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N J Trun

Publications and source records attributed to N J Trun.

11 recordsLinked to original sources
PubMed1997

Unfolding of the bacterial nucleoid both in vivo and in vitro as a result of exposure to camphor.

Both prokaryotic and eukaryotic cells are sensitive to killing by camphor; however, the mechanism by which camphor kills has not been elucidated. We report here that camphor unfolds the nucleoid of Escherichia coli and that unfolding does not require DNA replication, translation, or cell division. We show that exposure of isolated nucleoids to camphor results in unfolding of the chromosome.

Camphor↗
PubMed1996

Overproduction of three genes leads to camphor resistance and chromosome condensation in Escherichia coli.

We isolated and characterized three genes, crcA, cspE and crcB, which when present in high copy confer camphor resistance on a cell and suppress mutations in the chromosomal partition gene mukB. Both phenotypes require the same genes. Unlike chromosomal camphor resistant mutants, high copy number crcA, cspE and crcB do not result in an increase in the ploidy of the cells. The cspE gene has been previously identified as a cold shock-like protein with homologues in all organisms tested. We also demonstrate that camphor causes the nucleoids to decondense in vivo and when the three genes are present in high copy, the chromosomes do not decondense. Our results implicate camphor and mukB mutations as interfering with chromosome condensation and high copy crcA, cspE and crcB as promoting or protecting chromosome folding.

Amino Acid Sequence↗
PubMed1992

Escherichia coli prlC encodes an endopeptidase and is homologous to the Salmonella typhimurium opdA gene.

Mutations at the Escherichia coli prlC locus suppress the export defect of certain lamB signal sequence mutations. The Salmonella typhimurium opdA gene encodes an endoprotease that can participate in the catabolism of certain peptides and is required for normal development of phage P22. Plasmids carrying either the wild-type (pTC100 prlC+) or suppressor alleles of prlC complemented all phenotypes associated with an S. typhimurium opdA mutation. A plasmid carrying an amber mutation in prlC [prlC31(AM)] was unable to complement except in an amber suppressor background. Tn1000 insertions which eliminated the ability of pTC100 (prlC+) to complement opdA mapped to the region of the plasmid shown by deletion analysis and subcloning to contain prlC. The nucleotide sequence of a 2.7-kb fragment including this region was determined, revealing an open reading frame encoding a 77-kDa protein. The sequences of this open reading frame and its putative promoter region were very similar (84% nucleotide sequence identity and 95% amino acid identity) to those of S. typhimurium opdA, showing that these genes are homologs. The nucleotide sequence of the prlC1 suppressor allele was determined and predicts an in-frame duplication of seven amino acids, providing further confirmation that the prlC suppressor phenotype results from changes in the endopeptidase OpdA.

Alleles↗
PubMed1990

On the bacterial cell cycle: Escherichia coli mutants with altered ploidy.

We describe a scheme for isolation of new classes of mutants in the cell cycle of Escherichia coli. The mutants were selected as resistant to camphor vapors, which results in increased ploidy, and were subsequently screened for an increase in cell density and an increase in the gene dosage of the lac operon. Our mutations are located at four different places in the chromosome; we have named these loci mbr (moth ball resistant). mbrA maps to 68 min on the E. coli chromosome, mbrB to 88.5 min, mbrC to 89.5 min, and mbrD to 90 min. mbrD mutations may be alleles of rpoB (a subunit of RNA polymerase). In addition to the selected or screened phenotypes, most of the mutants fail to grow on rich media or at high temperatures. We have examined the nine mutants under nonpermissive conditions, using several techniques to determine the cause of death. We have also coupled our mutations with lesions in dnaA, which is required for cell-cycle-specific DNA replication, and rnh (the gene for RNase H), which is required for specificity in the DNA initiation reaction, and determined the effects of the double and triple mutants under permissive and nonpermissive conditions. These tests have shown that bacteria mutated at mbrA do not tolerate a null mutation in rnh, indicating that they are dependent on DNA replication initiating at oriC. In contrast, mutations at mbrB, mbrC, and mbrD exhibit their phenotypes independent of oriC initiation of DNA replication, suggesting that the mutations affect factors that influence the DNA/cell ratio regardless of the origin of DNA replication. Based on our results, the mbr mutations appear to have defects in cell-cycle timing and/or defects in chromosomal partitioning.

