R Factors Improving Survival of Escherichia coli K-12 After Ultraviolet Irradiation

Two R factors which have the capacity to improve survival of some strains of Escherichia coli K-12 by approximately 60% after ultraviolet light have been identified and characterized. Both are fi(-), but neither produce colicins. The ability to enhance survival can be separated from all other identified R-factor functions. Improved survival does not result from improved excisional capacity, but does require an intact host capacity for genetic recombination. No effects on host cell growth or postirradiation lag were observed. A proposed mechanism of action is described.     

Partial characterization of a lysU mutant of Escherichia coli K-12.

The Escherichia coli K-12 strain GNB10181 shows no inducible lysyl-tRNA synthetase (LysRS) activity. Two-dimensional gel electrophoretic analysis of the polypeptides synthesized by this strain indicates that the normal lysU gene product, LysU, is absent. When both GNB10181 and its parent, MC4100, were grown at elevated temperatures (42 to 45 degrees C) no significant difference between their growth rates was observed. The lysU mutation was transferred to other E. coli K-12 backgrounds by using P1 transduction. The lysU transductants behaved comparably to their lysU+ parents at different growth temperatures. Therefore, the LysU proteins does not appear to be essential for growth at high temperatures, at least under the conditions examined here. In addition, lysU transductants were found to be defective for inducible lysine decarboxylase, (LDC), inducible arginine decarboxylase (ADI), and melibiose utilization (Mel), which are all missing in GNB10181. Complementation of the above missing functions was achieved by using the Clarke-Carbon plasmids pLC4-5 (LysU LDC) and pLC17-38 (LysU Mel ADI). From these experiments, it appears that GNB10181 has suffered a chromosomal deletion between 93.4 and 93.7 min, which includes the lysU gene. By using plasmid pLC17-38, the position of ADI on two-dimensional gels was identified. Finally, lysS delta lysU double mutants were constructed which can potentially be used as positive selection agents for the isolation of LysRS genes from other sources.     

Dominant Mutations (lex) in Escherichia coli K-12 Which Affect Radiation Sensitivity and Frequency of Ultraviolet Light-Induced Mutations

Three mutations, denoted lex-1, -2 and -3, which increase the sensitivity of Escherichia coli K-12 to ultraviolet light (UV) and ionizing radiation, have been found by three-factor transduction crosses to be closely linked to uvrA on the E. coli K-12 linkage map. Strains bearing these mutations do not appear to be defective in genetic recombination although in some conjugational crosses they may fail to produce a normal yield of genetic recombinants depending upon the time of mating and the marker selected. The mutagenic activity of UV is decreased in the mutant strains. After irradiation with UV, cultures of the strains degrade their deoxyribonucleic acid at a high rate, similar to recA(-) mutant strains. Stable lex(+)/lec(-) heterozygotes are found to have the mutant radiation-sensitive phenotype of haploid lex(-) strains.     

Dominance studies with stable merodiploids in the D-serine deaminase system of Escherichia coli K-12

An episome, F32, which carries the genetic markers dsdA(+), the presumed structural gene for d-serine deaminase, dsdC(+), a regulatory locus governing the synthesis of d-serine deaminase, aroC(+), and purC(+) was obtained from strain AB311 of Escherichia coli K-12, and was used to construct appropriate merodiploids with dsdC markers. In all dsdC / dsdC(+) diploids examined, dsdC was found to be cis dominant, trans recessive, to dsdC(+). In two cases, however, the cis dominance was only partial. Moreover, complementation was observed between one of the dsdC markers which is fully cis dominant and one which is partially cis dominant. Because of the size of the dsdC region, the phenotypes of the mutants, and the partial trans dominance of dsdC(+) over some of the dsdC mutations, it is suggested that the dsdC region specifies a product, but that this product does not move with facility through the cytoplasm     

Ultraviolet light-induced mutation in UV-resistant, thermosensitive derivatives of lexA-strains of Escherichia coli K-12

