Cloning and nucleotide sequences of the topoisomerase IV parC and parE genes of Mycoplasma hominis. - Related Documents




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45601.0000Cloning and nucleotide sequences of the topoisomerase IV parC and parE genes of Mycoplasma hominis. The topoisomerase IV parC and parE genes from the wall-less organism Mycoplasma hominis PG21 were cloned and sequenced. The coupled genes are located far from the DNA gyrase genes gyrA and gyrB. They encode proteins of 639 and 866 amino acids, respectively. As expected, the encoded ParE and ParC proteins exhibit higher homologies with the topoisomerase IV subunits of the gram-positive bacteria Staphylococcus aureus and Streptococcus pneumoniae than with their Escherichia coli counterparts. The conserved regions include the Tyr residue of the active site and the region involved in quinolone resistance (quinolone resistance-determining region [QRDR]) in ParC and the ATP-binding site and the QRDR in ParE.19989687401
45710.9998Molecular characterization of the genes encoding DNA gyrase and topoisomerase IV of Listeria monocytogenes. The genes encoding subunits A and B of DNA gyrase and subunits C and E of topoisomerase IV of Listeria monocytogenes, gyrA, gyrB, parC and parE, respectively, were cloned and sequenced. Compared with the sequences of quinolone-susceptible bacteria, such as Escherichia coli and Bacillus subtilis, the quinolone resistance-determining region (QRDR) of DNA gyrase subunit A was altered; the deduced amino acid sequences revealed the substitutions Ser-84-->Thr and Asp/Glu-88-->Phe, two amino acid variations at hot spots, commonly associated with resistance to quinolones. No relevant divergences from QRDR consensus sequences were observed in GyrB or both topoisomerase IV subunits. Thus, it could be argued that the amino acid substitutions in GyrA would explain the intrinsic resistance of L. monocytogenes to nalidixic acid. In order to analyse the actual role of the GyrA alterations, a plasmid-encoded gyrA allele was mutated and transformed into L. monocytogenes. However, these heterodiploid strains were not affected in their resistance to nalidixic acid. The effects of the mutant plasmids on ciprofloxacin and sparfloxacin susceptibility were only modest.200212039883
625620.9996Conjugation between quinolone-susceptible bacteria can generate mutations in the quinolone resistance-determining region, inducing quinolone resistance. Quinolones are an important group of antibacterial agents that can inhibit DNA gyrase and topoisomerase IV activity. DNA gyrase is responsible for maintaining bacteria in a negatively supercoiled state, being composed of subunits A and B. Topoisomerase IV is a homologue of DNA gyrase and consists of two subunits codified by the parC and parE genes. Mutations in gyrA and gyrB of DNA gyrase may confer resistance to quinolones, and the majority of resistant strains show mutations between positions 67 and 106 of gyrA, a region denoted the quinolone resistance-determining region (QRDR). The most frequent substitutions occur at positions 83 and 87, but little is known about the mechanisms promoting appearance of mutations in the QRDR. The present study proposes that some mutations in the QRDR could be generated as a result of the natural mechanism of conjugation between bacteria in their natural habitat. This event was observed following conjugation in vitro of two different isolates of quinolone-susceptible Pseudomonas aeruginosa, which transferred plasmids of different molecular weights to a recipient strain of Escherichia coli (HB101), also quinolone-susceptible, generating two different transconjugants that presented mutations in DNA gyrase and acquisition of resistance to all quinolones tested.201525262036
597930.9995Mutations in gyrA, gyrB, parC, and parE in quinolone-resistant strains of Neisseria gonorrhoeae. Mutations in the genes for the subunits GyrA and ParC of the target enzymes DNA gyrase and topoisomerase IV are important mechanisms of resistance in quinolone-resistant bacteria, including Neisseria gonorrhoeae. The target enzymes also consist of the subunits GyrB and ParE, respectively, though their role in quinolone-resistance has not been fully investigated. We sequenced the quinolone-resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE in 25 ciprofloxacin-resistant strains from Bangladesh (MIC 4-->32 mg/l) and 5 susceptible strains of N. gonorrhoeae. All the resistant strains had three or four mutations. Two of these were at positions 91 and 95 of gyrA. Fourteen strains had an additional mutation in parC at position 91, and 17 strains had an additional mutation in parE in position 439. No alterations were found in gyrB. The five susceptible strains had identical DNA sequences. Data indicate that the mutations detected in the QRDR of gyrA and parC may be important in the development of quinolone resistance. According to transformation experiments we assume that the alteration in parE is not related to a high degree of quinolone resistance. There was no correlation between ciprofloxacin MICs and pattern or number of mutations in the target genes.200212529019
