Genetic Location in Escherichia coli K-12 of the Ultraviolet-Sensitive Mutation uvrD3

A host cell reactivation-negative mutation, uvrD3, in Escherichia coli K-12 is located between ilv and metE.     

Isolation of an Escherichia coli K-12 dnaE mutation as a mutator.

Using a papillation method, a large number of Escherichia coli K-12 mutator mutations have been isolated. Only one of these (out of 1,250) mutator mutations has proved to be conditionally lethal at high temperatures. In vivo complementation tests indicated that this mutation, dnaE9, lies in dnaE, the structural gene for DNA polymerase III. The dnaE9 polymerase was not thermolabile in vitro; however, it showed a slow decline in specific activity in vivo at the nonpermissive temperature. Cultures of this mutant exhibited a comparably slow shutoff of DNA synthesis on shift to a nonpermissive temperature. dnaE9 showed temperature-sensitive mutator activity, which is not dependent on recA.     

Chromosomal mutation for citrate utilization by Escherichia coli K-12.

A mutant strain of Escherichia coli K-12 that utilizes citrate as a sole source of carbon and energy was isolated. Citrate utilization arose as the consequence of two mutations in genes citA and citB, which are linked to the gal operon. The mutant strain expresses a semiconstitutive citrate transport system, and it utilizes both citrate and isocitrate as carbon and energy sources. It is capable of utilizing cis- and trans-aconitate, but only if it is preinduced by growth on citrate.     

tif-Stimulated deoxyribonucleic acid repair in Escherichia coli K-12

Bacterial survival is significantly increased after ultraviolet irradiation in tif sfi cells, provided that the thermosensitive tif mutation has been expressed at 41 degrees C before irradiation. This tif-mediated "reactivation of ultraviolet irradiated bacteria" needs de novo protein synthesis, as is the case for the tif-mediated reactivation of ultraviolet-irradiated phage lambda. However, in striking contrast to the phage reactivation process, this tif-mediated reactivation is no longer associated with mutagenesis. It also requires the presence of the uvrA+ excision function. These results strongly suggest the existence in Escherichia coli K-12 of a repair pathway acting on bacterial deoxyribonucleic acid which is inducible, error free, and uvr dependent.     

Indirect stimulation of recombination in Escherichia coli K-12: dependence on recJ, uvrA, and uvrD.

Direct and indirect UV-stimulated homologous genetic recombination was investigated in Escherichia coli strains blocked in several host-encoded functions. Genetic recombination was assayed by measuring beta-galactosidase produced after recombination between two noncomplementing lacZ ochre alleles. Both types of stimulation (direct and indirect) were found to be primarily RecF pathway-mediated. In a rec+ background, both direct and indirect stimulation were found to be dependent on uvrD (coding for helicase II). In a recB21 sbcB15 background, direct and indirect stimulation were uvrD dependent only when the strain was additionally deficient in the UvrABC excision repair pathway. Indirect but not direct stimulation was also dependent on recJ (coding for a 5'-to-3' exonuclease specific for single-stranded DNA) regardless of sbcA or sbcB configuration. The methyl-directed mismatch repair system (mutSLH) also appeared to play an important role in stimulation. On the basis of these findings, we suggest that excision of UV-induced DNA damage is a prelude to UV-mediated stimulation of genetic recombination.     

Influence of uvrD3, uvrE502, and recL152 mutations on the phenotypes of Escherichia coli K-12 dam mutants.

The recF143 allele did not alter the phenotypes of dam mutants of Escherichia coli. The uvrD3, uvrE502, and recL152 mutations did alter some of the phenotypes of dam bacteria. It was concluded that the uvrD, uvrE, and recL gene products are involved in the same deoxyribonucleic acid repair pathway as the dam gene product.     

Map location of the ssd mutation in Escherichia coli K-12.

A pleiotropic mutation at the ssd locus was mapped at 86 min near rha. A mutation at the ssd locus resulted in elevated L-serine deaminase activity, inability to grow with succinate as the carbon source, and inability to grow anaerobic conditions.     

Effect of tsl (thermosensitive suppressor of lex) mutation on postreplication repair in Escherichia coli K-12.

Cells of Escherichia coli K-12 carrying lexA or recA mutations are more sensitive to UV radiation than corresponding wild-type cells and are defective in postreplication repair. Supressor mutations (tsl) have been described previously which increase the UV resistance of lexA uvr+, lexA uvrA, and recAI uvr+ strains, but not the resistance of recA1 uvrA strains. We have studied the effect of the tsl-1 mutation on postreplication repair and find that the enhanced survival conferred by this mutation is correlated with an increased capacity for postreplication repair.     

