Effect of pH and temperature on nitrosamide-induced mutation in Escherichia coli

N-Nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) induced reversions in four mutant auxotropic strains of E. coli. Among other nitroso compounds tested only N-methyl-N′-nitro-N-nitrosoguanidine (MNG) was an active mutagen in the system used.     

An N-ethyl-N-nitrosourea-induced mutation in N-acetyltransferase 1 in mice

Genetic variation in human N-acetyltransferases (NAT) has been implicated in susceptibility to aromatic amine and hydrazine carcinogens and therapeutic drugs. There are mouse models for variability of human NAT1; however mice with genetic differences in Nat1 (corresponding to human NAT2), have not been available. N-Ethyl-N-nitrosourea (ENU) mutagenesis was used to create genetic variation in Nat1. Among a number of mutations identified, a base-pair change substituting threonine for isoleucine at position 95 was recovered and studied. Molecular models suggested that this substitution would alter substrate binding. Analysis of hepatic Nat1 activity with the selective substrate isoniazid showed that there was a significant reduction in enzymatic activity in the homozygous mutants compared to the parental strain.     

Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli

Previously, we described the production of N-acetylneuraminic acid (NeuAc) from N-acetylglucosamine (GlcNAc) in a system combining recombinant Escherichia coli expressing GlcNAc 2-epimerase (slr1975), E. coli expressing NeuAc synthetase (neuB), and Corynebacterium ammoniagenes. However, this system was unsuitable for large-scale production because of its complexity and low productivity. To overcome these problems, we constructed a recombinant E. coli simultaneously overexpressing slr1975 and neuB. This recombinant E. coli produced 81 mM (25 g/L) NeuAc in 22 h without the addition of C. ammoniagenes cells. For manufacturing on an industrial scale, it is preferable to use unconcentrated culture broth as the source of enzymes, and therefore, a high-density cell culture is required. An acetate-resistant mutant strain of E. coli (HN0074) was selected as the host strain because of its ability to grow to a high cell density. The NeuAc aldolase gene of E. coli HN0074 was disrupted by homologous recombination yielding E. coli N18-14, which cannot degrade NeuAc. After a 22 h reaction with 540 mM (120 g/L) GlcNAc in a 5 L jar fermenter, the culture broth of E. coli N18-14 overexpressing slr1975 and neuB contained 172 mM (53 g/L) NeuAc.     

Phenotypes of dnaA mutants of Escherichia coli sensitive to phenothiazine derivatives

The activation of DnaA protein by cardiolipin is inhibited by fluphenazinein vitro. We therefore examined the sensitivity of temperature-sensitivednaA mutants ofEscherichia coli to fluphenazine and other phenothiazine derivatives. Among the eightdnaA mutants tested,dnaA5, dnaA46 dnaA602, anddnaA604, mutants with mutations in the putative ATP binding site of DnaA protein, showed higher sensitivities to phenothiazine derivatives than did the wild-type strain. ThednaA508 anddnaA167 mutants, which have mutations in the N-terminal region of DnaA protein, also showed higher sensitivities to phenothiazine derivatives. On the other hand, thednaA204 anddnaA205 mutants, with lesions in the C-terminal region of the DnaA protein, showed the same sensitivity to phenothiazine derivatives as the wild-type strain. Complementation analysis with a plasmid containing the wild-typednaA gene and phage P1-mediated transduction confirmed thatdnaA mutations are responsible for these sensitivity phenotypes.     

Escherichia coli tatC mutations that suppress defective twin-arginine transporter signal peptides

In vitro studies have suggested that the TatBC complex serves as the receptor for signal peptides targeted for export via the twin-arginine translocation (Tat) pathway. Substitution of the hallmark twin-arginine dipeptide with two lysines abrogates export of physiological substrates in all organisms. We report the isolation and characterization of suppressor mutations that allow export of an ssTor(KK)-GFP-SsrA tripartite fusion. We identified two amino acid suppressor mutations in the first cytoplasmic loop of TatC. In addition, two other amino acids in the first cytoplasmic loop exhibit epistatic suppression. Surprisingly, we also identified a suppressor mutation predicted to lie within the second periplasmic loop of TatC, a region that is not expected to interact directly with the signal peptide. The suppressor mutations allowed export of the native Esherichia coli Tat substrate trimethylamine N-oxide reductase with a twin-lysine substitution in its signal sequence. The cytoplasmic suppressor mutations conferred SDS sensitivity and partial filamentation, indicating that Tat export of authentic substrates was impaired.     

