Molecular cloning of the DNA gyrase genes from Methylovorus sp. strain SS1 and the mechanism of intrinsic quinolone resistance in methylotrophic bacteria

The genes encoding the DNA gyrase A (GyrA) and B subunits (GyrB) of Methylovorus sp. strain SS1 were cloned and sequenced. gyrA and gyrB coded for proteins of 846 and 799 amino acids with calculated molecular weights of 94,328 and 88,714, respectively, and complemented Escherichia coli gyrA and gyrB temperature sensitive (ts) mutants. To analyze the role of type II topoisomerases in the intrinsic quinolone resistance of methylotrophic bacteria, the sequences of the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase and the C subunit (ParC) of topoisomerase IV (Topo IV) of Methylovorus sp. strain SS1, Methylobacterium extorquens AM1 NCIB 9133, Methylobacillus sp, strain SK1 DSM 8269, and Methylophilus methylotrophus NCIB 10515 were determined. The deduced amino acid sequences of the QRDRs of the ParCs in the four methylotrophic bacteria were identical to that of E. coli ParC. The sequences of the QRDR in GyrA were also identical to those in E. coli GyrA except for the amino acids at positions 83, 87, or 95. The Ser83 to Thr substitution in Methylovorus sp. strain SS1, and the Ser83 to Leu and Asp87 to Asn substitutions in the three other methylotrophs, agreed well with the minimal inhibitory concentrations of quinolones in the four bacteria, suggesting that these residues play a role in the intrinsic susceptibility of methylotrophic bacteria to quinolones.     

The structural basis for substrate specificity in DNA topoisomerase IV

Most bacteria possess two type IIA topoisomerases, DNA gyrase and topo IV, that together help manage chromosome integrity and topology. Gyrase primarily introduces negative supercoils into DNA, an activity mediated by the C-terminal domain of its DNA binding subunit (GyrA). Although closely related to gyrase, topo IV preferentially decatenates DNA and relaxes positive supercoils. Here we report the structure of the full-length Escherichia coli ParC dimer at 3.0 A resolution. The N-terminal DNA binding region of ParC is highly similar to that of GyrA, but the ParC dimer adopts a markedly different conformation. The C-terminal domain (CTD) of ParC is revealed to be a degenerate form of the homologous GyrA CTD, and is anchored to the top of the N-terminal domains in a configuration different from that thought to occur in gyrase. Biochemical assays show that the ParC CTD controls the substrate specificity of topo IV, likely by capturing DNA segments of certain crossover geometries. This work delineates strong mechanistic parallels between topo IV and gyrase, while explaining how structural differences between the two enzyme families have led to distinct activity profiles. These findings in turn explain how the structures and functions of bacterial type IIA topoisomerases have evolved to meet specific needs of different bacterial families for the control of chromosome superstructure.     

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.     

Binding of two DNA molecules by type II topoisomerases for decatenation

Topoisomerases (topos) maintain DNA topology and influence DNA transaction processes by catalysing relaxation, supercoiling and decatenation reactions. In the cellular milieu, division of labour between different topos ensures topological homeostasis and control of central processes. In Escherichia coli, DNA gyrase is the principal enzyme that carries out negative supercoiling, while topo IV catalyses decatenation, relaxation and unknotting. DNA gyrase apparently has the daunting task of undertaking both the enzyme functions in mycobacteria, where topo IV is absent. We have shown previously that mycobacterial DNA gyrase is an efficient decatenase. Here, we demonstrate that the strong decatenation property of the enzyme is due to its ability to capture two DNA segments in trans. Topo IV, a strong dedicated decatenase of E. coli, also captures two distinct DNA molecules in a similar manner. In contrast, E. coli DNA gyrase, which is a poor decatenase, does not appear to be able to hold two different DNA molecules in a stable complex. The binding of a second DNA molecule to GyrB/ParE is inhibited by ATP and the non-hydrolysable analogue, AMPPNP, and by the substitution of a prominent positively charged residue in the GyrB N-terminal cavity, suggesting that this binding represents a potential T-segment positioned in the cavity. Thus, after the GyrA/ParC mediated initial DNA capture, GyrB/ParE would bind efficiently to a second DNA in trans to form a T-segment prior to nucleotide binding and closure of the gate during decatenation.     