Genetic and biochemical characterization of the Escherichia coli K-12 fhuB mutation.

The fhuB region of Escherichia coli K-12 was subcloned from pLC4-44 into pP lac to obtain pCPN1. Deletions of this recombinant plasmid were made, and a 1.4-kilobase PstI fragment was further subcloned into the vector plasmid pKK177-2 to obtain pCPN12. The response of tonA and tonB strains and fhuB strains containing the plasmids to 15 hydroxamate siderophores were assayed. Results showed that tonA strains were deficient only in the utilization of ferrichrome-type siderophores, whereas fhuB strains were deficient in the utilization of all hydroxamate-type siderophores. The response of the plasmid-containing fhuB strains to the siderophores showed that the fhuB gene resides on a 1.4-kilobase PstI fragment of DNA. The proteins synthesized by these plasmids were examined in maxicells of strain CSR603. Plasmid pCPN1 expressed five proteins of molecular weights 78,000, 40,000, 30,000, 24,000, and 13,700. By the use of deletions of pCPN1, the approximate order of the genes for these proteins was determined. Plasmid pCPN12 expressed no proteins other than the beta-lactamase proteins in maxicell strain CSR603. However, in maxicell strain BN660, a lon mutant, it expressed a 20,000-molecular-weight protein. Inner membrane vesicles made from tonB and fhuB strains were able to transport [55Fe]ferrichrome and [55Fe]rhodotorulate at rates similar to those obtained in vesicles from tonB+ and fhuB+ strains.     

Purine-mediated growth inhibition caused by a pyrE mutation in Escherichia coli K-12.

A purine-sensitive phenotype results from a previously described mutation in the structural gene (pyrE) for orotate phosphoribosyltransferase (OPT) in Escherichia coli K-12. OPT from both the mutant and the wild-type was partially inhibited by adenine and adenosine, although other purine derivatives were not effective for this inhibition. The Km values of the mutant OPT were 580 and 760 microM for orotate and 5'-phosphoribosyl-1'-pyrophosphate (PRib-PP), respectively, whereas the corresponding values for the wild-type OPT were 40 and 60 microM. The intracellular level of PRib-PP was decreased to less than 15% of the normal level when purine derivatives were added to exponentially growing cultures of both the parent and mutant strains. However, this decrease of the PRib-PP level was not found in strains derived from the mutant, in which the purine-sensitive phenotype was suppressed by a secondary mutation. The purine-sensitive phenotype was caused by retardation of the pyrimidine de novo pathway, when the intracellular level of PRib-PP was diminished by exogenously supplied purine derivatives.     

Influence of recC and uvrD on ultraviolet-induced mutation to valine resistance in E. coli K12

The production of mutants in E. coli exposed to ultraviolet light is initiated by photochemical reactions, and completed by metabolic processes controlled by recA and other genes. Ultraviolet-induced mutagenesis to valine resistance was measured in cells carrying recC, uvrD, or both recC and uvrD. The spontaneous and UV-induced mutagenesis was slightly greater in those carrying uvrD, as compared to recC or wild-type. At low doses, UV mutagenesis in the recC uvrD double mutant was greater than in either recC or wild-type, and was comparable to that in the uvrD strain, although this double mutant was very UV-sensitive and showed poor survival at doses above 2 J/m².     

Trans-dominant mutation affecting restriction and modification in Escherichia coli K-12

Summary We have analysed the mechanism of action of a ts mutation in E. coli, which has an effect on the expression of the restriction and modification phenotype. The frequencies of recombinants obtained in transduction experiments support the idea that the temperature sensitive mutation is located outside the hsd operon in the gene denoted hsd. X. Complementation experiments demonstrated the trans-dominant nature of the temperature sensitive mutation. The possible role of the hsd.X product in the formation of EcoR.K and EcoM.K complexes and their interaction with the recognition site on the DNA is discussed.     

Long repair replication patches are produced by the short-patch pathway in a uvrD252 (recL152) mutant of Escherichia coli K-12

The uvrD252 mutation leads to increased UV sensitivity, diminished dimer excision and host cell reactivation capacity, and an increase in the average patch size after repair replication. A recA56 uvrD252 double mutant was far more resistant to UV than was a recA56 uvrB5 double mutant. Its host cell reactivation capacity was identical to that of uvrD252 single mutant and was far greater than that of the uvrB5 single mutant. The strain showed no Weigle reactivation. From these results, we concluded that the double mutant has no inducible DNA repair (including long-patch excision repair) but retains dimer excision capabilities comparable to the uvrD252 single mutant. It appears, therefore, that the long patches detected in the uvrD mutant were not identical to the recA-dependent patches seen in wild-type cells.     