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.     

Genetics and function of DNA ligase in Escherichia coli

The characterization of two classes of DNA ligase mutants in Escherichia coli is described. The first class consists of three mutations coding for a temperature-sensitive ligase and defines the structural gene for DNA ligase (lig). The second class of mutants (lop) overproduces an apparently wild-type enzyme; a genetic diploid analysis implies that these are promoter or operator mutations, lig and lop are cotransduced by phage P1 and map at 46 minutes on the E. coli map. Detailed studies of two lig mutants (lig4 and lig ts7) are reported, lig ts7 is a conditionally lethal mutation, proving the essential nature of the ligase gene product. Neither mutant has a major defect in recombination or ultraviolet-repair, but both show retarded sealing of 10 S pulse-labeled DNA (Okazaki fragments).     

Rates of transposition in Escherichia coli

The evolutionary role of transposable elements (TEs) is still highly controversial. Two key parameters, the transposition rate (u and w, for replicative and non-replicative transposition) and the excision rate (e) are fundamental to understanding their evolution and maintenance in populations. We have estimated u, w and e for six families of TEs (including eight members: IS1, IS2, IS3, IS4, IS5, IS30, IS150 and IS186) in Escherichia coli, using a mutation accumulation (MA) experiment. In this experiment, mutations accumulate essentially at the rate at which they appear, during a period of 80 500 (1610 generations × 50 lines) generations, and spontaneous transposition events can be detected. This differs from other experiments in which insertions accumulated under strong selective pressure or over a limited genomic target. We therefore provide new estimates for the spontaneous rates of transposition and excision in E. coli. We observed 25 transposition and three excision events in 50 MA lines, leading to overall rate estimates of u ∼ 1.15 × 10^–5, w ∼ 4 × 10⁻⁸ and e ∼ 1.08 × 10⁻⁶ (per element, per generation). Furthermore, extensive variation between elements was found, consistent with previous knowledge of the mechanisms and regulation of transposition for the different elements.     

Density Gradient Enrichment of Escherichia coli Conditional msbA Mutants

Insight into the mechanism of lipid transport to the outer membrane of gram-negative bacteria has been hampered by the lack of an effective genetic screen for defective mutants. This work demonstrates an enrichment of conditional mutants defective in lipopolysaccharide export by Ludox density gradient centrifugation and selection for detergent resistance. New temperature-sensitive mutants with lipid export defects were isolated with single missense mutations in msbA. The results demonstrate the power of this approach for the study of lipid export in Escherichia coli.     

Novel Mutations Affecting a Signaling Component for Chemotaxis of Escherichia coli

The genetic relationship between tsr and cheD mutations, which affect chemotactic ability and map at approximately 99 min on the Escherichia coli chromosome, was investigated. Mutants defective in tsr function typically exhibited wild-type swimming patterns, but were unable to carry out chemotactic responses to a number of attractant and repellent chemicals. In contrast, cheD mutants swam smoothly, with few spontaneous directional changes, and were generally nonchemotactic. In complementation tests, cheD mutations, unlike tsr, proved to be dominant to wild type, suggesting that the cheD defect might be due to an active inhibitor of chemotaxis. Mutations that inactivated the putative inhibitor were obtained by selecting for restoration of chemotactic ability or for loss of cheD dominance. The resultant double mutants were shown to carry the original cheD mutation and a second tightly linked mutation, some of which exhibited nonsense or temperature-sensitive phenotypes, implying that they had occurred in a structural gene for a protein. All such double mutants behaved like typical tsr mutants in all other respects, including complementation pattern, swimming behavior, and chemotactic ability. These findings implied that either overproduction of tsr product or synthesis of an aberrant tsr product was responsible for the chemotaxis defect of cheD strains. Such mutants should be useful in analyzing the role of the tsr product in chemotactic responses.     

