates were sequenced. Of 12 different unmixed allelic combinations, the triple dhfr mutation Asn-108/Arg-59/Ile-51 was observed in all patients responding with early treatment failure. Some, but not all, patients with an adequate clinical response also harbored isolates with the triple dhfr mutation. Higher initial parasitemia and fever distinguished these 2 patient groups. The dhps genotype apparently had no influence on the clinical outcome. The other dhfr alleles with 1 or 2 mutations and the wild-type allele were found in patients with an adequate clinical response. The triple dhfr mutation is one of the genetic determinants associated with in vivo resistance to sulfadoxine-pyrimethamine.

Dapsone resistance inMycobacterium leprae

The folP1 gene of Mycobacterium leprae, which encodes dihydropteroate synthase (DHPS), was studied for the presence of mutations associated with resistance to dapsone (DDS). When the folP1 of several DDS-resistant clinical isolates of M. leprae were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting isoleucine for threonine in DHPS-1, and a second mutation occurred in codon 55, substituting arginine for proline. DNA sequencing of strains of M. leprae resistant to 0.01 g% DDS in the mouse diet revealed that 13 of 14 strains contained either the 53 or 55 folP1 mutation. None of the susceptible strains and only one of five strains resistant to 0.001 g% DDS revealed a mutation in folP1, suggesting that only high-level DDS resistance is associated with the mutations identified in folP1. Development and application of simple molecular tests to assess drug-related mutations in M. leprae could establish current levels of drug resistance in leprosy as a reference point for future monitoring of drug resistance at the global level.

Novel expansions of the gene encoding dihydropteroate synthase in trimethoprim-sulfamethoxazole-resistant Streptococcus pneumoniae.

A study of eight sulfonamide-resistant clinical isolates of Streptococcus pneumoniae revealed chromosomal mutations within the gene encoding dihydropteroate synthase that play a role in conferring resistance to sulfamethoxazole. The presence of the suld mutation, found previously only in a laboratory mutant, was shown to occur in three of the wild-type clinical isolates. The duplication of Ser(61), the other previously defined mutation in the dihydropteroate synthase gene of S. pneumoniae, was observed in only one of the isolates characterized. We report two previously unidentified amino acid alterations, namely, a duplication of Arg(58) and Pro(59) and an insertion of an arginine residue between Gly(60) and Ser(61) in trimethoprim-sulfamethoxazole-resistant strains. The significance of these mutations was confirmed by site-directed mutagenesis and by the transformation of a susceptible strain of S. pneumoniae to sulfamethoxazole resistance. Two resistant isolates did not contain any mutations within the gene encoding dihydropteroate synthase. The results presented suggest the independent generation of resistant mutations among South African clinical isolates. It is also proposed that the mechanism of sulfonamide resistance in S. pneumoniae involves the expansion of a specific region within dihydropteroate synthase, which probably forms part of the sulfonamide binding site.

Mechanism of sulfonamide resistance in clinical isolates of Streptococcus pneumoniae

The genetic basis of sulfonamide resistance in six clinical isolates of Streptococcus pneumoniae was demonstrated to be 3- or 6-bp duplications within sulA, the chromosomal gene encoding dihydropteroate synthase. The duplications all result in repetition of one or two amino acids in the region from Arg58 to Tyr63, close to but distinct from the sul-d mutation, a duplication previously reported in a resistant laboratory strain (P. Lopez, M. Espinosa, B. Greenberg, and S. A. Lacks, J. Bacteriol. 169:4320-4326, 1987). Six sulfonamide-susceptible clinical isolates lacked such duplications. The role of the duplications in conferring sulfonamide resistance was confirmed by transforming 319- or 322-bp PCR fragments into the chromosome of a susceptible recipient. Two members of a clone of serotype 9V, one susceptible and one resistant to sulfonamide, which are highly related by other criteria, were shown to have sulA sequences that differ in 7.2% of nucleotides in addition to the duplication responsible for resistance. It is postulated that horizontal gene exchange has been involved in the acquisition (or loss) of resistance within this clone. However, five of the six resistant isolates have distinct duplications and other sequence polymorphisms, suggesting that resistance has arisen independently on many occasions.

An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene

McDonald and Burke (J. Bacteriol. 149:391-394, 1982) previously cloned a sulfanilamide-resistance gene, sul, residing on a 4.9-kb segment of Bacillus subtilis chromosomal DNA, into plasmid pUB110. In this study we determined the nucleotide sequence of the entire 4.9-kb fragment. Genes identified on the fragment include pab, trpG, pabC, sul, one complete unidentified open reading frame, and one incomplete unidentified open reading frame. The first three of these genes, pab, trpG, and pabC, are required for synthesis of p-aminobenzoic acid. The trpG gene encodes an amphibolic glutamine amidotransferase required for synthesis of both p-aminobenzoate and anthranilate, the latter an intermediate in the tryptophan biosynthetic pathway. The pabC gene may encode a B. subtilis analog of enzyme X, an enzyme needed for p-aminobenzoate synthesis in Escherichia coli. The sul gene probably encodes dihydropteroate synthase, the enzyme responsible for formation of 7,8-dihydropteroate, the immediate precursor of folic acid. All six of the cloned genes are arranged in a single operon. Since all four of the identified genes are needed for folate biosynthesis, we refer to this operon as a folic acid operon. Expression of the trpG gene is known to be negatively controlled by tryptophan. We propose that this regulation is at the level of translation. This hypothesis is supported by the finding of an apparent Mtr-binding site which overlaps with the trpG ribosome-binding site.

Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme.

A chromosomal gene of Streptococcus pneumoniae carrying a spontaneous mutation to sulfonamide resistance was identified. Comparison of its DNA sequence with the wild-type sequence showed that the mutation, sul-d, consisted of an insert of 6 base pairs, a repeat of an adjacent 6-base-pair segment. The gene encoded a 34-kilodalton polypeptide, SulA, which as a dimer or trimer constituted the enzyme dihydropteroate synthase. This was shown by enzyme activity measurements, expression in minicells of Bacillus subtilis, and the amino-terminal sequence of the polypeptide product. Subcloning of the gene in an Escherichia coli expression vector allowed purification of the enzyme to 80% homogeneity in a single step and at high yield. Although a deleted plasmid, pLS83, produced the mutant dihydropteroate synthase, it did not confer sulfonamide resistance in vivo. It is suggested that the SulA polypeptide is also a component of an enzyme that acts in another step of folate biosynthesis and that this step is inhibited in vivo by either free or conjugated sulfonamides.