Suppressors of a genetic regulatory mutation affecting isoleucine-valine biosynthesis in Escherichia coli K-12.

Escherichia coli K-12 mutant PS187 carries a mutation, ilvA538, in the structural gene for the biosynthetic L-threonine deaminase that leads to a leucine-sensitive growth phenotype, an isoleucine- and leucine-hypersensitive L-threonine deaminase, and pleiotropic effects resulting in abnormally low and invariant expression of some of the isoleucine-valine biosynthetic enzymes. Fifty-eight derivatives of strain PS187 were isolated as resistant to growth inhibition by leucine, by valine, or by valine plus glycly-valine and were biochemically, genetically, and physiologically characterized. All of these derivatives produced the feedback-hypersensitive L-threonine deaminase, and thus presumably possess the ilvA538 allele of the parent strain. Elevated synthesis of L-threonine deaminase was observed in 41 of the 58 isolates. Among 18 strains analyzed genetically, only those with mutations linked to the ilv gene clusters at 83 min produced elevated levels of L-threonine deaminase. One of the strains, MSR91, isolated as resistant to valine plus glycyl-valine, was chosen for more detailed study. The locus in strain MSR91 conferring resistance was located in four factor crosses between ilvE and rbs, and is in or near the ilvO gene postulated to be a site controlling the expression of the ilvEDA genes. Synthesis of the ilvEDA gene products in strain MSR91 is constitutive and derepressed approximately 200-fold relative to the parent strain, indicating that the genetic regulatory effects of the ilvA538 allele have been suppressed. Strain MSR91 should be suitable for use in purification of the ilvA538 gene product, since enzyme synthesis is fully derepressed and the suppressor mutation is clearly not located within the ilvA gene.     

Identification of a mutation affecting an alanine-alpha-ketoisovalerate transaminase activity in Escherichia coli K-12.

Summary A mutation affecting alanine--ketoisovalerate transaminase activity has been shown to be cotransducibe with the ilv gene cluster. The transaminase deficiency results in conditional isoleucine auxotrophy in the presence of alanine.     

Effect of triethyltin on Escherichia coli K-12.

Triethyltin (TET) stimulated the basal respiration of Escherichia coli K-12 membrane vesicles in chloride (Cl-) medium but it had little effect on respiration in sulphate (SO4(2-)) medium. Since this uncoupling activity was Cl- dependent it was attributed to the Cl-/hydroxide (OH-) exchange reaction known to be mediated by TET [1,2]. TET inhibited the oxidation of succinate by intact E. coli in both Cl- and SO4(2-) medium, but at the same concentration of TET, inhibition was always more extensive in Cl- than SO4(2-) medium. In Cl- medium uncoupling in membrane vesicles and inhibition of succinate oxidation in intact bacteria occurred over the same concentration range and it appeared that the same mechanism, i.e. Cl-/OH- exchange, was responsible for both effects. Inhibition of succinate oxidation in SO4(2-) medium was not substantial until the concentration of TET was greater than 10(-5) M. Although the nature of this inhibition could not be determined by experiments with membrane vesicles indirect evidence from growth experiments indicated that it was due to impairment of oxidative phosphorylation. The relationship between these biochemical findings and the bacteriocidal action of TET was examined by using various concentrations of anion and substrate in the growth medium. Growth was inhibited in media containing either Cl- or SO4(2-) as the main anion but at a particular concentration of TET, inhibition was greater in Cl- medium. Growth was also inhibited to a greater extent in succinate than glucose medium. Furthermore in either Cl- or SO4(2-) glucose medium, lactic acid production increased as the concentration of TET was increased. These findings imply that the bacteriocidal action of TET is related to its effect(s) on oxidative phosphorylation.     

Acetohydroxy acid synthase activity from a mutation at ilvF in Escherichia coli K-12.

Examination of the ilvF locus at 54 min on the Escherichia coli K-12 chromosome revealed that it is a cryptic gene for expression of a valine-resistant acetohydroxy acid synthase (acetolactate synthase; EC 4.1.3.18) distinct from previously reported isozymes. A spontaneous mutation, ilvF663, yielded IlvF+ enzyme activity that was multivalently repressed by all three branched-chain amino acids, was completely insensitive to feedback inhibition, was highly stable at elevated temperatures, and expressed optimal activity at 50 degrees C. The IlvF+ enzyme activity was expressed in strains in which isozyme II was inactive because of the ilvG frameshift in the wild-type strain K-12 and isozymes I and III were inactivated by point mutations or deletions. Tn5 insertional mutagenesis yielded two IlvF- mutants, with the insertion in ilvF663 in each case. These observations suggest that the ilvF663 locus may be a coding region for a unique acetohydroxy acid synthase activity.