Catechins are not major components responsible for anti-genotoxic effects of tea extracts against nitroarenes

A plasmid called pMucA, from a piece of the plasmid pKM101 (Mol. Gen. Genet 167 (1979) 317) cloned in the vector pDM2 (J. Bacteriol. 151 (1982) 1605), caused higher mutation in a local region of Haemophilus influenzae and caused even more mutation there in a strain also containing novC, the latter causing an increase in supercoiling (J. Bacteriol 164 (1985) 525). The novD mutation depressed supercoiling, and also depressed the mutation by pMucA in the local region of the chromosome. Thus, it is clear that supercoiling is an important phenomenon in spontaneous mutation of H. influenzae. The pMucA plasmid caused a number of other phenomena in H. influenzae, induced UV mutation (Proc. Natl. Acad. Sci. USA 82 (1985) 7753), decreased UV sensitivity of transforming DNA, but not cells, and UV-induced recombination of mutants of phage HP1c1. The effect of the MucA protein in mutagenesis of H. influenzae we consider to be due to the introduction of some of the E. coli functions from pKM101. We postulate that the localized mutation caused by the MucA plasmid also involved localization of the plasmid or its coded protein in the same area, resulting from binding to a homologous gene, probably rec-1, very close to the localized region.     

Mutations affecting gyrase in Haemophilus influenzae.

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch.     

DNA gyrase complex with DNA: determinants for site-specific DNA breakage.

DNA gyrase catalyses DNA supercoiling by making a transient double-stranded DNA break within its 120-150 bp binding site on DNA. Addition of the inhibitor oxolinic acid to the reaction followed by detergent traps a covalent enzyme-DNA intermediate inducing sequence-specific DNA cleavage and revealing potential sites of gyrase action on DNA. We have used site-directed mutagenesis to examine the interaction of Escherichia coli gyrase with its major cleavage site in plasmid pBR322. Point mutations have been identified within a short region encompassing the site of DNA scission that reduce or abolish gyrase cleavage in vitro. Mapping of gyrase cleavage sites in vivo reveals that the pBR322 site has the same structure as seen in vitro and is similarly sensitive to specific point changes. The mutagenesis results demonstrate conclusively that a major determinant for gyrase cleavage resides at the break site itself and agree broadly with consensus sequence studies. The gyrase cleavage sequence alone is not a good substrate, however, and requires one or other arm of flanking DNA for efficient DNA breakage. These results are discussed in relation to the mechanism and structure of the gyrase complex.     

Bacterial DNA supercoiling and [ATP]/[ADP]. Changes associated with a transition to anaerobic growth.

Shifting Escherichia coli from aerobic to anaerobic growth caused changes in the ratio of [ATP]/[ADP] and in negative supercoiling of chromosomal and plasmid DNA. Shortly after lowering oxygen tension, both [ATP]/[ADP] and supercoiling transiently decreased. Under conditions of exponential anaerobic growth, both were higher than under aerobic conditions. These correlations may reflect an effect of [ATP]/[ADP] on DNA gyrase, since in vitro [ATP]/[ADP] influences the level of plasmid supercoiling attained when gyrase is either introducing or removing supercoils. When the supercoiling activity of gyrase was perturbed by a mutation in gyrB, a shift to anaerobic conditions resulted in plasmid supercoil relaxation similar to that seen with wild-type. However, the low level of supercoiling in the mutant persisted during a time when supercoiling in wild-type recovered and then exceeded aerobic levels. Thus, changes in oxygen tension can alter DNA supercoiling through an effect on gyrase, and correlations exist between changes in supercoiling and changes in the intracellular ratio of [ATP]/[ADP].     

Structural maintenance of chromosomes protein of Bacillus subtilis affects supercoiling in vivo

Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo. We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant. Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.     

DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the tolC locus

The level of DNA supercoiling is crucial for many cellular processes, including gene expression, and is determined, primarily, by the opposing actions of two enzymes: topoisomerase I and DNA gyrase. Escherichia coli strains lacking topoisomerase I (topA mutants) normally fail to grow in the absence of compensatory mutations which are presumed to relax DNA. We have found that, in media of low osmolarity, topA mutants are viable in the absence of any compensatory mutation, consistent with the view that decreased extracellular osmolarity causes a relaxation of cellular DNA. At higher osmolarity most compensatory mutations, as expected, are in the gyrA and gyrB genes. The only other locus at which compensatory mutations arise, designated toc, is shown to involve the amplification of a region of chromosomal DNA which includes the tolC gene. However, amplification of tolC alone is insufficient to explain the phenotypes of toc mutants. tolC insertion mutations alter the distribution of plasmid topoisomers in vivo. This effect is probably indirect, possibly a result of altered membrane structure and an alteration in the cell's osmotic barrier. As tolC is a highly pleiotropic locus, affecting the expression of many genes, it is possible that some of the TolC phenotypes are a direct result of this topological change. The possible relationship between toc and tolC mutations, and the means by which tolC mutations might affect DNA supercoiling, are discussed.     

An Escherichia coli DNA topoisomerase I mutant has a compensatory mutation that alters two residues between functional domains of the DNA gyrase A protein.

Nucleotide sequence analysis revealed that the compensatory gyrA mutation in Escherichia coli DM750 affects DNA supercoiling by interchanging the identities of Ala-569 and Thr-586 in the DNA gyrase A subunit. These residues flank Arg-571, a site for trypsin cleavage that splits gyrase A protein between DNA breakage-reunion and DNA-binding domains. The putative interdomain locations of the DM750 mutation and that of E. coli DM800 (in gyrase B protein) suggests that these compensatory mutations may reduce DNA supercoiling activity by altering allosteric interactions in the gyrase complex.     

A novel locus conferring fluoroquinolone resistance in Staphylococcus aureus.

Fluoroquinolones such as ciprofloxacin and ofloxacin are potent antimicrobial agents that antagonize the A subunit of DNA gyrase. We selected and mapped a novel fluoroquinolone resistance gene on the Staphylococcus aureus chromosome. Resistant mutants were selected with ciprofloxacin or ofloxacin and were uniformly localized to the A fragment of chromosomal DNA digested with SmaI and arrayed by pulsed-field gel electrophoresis. Several mutants (cfxB, ofxC) were genetically mapped between the thr and trp loci in the A fragment. A majority of A fragment fluoroquinolone resistance mutations were associated with reduced susceptibility to novobiocin, an antagonist of the B subunit of DNA gyrase. Two genes previously associated with fluoroquinolone resistance, the gyrA gene of DNA gyrase and the norA gene (associated with decreased drug accumulation), were localized to the G and D fragments, respectively. Thus, the fluoroquinolone resistance mutations in the A fragment are distinct from previously identified fluoroquinolone resistance mutations in gyrA and norA. Whether mutations in the A fragment after a second topoisomerase or another gene controlling supercoiling or affect drug permeation is unknown.     

Increase in negative supercoiling of plasmid DNA in Escherichia coli exposed to cold shock

Negative supercoiling of plasmid DNA in Escherichia coli cells can decrease transiently when exposed to heat shock. The effect of cold shock on DNA supercoiling was examined, and analysis by agarose gel electrophoresis in the presence of chloroquine revealed that negative supercoiling of plasmid DNA in cells increased when cells were exposed to cold shock. This increase was transient and was nil when the cells were pretreated with nalidixic acid, an inhibitor of DNA gyrase. In a mutant deficient in expression of HU protein, the increase in negative supercoiling of DNA by cold shock is less apparent than in wild-type cells. It is proposed that DNA gyrase and HU protein have a role in the DNA supercoiling reaction seen with cold shock.     

DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling

Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon.     

The genetic control of DNA supercoiling in Salmonella typhimurium

We have elucidated the genetic control of DNA supercoiling in Salmonella typhimurium. The level of superhelix density is controlled by two classes of genes. The only member of the first class is topA, the structural gene for topoisomerase I. The second class, tos, (topoisomerase one suppressor) consists of at least two genes, one of which is linked to gyrA, the structural gene for the topoisomerase subunit of DNA gyrase. Deletions of topA result in oversupercoiling of plasmid DNA. These mutations do not require the acquisition of second-site compensatory mutations to allow cell growth, in contrast to the situation in Escherichia coli. However, tos mutations, unlinked to topA, have been isolated which reduce plasmid superhelix density. We conclude that the level of DNA supercoiling in S. typhimurium is a dynamic balance between the effects of the gene products of topA (relaxation) and tos (supercoiling) which act independently of each other. Using a variety of combinations of these mutations we have constructed a series of isogenic strains, each of which has a different but precisely defined level of plasmid supercoiling; the series as a whole provides a wide range of supercoiling both above and below the wild-type level.     

