Identification of a central regulator of stationary-phase gene expression in Escherichia coli

During carbon-starvation-induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30-50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C-starvation-regulated genes we isolated and characterized chromosomal C-starvation-induced csi::lacZ fusions using the lambda placMu system. One operon fusion (csi2::lacZ) has been studied in detail. csi2::lacZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two-dimensional gel electrophoresis, cells carrying csi2::lacZ did not synthesize at least 16 proteins present in an isogenic csi2+ strain. Cells containing csi2::lacZ or csi2::Tn10 did not produce glycogen, did not develop thermotolerance and H2O2 resistance, and did not induce a stationary-phase-specific acidic phosphatase (AppA) as well as another csi fusion (csi5::lacZ). Moreover, they died off much more rapidly than wild-type cells during prolonged starvation. We conclude that csi2::lacZ defines a regulatory gene of central importanc e for stationary phase E. coli cells. These results and the cloning of the wild-type gene corresponding to csi2 demonstrated that the csi2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi2/katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose 'rpoS' ('sigma S') as appropriate designations.     

The Bacillus subtilis outB gene is highly homologous to an Escherichia coli ntr-like gene.

The Bacillus subtilis outB gene was found to have strong similarities to an Escherichia coli gene complementing ntr-like mutations in Rhodobacter capsulatus. The deduced gene products had 52% identical amino acids (65% similar residues). The phenotype of strains affected in the OutB function indicates that this B. subtilis gene may be involved in nitrogen utilization.     

Evidence for a Micrococcus luteus gene homologous to uvrB of Escherichia coli

Summary Restriction fragments of Micrococcus luteus DNA that contained the gene defined by the mutation of an excision repair-deficient mutant, UV^sN1, were cloned from both the parental and mutant strains with the Escherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to ultraviolet, mitomycin C, and 4-nitroquinoline-1-oxide by one-step transformation. Determination of the nucleotide sequences revealed an open reading frame potentially coding for a protein of 709 amino acid residues, within which the mutation was identified as a CGTA transition causing a change from serine to phenylalanine. The putative product of the open reading frame showed an extensive amino acid sequence homology to the E. coli UvrB protein comprising 673 residues; the homologous region extended over the greater parts of both polypeptides, in which 55% and 17% of the 659 pairs of aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. This indicates that the gene defined by the UV^sN1 mutation represents a homolog of the E. coli uvrB gene, implying the presence in M. luteus of an enzyme complex homologous to the E. coli UvrABC excinuclease.Abbreviations Ap ampicillin - AP apurinic-apyrimidinic - MC mitomycin - C: 4NQO 4-nitroquinoline-1-oxide - r resistant - s sensitive - UV ultraviolet Dedicated to the memory of Shunzo Okubo (1930–1978) who played the pivotal role in our earlier studies on the M. luteus repair system     

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.     

Identification of the hopG gene, a component of Escherichia coli K-12 type II export system, and its conservation among different pathogenic Escherichia coli and Shigella isolates.

The Escherichia coli K-12 gene coding for a component of a type II export system was identified and characterized. The HopG protein contains a typical prepilin peptidase cleavage site and has a high degree of homology with proteins PulG, OutG, and ExeG, which are components of type II secretion systems from Klebsiella pneumoniae, Erwinia carotovora, and Aeromonas hydrophila.     

Microbial genes homologous to the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli

We have cloned and determined the nucleotide (nt) sequences of the genes encoding peptidyl-tRNA hydrolase (Pth) homologues of Salmonella typhi (St) and the Lyme disease spirochaete, Borrelia burgdorferi (Bb). We also completed the nt sequence of a pth homologous gene contained in a Chlamydia trachomatis (Ct) clone identified in the databanks. The open reading frames (ORFs) of the Pth homologues encode putative polypeptides of 194 (St), 188 (Bb) and 194 (Ct) amino acids exhibiting significant identity with Escherichia coli (Ec) Pth. Together with the products of two previously unidentified ORFs from Bacillus subtilis and Saccharomyces cerevisiae, and the recently recognized Haemophilus influenzae and Mycoplasma genitalium pth genes, these seven putative polypeptides and the Ec Pth form a group of homologous basic proteins spanning eubacteria and eukaryota which can be defined by at least three conserved regions. Previously known Ec pth mutations were located in highly conserved residues     

Identification and characterization of a chromosomal virulence gene, vacJ, required for intercellular spreading of Shigella flexneri

Intercellular spreading of shigellae Is a prerequisite for shigellosis, although the molecular mechanisms underlying the phenomenon are still largely obscure. To elucidate some of these mechanisms, we performed random TniO insertion mutagenesis in Shigella flexneri YSH6000T and found a chromosomal locus in the Notl-J segment responsible for bacterial spreading. The locus affected in the mutant, designated vacJ, was neither involved in the invasion of epithelial cells nor in intracellular movement, but was required for intercellular spread. The vacJ mutant was capable of forming bacterium-containing membranous protrusions within the infected cell, but had diminished ability to move from the protrusions into the cytoplasm of the adjacent epithelial cells. Cloning and sequencing of the vacJ region Indicated that the vacJ gene encoded a 28.0 kDa protein possessing a signal peptide at the N-terminus, which contained the motif characteristic of lipoproteins. The analysis of the vacJ product indicated that VacJ was exposed on the bacterial surface. The vacJ gene was distributed among shigellae and enteroinvasive Escherichia coli, and the constructed vacJ mutants failed to spread intercellularly, indicating that vacJ is a chromosomal gene essential for the pathogenicity of shigeiiae.