Mutations in the gyrB, parC, and parE genes of quinolone-resistant isolates and mutants of Edwardsiella tarda

The full length genes gyrB (2,415 bp), parC (2,277 bp), and parE (1,896 bp) in Edwardsiella tarda were cloned by PCR with degenerate primers based on the sequence of the respective quinolone resistance-determining region (QRDR), followed by elongation of 5' and 3' ends using cassette ligation-mediated PCR (CLMP). Analysis of the cloned genes revealed open reading frames (ORFs) encoding proteins of 804 (GyrB), 758 (ParC), and 631 (ParE) amino acids with conserved gyrase/topoisomerase features and motifs important for enzymatic function. The ORFs were preceded by putative promoters, ribosome binding sites, and inverted repeats with the potential to form cruciform structures for binding of DNA-binding proteins. When comparing the deduced amino acid sequences of E. tarda GyrB, ParC, and ParE with those of the corresponding proteins in other bacteria, they were found to be most closely related to Escherichia coli GyrB (87.6% identity), Klebsiella pneumoniae ParC (78.8% identity) and Salmonella typhimurium ParE (89.5% identity), respectively. The two topoisomerase genes, parC and parE, were found to be contiguous on the E. tarda chromosome. All 18 quinoloneresistant isolates obtained from Korea thus far did not contain subunit alternations apart from a substitution in GyrA (Ser83→Arg). However, an alteration in the QRDR of ParC (Ser84→Ile) following an amino acid substitution in GyrA (Asp87→Gly) was detected in E. tarda mutants selected in vitro at 8 microng/ml ciprofloxacin (CIP). A mutant with a GyrB (Ser464→Leu) and GyrA (Asp87→Gly) substitution did not show a significant increase in the minimum inhibitory concentration (MIC) of CIP. None of the in vitro mutants exhibited mutations in parE. Thus, gyrA and parC should be considered to be the primary and secondary targets, respectively, of quinolones in E. tarda.     

Role of mutations in parC and gyrA in forming resistance of Mycoplasma hominis to fluoroquinolones

The set of the laboratory strain M. hominis H-34 mutants resistant to fluoroquinolones (ciprofloxacin-Cfl, lomefloxacin-Lfl, ofloxacin-Ofl) was obtained by selection in broth medium. The mutation was found in the quinolone resistance-determining region (QRDR) of A subunit of topoisomerase IV gene (parC) and new mutations were found in QRDR of genes encoding the A subunit of DNA gyrase (gyrA) in M. hominis mutants resistant to various concentrations of the Cfl, Lfl and Ofl. After multistep selection of the obtained mutants at constant concentrations of Cfl additional mutation Ser83 to Trp was revealed. No mutations in parE and gyrB were found. Mutations in parC for laboratory strain M. hominis H34 appeared at lower antibiotic concentrations than in gyrA. All mutations in gyr A were associated with mutations in parC. This confirms the previous data that topoisomerase IV is the primary target of Cfl and Ofl and suggests that it is the primary target of Lfl. Some M. hominis mutants selected at Ofl without any substitution in QRDRs were shown to be insensitive to Cfl and of Lfl. Studies of cross-resistance of the selected M. hominis mutants showed that their resistance to various fluoroquinolone concentrations could not depend on any mutations in QRDR of topoisomerase IV and DNA gyrase genes and suggests involvement of other unknown molecular mechanisms specific for Mycoplasmas.     

A cluster of genes that affects nucleoid segregation in Salmonella typhimurium.

