Anti-mutagenic effect of ultraviolet light on spontaneous tyrosine tRNA ochre suppressor mutations in Escherichia coli

Summary The numbers of tyrosine tRNA ochre suppressor mutations arising spontaneously or after UV irradiation in different strains of Escherichia coli K12 are considered. The DNA sequence change requisite for this type of mutation would be a transversion at a cytosine between two purines, where pyrimidine-pyrimidine photoproducts could not form. We find that UV mutagenesis does not produce these tyrosine tRNA ochre suppressor mutations. With lexA51 recA441 defective cells, the spontaneous yield of these mutations is elevated and UV irradiation produces a significant decrease in the numbers of this particular mutation. As explanation we suggest that the spontaneous appearance of these mutations reflects mutation at apurinic sites, the efficiency of which is elevated in lexA51 recA441 cells (with derepressed SOS functions and an activated form of RecA protein). The addition of UV damage in the DNA of these cells cannot further stimulate the positive functions that are required for the production of these mutations and are typically associated with UV mutagenesis (induction of SOS functions, activation of RecA protein and introduction of a targeting photoproduct) but apparently can have a negative effect on mutagenesis, hitherto not realized.     

Specificity of mutations induced by riboflavin mediated photosensitization in the supF gene of Escherichia coli

Riboflavin-mediated photosensitization has been shown to produce 8-hydroxyguanine (oh⁸Gua) in DNA. We investigated the specificity of mutation of photosensitized supF gene induced in Escherichia coli. The oh⁸Gua repair deficient E. coli mutant mutM and mutY were transformed with plasmid pUB3 carrying the supF gene irradiated with white light in the presence of riboflavin. Under these conditions, riboflavin photosensitization increased the amounts of oh⁸Gua in pUB3 DNA. Three types of a single base substitution occurring at G:C pairs were detected in both wild-type and mutM mutant strains. Almost all base substitutions were transversions to T:A or C:G pairs occurring at a similar extent in both wild-type and mutM strains. Mutations derived from mutY strain transformed with photosensitized DNA were only G:C to T:A transversions. These G:C to T:A transversions observed in the mutY strain were suggested to be the result of mispairing of oh⁸Gua with adenine. Riboflavin-mediated photosensitization may also produce lesions on DNA causing G:C to C:G changes by unknown mechanisms.     

Analysis of rpsD mutations in Escherichia coli

Summary A certain proportion of protein S7 exists in an altered form in E. coli rpsD (S4) mutants. Depending on the type of S4 mutation involved, two different forms of the altered S7 can be distinguished. The unusual form is longer than normal S7 by about 500 daltons due to extra material at the carboxyl end of the protein. It is suggested that a mutationally altered S4 might lower the efficiency of termination during translation of the messenger for S7. This results in an increased frequency of translational read-through, which gives the observed longer forms of S7. Data are interpreted to mean that one class of S4 mutants might suppress UGA and UAG whereas another class only suppresses UGA.     

The frequency of MMS-induced, umuDC-dependent, mutations declines during starvation in Escherichia coli

It has been found that the level of methyl methanesulfonate (MMS)-induced mutation in Escherichia coli is dependent on the level of UmuD(D)C proteins. The frequency of argE(ochre)Arg⁺ mutations (which occur predominantly by ATTA transversions) and Rif^SRif^R mutations is much higher when UmuDC or UmuD'C are overproduced in the cell. When MMS-treated bacteria were starved for progressively longer times and hence the expression of mutations delayed, the level of mutations observed progressively declined. This same treatment had no effect on the degree of SOS induction. Examination of plasmid DNAs, isolated from MMS-treated cells, for their sensitivity to the specific endonucleases Fpg and Nth revealed that MMS causes formation of abasic sites, which are repaired during cell starvation. It is assumed that, in non-dividing cells, apurinic sites are mostly repaired by RecA-mediated recombinational repair. This pathway, which is error-free, is compared with the processing pathway in metabolically active cells, where translesion synthesis by the UmuD₂C-RecA-DNA polymerase III holoenzyme complex occurs; this latter pathway is error-prone.     

