Identification of a umuDC locus in Salmonella typhimurium LT2.

The umuDC operon of Escherichia coli is required for efficient mutagenesis by UV light and many other DNA-damaging agents. The existence of a umuDC analog in Salmonella typhimurium has been questioned. With DNA probes to the E. coli umuD and umuC genes, we detected, by Southern blot hybridization, sequences similar to both of these genes in S. typhimurium LT2. We also confirmed that the presence of cloned E. coli umuD enhances the UV mutability and resistance of S. typhimurium. Our data strongly suggest that S. typhimurium contains a functional umuDC operon.     

Salmonella typhimurium has two homologous but different umuDC operons: cloning of a new umuDC-like operon (samAB) present in a 60-megadalton cryptic plasmid of S. typhimurium.

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The DNA which can restore UV mutability to a umuD44 strain and to a umuC122::Tn5 strain of E. coli has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the cloned DNA potentially encoded proteins with calculated molecular weights of 15,523 and 47,726 and was an analog of the E. coli umuDC operon. We have termed this cloned DNA the samAB (for Salmonella mutagenesis) operon and tentatively referred to the umuDC operon of S. typhimurium LT2 (C. M. Smith, W. H. Koch, S. B. Franklin, P. L. Foster, T. A. Cebula, and E. Eisenstadt, J. Bacteriol. 172:4964-4978, 1990; S. M. Thomas, H. M. Crowne, S. C. Pidsley, and S. G. Sedgwick, J. Bacteriol. 172:4979-4987, 1990) as the umuDCST operon. The samAB operon is 40% diverged from the umuDCST operon at the nucleotide level. Among five umuDC-like operons so far sequenced, i.e., the samAB, umuDCST, mucAB, impAB, and E. coli umuDC operons, the samAB operon shows the highest similarity to the impAB operon of TP110 plasmid while the umuDCST operon shows the highest similarity to the E. coli umuDC operon. Southern hybridization experiments indicated that (i) S. typhimurium LT2 and TA1538 had both the samAB and the umuDCST operons and (ii) the samAB operon was located in a 60-MDa cryptic plasmid. The umuDCST operon is present in the chromosome. The presence of the two homologous but different umuDC operons may be involved in the poor mutability of S. typhimurium by UV and chemical mutagens.     

Sequence analysis and mapping of the Salmonella typhimurium LT2 umuDC operon.

In Escherichia coli, efficient mutagenesis by UV requires the umuDC operon. A deficiency in umuDC activity is believed to be responsible for the relatively weak UV mutability of Salmonella typhimurium LT2 compared with that of E. coli. To begin evaluating this hypothesis and the evolutionary relationships among umuDC-related sequences, we cloned and sequenced the S. typhimurium umuDC operon. S. typhimurium umuDC restored mutability to umuD and umuC mutants of E. coli. DNA sequence analysis of 2,497 base pairs (bp) identified two nonoverlapping open reading frames spanning 1,691 bp that were were 67 and 72% identical at the nucleotide sequence level to the umuD and umuC sequences, respectively, from E. coli. The sequences encoded proteins whose deduced primary structures were 73 and 84% identical to the E. coli umuD and umuC gene products, respectively. The two bacterial umuDC sequences were more similar to each other than to mucAB, a plasmid-borne umuDC homolog. The umuD product retained the Cys-24--Gly-25, Ser-60, and Lys-97 amino acid residues believed to be critical for RecA-mediated proteolytic activation of UmuD. The presence of a LexA box 17 bp upstream from the UmuD initiation codon suggests that this operon is a member of an SOS regulon. Mu d-P22 inserts were used to locate the S. typhimurium umuDC operon to a region between 35.9 and 40 min on the S. typhimurium chromosome. In E. coli, umuDC is located at 26 min. The umuDC locus in S. typhimurium thus appears to be near one end of a chromosomal inversion that distinguishes gene order in the 25- to 35-min regions of the E. coli and S. typhimurium chromosomes. It is likely, therefore, that the umuDC operon was present in a common ancestor before S. typhimurium and E. coli diverged approximately 150 million years ago. These results provide new information for investigating the structure, function, and evolutionary origins of umuDC and for exploring the genetic basis for the mutability differences between S. typhimurium and E. coli.     