Thirteen conditional lethal mutations in genes of Salmonella typhimurium map at the clmF locus and affect both viability and the faithful partitioning of daughter nucleoids. These mutations have now been divided into three complementation groups by using cloned fragments of S. typhimurium DNA and renamed parC, parE, and parF. The proteins produced from the cloned fragments predict that ParC is an 85-kD protein, ParE is 75 kD in size, and ParF, 27 kD. The parE gene is about 5 kb upstream of the parC gene, and parC is just upstream of parF. Genes situated between parC and parE produce at least two proteins of unknown function. The DNA sequence of the S. typhimurium parC gene was determined and has 56% homology with the first 1400 base pairs of the Escherichia coli gryA gene, which encodes the A subunit of DNA gyrase, and 85% homology with the E. coli parC gene. Despite the strong homology between gryA and parC, these two genes cannot substitute for one another. The DNA sequence of the S. typhimurium parF gene was determined and predicts a protein with a hydrophobic N terminus. The ParF protein may interact with ParC and ParE to anchor these proteins to the membrane. These results raise questions about the relative roles of gyrase and ParCEF in nucleoid decatenation. In addition, the parC and gyrA genes provide an example of the evolution of essential functions by gene duplication.     

A ParE-ParC fusion protein is a functional topoisomerase.

Type II topoisomerases are responsible for DNA unlinking during DNA replication and chromosome segregation. Although eukaryotic enzymes are homodimers and prokaryotic enzymes are heterotetramers, both prokaryotic and eukaryotic type II topoisomerases belong to a single protein family. The amino- and carboxyl-terminal domains of eukaryotic enzymes are homologous to the ATP-binding and catalytic subunits of prokaryotic enzymes, respectively. Topoisomerase IV, a prokaryotic type II topoisomerase, consists of the ATP-binding subunit, ParE, and the catalytic subunit, ParC. We have joined the coding regions of parE and parC in frame and constructed a fusion protein of the two subunits of topoisomerase IV. This fusion protein, ParEC, can catalyze both decatenation and relaxation reactions. The ParEC protein is also capable of decatenating replicating daughter DNA molecules during oriC DNA replication in vitro. Furthermore, the fusion gene, parEC, complements the temperature-sensitive growth of both parC and parE strains, indicating that the ParEC protein can substitute for topoisomerase IV in vivo. These results demonstrate that a fusion protein of the two subunits of topoisomerase IV is a functional topoisomerase. Thus, a heterotetrameric type II topoisomerase can be converted into a homodimeric type II topoisomerase by gene fusion.     

Topoisomerase III can serve as the cellular decatenase in Escherichia coli

topB, encoding topoisomerase III, was identified as a high copy suppressor of the temperature-sensitive parC1215 allele, encoding one of the subunits of topoisomerase IV. Overexpression of topoisomerase III at the nonpermissive temperature was shown subsequently to restore timely chromosome decatenation and suppress lethality in strains carrying either temperature-sensitive parE or parC alleles. By developing an assay in vitro for precatenane unlinking, we demonstrated directly that both topoisomerase III and topoisomerase IV were efficient at this task, whereas DNA gyrase was very inefficient at precatenane removal. These observations suggest that precatenane unlinking is sufficient to sustain decatenation of replicating daughter chromosomes in the cell.     

DNA gyrase and DNA topoisomerase of Bacillus subtilis: expression and characterization of recombinant enzymes encoded by the gyrA,gyrB and parC,parE genes

Bacillus subtilis Bs gyrA and gyrB genes specifying the DNA gyrase subunits, and parC and parE genes specifying the DNA topoisomerase IV subunits, have been separately cloned and expressed in Escherichia coli as hexahistidine (his6)-tagged recombinant proteins. Purification of the gyrA and gyrB subunits together resulted in predominantly two bands at molecular weights of 94 and 73kDa; purification of the parC and parE subunits together resulted in predominantly two bands at molecular weights of 93 and 75kDa, as predicted by their respective sequences. The ability of the subunits to complement their partner was tested in an ATP-dependent decatenation/supercoiling assay system. The results demonstrated that the DNA gyrase and the topoisomerase IV subunits produce the expected supercoiled DNA and relaxed DNA products, respectively. Additionally, inhibition of these two enzymes by fluoroquinolones has been shown to be comparable to those of the DNA gyrases and topoisomerases of other bacterial strains. In sum, the biological and enzymatic properties of these products are consistent with their authenticity as DNA gyrase and DNA topoisomerase IV enzymes from B. subtilis.     