A novel bicistronic vector for overexpressing Mycobacterium tuberculosis proteins in Escherichia coli

A putative DNA glycosylase encoded by the Rv3297 gene (MtuNei2) has been identified in Mycobacterium tuberculosis. Our efforts to express this gene in Escherichia coli either by supplementing tRNAs for rare codons or optimizing the gene with preferred codons for E. coli resulted in little or no expression. On the other hand, high-level expression was observed using a bicistronic expression vector in which the target gene was translationally coupled to an upstream leader sequence. Further comparison of the predicted mRNA secondary structures supported the hypothesis that mRNA secondary structure(s) surrounding the translation initiation region (TIR), rather than codon usage, played the dominant role in influencing translation efficiency, although manipulation of codon usage or tRNA supplementation did further enhance expression in the bicistronic vector. Addition of a cleavable N-terminal tag also facilitated gene expression in E. coli, possibly through a similar mechanism. However, since cleavage of N-terminal tags is determined by the amino acid at the P₁′ position downstream of the protease recognition sequence and results in the addition of an extra amino acid in front of the N-terminus of the protein, this strategy is not particularly amenable to Fpg/Nei family DNA glycosylases which carry the catalytic proline residue at the P₁′ position and require a free N-terminus. On the other hand, the bicistronic vector constructed here is potentially valuable particularly when expressing proteins from G/C rich organisms and when the proteins carry proline residues at the N-terminus in their native form. Thus the bicistronic expression system can be used to improve translation efficiency of mRNAs and achieve high-level expression of mycobacterial genes in E. coli.     

Strand targeting signal(s) for in vivo mutation avoidance by post-replication mismatch repair in Escherichia coli

Summary The involvement of GATC sites in directing mismatch correction for the elimination of replication errors in Escherichia coli was investigated in vivo by analyzing mutation rates for a gene carried on a series of related plasmids that contain 2, 1 and 0 such sites. This gene encoding chloramphenicol acetyl transferase (Cat protein) was inactivated by a point mutation. In vivo mutations restoring resistance to chloramphenicol were scored in mismatch repair proficient (mut ⁺) and deficient (mutHLS^-) strains. In mut ⁺ cells, reduction of GATC sites from 2 to 0 increased mutation rates approximately 10-fold. Removal of the GATC site distal to the cat ^- mutation increased the rate of mutation less than 2-fold, indicating that mismatch repair can proceed normally with a single site. The mutation rate increased 3-fold after removal of the GATC site proximal to the mutation. In the absence of a GATC site, mutL^- and mutS^- strains exhibited a 2- to 3-fold increased mutation rate as compared to isogenic mutH^- and mut ⁺ strains. This indicates that 50%–70% of replication errors can be corrected in a mutLS-dependent way in the absence of any GATC site to target mismatch correction to newly synthesized DNA strands. Other strand targeting signals, possibly single strand discontinuities, might be used in mutLS-dependent repair     

Mechanisms of spontaneous mutation in DNA repair-proficient Escherichia coli

This paper describes the DNA sequence analysis of 729 independent spontaneous lacI⁻ mutation This total is comprised of 478 novel mutations and 251 previously described events, and therefore should allow a more comprehensive view of spontaneous mutation in Escherichia coli. The spectrum is dominated by a hotspot (71% of all events). Mutations at this site consist of related addition and deletion events involving a number of repetitive sequences. Here we discuss how the frequency and proportion of these events vary in different DNA repair-deficient genetic backgrounds. The distribution of non-hotspot events includes base substitutions (38%), deletions (35%), frameshifts (14%), duplications (4%) and insertion elements (4%). G:C → A:T events dominate among base substitutions, while G:C → C:G events are the least common; the remaining types of base substitution are equally represented. Among deletions, a significant number do not display repeated sequences at their endpoints (26/72). However, almost all multiply recovered events (15/17) possess repeated sequences capable of accounting for the deletion endpoints. Similarily, over of all duplications recovered (5/7) display repeated endpoints. Single-base frameshifts are equally divided between A:T and G:C sites, in each case (−) 1 events occur 3-fold more frequently that (+)1 events. A comparative analysis of each mutational class recovered to lacI⁻ spectra available in a variety of DNA repair/metabolism-deficient strains is presented here in an attempt to assess possible contributions from chemical, physical and enzymic sources of damage.     

