Molecular analysis of the Escherichia coli phoP-phoQ operon.

The phoP-phoQ operon of Salmonella typhimurium is a member of the family of two-component regulatory systems and controls expression of the phoN gene that codes for nonspecific acid phosphatase and the genes involved in the pathogenicity of the bacterium. The phoP-phoQ operon of Escherichia coli was cloned on a plasmid vector by complementation of a phoP mutant, and the 4.1-kb nucleotide sequence, which includes the phoP-phoQ operon and its flanking regions, was determined. The phoP-phoQ operon was mapped at 25 min on the standard E. coli linkage map by hybridization with the Kohara mini set library of the E. coli chromosome (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). The predicted phoP and phoQ gene products consist of 223 and 486 amino acids with estimated molecular masses of 25,534 and 55,297 Da, respectively, which correspond well with the sizes of the PhoP and PhoQ proteins identified by the maxicell method. The amino acid sequences of PhoP and PhoQ of E. coli were 93 and 86% identical, respectively, to those of S. typhimurium.     

Genetic analysis of the rnc operon of Escherichia coli.

RNase III, an Escherichia coli double-stranded endoribonuclease, is known to be involved in maturation of rRNA and regulation of several bacteriophage and Escherichia coli genes. Clones of the region of the E. coli chromosome containing the gene for RNase III (rnc) were obtained by screening genomic libraries in lambda with DNA known to map near rnc. A phage clone with the rnc region was randomly mutagenized with a delta Tn10 element, and the insertions were recombined onto the chromosome, generating a series of strains with delta Tn10 insertions in the rnc region. Two insertions that had Rnc- phenotypes were located. One of them lay in the rnc gene, and one was in the rnc leader sequence. Polarity studies showed that rnc is in an operon with two other genes, era and recO. The sequence of the recO gene beyond era indicated it could encode a protein of approximately 26 kilodaltons and, like rnc and era, had codon usage consistent with a low level of expression. Experiments using antibiotic cassettes to disrupt the genes rnc, era, and recO showed that era is essential for E. coli growth but that rnc and recO are dispensable.     

Membrane Homeostasis Requires Intact pst in Extraintestinal Pathogenic Escherichia coli

In extraintestinal pathogenic Escherichia coli (ExPEC) strains, mutation in the PstSCAB inorganic phosphate transporter results in multiple sensitivity phenotypes and loss of virulence. Here, we show that a pst mutant is subject to an increased outer membrane permeability and that pst copy number influences fatty acids regulation. Such perturbations are likely to participate in the impaired response of pst mutants to their environment.     

gltBDF operon of Escherichia coli.

A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega.     

Regulation of the L-arabinose operon of Escherichia coli

Over forty years of research on the L-arabinose operon of Escherichia coli have provided insights into the mechanism of positive regulation of gene activity. This research also discovered DNA looping and the mechanism by which the regulatory protein changes its DNA-binding properties in response to the presence of arabinose. As is frequently seen in focused research on biological subjects, the initial studies were primarily genetic. Subsequently, the genetic approaches were augmented by physiological and then biochemical studies. Now biophysical studies are being conducted at the atomic level, but genetics still has a crucial role in the study of this system.