The "GyrA-box" is required for the ability of DNA gyrase to wrap DNA and catalyze the supercoiling reaction

DNA gyrase is the only topoisomerase that can introduce negative supercoils into DNA. It is thought that the binding of conventional type II topoisomerases, including topoisomerase IV, to DNA takes place at the catalytic domain across the DNA gate, whereas DNA gyrase binds to DNA not only at the amino-terminal catalytic domain but also at the carboxyl-terminal domain (CTD) of the GyrA subunit. The binding of the GyrA CTD to DNA allows gyrase to wrap DNA around itself and catalyze the supercoiling reaction. Recent structural studies, however, have revealed striking similarities between the GyrA CTD and the ParC CTD, as well as the ability of the ParC CTD to bind and bend DNA. Thus, the molecular basis of gyrase-mediated wrapping of DNA needs to be reexamined. Here, we have conducted a mutational analysis to determine the role of the "GyrA-box," a 7-amino acid-long motif unique to the GyrA CTD, in determining the DNA binding mode of gyrase. Either a deletion of the entire GyrA-box or substitution of the GyrA-box with 7 Ala residues abolishes the ability of gyrase to wrap DNA around itself and catalyze the supercoiling reaction. However, these mutations do not affect the relaxation and decatenation activities of gyrase. Thus, the presence of a GyrA-box allows gyrase to wrap DNA and catalyze the supercoiling reaction. The consequence of the loss of the GyrA-box during evolution of bacterial type II topoisomerases is discussed.     

猪源大肠杆菌耐药质粒图谱及耐药性分析

目的:探讨猪大肠杆菌的耐药质粒图谱、耐药性及耐药基因之间的关系。方法:从湖南省株洲、益阳的四个猪场分离出9株大肠杆菌,进行质粒电泳图谱分析、用PCR法检测耐喹诺酮类耐药基因Gyr A、Par C和耐四环素类耐药基因Tet A、Tet B,并采用Kirby-bauer法对这9株大肠杆菌进行药敏(18种抗生素)试验。结果:其中9株大肠杆菌含有三条或者三条以上的质粒条带,且其质粒谱型均不相同;9株大肠杆菌均检测出4种耐药基因Gyr A、Par C、Tet A和Tet B;9株大肠杆菌对所选用的抗生素存在不同程度的耐药性,其中7株大肠杆菌对10种或10种以上的抗生素耐药,最高对13种抗生素耐药,氨苄西林、青霉素、阿莫西林、红霉素的耐药率达100%,对四环素、多西环素的耐药率达到88.9%,而多粘菌素B、阿奇霉素、大观霉素耐药率较低。结论:耐药性与质粒条带数、耐药基因之间并无明显的相关性;猪大肠杆菌呈多重耐药之势,在治疗大肠杆菌病时最好根据药敏实验结果选用合适的抗生素。     

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.     

Lethal fragmentation of bacterial chromosomes mediated by DNA gyrase and quinolones

When DNA gyrase is trapped on bacterial chromosomes by quinolone antibacterials, reversible complexes form that contain DNA ends constrained by protein. Two subsequent processes lead to rapid cell death. One requires ongoing protein synthesis; the other does not. The prototype quinolone, nalidixic acid, kills wild-type Escherichia coli only by the first pathway; fluoroquinolones kill by both. Both lethal processes correlated with irreversible chromosome fragmentation, detected by sedimentation and viscosity of DNA from quinolone-treated cells. However, only fluoroquinolones fragmented purified nucleoids when incubated with gyrase purified from wild-type cells. A GyrA amino acid substitution (A67S) expected to perturb a GyrA-GyrA dimer interface allowed nalidixic acid to fragment chromosomes and kill cells in the absence of protein synthesis; moreover, it made a non-inducible lexA mutant hypersusceptible to nalidixic acid, a property restricted to fluoroquinolones with wild-type cells. The GyrA variation also facilitated immunoprecipitation of DNA fragments by GyrA antiserum following nalidixic acid treatment of cells. The ability of changes in both gyrase and quinolone structure to enhance protein synthesis-independent lethality and chromosome fragmentation is explained by drug-mediated destabilization of gyrase-DNA complexes. Instability of type II topoisomerase-DNA complexes may be a general phenomenon that can be exploited to kill cells.     

The additional 165 amino acids in the B protein of Escherichia coli DNA gyrase have an important role in DNA binding

DNA gyrase is the only enzyme known to negatively supercoil DNA. The enzyme is a heterotetramer of A(2)B(2) subunit composition. Alignment of the primary sequence of gyrase B (GyrB) from various species shows that they can be grouped into two classes. The GyrB of Gram-negative eubacteria has a stretch of about 165 amino acids in the C-terminal half, which is lacking in other GyrB subunits and type II topoisomerases. In Escherichia coli, no function has so far been attributed to this stretch. In this study, we have tried to assess the function of this region both in vivo and in vitro. A deletant (GyrBDelta160) lacking this region is non-functional in vivo. The holoenzyme reconstituted from gyrase A (GyrA) and GyrBDelta160 shows reduced but detectable supercoiling and quinolone-induced cleavage activity in vitro. GyrBDelta160 retains its ability to bind to GyrA and novobiocin. However, when reconstituted with GyrA, the deletant shows greatly impaired DNA binding. The intrinsic ATPase activity of the GyrBDelta160 is comparable to that of wild type GyrB, but this activity is not stimulated by DNA. These studies indicate that the additional stretch present in GyrB is essential for the DNA binding ability of E. coli gyrase.     

