Biomedical subjects

K Sekimizu

Publications and source records attributed to K Sekimizu.

At least 37 records · Page 2Linked to original sources

PubMed1998

A stimulation factor for hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli.

Hydrolysis of ATP bound to DnaA protein by its intrinsic ATPase activity negatively controls chromosomal DNA replication in Escherichia coli. We developed a new in vitro assay system for ATP hydrolysis, which makes feasible a search for factors affecting the ATPase activity of DnaA protein. A crude cell extract enhanced the hydrolysis of ATP bound to DnaA protein, in a dose-dependent manner. Gel-filtration analyses revealed a single entity of the stimulation factor for the ATP hydrolysis and an apparent molecular mass of 170 kDa. The stimulation activity for ATP hydrolysis coeluted with the inactivation activity for DnaA protein initiating an oriC DNA replication, as determined by anion-exchange and gel-filtration column chromatographies. Activity of the stimulation factor required DNA and ATP. These observations suggested that IdaA protein, a previously described negative factor for DnaA protein, inactivated DnaA protein through stimulation of the hydrolysis of ATP bound to DnaA protein.

Adenosine Diphosphate↗

PubMed1998

Effect of glycerol on the affinity of DnaA protein for ATP in the presence of cardiolipin.

Acidic phospholipids, such as cardiolipin, decrease the affinity of DnaA protein for adenine nucleotides and can activate the inactive form of DnaA protein in vitro. In this study, we examined the effect of glycerol on the affinity of DnaA protein for ATP in the presence of cardiolipin. High concentrations of glycerol (34%) restored the affinity of DnaA protein for ATP, which was decreased by cardiolipin. Glycerol inhibited the binding of cardiolipin with DnaA protein. Glycerol had little effect on membrane fluidity, which is essential for the interaction between cardiolipin and DnaA protein, whereas it increased the Kd value of DnaA protein for ATP in the absence of cardiolipin. These results suggest that glycerol causes DnaA protein to become insensitive as to the interaction with cardiolipin by changing the conformation of the protein without altering the physical nature of the phospholipid.

Adenosine Triphosphate↗

PubMed1998

Identification of proteins whose amounts are altered by mutation in the pgsA gene of Escherichia coli.

We identified proteins whose amounts were altered in an Escherichia coli pgsA3 mutant lacking the potential to synthesize phosphatidylglycerolphosphate, a precursor of phosphatidylglycerol. Proteins whose amounts were increased in the mutant were protease Do, periplasmic oligopeptide-binding protein, tryptophanase, and an unidentified protein, while the decreased one was flagellin. Transformation of the mutant with a plasmid containing the wild type pgsA gene complemented the phenotype, indicating that the pgsA3 mutation is responsible for the phenotype.

Amino Acid Sequence↗

PubMed1997

Heat shock-induced excessive relaxation of DNA in Escherichia coli mutants lacking the histone-like protein HU.

Plasmid DNA in exponentially growing Escherichia coli immediately relaxes after heat shock but the relaxed DNA re-supercoils rapidly, the despite continued presence of the heat shock conditions. We have now obtained genetic evidence indicating that the histone-like protein HU of E. coli is required for this re-supercoiling of DNA. Plasmid DNA in a hupA-hupB double gene-disruption mutant relaxed excessively after heat shock, while the relaxation of DNA in a himA-himD double gene-disruption mutant and in an hns insertion mutant was transient, thereby indicating that HU protein, but not IHF or H-NS proteins, is required for the re-supercoiling of DNA. Exposure of the hupA-hupB double mutant to a temperature of 50 degrees C led to both excessive relaxation of DNA and to a decrease in viable cell number but temperatures lower than 46 degrees C did not lead to these events. Based on these results, we propose that HU protein maintains the negative supercoiling of DNA during thermal stress and contributes to cellular thermotolerance in E. coli.

Bacterial Proteins↗

PubMed1997

Inhibition of thymidine transport in dnaA mutants of Escherichia coli.

DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. We report here our evidence that thymidine transport across cytoplasmic membranes in temperature-sensitive dnaA mutants is greatly decreased at a permissive temperature for growth of the mutants. Complementation analysis with a plasmid containing the wild type dnaA gene and P1 phage-mediated transduction confirmed that mutations in the dnaA gene were responsible for the phenotype. A low level of nucleoside transport in the dnaA mutant was specific for thymidine; transport activities for other nucleosides were much the same as those in wild type cells. Membrane vesicles prepared from the dnaA mutant showed much the same activity of thymidine transport as did those from the wild type cells. No significant difference in the activity of thymidine kinase, which is known to facilitate thymidine transport, was seen between the mutant and the wild type cells. An increase in the pool of dTTP, a negative regulator for thymidine kinase, was observed in the dnaA mutant. Based on these observations, we suggest that inhibition of thymidine transport in dnaA mutants is caused by increases in the dTTP pool.

Bacterial Proteins↗

PubMed1997

Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli.

