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Biomedical subjects

M Wachi

Publications and source records attributed to M Wachi.

At least 55 records · Page 3Linked to original sources
PubMed1995

Anucleate cell production by Escherichia coli delta hns mutant lacking a histone-like protein, H-NS.

Normal-sized anucleate cells were observed in the cultures of a delta hns mutant strain. Even in nucleate cells, some populations showed irregular intracellular localization of the nucleoids. The delta hns mutant showed reduced ploidy, although initiation of chromosome replication was essentially synchronous as defined by flow cytometric analysis. These results indicate that the delta hns mutant is defective in the mechanisms of chromosome partitioning and chromosome replication.

Bacterial Outer Membrane Proteins↗
PubMed1994

Cytoplasmic axial filaments in Escherichia coli cells: possible function in the mechanism of chromosome segregation and cell division.

Overproduction of CafA caused formation of chained cells and minicells. The cafA gene is located downstream from the mre region at 71 min on the Escherichia coli chromosome map and was previously called orfF. A long axial structure running through the chained cells, consisting of bundles of filaments assembled in a long hexagonal pillar several micrometers long and about 0.1 to 0.2 micron in diameter, was visible in both phase-contrast micrographs of the lysozyme-treated cells and electron micrographs of ultrathin sections. The CafA protein displays 34% amino acid similarity with the N terminus of the Ams protein of E. coli, which cross-reacts with antibody to a nonmuscle myosin heavy chain.

Bacterial Proteins↗
PubMed1993

Fully methylated oriC with negative superhelicity forms an oriC-membrane complex before initiation of chromosome replication.

In an in vitro assay, the oriC DNA has been shown to bind to the outer membrane fraction only when it is hemimethylated (G.B. Ogden et al., Cell, 54, 127-135,1988). In this report, however, we demonstrated that a significant amount of the oriC DNA was recovered from the cells just before initiation with the oriC DNA being fully methylated. Formation of this preinitiation oriC-membrane complex and following initiation of chromosome replication were strongly inhibited by novobiocin, a DNA gyrase B subunit inhibitor, which reduced the superhelicity of the reporter plasmid in the cells. On the other hand, both reactions proceeded in the presence of nalidixic acid, a DNA gyrase A subunit inhibitor, which did not have the effect of reducing the superhelicity. These results suggest that the negative superhelicity of the DNA is required for preinitiation oriC-membrane complex formation and following initiation event of replication.

Cell Compartmentation↗
PubMed1992

ATPase activity of SopA, a protein essential for active partitioning of F plasmid.

The sopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli. In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied. [alpha-32P]ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs. In contrast, no ATP binding activity was detected for the SopB protein. The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA. The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein. However, the SopB protein alone failed to stimulate the ATPase activity.

Adenosine Triphosphatases↗
PubMed1992

The 55-kilodalton protein in an oriC complex fraction is glycogen synthase.

Three proteins with molecular masses of 35, 55, and 75 kDa were found in an oriC complex fraction after purification through CsCl density gradient centrifugation (W. G. Hendrickson, T. Kusano, H. Yamaki, R. Balakrishnan, M. King, J. Murchie, and M. Schaechter, Cell 30:915-923, 1982). Of these three proteins, the 55-kDa protein was determined to be glycogen synthase on the basis of the N-terminal amino acid sequence and the molecular weight. The oriC complex was formed in glgA mutant cells, which produce no detectable glycogen, as well as in wild-type cells. None of the 35-, 55-, and 75-kDa proteins were detected in the fraction from this mutant. The results indicate that these proteins were not constituents of the oriC complex.

Bacterial Proteins↗
PubMed1992

Escherichia coli mraR gene involved in cell growth and division.

The mraR gene, which has a coding frame of 363 bp and lies close to and upstream of the ftsI gene of Escherichia coli, is involved in both cell division and cell lysis. It is thought to function in regulating the two distinct steps of the cell cycle, as two different one-base mutations in this unique gene caused different phenotypical changes in the cell. Comparison of nucleotide sequences of the mutant type mraR DNAs with the wild type suggested that filamentation of the cell was caused by a mutation in the putative start codon, whereas lysis of the cell was caused by a mutation which led to a change of one internal glutamate residue to lysine.

