Akiko Nishimura

Murein-lipoprotein of Escherichia coli: A protein involved in the stabilization of bacterial cell envelope

SummaryTwo independent mutants of Escherichia coli lacking murein-lipoprotein have been found. One mutant whose mutation was named lpo was subjected to detailed analyses. The absence of both found and unbound lipoproteins was shown by electrophoretic analysis of 14C-arginine labelled membrane proteins of the mutant. Nor was serologically cross-reacting material detected in the mutant by the Ouchterlony-method. Sequestering magnesium from mutant cell suspensions by ethylenediaminetetraacetic acid caused cell lysis, which was prevented in the presence of 0.5 M sucrose. Incubation in culture media at a very low level of magnesium resulted in the formation of blebs in the mutant. Examination of mutant cells by electron microscopy showed that the outer membrane of the mutant was uneven with small irregular protuberances, some of which pinched off forming vesicles of various sizes. Phosphotungstate used for negative-staining penetrated into the periplasmic space of the mutant cells. The mutants leaked a considerable fraction of their periplasmic enzymes. These physiological and morphological alterations in the lipoproteinless mutant suggest that murein-lipoprotein helps to maintain the outer envelope structure by connecting the outer membrane with murein so that the outer membrane may fulfil its physiological functions as a barrier to the environment.

Fractionation of Escherichia coli cell populations at different stages during growth transition to stationary phase.

Cultures of Escherichia coli could be separated into more than 15 cell populations, each forming a discrete band after Percoll gradient centrifugation. The cell separation was found to result from the difference in buoyant density but not the size difference. The cell density increases upon transition from exponential growth to stationary phase. Exponential phase cultures formed at least five discrete bands with lower densities, whereas stationary phase cultures formed more than 10 bands with higher densities. Two molecular markers characterizing each cell population were identified: the functioning promoter species, as identified by measuring the expression of green fluorescent protein under the control of test promoters; and the expressed protein species, as monitored by quantitative immunoblotting. These findings together suggest that the growth phase‐coupled transition of E. coli phenotype is discontinuous.

Growth Phase-Coupled Alterations in Cell Structure and Function of Escherichia coli

Escherichia coli cultures can be fractionated into more than 20 cell populations, each having a different bouyant density and apparently representing a specific stage of cell differentiation from exponential growth to stationary phase (H. Makinoshima, A. Nishimura, and A. Ishihama, Mol. Microbiol. 43:269-279, 2002). The density increase was found to be impaired at an early step for a mutant E. coli with the disrupted rpoS gene, which encodes the RNA polymerase RpoS (sigma-S) for stationary-phase gene transcription. This finding suggests that RpoS is need for the entire process of cell density increase. In the absence of RpoF sigma factor, the flagella are not formed as observed by electron microscopy, but the growth phase-coupled density increase takes place as in wild-type E. coli, confirming that the alteration in cell density is not directly correlated with the presence or absence of flagella. In the stationary-phase cells, accumulation of electron-dense areas was observed by electron microscopic observation of bacterial thin sections. By chemical determination, the increase in glycogen (or polysaccharides) was suggested to be one component, which contributes to the increase in weight-to-volume ratio of stationary-phase E. coli cells.

Diadenosine 5′,5′′′‐P1,P4‐tetraphosphate (Ap4A) controls the timing of cell division in Escherichia coli

The timing of the cell division in Escherichia coli is highly regulated, but its mechanism has not been identified. Previously we have found that the cfcA1 mutation uncouples DNA replication and cell division and elevates the frequency of cell division. We further analysed the structure and the role of the cfc genes of cfcA11, a derivative of cfcA1, and another cfc mutant, cfcB1.

A cell division regulatory mechanism controls the flagellar regulon in Escherichia coli

SummaryThe formation of flagella in various thermosensitive (Ts) cell division mutants of Escherichia coli was examined at the nonpermissive temperature. The number of flagella per unit cell length decreased sharply after shifting the culture temperature from 30° to 40° C in the following Ts mutants: ftsC108, ftsD1033, ftsE1181, ftsF1141, ftsG29, ftsZ84, parA110, dnaB42, nrdB, and dnaG. It was found that transcription of genes responsible for the formation and/or function of flagella (hag, fla, mot, che) decreased significantly at 40°C. However, in the ftsI730 mutant at the nonpermissive temperature, or in penicillin G treated wild-type cells, cell division was blocked but formation of flagella continued. Moreover, when the cfcA1 mutation, of a gene involved in coordinating DNA replication and cell division, was introduced into the dnaB42 mutant strain, inhibition of cell division and also of formation of flagella at 40°C was relaxed. These results indicate that the flagellar regulon is under the control of a cell division regulatory mechanism.

HscA is involved in the dynamics of FtsZ-ring formation in Escherichia coli K12

Background FtsZ, a homologue of eukaryotic tubulin, localizes throughout the cytoplasm in non‐dividing Escherichia coli. However, it assembles in cytokinetic rings at the early stages of septation. Factors controlling the dynamics of FtsZ ring formation are unknown, and the molecular mechanism governing these dynamics is yet to be determined.

