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.     

Characterization of a combined DNA initiation and cell division mutant of Bacillus subtilis.

Summary The temperature-sensitive mutation in Bacillus subtilis 168-134ts, a conditional lethal DNA initiation mutant, was transferred to the minicell producing strain, CU 403 div IV-B1, to study he relationship of DNA synthesis to cell division. Markers in the combined mutant were verified by transduction. DNA replication kinetics, genome location by autoradiography, and clonal analysis of cell division patterns during spore outgrowths were investigated. Growth of the double mutant at the restrictive temperature results in an impressive reduction of the percentage cell length covered by DNA grain clusters (60.2% at 30° C compared to 8.6% after 2 h at 45° C). The probability of a minicell producing division in double mutant clones is essentially the same at 30° C and during the initial 2–3 h growth at 45° C at which time lysis begins. Residual division at 45° C is attributable to processes initiated at 30° C. The CU 403 div IV-B1, 134ts, double mutant divides about 25% as frequently relative to growth as do wild type CU 403 clones when incubated at permissive temperature. This is approximately 15% greater division suppression than previously found in the CU 403 div IV-B1 mutant strain, and is presumably due to interactions of the mutant gene products both of which affect DNA.     

Thermoregulation in Yersinia enterocolitica is coincident with changes in DNA supercoiling

Yersinia enterocolitica is a facultative intracellular parasite, displaying the ability to grow saprophytically or invade and persist intracellularly in the mammalian reticuloendothelial system. The transition between such diverse environments requires the co-ordinated regulation of specific sets of genes on both the chromosome and virulence plasmid. Temperature has a profound pleiotropic effect on gene expression and phenotypically promotes alterations in cell morphology, outer-membrane protein synthesis, urease production, lipopolysaccharide synthesis, motility, and synthesis of genes involved in invasion of euKaryotic host cells. By examining thermoregulated flagella biosynthesis, we have determined that motility is repressed at 25° C (permissive temperature) with subinhibitory concentrations of novobiocin. These conditions also induce virulence gene expression suggesting novobiocin addition stimulates, at least partially, a high-temperature environment. Furthermore, temperature-shift experiments, using Y. enterocolitica containing pACYC184 as a reporter plasmid, indicate that thermo-induced alterations of DNA supercoiling coincide with temperature-induced phenotypic changes. A class of putative DNA gyrase mutant (novobiocin resistant) likewise demonstrates the 37° C phenotype when cultured at 25°C; it is non-motile, urease negative, calcium growth dependent, and positive for Yop expression. These results support a model implicating DNA topology as a contributing factor of Y. enterocolitica thermoregulation.     

Genetic recombination of homologous plasmids catalysed by cell-free extracts of topo-isomerase mutant strains of Saccharomyces cerevisiae

Cell-free extracts of the yeast Saccharomyces cerevisiae can be used to catalyse the recombination of bacterial plasmids in vitro. Recombination between homologous plasmids containing different mutations in the gene encoding tetracycline resistance is detectable by the appearance of tetracycline-resistance following transformation of the recombinant plasmid DNA into Escherichia coli DH5. This in vitro recombination system was used to determine the involvement of eukaryotic topo-isomerases in genetic recombination. Cell-free extracts prepared from a temperature-sensitive topo-isomerase II mutant (top2-1) of S. cerevisiae yielded tetracycline-resistant recombinants, when the recombination assays were performed at both a non-restrictive temperature (30°C) and the restrictive temperature (37°C). This result was obtained whether or not ATP was present in the recombination buffer. Extracts from a non-conditional topo-isomerase I mutant (top1-1) of S. cerevisiae yielded tetracycline-resistant recombinants, as did a temperature-sensitive double mutant (top2-1/top1-8) at the restrictive temperature. The results of this study indicate that neither topo-isomerase I nor topo-isomerase II was involved in the recombinational activity examined.     