AICAR is not an endogenous mutagen in Escherichia coli

A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen. The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E. coli. We constructed E. coli tester strains that had either a normal AICAR pool (pur ⁺ his ⁺ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine). Using a set of lacZ mutations, each of which can revert to Lac⁺ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool. We conclude that the normal AICAR pool in E. coli is not a significant source of spontaneous base substitution mutagenesis.     

Excess histidine enzymes cause AICAR-independent filamentation in Escherichia coli

High-level expression of the hisHAFI genes in Escherichia coli, cloned under the control of an IPTG-inducible promoter, caused filamentation, as previously reported in Salmonella typhimurium. We speculated that this filamentation might be produced by an action of the HisH and HisF enzymes on their product AICAR (amino-imidazole carboxamide riboside 5-phosphate), a histidine by-product and normal purine precursor, possibly by favouring the formation of ZTP, the triphosphate derivative of AICAR. However, filamentation occured even in the absence of carbon flow through the histidine and purine pathways, as observed in a hisG purF strain lacking the first enzyme in each pathway. Filamentation thus does not require either the normal substrate or products of the overproduced histidine enzymes and must reflect another activity.     

Conditionallethality of Escherichia coli strains carrying dnaE anddnaQ mutations

Summary A double mutant of Escherichia coli K12 which carries a conditional lethal mutator mutation, dnaQ49 (Horiuchi et al. 1978), and a DNA polymerase III-deficient mutation, dnaE486 (Wechsler and Gross 1971), was found to be more thermolabile than was either of the dnaQ49 or dnaE486 single mutants. The double mutant is able to grow at28° C but not at 30° C. Under the restrictive conditions DNA synthesis, but not protein synthesis, of the double mutant was suppressed. All the other combinations of dnaQ and dnaE mutation alleles tested so far rendered the cells thermolabile. a dnaZ mutation exerted a similar effect on the dnaQ strain. However, when non-specific temperaturesensitive graowth mutations were conbined with the dnaQ49 mutation, no such increase in thermosensitivity was observed. There is a possibility that the product of the dnaQ gene interacts directly with the DNA replicating enzyme complex.     

Characterization of a respiratory mutant of Escherichia coli with reduced uptake of aminoglycoside antibiotics

A strain of Escherichia coli (NSW77) which is partially resistant to streptomycin was isolated by selecting for growth on plates supplemented with 12.5 μg/ml streptomycin, a concentration which completely inhibits growth of wild-type strains. The low-level resistance of the mutant appears to result from a reduced ability to accumulate streptomycin intracellularly. In addition, the mutant strain is unable to use succinate for growth because of a defective respiratory chain. Thus, membranes of the mutant strain were found to have approximately half the NADH and D-lactate oxidase activity of the parent strain. Succinate oxidase activity was reduced more drastically, to a level of 7% that of the parent strain. Moreover, membranes of the mutant were found to contain demethyl-menaquinone and, in place of ubiquinone, a structural analogue, 2-octaprenyl-3-methyl-6-methoxy-1,4 benzoquinone. The mutation responsible for both the Suc⁻ phenotype and partial resistance to streptomycin was found to be located near minute 15 on the bacterial chromosome. Both the biochemical and genetic evidence suggests that the mutation in strain NSW77 resides in the ubi F gene. Another previously characterized ubi F strain was also found to have a reduced capacity to take up an aminoglycoside antibiotic (gentamicin). These results suggest that the respiratory defects in ubi F strains are responsible for the reduced capacity of such strains to accumulate aminoglycosides.     