C-terminal domain of gyrase A is predicted to have a beta-propeller structure

Two different type II topoisomerases are known in bacteria. DNA gyrase (Gyr) introduces negative supercoils into DNA. Topoisomerase IV (Par) relaxes DNA supercoils. GyrA and ParC subunits of bacterial type II topoisomerases are involved in breakage and reunion of DNA. The spatial structure of the C-terminal fragment in GyrA/ParC is not available. We infer homology between the C-terminal domain of GyrA/ParC and a regulator of chromosome condensation (RCC1), a eukaryotic protein that functions as a guanine-nucleotide-exchange factor for the nuclear G protein Ran. This homology, complemented by detection of 6 sequence repeats with 4 predicted beta-strands each in GyrA/ParC sequences, allows us to predict that the GyrA/ParC C-terminal domain folds into a 6-bladed beta-propeller. The prediction rationalizes available experimental data and sheds light on the spatial properties of the largest topoisomerase domain that lacks structural information.     

Ciprofloxacin affects conformational equilibria of DNA gyrase A in the presence of magnesium ions

The conformational equilibria of the A subunit of DNA gyrase (GyrA), of its 59 kDa N-terminal fragment (GyrA59) and of the quinolone-resistant Ser-Trp83 mutant (GyrATrp83), were investigated in the presence of mono- and divalent metal ions and ciprofloxacin, a clinically useful antibacterial quinolone. The stability of the proteins was estimated from temperature denaturation, monitoring unfolding with circular dichroism spectroscopy. Two transitions were observed in GyrA and GyrATrp83, which likely reflect unfolding of the N and C-terminal protein domains. Accordingly, one thermal transition is observed for GyrA59.The melting profile of the GyrA subunit is dramatically affected by monovalent and divalent metal ions, both transitions being shifted to lower temperature upon increasing salt concentration. This effect is much more pronounced with divalent ions (Mg(2+)) and cannot be accounted for by changes in ionic strength only. The presence of ciprofloxacin shifts the melting transitions of the wild-type subunit to higher temperatures when physiological concentrations of Mg(2+) are present. In contrast, both the mutant protein and the 59 kDa fragment do not show evidence for quinolone-driven changes. These data suggest that ciprofloxacin binds to the wild-type subunit in an interaction that involves Ser83 of GyrA and that both C and N-terminal domains may be required for effective drug-protein interactions. The bell-shaped dependence of the binding process upon Mg(2+) concentration, with a maximum centred at 3-4 mM [Mg(2+)], is consistent with a metal-ion mediated GyrA-quinolone-interaction. Affinity chromatography data fully support these findings and additionally confirm the requirement for a free carboxylate to elicit binding of the quinolone to GyrA.We infer that the Mg(2+)-GyrA interaction at physiological metal ion concentration could bear biological relevance, conferring more conformational flexibility to the active enzyme. The results obtained in the presence of ciprofloxacin additionally suggest that the Mg(2+)-mediated quinolone binding to the enzyme might be involved in the mechanism of action of this family of drugs.     

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity

DNA topoisomerases manage chromosome supercoiling and organization in all cells. Gyrase, a prokaryotic type IIA topoisomerase, consumes ATP to introduce negative supercoils through a strand passage mechanism. All type IIA topoisomerases employ a similar set of catalytic domains for function; however, the activity and specificity of gyrase are augmented by a specialized DNA binding and wrapping element, termed the C-terminal domain (CTD), which is appended to its GyrA subunit. We have discovered that a nonconserved, acidic tail at the extreme C terminus of the Escherichia coli GyrA CTD has a dramatic and unexpected impact on gyrase function. Removal of the CTD tail enables GyrA to introduce writhe into DNA in the absence of GyrB, an activity exhibited by other GyrA orthologs, but not by wild-type E. coli GyrA. Strikingly, a "tail-less" gyrase holoenzyme is markedly impaired for DNA supercoiling capacity, but displays normal ATPase function. Our findings reveal that the E. coli GyrA tail regulates DNA wrapping by the CTD to increase the coupling efficiency between ATP turnover and supercoiling, demonstrating that CTD functions can be fine-tuned to control gyrase activity in a highly sophisticated manner.     