Initiation of DNA replication in Bacillus subtilis. V. Role of DNA gyrase and superhelical structure in initiation

Summary When spores of a thymine-requiring mutant of Bacillus subtilis were germinated in a medium lacking thymine, an initiation potential (an ability to initiate and complete one round of replication in the presence of thymine and in the absence of protein and RNA synthesis) was formed for both chromosomal and plasmid replication. The effect of two inhibitors of DNA gyrase, novobiocin (Nov) and nalidixic acid (Nal), on the initiation potential formed during germination for chromosomal and plasmid replication was examined.Nov and Nal inhibited formation of the initiation potential completely if the drug was added at the onset of germination. In contrast, initiation of chromosomal and plasmid replication occurred in the presence of DNA gyrase inhibitors when the drug was added after the initiation potential had been fully formed. However, chromosomal replication initiated in the presence of the inhibitors ceased after a fragment of approximately 15 MD (15×10⁶ daltons) had been replicated, and plasmid replication was limited to one round of replication in approximately half of the plasmid molecules present in the spores.Furthermore the initiation potential for both chromosomal and plasmid replication though established was destroyed gradually but steadily by prolonged incubation with Nov in the absence of thymine. In addition, relaxation of the superhelical structure of plasmid DNA during incubation with Nov was observed in vivo. This relaxation was blocked by ethidium bromide, which dissociated the S-complex. On the other hand, incubation with Nal did not reduce the initiation potential nor did it change the superhelicity of the plasmid DNA in vivo. This is consistent with the known effect of gyrase inhibitors on the enzymatic activity of DNA gyrase.These results clearly demonstrate that both the action of DNA gyrase and the superhelical structure of the DNA are essential for the initiation of chromosomal and plasmid replication. The specific chromosome organization essential for initiation and elongation and the role of DNA gyrase are discussed.IV of this series is Yoshikawa et al. 1980     

Enhancement of ribosomal ribonucleic acid synthesis by deoxyribonucleic acid gyrase activity in Escherichia coli.

The effect of the deoxyribonucleic acid (DNA) gyrase inhibitors coumermycin A1, novobiocin, and oxolinic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was studied in vivo and in vitro. Preferential inhibition of ribosomal RNA (rRNA) synthesis was observed. No effect of oxolinic acid and coumermycin on rRNA synthesis was seen in mutants having a DNA gyrase which is resistant to these inhibitors. In a temperature-sensitive DNA gyrase mutant rRNA synthesis was decreased at nonpermissive temperatures. Thus, a functional DNA gyrase is required for rRNA synthesis. Purified DNA gyrase had no effect on rRNA synthesis in a purified system. However, DNA gyrase does show preferential stimulation of rRNA synthesis in a system supplemented with other proteins. Apparently, DNA gyrase stimulation of rRNA synthesis requires another protein.     

A plasmid vector with a selectable marker for halophilic archaebacteria.

A mutant resistant to the gyrase inhibitor novobiocin was selected from a halophilic archaebacterium belonging to the genus Haloferax. Chromosomal DNA from this mutant was able to transform wild-type cells to novobiocin resistance, and these transformants formed visible colonies in 3 to 4 days on selective plates. The resistance gene was isolated on a 6.7-kilobase DNA KpnI fragment, which was inserted into a cryptic multicopy plasmid (pHK2) derived from the same host strain. The recombinant plasmid transformed wild-type cells at a high efficiency (greater than 10(6)/micrograms), was stably maintained, and could readily be reisolated from transformants. It could also transform Halobacterium volcanii and appears to be a useful system for genetic analysis in halophilic archaebacteria.     

Effect of nalidixic acid and novobiocin on pBR322 genetic expression in Escherichia coli minicells.