625840.9995Alterations in GyrA and ParC associated with fluoroquinolone resistance in Enterococcus faecium. High-level quinolone resistance in Enterococcus faecium was associated with mutations in both gyrA and parC genes in 10 of 11 resistant strains. On low-level resistant strain without such mutations may instead possess an efflux mechanism or alterations in the other subunits of the gyrase or topoisomerase IV genes. These findings are similar to those for other gram-positive bacteria, such as Enterococcus faecalis.199910103206
598150.9995Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus. A 4.2-kb DNA fragment conferring quinolone resistance was cloned from a quinolone-resistant clinical isolate of Staphylococcus aureus and was shown to possess a part of the grlB gene and a mutated grlA gene. S-80-->F and E-84-->K mutations in the grlA gene product were responsible for the quinolone resistance. The mutated grlA genes responsible for quinolone resistance were dominant over the wild-type allele, irrespective of gene dosage in a transformation experiment with the grlA gene alone. However, dominance by mutated grlA genes depended on gene dosage when bacteria were transformed with the grlA and grlB genes in combination. Quinolone-resistant gyrA mutants were easily isolated from a strain, S. aureus RN4220, carrying a plasmid with the mutated grlA gene, though this was not the case for other S. aureus strains lacking the plasmid. The elimination of this plasmid from such quinolone-resistant gyrA mutants resulted in marked increases in quinolone susceptibility. These results suggest that both DNA gyrase and DNA topoisomerase IV may be targets of quinolones and that the quinolone susceptibility of organisms may be determined by which of these enzymes is most quinolone sensitive.19968723458
598460.9995First characterization of fluoroquinolone resistance in Streptococcus suis. We have identified and sequenced the genes encoding the quinolone-resistance determining region (QRDR) of ParC and GyrA in fluoroquinolone-susceptible and -resistant Streptococcus suis clinical isolates. Resistance is the consequence of single point mutations in the QRDRs of ParC and GyrA and is not due to clonal spread of resistant strains or horizontal gene transfer with other bacteria.200717116660
305270.9994Expression of antibiotic resistance genes from Escherichia coli in Bacillus subtilis. Bifunctional recombinant plasmids were constructed, comprised of the E. coli vectors pBR322, pBR325 and pACYC184 and different plasmids from Gram-positive bacteria, e.g. pBSU161-1 of B. subtilis and pUB110 and pC221 of S. aureus. The beta-lactamase (bla) gene and the chloramphenicol acetyltransferase (cat) gene from the E. coli plasmids were not transcribed and therefore not expressed in B. subtilis. However, tetracycline resistance from the E. coli plasmids was expressed in B. subtilis. Transcription of the tetracycline resistance gene(s) started in B. subtilis at or near the original E. coli promoter, the sequence of which is almost identical with the sequence recognized by sigma 55 of B. subtilis RNA polymerase.19836410152
625780.9994Mechanism of action of and resistance to quinolones. Fluoroquinolones are an important class of wide-spectrum antibacterial agents. The first quinolone described was nalidixic acid, which showed a narrow spectrum of activity. The evolution of quinolones to more potent molecules was based on changes at positions 1, 6, 7 and 8 of the chemical structure of nalidixic acid. Quinolones inhibit DNA gyrase and topoisomerase IV activities, two enzymes essential for bacteria viability. The acquisition of quinolone resistance is frequently related to (i) chromosomal mutations such as those in the genes encoding the A and B subunits of the protein targets (gyrA, gyrB, parC and parE), or mutations causing reduced drug accumulation, either by a decreased uptake or by an increased efflux, and (ii) quinolone resistance genes associated with plasmids have been also described, i.e. the qnr gene that encodes a pentapeptide, which blocks the action of quinolones on the DNA gyrase and topoisomerase IV; the aac(6')-Ib-cr gene that encodes an acetylase that modifies the amino group of the piperazin ring of the fluoroquinolones and efflux pump encoded by the qepA gene that decreases intracellular drug levels. These plasmid-mediated mechanisms of resistance confer low levels of resistance but provide a favourable background in which selection of additional chromosomally encoded quinolone resistance mechanisms can occur.200921261881