Oxidative phosphorylation in yeast X. Phosphorylation ability of mutants deficient in cytochromes a and b

1. Mitochondria from three non-allelic mutants of Saccharomyces cerevisiae, each lacking cytochrome aa₃ as a consequence of single nuclear gene mutation, exhibited oxidative phosphorylation with ferricyanide as electron acceptor with the same efficiency as wild-type yeast mitochondria.     

Residues Arg114 and Arg337 are critical for the proper function of Escherichia coli gamma-glutamyltranspeptidase

To evaluate the importance of conserved Arg114 and Arg337 residues of Escherichia coli γ-glutamyltranspeptidase (EcGGT), Lys, Leu, or Asp-substituted mutants were constructed by site-directed mutagenesis. The wild-type and mutant enzymes were overexpressed in the recombinant E. coli M15 and purified by nickel-chelate chromatography to near homogeneity. With the exception of R114K, all the other mutants significantly lost GGT activity, confirming the importance of these two residues in EcGGT. Kinetic analysis of R114L, R114D, R337K, and R337L revealed a significant increase in K_m with a minor change in k_cat, leading to more than an 8-fold decrease in k_cat/K_m values. Mutations of Arg337 impaired the capability of autocatalytic processing of the enzyme. In vitro maturation experiments revealed that EcGGT precursor mutants, pro-R337K and pro-R337L, could precede a time-dependent autocatalytic process to generate the small and large subunits, while no autocatalytic processing was observed in pro-R337D. Computer modeling showed that the critical bonding distance of Gln390 O-Thr391 HG1 and Gln390 C-Thr391 OG1 are significantly increased in Arg337 replacements, implying that these distance changes might be responsible for the lack of enzyme maturation.     

Effect of the recC gene in Escherichia coli on frequencies of ultraviolet-induced mutants

UV induction of Lac^? mutations was compared with UV induction of Mal⁺ mutations in E. coli B/r strains differing in the recC gene. The frequency of Lac^? mutants per survivor induced by the same dose was not significantly affected by the recC gene but the percentage of pure rather than sectored Lac^? colonies was greater when the recC gene was present. On the other hand, as reported previously, frequencies of Mal⁺ mutants induced by the same UV dose were lower when the strain was recC. The reduction factor was the same as for spontaneous Mal⁺ mutants. The difference in the effect of the recC gene on the yields of Lac^? and Mal⁺ mutants can be explained by taking into account the influence of lethal sectoring, which introduces an artifact when mutants arising in the recC strain are scored selectively as in the case for Mal⁺ mutants, but not when the scoring is non-selective as for Lac^? mutants. Lethal sectoring as indicated by a discrepancy between total cell counts and numbers of colony-formers, was observed for the recC strain growing in liquid minimal medium corresponding to the agar medium used to score Mal⁺ mutants but was not observed for the rec⁺ strain. Both strains showed lethal sectoring in the liquid medium corresponding to the agar medium to score Lac^? mutants. The hypothesis concerning the role of lethal sectoring in the selective scoring of mutants arising in a recC background is supported by evidence concerning the UV induction of mutants in a polA1 background. Like the recC gene, the polA1 gene did not affect yields of Lac^? mutants. However, unlike the recC gene, the polA1 gene has previously been shown not to influence UV yields of prototrophic mutations (scored selectively) and not to cause lethal sectoring except under irrelevant conditions.     