The effects of two deoxyribonucleic acid (DNA) gyrase inhibitors, nalidixic acid and novobiocin, on the gene expression of plasmid pBR322 in Escherichia coli minicells were studied. Quantitative estimates of the synthesis of pBR322-coded polypeptides in novobiocin-treated minicells showed that the synthesis of a polypeptide of molecular weight of 34,000 (the tetracycline resistance protein) was reduced to 11 to 20% of control levels, whereas the amount of a polypeptide of 30,500 (the beta-lactamase precursor) was increased to as much as 200%. Nalidixic acid affected the synthesis of the tetracycline resistance protein similarly to novobiocin, although to a lesser extent. The effects of nalidixic acid were not observed in a nalidixic-resistant mutant; those induced by novobiocin were only partially suppressed in a novobiocin-resistant mutant. The synthesis of one of the inducible tetracycline-resistant proteins (34,000) coded by plasmid pSC101 was also reduced in nalidixic acid- and novobiocin-treated minicells. These results suggest that the gyrase inhibitors modified the interaction of ribonucleic acid polymerase with some promoters, either by decreasing the supercoiling density of plasmid DNA or by altering the association constant of the gyrase to specific DNA sites.     

DNA Supercoiling and Osmoresistance in Bacillus subtilis 168

The importance of the DNA structure for the expression of the osmotic response (osmotolerance) was investigated in Bacillus subtilis 168. Plasmid pUB110 DNA was used as a reporter of the chromosomal DNA topology, and analyses were performed in chloroquine agarose gels. Plasmidic DNA obtained from cultures in Schaeffer medium (D) taken in those periods in which B. subtilis is able to express osmotolerance (early stationary phase or from germinating spores) or from adapted cultures to hyperosmotic medium (DN) presented a higher level of negative supercoiling than DNA samples from vegetative cultures, normally refractory to induction of osmotolerance. The involvement of the DNA gyrase was investigated through the sensitivity to novobiocin, an antibiotic inhibitor of its activity and the behavior of a gyrB1 mutant strain (RG1). In the wild-type strain, the addition of a sublethal concentration of novobiocin (0.5 μg/ml) to the hyperosmotic medium relaxed DNA and inhibited growth. Moreover, already growing cultures in DN medium and later submitted to the same antibiotic presented a relaxed DNA and stopped growing. The RG1 mutant strain submitted to similar novobiocin treatments displayed normal growth in DN novobiocin medium. These results pointed to the requirement of a highly negative supercoiled DNA structure involving the gyrase activity in osmotic response. Received: 9 May 1997 / Accepted: 18 June 1997     

Tn1 insertion mutagenesis in Escherichia coli K-12 using a temperature-sensitive mutant of plasmid RP4

Summary A method for Tn1 insertion mutagenesis in Escherichia coli has been developed using pTH10, a mutant plasmid of RP4 temperature-sensitive for maintenance. The mutagenesis involves three steps. Firstly, from strains carrying pTH10 showing resistance to the antibiotics kanamycin, tetracycline, and ampicillin at 30° C but not at 42° C, clones are isolated resistant to kanamycin at 42° C. Such temperature-independent, drug resistant clones probably carry pTH10 integrated into the host chromosome. Secondly, they are cultivated at 30° C. At this temperature segregants carrying pTH10, which has been excised from the host chromosome, accumulate. Thirdly, to cure such segregants of autonomous pTH10, they are cultivated at 42° C. By these procedures, clones free of pTH10, but carrying Tn1 insertions on the host chromosome, were obtained.About 3% of the clones carrying Tn1 insertions were auxotrophic. Distribution of auxotrophic mutations was not random, indicating the existence of preferential integration sites of Tn1 on the host chromosome. The frequency of precise excision of Tn1 was less than 10^-10.The pTH10 plasmid has a wide host range among Gram-negative bacteria and thus may serve as a excellent vector for insertion mutagenesis of Tn₁ in many Gram-negative bacterial species.     

Mutagenesis of plasmid DNA with hydroxylamine: Isolation of mutants of multi-copy plasmids

Summary An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described. The treated plasmid DNA was used to transform Escherichia coli K12. Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised. They were classified according to the reduction in level of their -lactamase activity. Hydroxylamine-induced mutants of NTP14 were also isolated. This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes. One class of mutants is lethal to the host strain at temperatures above 33° C, but carrier strains grow well at 28° C. There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures. The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.     

