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.     

Physical and genetic characterization of the glucitol operon in Escherichia coli.

The glucitol (gut) operon has been identified in the colony bank of Clark and Carbon (A. Sancar and W. D. Rupp, Proc. Natl. Acad. Sci. USA 76:3144-3148, 1979). We subcloned the gut operon by using paCYC184, pACYC177, and pBR322. The operon, which is encoded in a 3.3-kilobase nucleotide fragment, consists of the gutC, gutA, gutB, and gutD genes. The repressor of the gut operon seemed to be encoded in the region downstream from the operon. The gene products of the gut operon were identified by using maxicells. The apparent molecular weights of the glucitol-specific enzyme II (product of the gutA gene), enzyme III (product of the gutB gene), and glucitol-6-phosphate dehydrogenase (product of the gutD gene) were about 46,000, 13,500, and 27,000, respectively, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Physical and genetic characterization of the melibiose operon and identification of the gene products in Escherichia coli

We have identified hybrid plasmids carrying the melibiose operon of Escherichia coli in a colony bank of Clarke and Carbon (Tsuchiya, T., Ottina, K., Moriyama, Y., Newman, M., and Wilson, T. H. (1982) J. Biol. Chem. 257, 5125-5128). Using one of the plasmids as a starting material, the DNA fragments containing the melibiose operon were recloned in a vector pBR322. Restriction maps were prepared, and several DNA segments were subcloned into pBR322. Genetic complementation tests and recombination analyses using those plasmids and melA- and melB- mutants as well as biochemical analyses of mel mutants transformed with those plasmids enabled us to determine the physical location of promoter, melA, and melB on the DNA segment. The size of the melAB region was about 3,000 base pairs. Gene products were identified using maxicells harboring plasmids carrying the melibiose operon. The apparent molecular weight of the alpha-galactosidase (coded by melA) was about 50,000 and that of the melibiose carrier (coded by melB) was about 31,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The melibiose carrier was also identified as a 30,000-dalton protein in reconstituted proteoliposomes which possessed melibiose transport activity.     

Molecular cloning and characterization of the purE operon of Escherichia coli

The purE operon of Escherichia coli has been cloned and localized to a 1.7-kb HpaI fragment. The operon has been further characterized by subcloning into the lac fusion vector, pMC1403, and by the construction of BAL 31-generated deletions. The purE regulation region has been identified by assay of beta-galactosidase produced by pur-lac fusion plasmids and by RNA polymerase binding to end-labelled restriction fragments. Two purE promoters have been identified; one strong that is regulated by purines, the other weaker which is not regulated. The latter may be internal to the purE1 structural gene.     

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.     

Genetic regulation of the constitutive D-ribose operon in Escherichia coli B/r.

Merodiploid complementation analysis of the constitutive synthesis of the D-ribokinase and the D-ribose permease in Escherichia coli B/r has shown that the constitutive D-ribose operon is genetically controlled by a transdominant regulatory gene closely linked to the D-ribokinase and D-ribose permease structural genes. The regulatory mechanism for this operon shows no requirement for operator-repressor interaction, rather a truly positive control mechanism and thus suggests an extension of the operon model in its application to constitutive enzyme regulation in bacteria.     

Molecular characterization of the Salmonella typhimurium flhB operon and its protein products.

The flhB and flhA genes constitute an operon called flhB operon on the Salmonella typhimurium chromosome. Their gene products are required for formation of the rod structure of flagellar apparatus. Furthermore, several lines of evidence suggest that they, together with FliI and FliH, may constitute the export apparatus of flagellin, the component protein of flagellar filament. In this study, we determined the nucleotide sequence of the entire flhB operon from S. typhimurium. It was shown that the flhB and flhA genes encode highly hydrophobic polypeptides with calculated molecular masses of 42,322 and 74,848 Da, respectively. Both proteins have several potential membrane-spanning segments, suggesting that they may be integral membrane proteins. The flhB operon was found to contain an additional open reading frame capable of encoding a polypeptide with a calculated molecular mass of 14,073 Da. We designated this open reading frame flhE. The N-terminal 16 amino acids of FlhE displays a feature of a typical signal sequence. A maxicell labeling experiment enabled us to identify the precursor and mature forms of the flhE gene products. Insertion of a kanamycin-resistant gene cartridge into the chromosomal flhE gene did not affect the motility of the cells, indicating that the flhE gene is not essential for flagellar formation and function. We have overproduced and purified N-terminally truncated FlhB and FlhA proteins and raised antibodies against them. By use of these antibodies, localization of the FlhB and FlhA proteins was analyzed by Western blotting (immunoblotting) with the fractionated cell extracts. The results obtained indicated that both proteins are localized in the cytoplasmic membrane.     

Genetic and molecular analyses of Escherichia coli N-acetylneuraminate lyase gene.

Two plasmids containing the N-acetylneuraminate lyase (NALase) gene (nanA) of Escherichia coli, pNL1 and pNL4, were constructed. Immunoprecipitation analysis indicated that the 35,000-dalton protein encoded in pNL4 was NALase. The synthesis of NALase in E. coli carrying these plasmids was constitutive.