Escherichia coli cis- and trans-acting mutations that increaseglyA gene expression

We used anEscherichia coli strain blocked in serine biosynthesis and carrying a partialglyA deletion to isolate strains with altered regulation of theglyA gene. TheglyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increasedglyA expression were isolated on glycine supplemented plates. One class of mutations increasedglyA expression 10-fold by directly altering the – 35 consensus sequence of theglyA promoter. The two other classes increasedglyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of themetE gene of the folate branch of the methionine pathway, but notmetA in the nonfolate branch of the methionine pathway, or thegcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on theE. coli chromosome.     

Morphogenesis of Escherichia coli

Morphogenesis of the rod-shaped Escherichia coli is determined by controlled growth of an exoskeleton made of murein (peptidoglycan). Recent insights in the growth strategy of the stress-bearing murein sacculus has contributed to our understanding of how the required concerted action of murein polymerizing and hydrolyzing enzymes is achieved. The proteins involved are coordinated by the formation of multienzyme complexes. In this review, we summarize the recent results on murein structure and metabolism. On the basis of these findings, we present a model that explains maintenance of the specific rod shape of E. coli.     

recO and recR mutations delay induction of the SOS response in Escherichia coli

RecF, RecO and RecR, three of the important proteins of the RecF pathway of recombination, are also needed for repair of DNA damage due to UV irradiation. recF mutants are not proficient in cleaving LexA repressor in vivo following DNA damage; therefore they show a delay of induction of the SOS response. In this communication, by measuring the in vivo levels of LexA repressor using anti-LexA antibodies, we show that recO and recR mutant strains are also not proficient in LexA cleavage reactions. In addition, we show that recO and recR mutations delay induction of -galactosidase activity expressed from a lexA-regulated promoter following exposure of cells to UV, thus further supporting the idea that recF, recO and recR gene products are needed for induction of the SOS response.     

大肠杆菌转酮醇酶分子改造及催化酒石酸半醛合成

转酮醇酶(transketolase, TK, EC. 2.2.1.1)是一种焦磷酸硫胺素和二价阳离子依赖性酶,可催化二碳单位的转移,可逆形成C–C键,在多酶催化生产化学品、药物前体和不对称合成方面有广泛应用。文中以大肠杆菌(Escherichia coli) K12转酮醇酶TKTA为研究对象,通过定点饱和突变和组合突变提升对非磷酸化底物的反应活性,并探索突变酶TKTA＿M催化合成酒石酸半醛。结果表明：突变酶TKTA＿M (R358I/H461S/R520Q)最适反应温度为32℃,最适反应pH为7.0,以D-甘油醛为受体底物的比酶活为(6.57±0.14) U/mg,是野生型比酶活((0.71±0.02) U/mg)的9.25倍。在酶学性质研究的基础上,设计20 mL的反应体系,以50 mmol/L 5-酮基-D-葡萄糖酸和50 mmol/L非磷酸化乙醇醛为底物,TKTA＿M催化合成酒石酸半醛,最终酒石酸半醛的产量为3.71g,摩尔转化率为55.34%。研究结果为生物质制备L-(+)-酒石酸提供数据支撑,同时为转酮醇酶催化非磷酸化底物提供了借鉴。     

基因编辑技术对大肠杆菌yjjW基因点突变的两步法策略

[目的]为了实现对大肠杆菌靶基因的点突变,本研究将同源重组系统与CRISPR-Cas9技术相结合,探索一种高效、简捷的两步法策略.[方法]将靶基因的上下游同源臂和标记基因(amp)与pKOV质粒连接,获得pKOV-HR重组质粒.将pKOV-HR转化至大肠杆菌,借助其自身RecA重组系统,介导DNA发生同源重组,获得靶基...