Possible involvement of the ugpA gene product in the stable maintenance of mini-F plasmid in Escherichia coli

Summary The seg-3 mutant Escherichia coli does not support the maintenance of mini-F plasmid at 42° C. We cloned the chromosomal DNA segment of the wild-type strain W3110 that complements the Seg^– phenotype of this mutant. Cleavage mapping of this segment showed that it was derived from the 76-min region of the E. coli chromosome map. Complementation tests using plasmids carrying subcloned DNA segments suggested that the seg-3 mutant carried two mutations that additively affected the maintenance of mini-F plasmid; one was in the ugpA gene and the other was presumably in the rpoH gene. We generated a disrupted ugpA null mutant and found that the mini-F plasmid was unstable in this ugpA null mutant even at 30° C. This suggests that the ugpA gene product is required for the stable maintenance of mini-F plasmid.     

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.     

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.     

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.     

The RAD3 gene of Saccharomyces cerevisiae: isolation and characterization of a temperature-sensitive mutant in the essential function and of extragenic suppressors of this mutant

Summary Mutations in the RAD3 gene of Saccharomyces cerevisiae were generated by integration of a mutagenized incomplete copy of the cloned gene into wild-type cells. Integrants were mass screened for colonies with abnormal growth characteristics at 37°C. A single temperature-sensitive mutant (rad3ts-1) was isolated and was shown to result from a missense mutation at codon 73 of the RAD3 gene. When shifted from 30° C to 37° C the strain undergoes only 2–4 cell doublings. This phenotype can be rescued by plasmids in which the essential function of the cloned RAD3 gene is intact, but not plasmids in which this function is inactivated. The mutant strain is weakly sensitive to ultraviolet (UV) radiation at restrictive temperatures. Measurement of RNA, DNA and protein synthesis at various times after shifting to restrictive temperatures does not show preferential inactivation of any one of these parameters and the temperature-sensitive mutation does not cause arrest at any specific phase of the cell cycle. The rad3ts-1 strain was transformed with multicopy plasmids from a normal yeast genomic library and two plasmids that partially suppress the temperature-sensitive phenotype were isolated. These suppressor genes (designated SRE1 and SRE2) are distinct from RAD3 and do not suppress the phenotype of several other temperature-sensitive mutants tested. Mutant strains carrying disruptions of the SRE1 gene are viable and are not sensitive to UV or radiation.     

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.     

Variable expression of the ssb-1 allele in different strains of Escherichia coli K12 and B: Differential suppression of its effects on DNA replication, DNA repair and ultraviolet mutagenesis

Summary We have transduced the mutant allele ssb-1, which encodes a temperature-sensitive single-strand DNA binding protein (SSB), into several Escherichia coli strains, and have examined colony-forming ability, DNA replication, sensitivity to ultraviolet light (UV) and UV-induced mutability at the nonpermissive temperature. We have found: 1) that the degree of ssb-1-mediated temperature-sensitivity of colony-forming ability and of DNA replication is strain-dependent, resulting in plating efficiencies at 42° C (relative to 30° C) ranging from 100% to 0.002%; 2) that complete suppression of the temperature-sensitivity caused by ssb-1 occurs only on nutrient agar, and not in any other medium tested; 3) that strains in which ssb-1-mediated temperature-sensitivity is completely suppressed show moderate UV sensitivity and normal UV mutability at 30° C, but much more extreme UV sensitivity and drastically reduced UV mutability at 42° C; and 4) that defects in excision repair or in other Uvr⁺-dependent processes are not responsible for most of the UV sensitivity promoted by ssb-1. We discuss our results in relation to the known properties of SSB and its possible role in the induction of DNA damage-inducible (SOS) functions.