Camphor↗
PubMed1989

PrlC, a suppressor of signal sequence mutations in Escherichia coli, can direct the insertion of the signal sequence into the membrane.

The prlC gene product of Escherichia coli can be altered by mutation so that it restores export of proteins with defective signal sequences. The strongest suppressor, prlC8, restores processing of a mutant signal sequence to a rate indistinguishable from the wild-type. Data obtained by changing gene dosage of the dominant suppressor and its specificity for different signal sequence mutations suggest that PrlC8 interacts directly with the hydrophobic core of the signal sequence. Despite the fact that signal sequence processing appears to be mediated by leader peptidase, the processed mature protein is not translocated efficiently from the cytoplasm. Results obtained with various double mutants indicate that PrlC8-mediated processing of mutant signal sequences does not require components of the cellular export machinery such as SecA, SecB or PrlA (SecY) and that the block in translocation from the cytoplasm occurs because PrlA (SecY) fails to recognize the defective signal sequence. We suggest that PrlC8 directs insertion of the mutant signal sequence into the membrane bilayer to an extent that processing by leader peptidase can occur. This reaction is novel in that it has not been observed previously in vivo.

Bacterial Outer Membrane Proteins↗
PubMed1989

The genetics of protein targeting in Escherichia coli K12.

Genetic analysis of protein targeting in Escherichia coli has been facilitated by the use of lacZ gene fusions. Strains which contain a fusion between a gene specifying a noncytoplasmic protein and lacZ exhibit novel phenotypes that can be exploited to obtain export-defective mutations. Analysis of these mutations has demonstrated the importance of the signal sequence and provided some insight into the functions performed by this complex intragenic export signal. In addition, genes that specify components of the cellular protein export machinery have been identified. Three of these genes, secA, secB, and prlA/secY have been studied in some detail and results indicate that the protein products function in the translocation of exported proteins across the cytoplasmic membrane.

Bacterial Proteins↗
PubMed1988

Two cellular components, PrlA and SecB, that recognize different sequence determinants are required for efficient protein export.

We exploited the conditional-lethal phenotype of secB null mutations to demonstrate that SecB function was required for PrlA-mediated suppression of signal sequence mutations. The results of these experiments provide information about the functions performed and the sequence determinants recognized by each of these components of the protein export machinery of Escherichia coli.

Bacterial Outer Membrane Proteins↗
PubMed1987

Characterization and in vivo cloning of prlC, a suppressor of signal sequence mutations in Escherichia coli K12.

The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamB signal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using lambda placMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

Alleles↗
PubMedDate not supplied

Characterization of Escherichia coli mutants with altered ploidy.

We describe the isolation and characterization of new mutants in the cell cycle of Escherichia coli. The mutants were selected as gain of function mutants that are able to maintain more than the normal number of chromosomes. Our increased ploidy mutants were isolated as resistant to camphor vapours, which selects for cells with more chromosomes than normal. The mutants (called mbr for moth-ball-resistant) map to four chromosomal locations: mbrA at 68 min; mbrB at 88.5 min; mbrC at 89.5 min; and mbrD at 90 min. To investigate the nature of these cell cycle mutants, we have coupled them with defects in recA, to test for induction of the SOS response, and dam, to determine if methylation is required for mbr function. Based on the results of these and other tests, we have made a preliminary placement of the mbr mutants within the context of the cell cycle. mbrA mutations appear to be defective in the coupling of the DNA replication cycle to the cell division cycle, and as such, may define a new link between the two processes. mbrB does not seem to be able to coordinate the cell cycle and the growth rate of the cell. mbrC appears to be defective in partitioning of chromosomes. mbrD, which may be allelic to rpoB (a subunit of RNA polymerase), appears to be defective in either chromosomal partitioning or the later stages of DNA replication.

Camphor↗