Mechanisms of fluoroquinolone resistance in Escherichia coli isolates from food-producing animals

Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.     

Characterization of the interaction between DNA gyrase inhibitor and DNA gyrase of Escherichia coli.

Escherichia coli DNA gyrase is comprised of two subunits, GyrA and GyrB. Previous studies have shown that GyrI, a regulatory factor of DNA gyrase activity, inhibits the supercoiling activity of DNA gyrase and that both overexpression and antisense expression of the gyrI gene suppress cell proliferation. Here we have analyzed the interaction of GyrI with DNA gyrase using two approaches. First, immunoprecipitation experiments revealed that GyrI interacts preferentially with the holoenzyme in an ATP-independent manner, although a weak interaction was also detected between GyrI and the individual GyrA and GyrB subunits. Second, surface plasmon resonance experiments indicated that GyrI binds to the gyrase holoenzyme with higher affinity than to either the GyrA or GyrB subunit alone. Unlike quinolone antibiotics, GyrI was not effective in stabilizing the cleavable complex consisting of gyrase and DNA. Further, we identified an 8-residue synthetic peptide, corresponding to amino acids (89)ITGGQYAV(96) of GyrI, which inhibits gyrase activity in an in vitro supercoiling assay. Surface plasmon resonance analysis of the ITGGQYAV-containing peptide-gyrase interaction indicated a high association constant for this interaction. These results suggest that amino acids 89--96 of GyrI are essential for its interaction with, and inhibition of, DNA gyrase.     

脲原体拓扑异构酶基因突变与喹诺酮类耐药的相关性研究

脲原体对喹诺酮类抗生素的耐药性与拓扑异构酶的基因突变有关。本研究在前期提出的棋盘稀释法脲原体药敏试验的基础上,对拓扑异构酶基因进行序列分析。结合既往的文献资料,确认了ParCS83L是脲原体喹诺酮类耐药性相关突变;而GyrAE112D和ParCT125A属于菌株的多态性表现。同时,本研究还发现了一些新的可能和喹诺酮类耐药相关的基因突变,这些突变以及多个基因突变是否存在叠加效应尚需进一步研究。     

Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.

DNA topoisomerase IV mediates chromosome segregation and is a potential target for antibacterial agents including new antipneumococcal fluoroquinolones. We have used hybridization to a Staphylococcus aureus gyrB probe in concert with chromosome walking to isolate the Streptococcus pneumoniae parE-parC locus, lying downstream of a putative new insertion sequence and encoding 647-residue ParE and 823-residue ParC subunits of DNA topoisomerase IV. These proteins exhibited greatest homology respectively to the GrlB (ParE) and GrlA (ParC) subunits of S. aureus DNA topoisomerase IV. When combined, whole-cell extracts of Escherichia coli strains expressing S. pneumoniae ParC or ParE proteins reconstituted a salt-insensitive ATP-dependent decatenase activity characteristic of DNA topoisomerase IV. A second gyrB homolog isolated from S. pneumoniae encoded a 648-residue protein which we identified as GyrB through its close homology both to counterparts in S. aureus and Bacillus subtilis and to the product of the S. pneumoniae nov-1 gene that confers novobiocin resistance. gyrB was not closely linked to gyrA. To examine the role of DNA topoisomerase IV in fluoroquinolone action and resistance in S. pneumoniae, we isolated mutant strains stepwise selected for resistance to increasing concentrations of ciprofloxacin. We analysed four low-level resistant mutants and showed that Ser-79 of ParC, equivalent to resistance hotspots Ser-80 of GrlA and Ser-84 of GyrA in S. aureus, was in each case substituted with Tyr. These results suggest that DNA topoisomerase IV is an important target for fluoroquinolones in S. pneumoniae and establish this organism as a useful gram-positive system for resistance studies.     

Plasmid-mediated quinolone resistance in pseudomonas putida isolates from imported shrimp

Fourteen quinolone-resistant Pseudomonas putida isolates were recovered from imported frozen shrimp sold in the United States. Two isolates harbored plasmids with qnrA and qnrB genes. PCR and DNA sequencing of quinolone resistance-determining regions identified novel substitutions in GyrA (His139→Glu and Thr128→Ala) and GyrB (Thr442→Asn, Gly470→Ala, and Ile487→Pro) and previously reported substitutions in GyrB (Asp489→Glu) and ParC (Thr105→Pro).