A role for the umuDC gene products of Escherichia coli in increasing resistance to DNA damage in stationary phase by inhibiting the transition to exponential growth

The umuDC gene products, whose expression is induced by DNA-damaging treatments, have been extensively characterized for their role in SOS mutagenesis. We have recently presented evidence that supports a role for the umuDC gene products in the regulation of growth after DNA damage in exponentially growing cells, analogous to a prokaryotic DNA damage checkpoint. Our further characterization of the growth inhibition at 30 degrees C associated with constitutive expression of the umuDC gene products from a multicopy plasmid has shown that the umuDC gene products specifically inhibit the transition from stationary phase to exponential growth at the restrictive temperature of 30 degrees C and that this is correlated with a rapid inhibition of DNA synthesis. These observations led to the finding that physiologically relevant levels of the umuDC gene products, expressed from a single, SOS-regulated chromosomal copy of the operon, modulate the transition to rapid growth in E. coli cells that have experienced DNA damage while in stationary phase. This activity of the umuDC gene products is correlated with an increase in survival after UV irradiation. In a distinction from SOS mutagenesis, uncleaved UmuD together with UmuC is responsible for this activity. The umuDC-dependent increase in resistance in UV-irradiated stationary-phase cells appears to involve, at least in part, counteracting a Fis-dependent activity and thereby regulating the transition to rapid growth in cells that have experienced DNA damage. Thus, the umuDC gene products appear to increase DNA damage tolerance at least partially by regulating growth after DNA damage in both exponentially growing and stationary-phase cells.     

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.     

Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase

The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon. Expression of the cloned S. coelicolor glnA gene in E. coli cells was found to require an E. coli plasmid promoter. The nucleotide sequence of an S. coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced. Comparison of the derived S. coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S. coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.     

Construction of a umuDC operon substitution mutation in Escherichia coli.

Using a specialized transducing lambda phage, the umuDC operon of Escherichia coli was deleted and replaced with the chloramphenicol acetyltransferase gene. The delta (umuDC)595::cat mutation was subsequently transferred by generalized P1 transduction into a variety of genetic backgrounds. It is concluded that the UmuDC proteins, which are normally required for inducible mutagenesis, are not essential for cell survival.     

UV-light-induced mutability in Salmonella strains containing the umuDC or the mucAB operon: evidence for a umuC function

Multicopy plasmids carrying either the umuDC operon of Escherichia coli or its analog mucAB operon, were introduced into Ames Salmonella strains in order to analyze the influence of UmuDC and MucAB proteins on repair and mutability after UV irradiation. It was found that in uvr+ bacteria, plasmid pICV80:mucAB increased the frequency of UV-induced His+ revertants whereas pSE117:umuDC caused a smaller increase in UV mutagenesis. In delta uvrB bacteria, the protective role of pSE117 against UV killing was weak, and there was a great reduction in the mutant yield. In contrast, in these cells, pICV80 led to a large increase in both cell survival and mutation frequency. These results suggest that in Salmonella, as in E. coli, MucAB proteins mediate UV mutagenesis more efficiently than UmuDC proteins do. Plasmid pICV84:umuD+ C- significantly increased UV mutagenesis of TA2659: delta uvrB cells whereas in them, pICV77:mucA+ B- had no effect on mutability indicating the presence in Salmonella TA2659 of a gene functionally homologous to umuC.     

Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair.

Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, a umu-like function of S. typhimurium has been found; the phenotype of this function is weaker than that of its E. coli counterpart, which is consistent with the weak mutagenic response of S. typhimurium to UV compared with the response in E. coli.     

The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'non-replicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA-damaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a site-specific lesion is converted to circular double-stranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific epsilonC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C --> A mutations predominating over C --> T mutations, a specificity similar to that observed in vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.     

The genetic requirements for UmuDC-mediated cold sensitivity are distinct from those for SOS mutagenesis.

The umuDC operon of Escherichia coli, a member of the SOS regulon, is required for SOS mutagenesis. Following the posttranslational processing of UmuD to UmuD' by RecA-mediated cleavage, UmuD' acts in concert with UmuC, RecA, and DNA polymerase III to facilitate the process of translesion synthesis, which results in the introduction of mutations. Constitutive expression of the umuDC operon causes an inhibition of growth at 30 degrees C (cold sensitivity). The umuDC-dependent physiological phenomenon manifested as cold-sensitive growth is shown to differ from SOS mutagenesis in two respects. Intact UmuD, the form inactive in SOS mutagenesis, confers a significantly higher degree of cold sensitivity in combination with UmUC than does UmuD'. In addition, umuDC-mediated cold sensitivity, unlike SOS mutagenesis, does not require recA function. Since the RecA protein mediates the autodigestion of UnmD to UmuD', this finding supports the conclusion that intact UmuD is capable of conferring cold sensitivity in the presence of UmuC. The degree of inhibition of growth at 30 degrees C correlates with the levels of UmuD and UmuC, which are the only two SOS-regulated proteins required to observe cold sensitivity. Analysis of the cellular morphology of strains that exhibit cold sensitivity for growth led to the finding that constitutive expression of the umuDC operon causes a novel form of sulA- and sfiC-independent filamentation at 30 degrees C. This filamentation is observed in a strain constitutively expressing the single, chromosomal copy of umuDC and can be suppressed by overexpression of the ftsQAZ operon.     

