Phylogenetic structures of the genus Acinetobacter based on gyrB sequences: comparison with the grouping by DNA-DNA hybridization.
(1/2086)
The phylogenetic relationships of 49 Acinetobacter strains, 46 of which have previously been classified into 18 genomic species by DNA-DNA hybridization studies, were investigated using the nucleotide sequence of gyrB, the structural gene for the DNA gyrase B subunit. The phylogenetic tree showed linkages between genomic species 1 (Acinetobacter calcoaceticus), 2 (Acinetobacter baumannii), 3 and TU13; genomic species 6, BJ15, BJ16 and BJ17; genomic species 5, BJ13 (synonym of TU14) and BJ14; genomic species 7 (Acinetobacter johnsonii), 10 and 11; and genomic species 8 and 9. The phylogenetic grouping of Acinetobacter strains based on gyrB genes was almost congruent with that based on DNA-DNA hybridization studies. Consequently, gyrB sequence comparison can be used to resolve the taxonomic positions of bacterial strains at the level of genomic species. However, minor discrepancies existed in the grouping of strains of genomic species 8, 9 and BJ17. The phylogenetic tree for these strains was reconstructed from the sequence of rpoD, the structural gene for the RNA polymerase sigma 70 factor. The latter tree was 100% congruent with the grouping based on DNA-DNA hybridization. The reliability of DNA-DNA hybridization may be superior to that of sequence comparison of a single protein-encoding gene in resolving closely related organisms since the former method measures the homologies between the nucleotide sequences of total genomic DNAs. Three strains that have not been characterized previously by DNA-DNA hybridization seem to belong to two new genomic species, one including strain ATCC 33308 and the other including strains ATCC 31012 and MBIC 1332. (+info)
Cloning, expression, and enzymatic characterization of Pseudomonas aeruginosa topoisomerase IV.
(2/2086)
The topoisomerase IV subunit A gene, parC homolog, has been cloned and sequenced from Pseudomonas aeruginosa PAO1, with cDNA encoding the N-terminal region of Escherichia coli parC used as a probe. The homolog and its upstream gene were presumed to be parC and parE through sequence homology with the parC and parE genes of other organisms. The deduced amino acid sequence of ParC and ParE showed 33 and 32% identity with that of the P. aeruginosa DNA gyrase subunits, GyrA and GyrB, respectively, and 69 and 75% identity with that of E. coli ParC and ParE, respectively. The putative ParC and ParE proteins were overexpressed and separately purified by use of a fusion system with a maltose-binding protein, and their enzymatic properties were examined. The reconstituted enzyme had ATP-dependent decatenation activity, which is the main catalytic activity of bacterial topoisomerase IV, and relaxing activities but had no supercoiling activity. So, the cloned genes were identified as P. aeruginosa topoisomerase IV genes. The inhibitory effects of quinolones on the activities of topoisomerase IV and DNA gyrase were compared. The 50% inhibitory concentrations of quinolones for the decatenation activity of topoisomerase IV were from five to eight times higher than those for the supercoiling activities of P. aeruginosa DNA gyrase. These results confirmed that topoisomerase IV is less sensitive to fluoroquinolones than is DNA gyrase and may be a secondary target of new quinolones in wild-type P. aeruginosa. (+info)
Fluoroquinolone action against clinical isolates of Mycobacterium tuberculosis: effects of a C-8 methoxyl group on survival in liquid media and in human macrophages.
(3/2086)
When the lethal action of a C-8 methoxyl fluoroquinolone against clinical isolates of Mycobacterium tuberculosis in liquid medium was measured, the compound was found to be three to four times more effective (as determined by measuring the 90% lethal dose) than a C-8-H control fluoroquinolone or ciprofloxacin against cells having a wild-type gyrA (gyrase) gene. Against ciprofloxacin-resistant strains, the C-8 methoxyl group enhanced lethality when alanine was replaced by valine at position 90 of the GyrA protein or when aspartic acid 94 was replaced by glycine, histidine, or tyrosine. During infection of a human macrophage model by wild-type Mycobacterium bovis BCG, the C-8 methoxyl group lowered survival 20- to 100-fold compared with the same concentration of a C-8-H fluoroquinolone. The C-8 methoxyl fluoroquinolone was also more effective than ciprofloxacin against a gyrA Asn94 mutant of M. bovis BCG. In an M. tuberculosis-macrophage system the C-8 methoxyl group improved fluoroquinolone action against both quinolone-susceptible and quinolone-resistant clinical isolates. Thus, a C-8 methoxyl group enhances the bactericidal activity of quinolones with N1-cyclopropyl substitutions; these data encourage further refinement of fluoroquinolones as antituberculosis agents. (+info)
Effect of cellular ATP depletion on topoisomerase II poisons. Abrogation Of cleavable-complex formation by etoposide but not by amsacrine.
(4/2086)
Topoisomerase (topo) II poisons have been categorized into ATP-independent and -dependent drugs based on in vitro studies. We investigated drug-induced topoII-DNA complexes in intact cells almost completely depleted of ATP. Virtually no DNA single-strand breaks (SSBs), as measured by alkaline elution, were detected in energy-depleted cells treated with the topoII poisons etoposide, teniposide, daunorubicin, doxorubicin, mitoxantrone, or clerocidin. This inhibition was reversible; subsequent incubation with glucose restored the level of DNA SSBs. The effect of ATP depletion was specific for topoII, because topoI-mediated cleavable complexes induced by camptothecin were unaffected by ATP depletion. Furthermore, etoposide-induced DNA-protein complexes and DNA double-strand breaks, as measured by filter elution techniques, and topoIIalpha and -beta trapping, as measured by a band depletion assay, were completely inhibited by energy depletion. Differences in drug transport could not explain the effect of ATP depletion. The topoII poison amsacrine (m-AMSA) was unique with respect to ATP dependence. In ATP-depleted cells, m-AMSA-induced DNA SSBs, DNA double-strand breaks, DNA-protein complexes, topoIIalpha and -beta trapping were only modestly reduced. The accumulation of m-AMSA was reduced in ATP-depleted cells, which indicates that drug transport could contribute to the modest decrease in m-AMSA-induced cleavable complexes. In conclusion, drug-induced topoII-DNA complexes were completely antagonized in ATP-depleted cells, except in the case of m-AMSA. One possible interpretation is that m-AMSA mainly produces prestrand passage DNA lesions, whereas the other topoII poisons tested exclusively stabilize poststrand passage DNA lesions in intact cells. (+info)
Mutation of a conserved serine residue in a quinolone-resistant type II topoisomerase alters the enzyme-DNA and drug interactions.