Cloning and sequencing of the Escherichia coli gyrA gene coding for the A subunit of DNA gyrase

The gene gyrA of Escherichia coli, which encodes the A subunit of DNA gyrase (topoisomerase II), has been cloned and a region of approximately 3300 base-pairs sequenced. An open reading frame of 2625 nucleotides coding for a protein of 97,000 Mr is located. The peptide weight of the subunit predicted from this open reading frame is in close agreement with previously published estimates of that of the A subunit. There is a "TATAAT" promoter motif located 44 bases upstream from the first "ATG" of the open reading frame. The amino acid sequence derived from the nucleotide sequence is about 50% homologous with that derived from the Bacillus subtilis gyrA gene sequence, with several regions showing greater than 90% homology.     

Identification and characterization of the gyrB gene from Treponema pallidum subsp. pallidum

The nucleotide sequence of a DNA gyrase B subunit gene (gyrB) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The organization of genes flanking the gyrB gene is unique in comparison to that of other bacteria. The gyrB gene encodes a 637 amino acid protein whose deduced sequence has a high degree of homology with type-II topoisomerase ATPase subunits (GyrB and ParE). Five type-II topoisomerase motifs, an ATP-binding site (Walker A), and amino acid residues that putatively interact with ATP, are highly conserved in the T. pallidum GyrB protein.     

Role of mutations in the A-subunit of Acholeplasma laidlawii PG-8B topoisomerase IV in formation of resistance to fluoroquinolones

Nucleotide sequence of Acholeplasma laidlawii genome site PG-8B (1000 n.p.), containing topoisomerase IV subunit genes (parE and parC), has been determined. Sequenced genome site contains a gene fragment coding for the C-terminal region of ParE and gene fragment coding for N-terminal region of ParC. Topoisomerase IV subunite genes in A. laidlawii genome are situated near each other and overlapping by 4 nucleotides. Selection in liquid nutrient medium with ascending antibiotic concentrations resulted in derivation of A. laidlawii PG-8B cells resistant to ciprofloxacin, a fluoroquinolone. The resistant clones contain a mutation in the parC QRDR region determining fluoroquinolone resistance: Ser(91) (corresponding to Ser(80) in Escherichia coli ParC) replacement) for Leu.     

Purification and characterization of DNA topoisomerase IV in Escherichia coli.

The subunits of topoisomerase IV (topo IV), the ParC and ParE proteins in Escherichia coli, were purified to near homogeneity from the respective overproducers. They revealed type II topoisomerase activity only when they were combined with each other. In the presence of Mg2+ and ATP, topo IV was capable of relaxing a negatively or positively supercoiled plasmid DNA or converting the knotted P4 phage DNA, whether nicked or ligated, to a simple ring. However, supercoiling activity was not detected. The topoisomerase activity was not detectable when the purified ParC and ParE proteins were combined with the purified GyrB and GyrA proteins, respectively. This is consistent with the result that neither a parC nor a parE mutation was compensated by transformation with a plasmid carrying either the gyrA or the gyrB gene. Simultaneous introduction of both the gyrA and gyrB plasmids corrected the phenotypic defect of parC and parE mutants. The results suggest that DNA gyrase can substitute for topo IV at least in some part of the function for chromosome partitioning. Antisera were prepared against the purified ParC, ParE, GyrA, and GyrB proteins and used to investigate cellular localization of these gene products. ParC protein was found to be specifically associated with inner membranes only in the presence of DNA. This result suggests that one of the functions of topo IV might be to anchor chromosomes on membranes as previously proposed for eukaryotic topoisomerase II.     

Structure of the Drosophila DNA topoisomerase II gene. Nucleotide sequence and homology among topoisomerases II

We have determined the nucleotide sequence of the Drosophila DNA topoisomerase II gene. Data from primer extension and S1 nuclease protection experiments were combined with comparisons of genomic and cDNA sequences to determine the structure of the mature messenger RNA. This message has a large open reading frame of 4341 nucleotides. The length of the predicted protein is 1447 amino acids with a molecular weight of 164,424. Topoisomerase II can be divided into three domains: (1) an N-terminal region with homology to the B (ATPase) subunit of the bacterial type II topoisomerase, DNA gyrase; (2) a central region with homology to the A (breaking and rejoining) subunit of DNA gyrase; (3) a C-terminal region characterized by alternating stretches of positively and negatively charged amino acids. DNA topoisomerase II from the fruit fly shares significant sequence homology with those from divergent sources, including bacteria, bacteriophage T4 and yeasts. The location and distribution of homologous stretches in these sequences are analyzed.     