RNase ES of Streptomyces coelicolor A3(2) can complement the rne and rng mutations in Escherichia coli

The Streptomyces coelicolor gene SCC88.10c encodes a protein (RNase ES) which is homologous to endoribonucleases in the RNase E/G family. We expressed S. coelicolor RNase ES as a 6 x His-tagged protein in an Escherichia coli mutant carrying a rng (which encodes RNase G) or a rne (which encodes RNase E) mutation to study whether S. coelicolor RNase ES is able to complement these mutations in host E. coli cells. The results clearly indicated that the S. coelicolor RNase ES can partially abrogate either the rng::cat or rne-1 mutation, as measured by the ability to suppress the several aberrant phenotypes resulting from the rng or rne mutation. Thus, S. coelicolor RNase ES appears to have the dual ability to supplant the functions of both RNase G and RNase E in E. coli.     

Studies on mutagenesis and repair induced by platinum analogs

Mutagenesis and cytotoxicity were studied in Escherichia coli by iproplatin and carboplatin, two analogs of cisplatin (CDDP) currently undergoing clinical trial. As with CDDP, mutagenesis by these agents was mediated by the umuDC gene product. In contrast to CDDP, however, mismatch repair did not substantially contribute to survival of cells after exposure to these agents since dam-3 E. coli were not more sensitive than wild type E. coli. UvrA- E. coli, however were more sensitive to these analogs demonstrating that as with CDDP, uvr endonuclease-mediated excision contributes to the repair of DNA damage induced by platinum compounds.     

天蓝色链霉菌中长链3-酮脂酰ACP合成酶的功能

【背景】链霉菌属于放线菌科,在土壤环境中广泛分布。链霉菌具有复杂的形态分化和多样性的次生代谢网络,能产生大量具有生物活性的次级代谢产物,被广泛深入研究。【目的】天蓝色链霉菌是链霉菌的模式菌株,其脂肪酸合成代谢与次级代谢联系紧密,但目前脂肪酸合成代谢途径还不清楚,其长链3-酮脂酰ACP合成酶还未见报道。【方法】利用大肠杆菌FabF序列进行同源比对,发现天蓝色链霉菌A3(2)的基因组中,SCO2390(ScoFabF1)、SCO1266(ScoFabF2)、SCO0548(ScoFabF3)和SCO5886 (ScoRedR)具有较高的相似性,并具有保守的Cys-His-His催化活性中心,可能具有长链3-酮脂酰ACP合成酶活性。采用PCR扩增方法分别获得以上基因,连入表达载体pBAD24M后分别互补大肠杆菌fabB(ts)突变株和fabB(ts)fabF双突变株,并检测转化子的生长情况。以上基因与pET-28b连接后,在大肠杆菌BL21(DE3)中表达,并利用Ni-NTA纯化获得蛋白,体外测定其催化活性。将以上基因分别互补大肠杆菌fabF突变株后,GC-MS测定互补株的脂肪酸组成。【结果】4个同源基因中,只有ScofabF1能恢复fabB(ts)fabF双突变株42°C时在添加油酸条件下的生长,其他3个基因均不能恢复生长。而这4个基因都不能恢复fabB(ts)突变株42°C时生长。体外活性测定ScoFabF1具有长链3-酮脂酰ACP合成酶活性,其他3个蛋白都不具有该活性。仅ScofabF1能显著提高大肠杆菌fabF突变株的顺-11-十八碳烯酸(C18:1)比例,其他3个基因都不具有该功能。【结论】天蓝色链霉菌中ScofabF1编码长链3-酮脂酰ACP合成酶II,在脂肪酸利用过程中发挥重要作用。天蓝色链霉菌中没有发现编码长链3-酮脂酰ACP合成酶I的基因,其可能通过其他途径合成少量的不饱和脂肪酸。以上研究结果为进一步研究天蓝色链霉菌中脂肪酸合成机制奠定了基础。     

Induction of base substitution mutations by aflatoxin B1 is mucAB dependent in Escherichia coli.

Recovery of aflatoxin B1-induced base substitution mutations in Escherichia coli was almost completely dependent on the presence of the SOS-mutagenesis-enhancing operon mucAB+; the normal E. coli analog, umuDC+, was not sufficient. Yet aflatoxin B1 induced the SOS response, including the umuDC operon, as well as did UV light. Neither preinduction of the SOS response nor the presence of additional copies of umuDC+ allowed the recovery of aflatoxin B1-induced base substitutions. Thus, the premutagenic DNA lesions induced by aflatoxin B1 reveal a functional difference between UmuDC and MucAB. We estimate that in the presence of MucAB the probability that aflatoxin B1-induced DNA lesions will be converted into mutations is increased at least 10-fold.