Changed properties of the A subunit in DNA gyrase with a B subunit mutation

Summary To investigate the interaction of subunits A and B of DNA gyrase during DNA supercoiling, a Cou^r mutant of Escherichia coli was obtained and the effect of nalidixic acid on the supercoiling of DNA by wild-type and mutant enzymes was assayed. The enzyme of the Cou^r strain proved to be more sensitive to nalidixic acid than the wild-type DNA gyrase. Hence the mutation affecting the B subunit can also change the properties of the A subunit, which fact suggests that the two subunits of DNA gyrase are in contact during DNA supercoiling.     

Regulation of DNA supercoiling in Escherichia coli: genetic basis of a compensatory mutation in DNA gyrase

Bacterial DNA supercoiling is controlled by balancing the supercoiling activity of DNA gyrase and the relaxing activity of DNA topoisomerase I. We have characterized the gyrB gene from a top A deletion mutant of Escherichia coli (DM800) that has a compensatory mutation in gyrB, lowering the activity of gyrase 10-fold, and thereby redressing the intracellular level of supercoiling. The mutant gene differs from the wild type in carrying three rather than two direct tandem repeats of a 6 bp sequence encoding Ala-Arg. We suggest this novel mutation affects domain spacing and was generated by an unequal crossing over event, possibly involving gyrase.     

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].     

An allele of gyrA prevents Salmonella enterica serovar Typhimurium from using succinate as a carbon source

A mutant gyrA allele resulting in an A271E substitution in the DNA gyrase protein generated a strain unable to grow on the C(4)-dicarboxylates succinate, malate, and fumarate. Bacteria harboring gyrA751 displayed decreased negative supercoiling in cells. Expression of the dctA gene, which encodes the C(4)-dicarboxylate transporter, was reduced in a gyrA751 mutant, providing the first evidence that dctA expression is supercoiling sensitive and uncovering a simple metabolic screen for lesions in gyrase that reduce negative supercoiling.     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

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.     

Risk assessment: the importance of genetic polymorphisms in man

Spontaneous mutation was greatly increased in a localized region of the chromosome of Haemophilus influenzae, but not at other loci, by a nov gene mutation called novC that increased DNA supercoiling. Another nov gene mutation, called novD, decreased spontaneous mutation in the same localized region and depressed DNA supercoiling. Both mutations, which code for the gyrase B subunit, have been cloned, and the cloned versions also altered mutagenesis and supercoiling in a similar fashion as the two mutations on the chromosome, although novC on the plasmid caused somewhat less mutation than on the chromosome. We postulate that the effects of the gyrase B mutations on the chromosome result from their effects on supercoiling because of increased gyrase near its site of production. The fact that the novC on a plasmid does not cause mutagenesis except in the same localized region that is altered by this mutation on the chromosome, is difficult to explain. One possibility is that there is a complex of proteins in this region which is necessary for the effects on supercoiling and thus, also on mutagenesis.     

Role of the histone-like proteins OsmZ and HU in homologous recombination.

The HU protein of Escherichia coli has been implicated in various site-specific recombination reactions. Moreover, recent data suggest that HU may also participate in homologous recombination. In particular, it has been shown that P1 transduction is inhibited in the absence of HU [Kano and Imamoto, Gene 89 (1990) 133-137]. In contrast, we found that transductional recombination and conjugational recombination were almost normal in hupA hupB mutants. However, it appeared that the recombination proficiency of hupA hupB mutant bacteria was reduced tenfold in an intrachromosomal recombination assay. Moreover, we found that intrachromosomal recombination was reduced tenfold in a gyrB226 strain and by more than 100-fold in an osmZ205 strain. The gyrB226 mutation affects the DNA gyrase activity, while mutations in osmZ are highly pleiotropic, affecting the expression of a variety of genes and increasing the frequency of site-specific inversion events. Since it has been shown that the hupA hupB mutations, like the gyrB226 mutation, decrease the level of DNA supercoiling, whereas the osmZ205 mutation increases the level of DNA supercoiling, it appears that the histone-like proteins HU and OsmZ may play a key role in intrachromosomal recombination by affecting the DNA topology.     

Modulation of DNA supercoiling activity of Escherichia coli DNA gyrase by F plasmid proteins. Antagonistic actions of LetA (CcdA) and LetD (CcdB) proteins.

The letA (ccdA) and letD (ccdB) genes of F plasmid contribute to stable maintenance of the plasmid in Escherichia coli cells; a product of the latter has a lethal effect on the host cell and that of the former neutralizes functions of the letD. In cells that overproduce the LetD (CcdB) protein, the plasmid DNA is extensively relaxed. Correspondingly, DNA supercoiling activity in a cell-free extract of the overproducing strain decreases to a level of less than 1% of that seen in normal cells. However, the extract does not inhibit DNA gyrase reconstituted from purified subunits, thereby indicating that the intrinsic DNA gyrase is inactivated in the overproducing strain. Upon addition of purified LetA (CcdA) protein to the extract of LetD overproducing cells, the DNA supercoiling activity was fully restored. Using this rejuvenation as an assay, we purified the "inactivated gyrase" and obtained evidence that the LetD protein formed an isolable complex with the A subunit of DNA gyrase. Thus, the LetD and the LetA proteins constitute an opposing pair in modulating the DNA supercoiling activity of gyrase, probably by direct interaction.     

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.     

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.     

Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress

DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4–5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic‐membrane‐located inhibitor of proton‐driven F₁F₀ ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin‐resistant (Nov^R) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with Nov^R gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug‐treated bacteria. The Salmonella cytosol reaches pH 5–6 in response to an external pH of 4–5: the ATP‐dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP‐dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid‐mediated impairment of the negative supercoiling activity of gyrase.     

Role of HU proteins in forming and constraining supercoils of chromosomal DNA inEscherichia coli

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12–13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying bothgyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.