Escherichia coli DNA gyrase: genetic analysis of gyrA and gyrB mutations responsible for thermosensitive enzyme activity.

Escherichia coli gyrA43 and gyrB203 alleles conferring temperature-sensitive (ts) growth encoded Gly751-->Asp and Pro171-->Ser substitutions in the DNA gyrase A and B subunits, respectively. A plasmid-borne gyrA43 allele was genetically dominant over a chromosomal quinolone-resistant gyrA gene at 30 degrees C but not at 42 degrees C. These results and others confirm the ts phenotype of the mutation, the first to be identified in the C-terminal DNA binding/complex stabilizing domain of gyrase A protein. By contrast, the Pro171-->Ser mutation is located near the ATP-binding site of gyrase B protein and could interfere with energy coupling during DNA supercoiling. These data are discussed in regard to recently described gyrA(ts) mutations that affect the control of chromosome segregation.     

Mechanism of DNA gyrase-mediated illegitimate recombination: characterization of Escherichia coli gyrA mutations that confer hyper-recombination phenotype.

To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we isolated temperature-sensitive gyrA mutants that confer spontaneous illegitimate recombination and spontaneous induction of lambda prophage at higher frequencies than that in the wild-type. After reconstruction of single mutations by targeted mutagenesis, we confirmed that two single mutations, gyrAL492P and gyrAL488P, and a double mutation, gyrAI203V+gyrAI205V, show the same properties as those described above. With respect to the phenotypes of hyper-recombination and higher induction of lambda prophage, these mutations were dominant over the wild-type. Analysis of recombination junctions of lambdabio transducing phages formed spontaneously in these mutants showed that the parental E. coli bio and lambda recombination sites have a homologous sequence of only 0. 7 base-pair on average, indicating that homology is not required for this illegitimate recombination. Analysis of nucleotide sequences of mutant gyrA genes revealed that the gyrAL492P and gyrAL488P mutations contain amino acid substitutions of Leu492-->Pro and Leu488-->Pro, respectively, which correspond to the alpha18 helix in the breakage-reunion domain of DNA gyrase A subunit. The gyrAI203V and gyrAI205V mutations contain Ile203-->Val and Ile205-->Val, respectively, which correspond to the alpha10' helix, also in the breakage-reunion domain of DNA gyrase A subunit. Biochemical analysis indicated that the GyrA63 protein that contains the L492P mutation has an apparently normal supercoiling activity, but it also produces a small amount of linear DNA in the absence of DNA gyrase inhibitor during the supercoiling reaction, suggesting that the mutant DNA gyrase may have a defect at the step of religation or a defect in the subunit interaction. These results suggest that the recombination is induced by defects of religation and/or dimer formation in the mutant DNA gyrases, implying that two alpha helices, alpha10' and alpha18, of DNA gyrase A subunit have crucial roles in subunit interaction and/or resealing of DNA.     

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: mutations in the topA gene allow pSC101 replication in the absence of IHF.

Integration host factor (IHF), encoded by the himA and himD genes, is a histonelike DNA-binding protein that participates in many cellular functions in Escherichia coli, including the maintenance of plasmid pSC101. We have isolated and characterized a chromosomal mutation that compensates for the absence of IHF and allows the maintenance of wild-type pSC101 in him mutants, but does not restore IHF production. The mutation is recessive and was found to affect the gene topA, which encodes topoisomerase I, a protein that relaxes negatively supercoiled DNA and acts in concert with DNA gyrase to regulate levels of DNA supercoiling. A previously characterized topA mutation, topA10, could also compensate for the absence of IHF to allow pSC101 replication. IHF-compensating mutations affecting topA resulted in a large reduction in topoisomerase I activity, and plasmid DNA isolated from such strains was more negatively supercoiled than DNA from wild-type strains. In addition, our experiments show that both pSC101 and pBR322 plasmid DNAs isolated from him mutants were of lower superhelical density than DNA isolated from Him+ strains. A concurrent gyrB gene mutation, which reduces supercoiling, reversed the ability of topA mutations to compensate for a lack of him gene function. Together, these findings indicate that the topological state of the pSC101 plasmid profoundly influences its ability to be maintained in populations of dividing cells and suggest a model to account for the functional interactions of the him, rep, topA, and gyr gene products in pSC101 maintenance.