Synthetic ColE1 Plasmids carrying genes for penicillin-binding proteins in Escherichia coli.

Abstract Clarke and Carbons collection of 2000 Escherichia coli strains which harbor ColE1 plasmids carrying small random segments of the E. coli chromosome was screened for the correction of mutational defects in penicillin-binding proteins (PBPs): ponA (PBP-1a), ponB (PBP-1b), dacB (PBP-4), and pfv (PBP-5). We found plasmids carrying chromosomal segments containing ponA+-aroB+ (pLC29-47), ponB+-tonA+ (pLC4-43, pLC4-44, and pLC19-19), and argG+-dacB+ (pLC10-46 and pLC18-38). Characters of these plasmids were analyzed. Two other plasmids (pLC26-6 and pLC4-14) previously found to correct ftsI mutation ( Y. Nishimura, Y. Takeda, A. Nishimura, H. Suzuki, M. Inouye, and Y. Hirota (1977) Plasmid 1, 67–77) were also investigated further. Restriction maps of chromosomal DNAs carried by pLC29-47, pLC4-44, pLC19-19, pLC18-38, pLC26-6, and pLC4-14 were constructed. The regions of ponB-tonA on pLC4-44 and pLC19-19, and of leuA-ftsI-murE and F on pLC26-6 were located on the restriction maps. Although both pLC26-6 and pLC4-14 corrected a thermosensitive mutation, ftsI, which causes a defect in cell division due to abnormal PBP-3, only pLC26-6 led to restoration of PBP-3 production by an ftsI mutant, while pLC4-14 did not. Restriction and heteroduplex analyses of pLC26-6 and pLC4-14 have shown the absence of nucleotide sequence homology between them. The plasmids, pLC29-47 carrying ponA+ and pLC4-43, pLC4-44, and pLC19-19 carrying ponB+ led the host cell to overproduce the respective PBP.

Synthetic ColE1 plasmids carrying genes for cell division in Escherichia coli.

Abstract Clarke and Carbons collection of 2000 E. coli strains, which harbor ColE1 plasmids carrying small random segments of the E. coli chromosome, was screened for the correction of thermosensitive defects in the processes of cell division and in the synthesis of murein-lipoprotein. The genetic defects examined in this screening were those in partition of daughter nuclei ( par ), cleavage of cells ( fts ), determination of a cell shape ( rod ), and synthesis of murein-lipoprotein ( lpo ). We found plasmids carrying E. coli chromosomal segments containing fts B + , fts E + , fts I + , fts M + , and par A + . However, none was found to transfer fts A + , fts C + , fts F + , fts G + , fts J + , fts K + , fts L + , par B + , rod + , and lpo + . One of the donor strains transferring a gene that corrected thermosensitive cell cleavage in the fts I − mutant overproduced the penicillin-binding protein 3 by ca. 10-fold.

A new gene controlling the frequency of cell division per round of DNA replication in Escherichia coli

SummaryA novel mutant of Escherichia coli, named cfcA1, was isolated from a temperature-sensitive dnaB42 strain, and found to have the following characteristics. Division arrest and lethality induced by inhibition of DNA replication was reduced and delayed in the cfcA1 dnaB42 strain, as compared with the parental dnaB42 strain. Two types of inhibition of division induced by the addition of nalidixic acid or hydroxyurea were suppressed by the cfcA1 mutation. Under permissive conditions for DNA replication, the colony forming ability of cfcA1 cells was significantly reduced as compared with that of cfc+ cells; conversely the division rate of cfcA1 cells was higher than that of cfc+ cells. The cfcA1 mutation partially restored division arrest induced in the thermosensitive ftsZ84 mutant at the restrictive temperature and suppresed the UV sensitivity of the lon mutation. The mutation was mapped at 79.2 min on the E. coli chromosome. Taking these properties into account, it is hypothesized that the cfcA gene is involved in determining the frequency of cell division per round of DNA replication by interacting with the FtsZ protein which is essential for cell division.

The DNA Replication Origin (ori) of Escherichia coli: Structure and Function of the ori-Containing DNA Fragment

Publisher Summary This chapter discusses the extensive investigation of the structure and function of the replication origin of E.coli DNA based on the nucleotide sequence of the segment, and the chapter propose a structural model of the replication origin. Fragments of E. coli chromosomal DNA, obtained by digestion with Eco RI, were ligated to a selectable, nonself-replicating DNA fragment (bla) coding for ampicillin resistance and the mixture was used to transform E. coli cells for the selective marker. Using this procedure, cells carrying the E.coli origin region were isolated. Analyses of this DNA fragment by two independent groups were in good agreement. This approach facilitated the further reduction of the size of the origin region. The merging of the genetic analyses of E.coli , rapid nucleotide sequence analysis, and gene cloning technology in E .coli promises a major breakthrough in ones understanding of the regulation of DNA replication and cell division. The in vitro initiation of replication at the E.coli chromosome origin has not yet been successful, and the absence of an in vitro DNA initiation system does not allow us to dissect the detailed reactions involving DNA initiation reactions.