Tn10 generated deletions of the ompB locus of Escherichia coli K12

Abstract We have isolated a set of Tn 10 -generated deletions starting from the distal end of the ompR envZ operon of Escherichia coli K12. Most of the deletions removed both ompR and envZ genes or ended in ompR . These deletions exhibited an OmpC⁻ OmpF⁻ phenotype. One deletion removed only part of envZ and the strain was phenotypically OmpC⁻ OmpF^+/−. This deletion of the distal part of envZ did not affect osmoregulation of ompC . However, ompF osmoregulation appeared reversed. High osmolarity in the growth medium resulted in production of OmpF close to the wild-type level.     

The recN locus of Escherichia coli K12: molecular analysis and identification of the gene product

Summary The recN gene which is necessary for inducible DNA repair and recombination in Escherichia coli has been cloned into the low copy plasmid vector pHSG415. Analysis of the recombinant plasmid, pSP100, revealed a 5.6 Kb HindIII insert of chromosomal DNA. Transposon inactivation of recN function and analysis of a recN::Mu(Ap lac) fusion located the coding region to a 1.4 Kb region within a 2.1 Kb BglII-AvaI DNA fragment transcribed in a clockwise direction with respect to the chromosome map. The gene product was identified in maxicells as a 60,000 dalton protein. Synthesis of this protein was increased in cells lacking LexA activity or in strains carrying recN cloned into the multicopy vector pBR322. Multiple copies of recN increase resistance to ionizing radiation in recN mutants but reduce the survival of a wild-type strain.     

Loss of rac locus DNA in merozygotes of Escherichia coli K12.

Summary Two genes governing ribosomal protein methylation have been located on the map of Escherichia coli by conjugation and transduction crosses between wild-type and prm (protein methylation) mutants. The Prm phenotype of recombinants was determined by an in vitro assay of methylgroups incorporation into protein.Gene prmA, governing methylation of protein L11 is situated at minute 71 on the map and is cotransduced with aroE (30%) and with rpsL (5%). Gene prmB, governing methylation of protein L3 is at minute 50, very close to aroC (98.5% co-transduction). A cold-sensitive phenotype was found associated with mutation prmB and was used to score a large number of recombinants in a three factor cross. The results of this cross suggest the order aroC-prmB-purF.The striking symmetrical clustering of aro, prm and rim (ribosome maturation) genes is discussed.     

Regulation of the chlA locus of Escherichia coli K12; involvement of molybdenum cofactor

The chlA locus encodes functions required for the biosynthesis of the molybdopterin part of the molybdenum cofactor. Mutants, carrying gene fusions at the chlA locus, which place beta-galactosidase expression under the control of the chlA promoter, have been isolated employing lambda placMu1 as the mutagen. The mutants exhibited beta-galactosidase expression which was greatly enhanced when grown anaerobically. Secondary mutations at the chlB, D, E or G loci did not affect the high level of expression. The fnr gene product was not required for the anaerobic expression. Bacteriophage lambda transducing phages were isolated which carried the phi(chlA-lac) mutations and were used to construct chlA+/phi(clA-lac) merodiploids. The merodiploids exhibited a much lower level of expression but showed the same characteristics as strains carrying lac fusions to the single chromosomal chlA locus. Genetic evidence is presented which strongly suggests that the molybdenum cofactor is a repressor of chlA expression. The anaerobic enhancement of chlA expression is mediated via a mechanism that is distinct from the molybdenum cofactor effect.     

Nucleotide sequence of the hemC locus encoding porphobilinogen deaminase of Escherichia coli K12.

Porphobilinogen deaminase, the product of the hemC locus in Escherichia coli K12, catalyses the tetrapolymerisation of porphobilinogen (PBG) into the hydroxymethylbilane, preuroporphyrinogen. The hemC locus has been subcloned from the Clarke and Carbon plasmid pLC41-4. The sequence of the hemC structural gene and flanking DNA was determined by the dideoxy chain termination method of Sanger. The structural gene for hemC is located within a 942bp sequence encoding the monomeric PBG deaminase, molecular weight 33,857. The extent of the coding region was confirmed by sequencing the N-terminus of the purified enzyme and by determination of the molecular weight. The hemC locus is closely linked to the cyaA locus, the genes being transcribed in a divergent manner. Upstream of the hemC coding region, a possible promoter and three repeated GGATG sequences were identified. This is the first report of a complete DNA sequence for a structural gene specifying an enzyme of the heme biosynthetic pathway in prokaryotes.     