Amino Acid Sequence↗
PubMed1991

The Escherichia coli mraY gene encoding UDP-N-acetylmuramoyl-pentapeptide: undecaprenyl-phosphate phospho-N-acetylmuramoyl-pentapeptide transferase.

Amplification of the mraY gene, previously called open reading frame Y (ORF-Y, 1,080 bp), at 2 min in the chromosome map of Escherichia coli enhanced the activity of UDP-N-acetylmuramoyl-pentapeptide: undecaprenyl-phosphate phospho-N-acetylmuramoyl-pentapeptide transferase (EC 2.7.8.13). This enzyme catalyzes the formation of undecaprenyl-pyrophosphoryl-N-acetylmuramoyl-pentapeptide from UDP-N-acetylmuramoyl-pentapeptide and undecaprenyl-phosphate, the first step in the lipid cycle reactions in biosynthesis of bacterial cell wall peptidoglycans. The enhanced enzyme activity was sensitive to tunicamycin, and the amino tunicamycin-sensitive N-acetylglucosamine-1-phosphate transferase of Saccharomyces cerevisiae. Very probably mraY is the structural gene for the above enzyme.

Amino Acid Sequence↗
PubMed1990

Periodic formation of the oriC complex of Escherichia coli.

We examined formation of an oriC-membrane complex through the chromosome replication cycle by dot-blot hybridization using an oriC plasmid as a probe. In a wild-type culture synchronized for chromosome replication, oriC complex formation was observed periodically and transiently corresponding to the replication initiation event. Prior to initiation of replication the oriC complex was recovered in the outer membrane fraction as well as at the time of initiation of replication. Moreover, periodic formation of the oriC complex was observed even when further initiation of replication was suppressed by culturing an initiation ts mutant at the restrictive temperature. Similar periodic formation of the oriC complex was also observed when DNA elongation was inhibited by addition of nalidixic acid to the culture. However, the second periodic peak did not appear when rifampicin or chloramphenicol was added. Cells which formed the oriC complex at the restrictive temperature could immediately initiate chromosome replication when the cells were transferred to the permissive temperature. We conclude that the oriC region of Escherichia coli forms a specific complex periodically just before and at the time of initiation of chromosome replication and that oriC complex formation is a prerequisite for initiation of chromosome replication.

Bacterial Outer Membrane Proteins↗
PubMed1989

New mutations fts-36, lts-33, and ftsW clustered in the mra region of the Escherichia coli chromosome induce thermosensitive cell growth and division.

Three new mutants of Escherichia coli showing thermosensitive cell growth and division were isolated, and the mutations were mapped to the mra region at 2 min on the E. coli chromosome map distal to leuA. Two mutations were mapped closely upstream of ftsI (also called pbpB), in a region of 600 bases; the fts-36 mutant showed thermosensitive growth and formed filamentous cells at 42 degrees C, whereas the lts-33 mutant lysed at 42 degrees C without forming filamentous cells. The mutation in the third new thermosensitive, filament-forming mutant, named ftsW, was mapped between murF and murG. By isolation of these three mutants, about 90% of the 17-kilobase region from fts-36-lts-33 to envA could be filled with genes for cell division and growth, and the genes could be aligned.

Base Sequence↗
PubMed1989

Structural similarity among Escherichia coli FtsW and RodA proteins and Bacillus subtilis SpoVE protein, which function in cell division, cell elongation, and spore formation, respectively.

The Escherichia coli cell division gene ftsW (2 min) was cloned and sequenced. It encodes a hydrophobic protein(s) with 414 and/or 384 amino acid residues. The deduced amino acid sequence and the hydropathy profile of the protein showed high homology with those of the E. coli RodA protein functioning in determination of the cell shape and the Bacillus subtilis SpoVE protein functioning in spore formation. Probably similar functional membrane proteins are involved in these three cell cycle process.