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by ATP. ATP bound to DnaA protein is slowly hydrolyzed to ADP, but the physiological role of ATP hydrolysis is unclear. We constructed, by site-directed mutagenesis, mutated DnaA protein with lower ATPase activity, and we examined its function in vitro and in vivo. The ATPase activity of purified mutated DnaA protein (Glu204-->Gln) decreased to one-third that of the wild-type DnaA protein. The mutation did not significantly affect the affinity of DnaA protein for ATP or ADP. The mutant dnaA gene showed lethality in wild-type cells but not in cells growing independently of the function of oriC. Induction of the mutated DnaA protein in wild-type cells caused an overinitiation of DNA replication. Our results lead to the thesis that the intrinsic ATPase activity of DnaA protein negatively regulates chromosomal DNA replication in E. coli cells.

Adenosine Diphosphate↗

PubMed1997

Immotile phenotype of an Escherichia coli mutant lacking the histone-like protein HU.

The histone-like protein HU in Escherichia coli are encoded by the hupA and hupB genes. A hupA-hupB double deletion mutant has now been shown to express an immotile phenotype. The motility of hupA or hupB single mutants was similar to that of wild-type cells. SDS-polyacrylamide gel electrophoresis revealed that the amount of flagellin in the hupA-hupB double deletion mutant was markedly reduced compared with the wild-type strain suggesting that the immotile phenotype of the double deletion mutant is caused by a loss of flagella.

Bacterial Outer Membrane Proteins↗

PubMed1997

Amounts of proteins altered by mutations in the dnaA gene of Escherichia coli.

We identified proteins whose amounts were altered in a temperature-sensitive dnaA46 mutant of Escherichia coli. Proteins whose amounts were increased in the mutant were serine hydroxymethyltransferase, beta-ketoacyl [acyl carrier protein] synthase II, long-chain fatty acid transport protein, and UDP-glucose 4-epimerase, while the decreased ones were flagellin and D-ribose-binding protein. Transformation of the mutant with a plasmid containing the wild type dnaA gene complemented the phenotype. As pulse-labeling experiments revealed that the rates of synthesis of the proteins were altered in the mutant, DnaA protein may be involved in expression of these proteins.

Amino Acid Sequence↗

PubMed1997

Conformational transition of DnaA protein by ATP: structural analysis of DnaA protein, the initiator of Escherichia coli chromosome replication.

DnaA protein binds to the chromosomal origin (oriC) to initiate DNA replication. We developed an efficient system for purification of DnaA protein which will facilitate physicochemical analysis of the protein. The yield of DnaA protein was increased at least 6-fold compared to an available method being used, and over 22 mg of the protein were obtained from only 100 g of cells. DnaA protein purified by this procedure showed an indistinguishable affinity for ATP, and activity for in vitro replication of oriC plasmid. The process of denaturation of DnaA protein, which was blocked by ATP, was monitored by intrinsic fluorescence and circular dichroism. Analysis of circular dichroism revealed that DnaA protein is rich in alpha-helices, and that ATP-binding leads to a significant transition of protein conformation in that the content of alpha-helices is decreased. This is the first evidence indicating that ATP-binding profoundly affects conformation of DnaA protein.

Adenosine Triphosphate↗

PubMed1997

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.

Bacterial Proteins↗

PubMed1997

Overinitiation of chromosome replication in the Escherichia coli dnaAcos mutant depends on activation of oriC function by the dam gene product.

The activity of DnaA protein, the initiator of chromosome replication in Escherichia coli, is regulated by adenine nucleotide binding; the ATP-bound form, not the ADP-bound form, is active. DnaAcos is a mutant protein that is insensitive to negative regulation by ADP. Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30 degrees C, a restrictive temperature for growth. To determine the control factors that act independently of adenine nucleotide binding of DnaA, we analysed suppressors from the dnaAcos mutant isolated by Tn5 insertion mutagenesis. Three of the suppressors carried Tn5 in the aroK or aroB gene, the first two cistrons in the dam operon. Complementation tests revealed that the dam gene is responsible for the suppression. Over-replication of the chromosome was inhibited in the dnaAcos aroK::Tn5 double mutant, and initiation of chromosome replication in the dnaA+ aroK::Tn5 mutant was partially inhibited. The aroK(or B)::Tn5 cells contained DnaA molecules at a level similar to that in the parental aroBK+ strain. Moreover, dnaAcos suppression depended on the function of the seqA gene. Thus, Dam activity positively regulates initiation of chromosome replication in vivo. SeqA function seems to be distinguished from the control of DnaA protein by adenine nucleotide binding.

Adenosine Diphosphate↗

PubMed1997

CedA is a novel Escherichia coli protein that activates the cell division inhibited by chromosomal DNA over-replication.