Use of homocysteic acid for selecting mutants at the gltS locus of Escherichia coli K12

L-Homocysteic acid is toxic to Escherichia coli K12. Sensitivity to this compound is higher in cells which can utilize glutamate as sole carbon source via the Na+-dependent glutamate transport system. Such cells become resistant by mutation at the gltS locus. Sensitivity of both wild-type and glutamate-utilizing strains is greater if cells are growing on acetate as compared with glucose as major carbon source.     

Kinetic analysis of respiratory nitrate reductase from Escherichia coli K12

Purified respiratory nitrate reductase from Escherichia coli is able to use either reduced viologen dyes or quinols as the electron donor and nitrate, chlorate, or bromate as the electron acceptor. When reduced viologen dyes act as the electron donor, the enzyme follows a compulsory-order, "Theorell-Chance" mechanism, in which it is an enzyme-nitrate complex that is reduced rather than the free enzyme. In contrast, if quinols are used as the electron donor, then the enzyme operates by a two-site, enzyme-substitution mechanism. Partial proteolysis of the cytochrome b containing holoenzyme by trypsin results in loss of cytochrome b and in cleavage of one of the enzyme's subunits. The cytochrome-free derivative exhibits a viologen dye dependent activity that is indistinguishable from that of the holoenzyme, but it is incapable of catalyzing the quinol-dependent reaction. The quinol-dependent, but not the viologen dye dependent, activity is inhibited irreversibly by exposure to diethyl pyrocarbonate and reversibly by treatment with 2-n-heptyl-4-hydroxyquinoline N-oxide. We conclude that the holoenzyme has two independent and spatially distinct active sites, one for quinol oxidation and the other for nitrate reduction.     

Identification of the purI locus in Escherichia coli K-12

A genetic locus has been identified in Escherichia coli that is analogous to the purI locus in Salmonella.     

Mutants of Escherichia coli K12 accumulating porphobilinogen: a new locus, hemC.

Mutants of Escherichia coli K12 which accumulated the haem precursor porphobilinogen are described. The mutants grew very slowly on carbon and energy sources which K12 uses only oxidatively, and they had low catalase activities, suggesting that they were deficient in haem. Extracts had one-tenth of the parental activity of the enzyme porphobilinogen deaminase. In transduction, the mutation mapped close to genes ilvD and metE at minute 84. The gene was tentatively identified as hemC, coding for porphobilinogen deaminase. The gene symbol hemC replaces the earlier and temporary symbol popE.     

Termination troubles in Escherichia coli K12

In this issue of Molecular Microbiology, Schaub and Hayes report that, compared with other enterobacteria, Escherichia coli K12 carries two mutations - one in the prfB gene encoding the release factor RF2, and the other in the rpsG gene encoding r-protein S7 - that together concur in compromising translation termination at the essential rpsG gene. As a consequence, the growth of E. coli K12 is very sensitive to a further mutation (rluD(-) ) that depresses RF2 activity, whereas the growth of its close relative, E. coli B, is not. We tentatively discuss how the K12-specific mutations in RF2 and S7 might have occurred and why inefficient translation termination at rpsG inhibits growth. The work of Schaub and Hayes illustrates the fact that, due probably to its long history in the laboratory, E. coli K12 has accumulated mutations that sometimes limit its value as a model for studying basic steps in prokaryotic gene expression.     

In vivo cloning ad characterization of mutations of the regulatory locus ompR of Escherichia coli K12

Summary The product of the ompR gene of E. coli K12 is a positive regulatory protein, which is needed for the expression of the major outer membrane proteins OmpC and OmpF in E. coli K12. A simple in vivo technique was used to transfer three ompR mutations (ompR101, ompR472, ompR4) onto a multicopy plasmid carrying the wild-type ompR gene. The resulting clones were transformed into wild type and corresponding mutant back-grounds to analyze their effects on ompC and ompF expression. All of the cloned ompR mutant alleles exhibited a dominant OmpC^- phenotype in an ompR ⁺background. In addition negative complementation of ompF expression was observed between chromosomal ompR4 and multicopy ompR101 alleles. The results suggest an interaction between different OmpR molecules, and thereby support the idea that OmpR can exist as a multimeric protein.