It was shown previously that a major class of UV-resistant derivatives of lexA- strains of E. coli K-12 is defective in cell division at 42.5 degrees. The thermosensitive mutations, judging by genetic mapping and complementation tests, are believed to be intragenic suppressor mutations that lower the activity of the diffusible product that results in the LexA- phenotype (Mount et al., 1973). Several thermosensitive derivatives have been characterized in regard to their susceptibility to mutation induction by UV at the permissive growth temperature (30 degrees). Although the strains tested are approximately as resistant to UV as lexA+ strains, they showed a level of mutation induction that was considerably lower. By means of genetic complementation tests it was demonstrated that the low levels of UV mutagenesis in lexA- strains and their thermosensitive derivatives result from the synthesis of a diffusible product. One possible interpretation of these results is that a diffusible product in lexA- strains prevents the induction of error-prone repair. Altering the activity of this product by tsl mutations can lead to increased, but not normal, levels of error-prone repair.     

Novel UGA-suppressors in Escherichia coli K-12

UGA-specific nonsense suppressors from Escherichia coli K-12 were isolated and characterized. One of them (Su+UGA-11) was identified as a mutant of the prfB gene for the peptide releasing factor RF2. It appears that in this strain, while peptide release at sites of UGA mutations is retarded, the UGA stop codon is read through even in the absence of a tRNA suppressor, exhibiting a novel type of passive nonsense suppression. Three suppressors (Su+UGA-12, -16 and -34) were capable of restoring the streptomycin sensitive phenotype in resistant bacteria (strAr). Because of their drug-related phenotype, these are possibly mutations in the components of the ribosomal machinery, particularly those concerned with peptide release at UGA nonsense codons. A tRNA suppressor was also obtained which was derived from the tRNA(Trp) gene. In this strain, a long region between rrnC (84.5 min) and rrnB (89.5 min) was duplicated and one of the duplicated genes of tRNA(Trp) was mutated to the suppressor. The mechanism of UGA-suppression is discussed in terms of translation termination at the nonsense codon in both active and passive fashions.     

Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12

A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenase isoenzyme. By means of Tn5-mediated insertional mutagenesis, a class of mutants was isolated (class I) that had retained 20% of the overall hydrogenase activity. As determined by neutral polyacrylamide gel electrophoresis, the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2. Therefore, the hydrogenase activity affected seemed to be electrophoretically labile and was called hydrogenase L. The presence of such an activity was recently suggested in various papers and was called isoenzyme 3. Hydrogenase L might be identical or part of the latter isoenzyme. By DEAE ion-exchange chromatography it could be separated from hydrogenases 1 and 2. Hydrogenase activity in the parent strain HB101, determined manometrically with cell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity. However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in the assay. This suggested that the fast initial activity of HB101 was due to hydrogenase L. The enzyme or enzyme complex showed an Mr around 300,000 and a pH optimum between 7 and 8. Strong indications about its physiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogen lyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero. Complementation with F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized the mutation (hydL) close to metC at approximately 64.8 min.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol     

Partial purification and characterization of four endodeoxyribonuclease activities from Escherichia coli K-12

Four hitherto undescribed endodeoxyribonucleases, temporarily designated A₁, A₂, A₃, and B, have been isolated from E. coli K-12. Each requires Mg⁺⁺ and is not stimulated by ATP or S-adenosylmethionine. A₃ is strongly inhibited by Fe⁺⁺⁺ and weakly inhibited by ATP, S-adenosylmethionine, and DPN, whereas B is inhibited by caffeine. Each can be purified free of exonuclease or DNA-3′-phosphatase. A₁ (molecular weight approximately 72,000) cleaves single-stranded, circular fd DNA to form 3′-hydroxyl termini and introduces nicks and breaks in the closed, double-stranded replicative form DNA of fd (fd RFI). A₂ (molecular weight approximately 46,000) cleaves fd DNA and introduces nicks and breaks in RFI, forming 3′-hydroxyl- and 5′-phosphoryl termini. A₃ (molecular weight approximately 38,000) cleaves fd DNA to form 3′-hydroxyl termini and introduces only nicks in fd RFI. Irradiation of the RFI with ultraviolet light markedly increases the rate of hydrolysis by A₃. B appears to form 3′-phosphoryl termini with fd DNA, but its characterization is highly preliminary due to its instability.     