We isolated and characterized a new gene related to the control of cell division regulation in Escherichia coli. At 30 degrees C, the dnaAcos mutant causes over-replication of the chromosome, and colony formation is inhibited. We found that, at this temperature, the dnaAcos cells form filaments; therefore, septum formation is inhibited. This inhibition was independent of SfiA, an inhibitor of the septum-forming protein, FtsZ. To identify factors involved in this pathway of inhibition, we isolated seven multicopy suppressors for the cold-sensitive phenotype of the dnaAcos mutant. One of these proved to be a previously unknown gene, which we named cedA. This gene encoded a 12 kDa protein and resided at 38.9min on the E. coli genome map. A multicopy supply of the cedA gene to the dnaAcos cells did not repress over-replication of the chromosome but did stimulate cell division of the host, the result being growth of cells with an abnormally elevated chromosomal copy number. Therefore, the expression level of the cedA gene seems to be important for inhibiting cell division of the dnaAcos mutant at 30 degrees C. We propose that over-replication of the chromosome activates a pathway for inhibiting cell division and that the cedA gene modulates this division control. In the dnaA+ background, cedA also seems to affect cell division.

Amino Acid Sequence↗

PubMed1997

Induction of DnaK and GroEL heat shock proteins by fluoroquinolones in Escherichia coli.

Various fluoroquinolones (norfloxacin, enoxacin, ofloxacin, levofloxacin, and sparfloxacin) induce DnaK and GroEL heat shock proteins in Escherichia coli. The induction is transient, consistent with the kinetics of cellular DNA relaxation. The concentrations of fluoroquinolones required for induction are similar to those required for DNA relaxation and much higher than those required for cell death.

Anti-Infective Agents↗

PubMed1997

Psychotropic drugs and local anesthetics restore cardiolipin-inhibited mammalian DNA topoisomerase I activity.

The activity of mammalian topoisomerase I is inhibited by acidic phospholipids. We investigated the effect of psychotropic drugs and local anesthetics on cardiolipin-inhibited calf thymus DNA topoisomerase I activity. Chlorpromazine, promethazine, and imipramine, which interact with phospholipids, suppressed the inhibitory effect of cardiolipin. The present results suggest that relaxation of DNA supercoiling is involved in the cytotoxic action of these drugs.

Anesthetics, Local↗

PubMed1997

Decrease in expression of the master operon of flagellin synthesis in a dnaA46 mutant of Escherichia coli.

We examined the expression of the fliC, fliA and fihD genes in the dnaA46 mutant. In Northern blot analysis, the amounts of fliC, fliA, and flhD mRNA decreased in the dnaA46 mutant growing at 37 degrees C but not at 28 degrees C. The promoter activity of each gene also decreased in this mutant at 37 degrees C. The decrease in expression of the flh D gene was not related to the cAMP-catabolite activator protein (CAP) pathway. We tentatively conclude that DnaA protein is involved in the expression of the flhD gene by a mechanism independent of the cAMP-CAP pathway.

Blotting, Northern↗

PubMed1997

Transient DNA relaxation in Escherichia coli induced by nalidixic acid.

We report here the transient relaxation of plasmid DNA by inhibitors of DNA gyrase in Escherichia coli. Relaxation of plasmid DNA in the presence of nalidixic acid, an inhibitor of the A subunit of DNA gyrase, lasted less than 60 min. The effect of nalidixic acid was not observed in a nalA26 mutant, a strain resistant to nalidixic acid due to a mutation in the gyrA gene. Novobiocin, whose target is the B subunit of DNA gyrase, also caused transient DNA relaxation. Resupercoiling of relaxed DNA in the presence of nalidixic acid was inhibited by pretreatment of the cells with chloramphenicol. As mutations of both the rpoH and recA genes did not affect the resupercoiling reaction, this step seems to require protein synthesis that is independent of both heat shock and SOS responses.

Bacterial Proteins↗

PubMed1997

Alteration of fatty acid composition in a pgsA3 mutant of Escherichia coli.

We examined the fatty acid composition in an Escherichia coli pgsA3 mutant lacking the potential to synthesize phosphatidylglycerolphosphate, a precursor of phosphatidylglycerol. The contents of C18:1cis-9 (oleic acid) and C18:1cis-11 (cis-vaccenic acid) in the total phospholipids extracted from the pgsA3 mutant growing at 37 degrees C were higher and that of C14:0 was lower than the wild type cells, resulting in a higher level of unsaturation of fatty acids (ratio of unsaturated fatty acids to saturated ones) in the mutant. The higher level of the unsaturated fatty acids in the pgsA3 mutant was more obvious in cardiolipin than in phosphatidylethanolamine. On the other hand, at 28 degrees C, at which the pgsA3 mutant shows limited cell growth, the content of unsaturated fatty acids in cardiolipin decreased in the pgsA3 mutant compared with the wild type. We consider that the pgsA3 mutant maintains cellular homeostasis by altering the level of unsaturated fatty acids in cardiolipin, and the mechanism is influenced by temperature.

Escherichia coli↗

PubMed1997

Molecular cloning and sequence analysis of the dnaA gene of Staphylococcus aureus.

The dnaA gene of Staphylococcus aureus has now been cloned and sequenced. The deduced amino acid sequence of Staphylococcus aureus DnaA protein is 62% and 39% identical to those of the DnaA protein from Bacillus subtilis and Escherichia coli, respectively. Expression of the dnaA gene of Staphylococcus aureus in Escherichia coli caused lethality in an oriC-dependent manner.

Amino Acid Sequence↗