The first report of the qnrB19, qnrS1 and aac(6´)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.     

A topA intein in Pyrococcus furiosus and its relatedness to the r-gyr intein of Methanococcus jannaschii

A new intein coding sequence was found in a topA (DNA topoisomerase I) gene by cloning and sequencing this gene from the hyperthermophilic Archaeon Pyrococcus furiosus. The predicted Pfu topA intein sequence is 373 amino acids long and located two residues away from the catalytic tyrosine of the topoisomerase. It contains putative intein sequence blocks (C, E, and H) associated with intein endonuclease activity, in addition to intein sequence blocks (A, B, F, and G) that are necessary for protein splicing. This DNA topoisomerase I intein is most related to a reverse gyrase intein from the methanogenic Archaeon Methanococcus jannaschii. These two inteins share 31% amino acid sequence identity and, more importantly, have the same insertion sites in their respective host proteins. It is suggested that these two inteins are homologous inteins present in structurally related, but functionally distinct, proteins, with implications on intein evolution and intein homing.     

The nucleotide sequence of the fission yeast DNA topoisomerase II gene: structural and functional relationships to other DNA topoisomerases.

We have determined the complete nucleotide sequence of a 5.3-kb long genomic DNA fragment of the fission yeast Schizosaccharomyces pombe that encodes DNA topoisomerase II. It contains a 4293 bp long single open reading frame. The predicted polypeptide has 1431 residues (mol. wt 162,000) and shows three characteristic domains; the large C-terminal region, which consists of alternating acidic-basic stretches and might be a chromatin-binding domain, the NH2 half domain homologous to the ATP-binding gyrB subunit of bacterial gyrase and the central-to-latter part which is homologous to the NH2 domain of the catalytic gyrA subunit, suggesting a possible evolutionary consequence of the gene fusion of the bacterial gyrase subunits into the eucaryotic DNA topoisomerase II gene. We have found that the cloned fission yeast TOP2 gene can complement the budding yeast top2 mutation, although the fission yeast TOP2 protein sequence is only 50% homologous to the recently determined sequence of budding yeast (J.C. Wang, personal communication). Conversely, the budding yeast TOP2 gene can complement the fission yeast top2 mutations, indicating that their DNA topoisomerase II genes are functionally exchangeable.     

Requirement of topoisomerase IV parC and parE genes for cell cycle progression and developmental regulation in Caulobacter crescentus

We have identified the parC and parE genes encoding DNA topoisomerase IV (Topo IV) in Caulobacter crescentus . We have also characterized the effect of conditional Topo IV mutations on cell division and morphology. Topo IV mutants of C. crescentus are unlike mutants of Escherichia coli and S. typhimurium , which form long filamentous cells that are defective in nucleoid segregation and divide frequently to produce anucleate cells. Topo IV mutants of C. crescentus are highly pinched at multiple sites (cell separation phenotype) and they do not divide to produce cells lacking DNA. These results suggest unique regulatory mechanisms coupling nucleoid partitioning and cell division in this aquatic bacterium. In addition, distinctive nucleoid-partitioning defects are not apparent in C. crescentus Topo IV mutants as they are in E. coli and S. typhimurium . However, abnormal nucleoid segregation in parE mutant cells could be demonstrated in a genetic background containing a conditional mutation in the C. crescentus ftsA gene, an early cell division gene that is epistatic to parE for cell division and growth. We discuss these results in connection with the possible roles of C. crescentus Topo IV in the regulation of cell division, chromosome partitioning, and late events in polar morphogenesis. Although the ParC and ParE subunits of Topo IV are very similar in sequence to the GyrA and GyrB subunits of DNA gyrase, we have used DNA sequence analysis to identify a highly conserved 'GyrA box' sequence that is unique to the GyrA proteins and may serve as a hallmark of the GyrA protein family.     