Amino Acid Sequence↗
PubMed1989

New mre genes mreC and mreD, responsible for formation of the rod shape of Escherichia coli cells.

New shape-determining genes in the mre cluster at 71 min on the Escherichia coli chromosome map, named mreC and mreD, were identified by complementation experiments using delta mre-678 mutant cells, which have a 5-kilobase-pair deletion encompassing the mre region, and by DNA sequencing. The delta mre-678 mutant cells required three genes, the previously reported mreB gene and the two new genes, to restore the normal rod shape of the cells and normal sensitivity of growth to mecillinam. The mreC gene is preceded by the mreB gene and by a 65-base-pair spacing sequence containing a palindrome sequence and a possible Shine-Dalgarno sequence. The deduced amino acid sequence of the MreC protein consists of 367 amino acid residues with a molecular weight of 39,530. The initiation codon of the mreD gene overlaps the termination codon of the mreC gene by one nucleotide residue. The deduced amino acid sequence of the MreD protein consists of 162 amino acid residues with a molecular weight of 18,755. In vitro, the coding frames of mreC and mreD produced proteins with Mrs of 40,000 and 15,000, respectively, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Amdinocillin↗
PubMed1989

Negative control of cell division by mreB, a gene that functions in determining the rod shape of Escherichia coli cells.

Exponentially growing Escherichia coli cells containing additional copies of the shape-determining gene mreB were found to be elongated, whereas mreB mutant cells were spherical and overproduced penicillin-binding protein 3, a septum peptidoglycan synthetase. The effect of the mreB gene on expression of ftsI, the structural gene for penicillin-binding protein 3, was examined by using an ftsI-lacZ fusion gene on a plasmid. Formation of beta-galactosidase from the fusion gene was significantly increased in mreB129 mutant cells, and its overproduction was suppressed to a normal level by the presence of a plasmid containing the mreB gene. These results indicate a negative mechanism of control of cell division by this morphology gene and suggest that the gene functions in determining whether division or elongation of the cells occurs.

Cell Division↗
PubMed1988

Determinations of the DNA sequence of the mreB gene and of the gene products of the mre region that function in formation of the rod shape of Escherichia coli cells.

The 6.5-kilobase mre region at 71 min in the Escherichia coli chromosome map, where genes involved in formation of a rod-shaped cell form a gene cluster, was analyzed by in vivo protein synthesis in a maxicell system and by base sequencing of DNA. An open reading frame that may code for a protein with an Mr of about 37,000 on sodium dodecyl sulfate-polyacrylamide gels was found and was correlated with the mreB gene. N-terminal amino acid sequencing of the hybrid mreB-lacZ protein confirmed the production by mreB of a protein of 347 amino acid residues with a molecular weight of 36,958. The amino acid sequence of this protein deduced from the DNA sequence showed close similarity with that of a protein of the ftsA gene which is involved in cell division of E. coli. Three other contiguous genes that formed three proteins with Mrs of about 40,000, 22,000, and 51,000, respectively, were detected downstream of the mreB gene by in vivo protein synthesis. The mreB protein and some of these three proteins may function together in determination of cell shape.

Amino Acid Sequence↗
PubMed1987

Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion.

A new beta-lactam-inducible penicillin-binding protein (PBP) that has extremely low affinity to penicillin and most other beta-lactam antibiotics has been widely found in highly beta-lactam(methicillin)-resistant Staphylococcus aureus (MRSA). The gene for this protein was sequenced and the nucleotide sequence in its promoter and close upstream area was found to show close similarity with that of staphylococcal penicillinase, while the amino acid sequence over a wide range of the molecule was found to be similar to those of two PBPs of Escherichia coli, the shape-determining protein (PBP 2) and septum-forming one (PBP 3). Probably the MRSA PBP (Mr 76462) evolved by recombination of two genes: an inducible type I penicillinase gene and a PBP gene of a bacterium, causing the formation of a beta-lactam-inducible MRSA PBP.

Anti-Bacterial Agents↗