44390.9994Deletion mutant analysis of the Staphylococcus aureus plasmid pI258 mercury-resistance determinant. Deletion mutant analysis of the mercury-resistant determinant (mer operon) from the Staphylococcus aureus plasmid pI258 was used to verify the location of the merA and merB genes and to show the existence of mercuric ion transport gene(s). ORF5 was confirmed to be a transport gene and has an amino acid product sequence homologous to the merT gene products from several gram-negative bacteria and a Bacillus species. Deletion analysis established that inactivation of merA on a broad-spectrum mer resistance determinant resulted in a mercury-hypersensitive phenotype. Gene dosage had no apparent effect on the level of resistance conferred by the intact mer operon or on the expression of an inducible phenotype, except that when the intact pI258 mer operon was on a high copy number plasmid, uninduced cells possessed a volatilization rate that was at most only 3.5-fold less than that observed for induced cells. There was no need for mercury ion transport proteins for full resistance when the mer operon was expressed in a high copy number plasmid.19911954576
428100.9993Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. The streptococcal plasmid pMV158 and its derivative pLS1 are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLS1 were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLS1 doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLS1 revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 Mr protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 Mr protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLS1 was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin.19862438417
455110.9993An inducible tellurite-resistance operon in Proteus mirabilis. Tellurite resistance (Te(r)) is widespread in nature and it is shown here that the natural resistance of Proteus mirabilis to tellurite is due to a chromosomally located orthologue of plasmid-borne ter genes found in enteric bacteria. The P. mirabilis ter locus (terZABCDE) was identified in a screen of Tn5lacZ-generated mutants of which one contained an insertion in terC. The P. mirabilis terC mutant displayed increased susceptibility to tellurite (Te(s)) and complementation with terC carried on a multicopy plasmid restored high-level Te(r). Primer extension analysis revealed a single transcriptional start site upstream of terZ, but only with RNA harvested from bacteria grown in the presence of tellurite. Northern blotting and reverse transcriptase-PCR (RT-PCR) analyses confirmed that the ter operon was inducible by tellurite and to a lesser extent by oxidative stress inducers such as hydrogen peroxide and methyl viologen (paraquat). Direct and inverted repeat sequences were identified in the ter promoter region as well as motifs upstream of the -35 hexamer that resembled OxyR-binding sequences. Finally, the 390 bp intergenic promoter region located between orf3 and terZ showed no DNA sequence identity with any other published ter sequences, whereas terZABCDE genes exhibited 73-85 % DNA sequence identity. The ter operon was present in all clinical isolates of P. mirabilis and Proteus vulgaris tested and is inferred for Morganella and Providencia spp. based on screening for high level Te(r) and preliminary PCR analysis. Thus, a chromosomally located inducible tellurite resistance operon appears to be a common feature of the genus Proteus.200312724390
6188120.9993Quinolone mode of action. Physical studies have further defined interactions of quinolones with their principal target, DNA gyrase. The binding of quinolones to the DNA gyrase-DNA complex suggests 2 possible binding sites of differing affinities. Mutations in either the gyrase A gene (gyrA) or the gyrase B gene (gyrB) that affect quinolone susceptibility also affect drug binding, with resistance mutations causing decreased binding and hypersusceptibility mutations causing increased binding. Combinations of mutations in both GyrA and GyrB have further demonstrated the contribution of both subunits to the quinolone sensitivity of intact bacteria and purified DNA gyrase. A working model postulates initial binding of quinolones to proximate sites on GyrA and GyrB. This initial binding then produces conformational changes that expose additional binding sites, possibly involving DNA. Quinolones also inhibit the activities of Escherichia coli topoisomerase IV (encoded by the parC and parE genes), but at concentrations higher than those inhibiting DNA gyrase. The patterns of resistance mutations in gryA and parC suggest that topoisomerase IV may be a secondary drug target in E. coli and Neisseria gonorrhoeae. In contrast, in Staphylococcus aureus these patterns suggest that topoisomerase IV may be a primary target of quinolone action. Regulation of expression of membrane efflux transporters may contribute to quinolone susceptibility in both Gram-positive and Gram-negative bacteria. The substrate profile of the NorA efflux transporter of S. aureus correlates with the extent to which the activity of quinolone substrates is affected by overexpression of NorA. In addition, the Emr transporter of E. coli affects susceptibility to nalidixic acid, and the MexAB OprK transport system of Pseudomonas aeruginosa affects susceptibility to ciprofloxacin.(ABSTRACT TRUNCATED AT 250 WORDS)19958549276