Escherichia coli K-12 Mutants Resistant to Nalidixic Acid: Genetic Mapping and Dominance Studies

Escherichia coli K-12 strains tested so far (approximately 20) can be separated into three groups on the basis of their abilities to form colonies on nutrient agar supplemented with nalidixic acid (NAL): (i) Nal(s) or wild type (no growth at 1 to 2 mug/ml); (ii) NalA(r) (growth at 40 mug/ml or higher); and (iii) NalB(r) (growth at 4 mug/ml, but no growth at 10 mug/ml). The NalA(r) group has a spectrum of sensitivity ranging from 60 to over 100 mug/ml. All Hfr strains of the NalA(r) and NalB(r) groups transfer NAL resistance to recipient cells at genetic loci which are at 42.5 +/- 0.5 and 51 +/- 1 min, respectively, on the Taylor-Trotter map. Some members of the NalA(r) group also have the genetic locus for NalB(r). The nalA(s) allele is completely dominant to nalA(r) in a partial diploid configuration. In haploids, nalA(r)-nalB(r) is phenotypically NalA(r); nalA(r)-nalB(s) is NalA(r); and nalA(s)-nalB(r) is NalB(r). The map location of nalA and the easy differentiation between NalA(r) and NalA(s) allow this marker to be used as a counterselector in bacterial conjugation experiments.     

L-Serine-sensitive mutants of Escherichia coli K-12

While attempting to isolate d-serine-sensitive mutants of Escherichia coli K-12, we found a class of mutants sensitive to low concentrations of l-serine (10 to 25 mug/ml).     

Ozone-induced damage of Escherichia coli K-12

Escherichia coli K-12 transformed with pACYC184 plasmid DNA was exposed to ozone (O₃) in aqueous solution. The damage to the membrane, protein, plasmid DNA, and cell survival were investigated. Cell viability was unaffected by short-term O₃ exposure (1–5 min) but membrane permeability was compromised as indicated by protein and nucleic acid leakage and lipid oxidation. The intracellular components, protein and DNA, remained intact. With longer durations of O₃ exposure (up to 30 min) cell viability decreased with a more significant increase in lipid oxidation and protein and nucleic acid leakage. The proteins leaking out were further oxidized by O₃. The total intracellular proteins run on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and plasmid DNA run on agarose gel, showed progressive degradation corresponding to the decrease in cell viability. The data indicate that membrane components are the primary targets of O₃ damage with subsequent reactions involving the intracellular components, protein and DNA. Received: 18 Apirl 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Potassium-dependant mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 that grow more slowly in media containing low concentrations of K have been isolated. All independent mutants of this type which have been studied carry a mutation in a small region of the bacterial chromosome between the supE and gal loci. The growth rate of the mutants is the same as that of the parental strains in medium containing more than 1 mm K, but is only 50% that of the parent when the K concentration is reduced to 0.1 mm. The mutants do not appear to have a primary alteration in K transport, and are therefore referred to as K-dependent. The abbreviation kdp is proposed for this class of mutant.     

Flavodoxin mutants of Escherichia coli K-12

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.     

Phosphoglucomutase Mutants of Escherichia coli K-12

Bacteria with strongly depressed phosphoglucomutase (EC 2.7.5.1) activity are found among the mutants of Escherichia coli which, when grown on maltose, accumulate sufficient amylose to be detectable by iodine staining. These pgm mutants grow poorly on galactose but also accumulate amylose on this carbon source. Growth on lactose does not produce high amylose but, instead, results in the induction of the enzymes of maltose metabolism, presumably by accumulation of maltose. These facts suggest that the catabolism of glucose-1-phosphate is strongly depressed in pgm mutants, although not completely abolished. Anabolism of glucose-1-phosphate is also strongly depressed, since amino acid- or glucose-grown pgm mutants are sensitive to phage C21, indicating a deficiency in the biosynthesis of uridine diphosphoglucose or uridine diphosphogalactose, or both. All pgm mutations isolated map at about 16 min on the genetic map, between purE and the gal operon.