Interaction of Colicins with Bacterial Cells III. Colicin-tolerant Mutations in Escherichia coli

Mutants that adsorb certain colicins without being killed, i.e., tolerant mutants (tol), were isolated from Escherichia coli K-12 strains. Selection was done either with colicin K or E2. Several groups of mutants showing different phenotypes were found, and some of them showed tolerance to both K and E colicins, which have different receptors. Many of these mutants mapped near gal. Typical mutants from group II, III, and IV were studied in more detail. The mutant loci were contransducible with gal by phage P1. The linkage order was deduced to be tol-gal-λ. In partially diploid strains, these mutant loci are recessive to wild-type alleles. Temperature-dependent conditionally tolerant mutants were also isolated. Two groups were found: the first was tolerant to E2 and E3 at 40 C, but sensitive at 30 C; the second was tolerant to E2 at 30 C, but sensitive at 40 C. Experiments done with these mutants suggest that these mutations affect the heat lability of some protein that is necessary for the response of cells to colicins. Conditionally lethal tolerant mutants were isolated which at 40 C were tolerant to E2 and E3 and could not grow, but which at 30 C were fully sensitive and grew normally. The mutation mapped near malA. The tolerance at 40 C is not due to a consequence of an inactivation of general cellular metabolism, but presumably is a cause of the subsequent inhibition of cellular growth. The results suggest that some protein components involved in the response to colicin are also vital to normal cellular growth.     

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc₁ complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8^m). Subsequently replacing ARG8^m with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit⁻). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.     

Mutants of Escherichia coli with altered deoxyribonucleases. II. Isolation and characterization of mutants for exonuclease I

Mutants of Escherichia coli K12 deficient in exonuclease I (xon^?)³ were identified by enzymic assay of randomly selected, heavily mutagenized clones. From one of the six mutants of independent origin a thermolabile variant of exonuclease I was partially purified and identified, indicating that the mutation is probably in a structural gene for the enzyme. Transduction of this mutation into a recB^? recC^? strain did not result in the suppression of any of the phenotypic traits of the recipient. Although the five other mutants also appear to have temperature-sensitive exonuclease I activities in crude extracts, these enzymes were not sufficiently stable to permit purification. These latter mutations were of the xonA^? type; they produced a temperature-dependent suppression of the sensitivity to ultraviolet light and to mitomycin C manifested by a recB^? recC^? strain. None of the six mutations were of the sbcB^? type; that is, they did not suppress the recombination deficiency of a recB^? recC^? strain.In experiments with bacteriophage Plke, the six mutations were 41 to 62% cotransducible with the his region of E. coli. Heterozygous F′-merodiploids were constructed and studied for possible complementation of exonuclease I activity. All six mutations and an sbcB^? mutation were recessive to the wild-type alleles, and all were found to belong to a single complementation group. The results suggest that alterations of a structural gene for exonuclease I may result in the indirect suppression of the ultraviolet and mitomycin sensitivity manifested by recB^? recC^? strains.     

Novel mutations of Escherichia coli that produce recombinogenic lesions in DNA. I. Identification and mapping of arl mutations

Hyper-rec mutants of Escherichia coli were originally identified as lac-diploid strains whose colonies exhibited unusually high numbers of Lac⁺ papillae during growth on indicator plates (Konrad, 1977). For this work, 38 hyper-rec strains with particularly high frequencies of papillation were selected and screened further, in order to identify those unusually proficient in recombination of bacteriophage λ. The screening procedure, plate-stock growth of λ duplication phages, yielded four strains that exhibited both enhanced recombination of λ and normal (or higher) yields of progeny phage. The mutants displayed the same novel phenotype: phage recombination was normal during the first lytic infection, but was stimulated four- to sixfold if the phages had previously been propagated for several cycles in the mutants. Phages thus appeared to accumulate an enhanced potential for recombination during growth in these four strains. The mutations responsible were designated arl. Enhanced recombination of the phages propagated on arl strains occurred in subsequent test infections of both arl and arl⁺ bacteria, but not in recA cells. Both the high frequency of Lac⁺ papillae and the effects on λ recombination appeared to result from the same mutations. The former phenotype was used for genetic analysis of two arl mutants; their location is near 2 minutes on the E. coli map. Known alleles of two nearby genes, polB and mutT, do not confer a hyper-rec phenotype (by the lac-diploid assay). High-level RecA-constitutive strains do not exhibit enhanced recombination of duplication phages.