(5/2086)
A Ser740 --> Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser83 in gyrase results in resistance to quinolones and confers hypersensitivity to etoposide (VP-16). We characterized the cleavage complexes induced by the top2(S740W) in the human c-myc gene. In addition to resistance to the fluoroquinolone CP-115,953, top2(S740W) induced novel DNA cleavage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone. Analysis of the VP-16 sites indicated that the changes in the cleavage pattern were reflected by alterations in base preference. C at position -2 and G at position +6 were observed for the top2(S740W) in addition to the previously reported C-1 and G+5 for the wild-type top2. The VP-16-induced top2(S740W) cleavage complexes were also more stable. The most stable sites had strong preference for C-1, whereas the most reversible sites showed no base preference at positions -1 or -2. Different patterns of DNA cleavage were also observed in the absence of drug and in the presence of calcium. These results indicate that the Ser740 --> Trp mutation alters the DNA recognition of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors. Thus, residue 740 of top2 appears critical for both DNA and drug interactions. (+info)
DNA topoisomerase IIalpha and -beta expression in human ovarian cancer.
(6/2086)
To study DNA topoisomerase IIalpha (Topo-IIalpha) and -beta expression and regulation in human ovarian cancer, 15 ovarian tumour samples were investigated. To compare different levels of expression, the samples were screened for topo IIalpha and -beta mRNA with Northern blotting and a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay for Topo-IIalpha mRNA. Additionally, protein levels were determined with Western blotting and topoisomerase II activity levels with the decatenation assay. The results obtained were compared with each other and with the tumour volume index of the samples. In tumours with a tumour volume index > or = 50%, the mRNA levels (as determined by Northern blotting) and protein levels for each isozyme were in accordance. Additionally, correlations were found between Topo-IIalpha RT-PCR data and Topo-IIalpha Northern blot results, and between Topo-IIalpha RT-PCR data and Topo-IIalpha protein levels. Interestingly, Topo-IIbeta protein levels correlated better with Topo-II activity than Topo-IIalpha protein levels. In eight ovarian cystadenoma samples, no Topo-IIalpha protein could be found. In only three out of eight of these cystadenomas, Topo-IIbeta protein could be detected. These findings suggest that Topo-IIalpha and Topo-IIbeta protein levels are up-regulated in ovarian cancer and may indicate that Topo-IIbeta is an interesting target for chemotherapy in ovarian tumours. (+info)
Analysis of DNA cleavage by reverse gyrase from Sulfolobus shibatae B12.
(7/2086)
Reverse gyrase is a type I-5' topoisomerase, which catalyzes a positive DNA supercoiling reaction in vitro. To ascertain how this reaction takes places, we looked at the DNA sequences recognized by reverse gyrase. We used linear DNA fragments of its preferred substrate, the viral SSV1 DNA, which has been shown to be positively supercoiled in vivo. The Sulfolobus shibatae B12 strain, an SSV1 virus host, was chosen for production of reverse gyrase. This naturally occurring system (SSV1 DNA-S. shibatae reverse gyrase) allowed us to determine which SSV1 DNA sequences are bound and cleaved by the enzyme with particularly high selectivity. We show that the presence of ATP decreases the number of cleaved complexes obtained whereas the non-hydrolyzable ATP analog adenosine 5'-[beta, gamma-imido]triphosphate increases it without changing the sequence specificity. (+info)
Molecular cloning and characterization of the human topoisomerase IIalpha and IIbeta genes: evidence for isoform evolution through gene duplication.
(8/2086)
Human DNA topoisomerase II is essential for chromosome segregation and is the target for several clinically important anticancer agents. It is expressed as genetically distinct alpha and beta isoforms encoded by the TOP2alpha and TOP2beta genes that map to chromosomes 17q21-22 and 3p24, respectively. The genes display different patterns of cell cycle- and tissue-specific expression, with the alpha isoform markedly upregulated in proliferating cells. In addition to the fundamental role of TOP2alpha and TOP2beta genes in cell growth and development, altered expression and rearrangement of both genes are implicated in anticancer drug resistance. Here, we report the complete structure of the human topoisomerase IIalpha gene, which consists of 35 exons spanning 27.5 kb. Sequence data for the exon-intron boundaries were determined and examined in the context of topoisomerase IIalpha protein structure comprising three functional domains associated with energy transduction, DNA breakage-reunion activity and nuclear localization. The organization of the 3' half of human TOP2beta, including sequence specifying the C-terminal nuclear localization domain, was also elucidated. Of the 15 introns identified in this 20 kb region of TOP2beta, the first nine and the last intron align in identical positions and display the same phases as introns in TOP2alpha. Though their extreme 3' ends differ, the striking conservation suggests the two genes diverged recently in evolutionary terms consistent with a gene duplication event. Access to TOP2alpha and TOP2beta gene structures should aid studies of mutations and gene rearrangements associated with anticancer drug resistance. (+info)