441130.9993Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities. A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.19853994373
5963140.9993Expression of the mphB gene for macrolide 2'-phosphotransferase II from Escherichia coli in Staphylococcus aureus. The genes mphA and mphB encode macrolide 2'-phosphotransferases I and II, respectively, and they confer resistance to macrolide antibiotics in Escherichia coli. To study the expression of these genes in Gram-positive bacteria, we constructed recombinant plasmids that consisted of an mph gene and the pUB110 vector in Bacillus subtilis. When these plasmids were introduced into Staphylococcus aureus, the mphB gene was active and macrolide 2'-phosphotransferase II was produced. The gene endowed S. aureus with high-level resistance to spiramycin, a macrolide antibiotic with a 16-membered ring. Moreover, transcription of the mphB gene in S. aureus began at the promoter that was active in E. coli.19989503630
5851150.9993Arsenic resistance determinants from environmental bacteria. Arsenic resistance determinants from 42 environmental bacterial isolates (32 Gram negative) were analyzed by DNA: DNA hybridization using probes derived from Escherichia coli and Staphylococcus plasmid or chromosomal arsenic resistance (ars) genes. In colony hybridization assays, 11 and 1 Gram negative strains hybridized with the E. coli chromosome and plasmid probes, respectively. No hybridization was detected using a probe containing only the arsA (ATPase) gene from E. coli plasmid or with a Staphylococcus plasmid ars probe. From Southern hybridization tests of some of the positive strains it was concluded that homology to ars chromosomal genes occurred within chromosome regions, except in an E. coli isolate where hybridization occurred in both the chromosome and a 130-kb plasmid. Our results show that DNA sequences homologous to E. coli ars chromosomal genes are commonly present in the chromosomes of environmental arsenic-resistant Gram negative isolates.199810932734
5983160.9993Analysis of mutational patterns in quinolone resistance-determining regions of GyrA and ParC of clinical isolates. Fluoroquinolone (FQ)-resistant bacteria pose a major global health threat. Unanalysed genomic data from thousands of sequenced microbes likely contain important hints regarding the evolution of FQ resistance, yet this information lies fallow. Here we analysed the co-occurrence patterns of quinolone resistance mutations in genes encoding the FQ drug targets DNA gyrase (gyrase) and topoisomerase IV (topo-IV) from 36,402 bacterial genomes, representing 10 Gram-positive and 10 Gram-negative species. For 19 species, the likeliest routes toward resistance mutations in both targets were determined, and for 5 species those mutations necessary and sufficient to predict FQ resistance were also determined. Target mutation hierarchy was fixed in all examined Gram-negative species, with gyrase being the primary and topo-IV the secondary quinolone target, as well as in six of nine Gram-positive species, with topo-IV being the primary and gyrase the secondary target. By contrast, in three Gram-positive species (Staphylococcus haemolyticus, Streptococcus pneumoniae and Streptococcus suis), under some conditions gyrase became the primary and topo-IV the secondary target. The path through individual resistance mutations varied by species. Both linear and branched paths were identified in Gram-positive and Gram-negative organisms alike. Finally, FQ resistance could be predicted based solely on target gene quinolone resistance mutations for Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus, but not Klebsiella pneumoniae or Pseudomonas aeruginosa. These findings have important implications both for sequence-based diagnostics and for understanding the emergence of FQ resistance.201930582984
5850170.9993Gram-positive merA gene in gram-negative oral and urine bacteria. Clinical mercury resistant (Hg(r)) Gram-negative bacteria carrying Gram-positive mercury reductase (merA)-like genes were characterized using DNA-DNA hybridization, PCR and sequencing. A PCR assay was developed which discriminated between the merA genes related to Staphylococcus and those related to the Bacillus/Streptococcus merA genes by the difference in size of the PCR product. DNA sequence analysis correlated with the PCR assay. The merA genes from Acinetobacter junii, Enterobacter cloacae and Escherichia coli were sequenced and shared 98-99% identical nucleotide (nt) and 99.6-100% amino acid identity with the Staphylococcus aureus MerA protein. A fourth merA gene, from Pantoeae agglomerans, was partially sequenced (60%) and had 99% identical nt and 100% amino acid identity with the Streptococcus oralis MerA protein. All the Hg(r) Gram-negative bacteria transferred their Gram-positive merA genes to a Gram-positive Enterococcus faecalis recipient with the resulting transconjugants expressing mercury resistance. These Gram-positive merA genes join Gram-positive tetracycline resistance and Gram-positive macrolide resistance genes in their association with mobile elements which are able to transfer and express in Gram-negative bacteria.200415358427