The tip of the iceberg: quinolone-resistance conferred by mutations in gyrA gene in non-typhoidal Salmonella strains

Food-borne infections due to Salmonella spp. seldom require antimicrobial therapy, but this is compulsory in systemic salmonellosis. Salmonella resistance to a large panel of antibiotics has been described worldwide. Since the introduction of nalidixic acid in therapy, Salmonella spp. have steadily developed resistance, especially over the last three decades. The source of quinolone resistance is thought to be the selective pressure determined by the use of quinolones in both human and veterinary practices. Resistance acquisition of Salmonella strains is a stepwise process. Several mechanisms are described, which can lead to the development of quinolone resistance. The main mechanism is considered to be linked with mutations in the quinolone-resistance determining region (QRDR) of the target genes (gyrA and gyrB encoding DNA gyrase, and parC and parE encoding topoisomerase IV). This first step in mutational resistance usually determines a rise in the nalidixic acid minimal inhibitory concentration (MIC). The most common amino acid substitutions in the GyrA subunit, resulting in varied degrees of quinolone resistance, occur at codons Ser83 and Asp87. Higher levels of resistance may occur by further mutational steps, with amino acid changes in the same or a different target enzyme. Other mechanisms are as well involved, like increased efflux or plasmid-mediated resistance. Acknowledgement of the epidemiology and the onset mechanisms of quinolone resistance in Salmonella spp. is compulsory, and surveillance for resistant bacteria among human, animal and food sources remains critical.     

Nucleotide sequence of a type II DNA topoisomerase gene. Bacteriophage T4 gene 39.

T4 DNA topoisomerase is a type II enzyme and is thought to be required for normal T4 DNA replication T4 gene 39 codes for the largest of the three subunits of T4 DNA topoisomerase. I have determined the nucleotide sequence of a region of 2568 nucleotides of T4 DNA which includes gene 39. The location of the gene was established by the identification of the first fifteen amino acids in the large open reading frame in the DNA sequence as those found at the amino-terminus of the purified 39-protein. The coding region of gene 39 has 1560 bases, and it is followed by two in-frame stop codons. The gene is preceded by a typical Shine-Dalgarno sequence as well as possible promoter sequences for E. coli RNA polymerase. T4 39-protein consists of 520 amino acids, and it has a calculated molecular weight of 58,478. By comparing the amino acid sequences, T4 39-protein is found to share homology with the gyrB subunit of DNA gyrase. This suggests that these topoisomerase subunits may be equivalent functionally. Some of the characteristics of the 39-protein and its structural features predicted from the DNA sequence data are discussed.     

A DNA fragment homologous to F1-ATPase beta subunit was amplified from genomic DNA of Methanosarcina barkeri. Indication of an archaebacterial F-type ATPase.

A 490 bp DNA fragment was amplified from Methanosarcina barkeri genomic DNA by the polymerase chain reaction (PCR) using oligonucleotide primers designed based on conserved amino acid sequences of the F1-ATPase beta subunits. The amino acid sequence deduced from the DNA sequence of this fragment was highly homologous to a portion of the F1-ATPase beta subunit. This indicates that this archaebacterium has a gene of F-type ATPase in addition to a gene of V-type ATPase.     

A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase

A hyper-recombination mutation was isolated that causes an increase in recombination between short repeated delta sequences surrounding the SUP4-omicron gene in S. cerevisiae. The wild-type copy of this gene was cloned by complementation of one of its pleiotropic phenotypes, slow growth. DNA sequence of the clone revealed a 656 amino acid open reading frame capable of encoding a protein homologous to the bacterial type I topoisomerase. No homology was detected with previously identified eukaryotic topoisomerases. Construction of double mutants with either of the two known yeast topoisomerase genes revealed synergistic effects on growth suggesting overlapping functions. Expression of bacterial topoisomerase I in yeast can fully complement the slow growth defect of a null mutation. We have named this locus TOP3 and suggest that it defines a novel eukaryotic topoisomerase gene.