Yukinobu Nishimura

New topoisomerase essential for chromosome segregation in E. coli

The nucleotide sequence of the parC gene essential for chromosome partition in E. coli was determined. The deduced amino acid sequence was homologous to that of the A subunit of gyrase. We found another new gene coding for about 70 kd protein. The gene was sequenced, and the deduced amino acid sequence revealed that the gene product was homologous to the gyrase B subunit. Mutants of this gene were isolated and showed the typical Par phenotype at nonpermissive temperature; thus the gene was named parE. Enhanced relaxation activity of supercoiled plasmid molecules was detected in the combined crude cell lysates prepared from the ParC and ParE overproducers. A topA mutation defective in topoisomerase I could be compensated by increasing both the parC and the parE gene dosage. It is suggested that the parC and parE genes code for the subunits of a new topoisomerase, named topo IV.

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.

Polyprenyl diphosphate synthase essentially defines the length of the side chain of ubiquinone

Ubiquinone, known as a component of the electron transfer system in many organisms, has a different length of the isoprenoid side chain depending on the species, e.g., Escherichia coli, Saccharomyces cerevisiae and humans have 8, 6, and 10 isoprene units in the side chain, respectively. No direct evidence has yet shown what factors define the length of the side chain of ubiquinone. Here we proved that the polyprenyl diphosphate that was available in cells determined the length of the side chain of ubiquinone. E. coli octaprenyl diphosphate synthase (IspB) was expressed with the mitochondrial import signal in S. cerevisiae. Such cells produced ubiquinone-8 in addition to the originally existing ubiquinone-6. When IspB was expressed in a S. cerevisiae COQ1 defective strain. IspB complemented the defect of the growth on the non-fermentable carbon source. Those cells had the activity of octaprenyl diphosphate synthase and produced only ubiquinone-8. These results opened the possibility of producing the type of ubiquinone that we need in S. cerevisiae simply by expressing the corresponding polyprenyl diphosphate synthase.

Yeast Saccharomyces cerevisiae has two cis‐prenyltransferases with different properties and localizations. Implication for their distinct physiological roles in dolichol synthesis

Background Dolichol is a family of long‐chain polyprenols, which is utilized as a sugar carrier in protein glycosylation in the endoplasmic reticulum (ER). We have identified a key enzyme of the dolichol synthesis, cis‐prenyltransferase, as Rer2p from Saccharomyces cerevisiae. We have also isolated a multicopy suppressor of an rer2 mutant and named it SRT1. It encodes a protein similar to Rer2p but its function has not been established.

Osmoregulation of the fatty acid receptor gene fadL in Escherichia coli

The fadL gene of Escherichia coli codes for an outer membrane protein that is involved in the uptake of long-chain fatty acids. Uptake is regulated by environmental osmolarity, and decreases when the cells are grown under conditions of high osmolarity. A temperature-sensitive mutant that requires fatty acid for growth at 42° C was unable to grow at the high temperature even in the presence of fatty acid if the medium contained 10% sucrose. Promoter activity of the fadL gene in vivo was repressed by high osmolarity in a FadR repressor null mutant. Furthermore, in vitro transcription of the fadL gene was strongly repressed by the addition of OmpR and EnvZ proteins. The results of gel retardation and DNase I protection experiments indicated that OmpR, after incubation with the protein kinase EnvZ, specifically binds to at least four sites around the fadL promoter, two upstream and two downstream from the transcriptional start site. These results suggest that transcription of the fadL gene is osmotically regulated by the OmpREnvZ two-component system.

Cloning of a gene from Escherichia coli that confers resistance to fosmidomycin as a consequence of amplification

A gene conferring resistance to fosmidomycin (Fs) was cloned from the gene pool of a wild-type strain of Escherichia coli. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 406 amino acids (aa) with a molecular weight of 43303. The gene mapped at 10.9 min on the E. coli chromosome and was designated fsr (fosmidomycin resistance). Maxicell analysis revealed that the Fsr protein migrated in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis as a broad band of 35 kDa. A comparison between the aa sequence of Fsr and sequences in a protein database revealed 18% homology to the bacterial drug-export proteins that mediate resistance to tetracycline and chloramphenicol. Hydropathy analysis of the Fsr protein revealed twelve putative transmembrane segments. The degree of FsR of transformants depended on the number of copies of the plasmid that contained fsr. The levels of ubiquinone-8 and undecaprenyl phosphate in cells that harbored a high-copy-number plasmid that included fsr were almost the same as those in the cells without the plasmid. These results suggest that Fsr does not have any direct effect on the biosynthesis of isoprenoid in E. coli, and that the mechanism for FsR involves the efflux of the drug by a process that is facilitated by Fsr.

Escherichia coli parA is an allele of the gyrB gene

SummaryA thermosensitive (ts) parA mutant, MFT110, of Escherichia coli carried at least two ts mutations. The major ts defect, resulting from a mutation mapped originally at 95 min and complemented by pLC8-47, was most probably due to psd. A plasmid carrying the 1.6 kb BamHI-PvuII fragment recloned from pLC8-47 complemented the major ts mutation in MFT110 and psd(ts) in two mutants, but did not correct the Par phenotype of MFT110. The second ts mutation was salt-repairable and mapped at 83 min close to recF and tnaA. This mutation was linked with the Par phenotype as shown unambiguously by 4′,6-diamidino-2-phenylindole stained nucleoids in parA mutant cells with the W3110 genetic background. Both salt-repairable ts and Par traits were corrected concomitantly by a plasmid carrying the chromosomal region solely for the gyrB gene. This strongly suggests that parA is an allele of gyrB.

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 cryptic lysis gene near the start of the Qβ replicase gene in the +1 frame

The maturation/lysis (A2) protein encoded by the group B single‐stranded RNA bacteriophage Qβ mediates lysis of host Escherichia coli cells. We found a frameshift mutation in the replicase (β‐subunit) gene of Qβ cDNA causes cell lysis. The mutant has a single base deletion 73 nucleotides (nt) 3′ from the start of the replicase gene with consequent translation termination at a stop codon 129–131 nt further 3′. The 43‐amino acid C‐terminal part of the 67‐amino acid product encoded by what in WT (wild‐type) is the +1 frame, is rich in basic amino acids This 67‐aa protein can mediate cell lysis whose characteristics indicate that the protein may cause lysis by a different mechanism and via a different target, than that caused by the A2 maturation/lysis protein. Synthesis of a counterpart of the newly discovered lysis product in wild‐type phage infection would require a hypothetical ribosomal frameshifting event. The lysis gene of group A RNA phages is also short, 75 codons in MS2, and partially overlaps the first part of their equivalently located replicase gene, raising significant evolutionary implications for the present finding.