486180.9993Detection of heavy metal ion resistance genes in gram-positive and gram-negative bacteria isolated from a lead-contaminated site. Resistance to a range of heavy metal ions was determined for lead-resistant and other bacteria which had been isolated from a battery-manufacturing site contaminated with high concentration of lead. Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) and Gram-negative (Alcaligenes species) isolates were resistant to lead, mercury, cadmium, cobalt, zinc and copper, although the levels of resistance to the different metal ions were specific for each isolate. Polymerase chain reaction, DNA-DNA hybridization and DNA sequencing were used to explore the nature of genetic systems responsible for the metal resistance in eight of the isolates. Specific DNA sequences could be amplified from the genomic DNA of all the isolates using primers for sections of the mer (mercury resistance determinant on the transposon Tn501) and pco (copper resistance determinant on the plasmid pRJ1004) genetic systems. Positive hybridizations with mer and pco probes indicated that the amplified segments were highly homologous to these genes. Some of the PCR products were cloned and partially sequenced, and the regions sequenced were highly homologous to the appropriate regions of the mer and pco determinants. These results demonstrate the wide distribution of mercury and copper resistance genes in both Gram-positive and Gram-negative isolates obtained from this lead-contaminated soil. In contrast, the czc (cobalt, zinc and cadmium resistance) and chr (chromate resistance) genes could not be amplified from DNAs of some isolates, indicating the limited contribution, if any, of these genetic systems to the metal ion resistance of these isolates.19979342884
4505190.9993Origin and evolution of genes specifying resistance to macrolide, lincosamide and streptogramin antibiotics: data and hypotheses. Resistance to macrolide, lincosamide and streptogramin antibiotics is due to alteration of the target site or detoxification of the antibiotic. Postranscriptional methylation of 23S ribosomal rRNA confers resistance to macrolide (M), lincosamide (L) and streptogramin (S) B-type antibiotics, the so-called MLSB phenotype. Several classes of rRNA methylases conferring resistance to MLSB antibiotics have been characterized in Gram-positive cocci, in Bacillus spp, and in strains of actinomycetes producing erythromycin. The enzymes catalyze N6-dimethylation of an adenine residue situated in a highly conserved region of prokaryotic 23S rRNA. In this review, we compare the amino acid sequences of the rRNA methylases and analyze the codon usage in the corresponding erm (erythromycin resistance methylase) genes. The homology detected at the protein level is consistent with the notion that an ancestor of the erm genes was implicated in erythromycin resistance in a producing strain. However, the rRNA methylases of producers and non-producers present substantial sequence diversity. In Gram-positive bacteria the preferential codon usage in the erm genes reflects the guanosine plus cytosine content of the chromosome of the host. These observations suggest that the presence of erm genes in these micro-organisms is ancient. By contrast, it would appear that enterobacteria have acquired only recently an rRNA methylase gene of the ermB class from a Gram-positive coccus since the genes isolated in Escherichia coli and in Gram-positive cocci are highly homologous (homology greater than 98%) and present a codon usage typical of the latter micro-organisms. As opposed to the MLSB phenotype which results from a single biochemical mechanism, inactivation of structurally related antibiotics of the MLS group involves synthesis of various other enzymes. In enterobacteria, resistance to erythromycin and oleandomycin is due to production of erythromycin esterases which hydrolyze the lactone ring of the 14-membered macrolides. We recently reported the nucleotide sequence of ereA and ereB (erythromycin resistance esterase) genes which encode erythromycin esterases type I and II, respectively. The amino acid sequences of the two isozymes do not exhibit statistically significant homology. Analysis of codon usage in both genes suggests that esterase type I is indigenous to E. coli, whereas the type II enzyme was acquired by E. coli from a phylogenetically remote micro-organism. Inactivation of lincosamides, first reported in staphylococci and lactobacilli of animal origin, was also recently detected in Gram-positive cocci isolated from humans.(ABSTRACT TRUNCATED AT 400 WORDS)19873326871