Rescue by chloramphenicol and rifampicin of UV-irradiated lon, exrA and recA mutants of Escherichia coli K12

The effect of temporary treatment with chloramphenicol or rifampicin on the survival of UV irradiated cells of selected Escherichia coli K12 radiation sensitive mutants was examined. Increased survival resulted for both exrA and recA mutants, and also for the unsuppressed lon mutant, but cells of the parent strain and the recB mutant were not rescued. This contrasts with our earlier finding that after exposure of the bacteria to γ-rays, chloramphenicol treatment rescued the exrA and lon mutants but not the recA mutant. We now report that an exrA recA double mutant was rescued by chlramphenicol after UV radiation, but not after anaerobic ionizing radiation. Inclusion of inhibitors of uvrA governed repair, caffeine and 8-methoxypsoralen (8-MOP), in the incubation medium containing chloramphenicol, did not reduce the rescue of the exrA or recA mutants, although caffeine eliminated rescue of the lon mutant, which was itself unaffected by 8-MOP. However it is concluded that chlormaphenicol rescue of the exrA and recA mutants after UV radiation was not entirely independent of the excision-repair process, since the uvrA recA and uvrA exrA double mutants were not rescued by this treatment.     

Morphological mutants of Escherichia coli K12. Mapping of the env C mutation

Summary Morphological env C mutation affects the septum and leads to chain formation. Genetic analysis of a non-conditional env C mutant is performed; the gene order found is xyl-mtl-env C-pyr E.     

Microcin-E492-insensitive mutants of Escherichia coli K12

Mutations in three Escherichia coli K12 genes, tonB, exbB and the newly discovered semA, reduce sensitivity to the low Mr polypeptide antibiotic microcin E492. The products of the tonB and exbB genes were previously shown to be involved in the uptake of siderophore-complexed iron and in the action of a number of colicins. Strains mutated at or close to semA (collectively referred to as sem mutations) remained fully sensitive to these colicins, and grew as well as wild-type strains under conditions of iron starvation. Expression of a number of sem-lacZ operon fusions was not affected by iron limitation, and sem mutations did not affect the production of iron-regulated outer membrane proteins which are known or thought to be involved in iron uptake. Hfr conjugation and P1 phage transduction experiments indicated that semA is located close to pabB at 40 min on the E. coli K12 chromosome. This places semA close to the mng locus, wherein mutations result in decreased manganese sensitivity. However, strains carrying the semA mutation exhibited increased manganese sensitivity.     

Ferrous iron transport mutants in Escherichia coli K12

A ferrous iron transport system in Escherichia coli is described. Mutants in this transport system were isolated using the antibiotic streptonigrin. The gene locus feo (for ferrous iron transport) was mapped near pncA at 38.5 min on the genetic map of E. coli K12. The transport of ferrous iron was regulated by fur as the siderophore transport systems.     

Isolation of haemin-requiring mutants of Escherichia coli K12.

Fifty-five haemin-requiring mutants were isolated from haemin-permeable mutants. According to their growth responses to haem precursors and their patterns of porphyrin accumulation, the 55 mutants fell into three groups which were judged to have defects in 5-aminolaevulinate dehydratase, ferrochelatase, and uroporphyrinogen III cosynthase or uroporphyrinogen decarboxylase. In mutants of the group deficient in 5-aminolaevulinate dehydratase, the mutations were adjacent to lac, and evidence is presented that the mutations were in hemB and were commonly deletions extending into proC.     

Properties of cysK mutants of Escherichia coli K12.

Triazole and azaserine resistant mutants of E. coli K12 affecting cysK gene coding for O-acetylserine sulphydrylase were isolated. The cysK gene in E. coli is located in the same region of chromosome as the cycK gene in Salmonella typhimurium. All azaserine and some triazole resistant mutants require cysteine for growth at a normal rate. The cysK mutants have reduced sulphate uptake. Stability and transfer by conjugation of triazole resistant phenotype were checked. Differences in sulphate metabolism between closely related organisms E. coli and S. typhimurium are discussed.     

Escherichia coli K12 arabinose-binding protein mutants with altered transport properties.

The arabinose-binding protein (ABP) of Escherichia coli binds L-arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell. To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose-binding cleft were subjected to site-directed mutagenesis. Thirty-eight ABP variants containing one to three amino acid substitutions were recovered. ABP variants were expressed with wild-type AraG and AraH from a plasmid, in a strain lacking the chromosomal araFGH operon, and the whole cell uptake parameters, Ven (maximum initial velocity of arabinose entry) and K(en) (concentration of arabinose yielding half-maximal entry) were determined. Twenty-four mutants had normal Ven values, 3 mutants had Ven and K(en) values twice wild type, and 11 mutants had Ven and K(en) values 20-50% of wild type. Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type. The mutant binding proteins behaved the same as wild type during purification, and each had a Kd (dissociation constant for bound arabinose) comparable to that of wild-type ABP. Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose-binding cleft, all of which are charged in the wild-type protein, and all of whose side chains project outward from the cleft. The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex.