Construction and characterization of mutants of Salmonella typhimurium deficient in DNA repair of O6-methylguanine.

Escherichia coli has two O6-methylguanine DNA methyltransferases that repair alkylation damage in DNA and are encoded by the ada and ogt genes. The ada gene of E. coli also regulates the adaptive response to alkylation damage. The closely related species Salmonella typhimurium possesses methyltransferase activities but does not exhibit an adaptive response conferring detectable resistance to mutagenic methylating agents. We have previously cloned the ada-like gene of S. typhimurium (adaST) and constructed an adaST-deletion derivative of S. typhimurium TA1535. Unexpectedly, the sensitivity of the resulting strain to the mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was similar to that of the parent strain. In this study, we have cloned and sequenced the ogt-like gene of S. typhimurium (ogtST) and characterized ogtST-deletion derivatives of TA1535. The ogtST mutant was more sensitive than the parent strain to the mutagenicity of MNNG and other simple alkylating agents with longer alkyl groups (ethyl, propyl, and butyl). The adaST-ogtST double mutant had a level of hypersensitivity to these agents similar to that of the ogtST single mutant. The ogtST and the adaST-ogtST mutants also displayed a two to three times higher spontaneous mutation frequency than the parent strain and the adaST mutant. These results indicate that the OgtST protein, but not the AdaST protein, plays a major role in protecting S. typhimurium from the mutagenic action of endogenous as well as exogenous alkylating agents.     

Complementation of sensitivity to alkylating agents in Escherichia coli and Chinese hamster ovary cells by expression of a cloned bacterial DNA repair gene.

Dual expression vectors derived from pSV2gpt and encoding all or part of the Escherichia coli ada+ gene have been constructed. Following transformation into an E. coli ada strain or transfection and stable integration into the genome of Chinese hamster ovary (CHO) cells, plasmid vectors containing the whole ada+ gene conferred resistance to both killing and mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Thus, the bacterial DNA repair gene was functionally expressed in the mammalian cells. Plasmids containing an N-terminal fragment of the ada+ gene which encoded only one of the two methyltransferase activities of the Ada protein did not significantly protect E. coli or CHO cells against MNNG. These results are consistent with the central role of the intact ada+ gene in controlling the adaptive response to alkylating agents in E. coli. However, the data further suggest that some alkylation lesions in DNA, such as O6-methylguanine, may exert partly different biological effects in E. coli and mammalian cells.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance.

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Molecular analysis of the Salmonella typhimurium phoN gene, which encodes nonspecific acid phosphatase.

The phoN gene of Salmonella typhimurium encodes nonspecific acid phosphatase (EC 3.1.3.2), which is regulated by a two-component regulatory system consisting of the phoP and phoQ genes. We cloned the phoN region into a plasmid vector by complementation of a phoN mutant strain and determined the nucleotide sequence of the phoN gene and its flanking regions. The phoN gene could encode a 26-kDa protein, which was identified by the maxicell method as the product of phoN. Results of the enzyme assay and Southern hybridization with chromosomal DNA of Escherichia coli K-12 suggests that there is no phoN gene in E. coli. The regulatory pattern of phoN in E. coli and Southern hybridization analysis of the E. coli chromosome with the S. typhimurium phoP gene suggest that E. coli K-12 also harbors the phoP and phoQ genes.     

Cloning of the pullulanase gene and overproduction of pullulanase in Escherichia coli and Klebsiella aerogenes.

The pullulanase gene (pul) of Klebsiella aerogenes was cloned into a pBR322 vector in Escherichia coli. Deletion analysis of the recombinant plasmid showed that the pul coding sequence, probably with the regulator gene, was located entirely within a 4.2-kilobase segment derived from the chromosomal DNA of K. aerogenes. E. coli cells carrying the recombinant plasmids produced about three- to sevenfold more pullulanase than did the wild-type strain of K. aerogenes W70. When the cloned cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Transfer of the plasmid containing the pul gene into K. aerogenes W70 resulted in about a 20- to 40-fold increase in total production of pullulanase, and the intracellular enzyme level was about 100- to 150-fold higher than that of the parent strain W70. The high level of pullulanase activity in K. aerogenes cells carrying the recombinant plasmid was maintained for at least 2 weeks.     

Cloning of the pullulanase gene and overproduction of pullulanase in Escherichia coli and Klebsiella aerogenes

The pullulanase gene (pul) of Klebsiella aerogenes was cloned into a pBR322 vector in Escherichia coli. Deletion analysis of the recombinant plasmid showed that the pul coding sequence, probably with the regulator gene, was located entirely within a 4.2-kilobase segment derived from the chromosomal DNA of K. aerogenes. E. coli cells carrying the recombinant plasmids produced about three- to sevenfold more pullulanase than did the wild-type strain of K. aerogenes W70. When the cloned cells of E. coli were grown with pullulan or maltose, most pullulanase was produced intracellularly, whereas K. aerogenes produced pullulanase extracellularly. Transfer of the plasmid containing the pul gene into K. aerogenes W70 resulted in about a 20- to 40-fold increase in total production of pullulanase, and the intracellular enzyme level was about 100- to 150-fold higher than that of the parent strain W70. The high level of pullulanase activity in K. aerogenes cells carrying the recombinant plasmid was maintained for at least 2 weeks.     

High-level expression of Escherichia coli NADPH-sulfite reductase: requirement for a cloned cysG plasmid to overcome limiting siroheme cofactor.

The flavoprotein and hemoprotein components of Escherichia coli B NADPH-sulfite reductase are encoded by cysJ and cysI, respectively. Plasmids containing these two genes overexpressed flavoprotein catalytic activity and apohemoprotein by 13- to 35-fold, but NADPH-sulfite reductase holoenzyme activity was increased only 3-fold. Maximum overexpression of holoenzyme activity was achieved by the inclusion in such plasmids of Salmonella typhimurium cysG, which encodes a uroporphyrinogen III methyltransferase required for the synthesis of siroheme, a cofactor for the hemoprotein. Thus, cofactor deficiency, in this case siroheme, can limit overexpression of a cloned enzyme. Catalytically active holoenzyme accounted for 10% of total soluble protein in a host containing cloned cysJ, cysI, and cysG. A 5.3-kb DNA fragment containing S. typhimurium cysG was sequenced, and the open reading frame corresponding to cysG was identified by subcloning and by identifying plasmid-encoded peptides in maxicells. Comparison with the sequence reported for the E. coli cysG region (J. A. Cole, unpublished data; GenBank sequence ECONIRBC) indicates a gene order of nirB-nirC-cysG in the cloned S. typhimurium fragment. In addition, two open reading frames of unknown identity were found immediately downstream of cysG. One of these contains 11 direct repeats of 33 nucleotides each, which correspond to the consensus amino acid sequence Asp-Asp-Val-Thr-Pro-Pro-Asp-Asp-Ser-Gly-Asp.     

Sequence of an osmotically inducible lipoprotein gene.

The osmB gene of Escherichia coli, whose expression is induced by elevated osmolarity, was cloned and physically mapped to a 0.65-kilobase-pair NsiI-HincII DNA fragment at 28 min on E. coli chromosome. The OsmB protein was identified in minicells expressing the cloned gene. The nucleotide sequence of a 652-base-pair chromosomal DNA fragment containing the osmB gene was determined. The open reading frame encodes a 72-residue polypeptide with an Mr of 6,949. This reading frame was confirmed by sequencing the fusion joint of an osmB::TnphoA gene fusion. The amino-terminal amino acid sequence of the open reading frame is consistent with reported signal sequences of exported proteins. The sequence around the putative signal sequence cleavage site, Leu-Ser-Ala-Cys-Ser-Asn, is highly homologous to the consensus sequence surrounding the processing site of bacterial lipoproteins. The presence of a lipid moiety on the protein was confirmed by demonstrating the incorporation of radioactive palmitic acid and inhibition of processing by globomycin. Preliminary localization of the authentic OsmB protein was determined in minicells harboring a plasmid that carries the NsiI-HincII fragment; it was primarily in the outer membrane. Surprisingly, an osmB mutant carrying the osmB::TnphoA insertion mutation was more resistant to the inhibition of metabolism by high osmolarity than the parent strain was.     

嗜麦芽假单胞菌酪氨酸酶基因在大肠杆菌中的克隆与表达

酪氨酸酶基因（ｍｅｌ）编码的酪氨酸酶是合成黑色素的关键酶。用鸟枪法分离嗜麦芽假单胞菌的ｍｅｌ基因：以ｐＵＣ１８为载体,Ｅ．ｃｏｌｉ ＨＢ１０１为受体菌,在加有一定量的Ａｍｐ和Ｌ－ｔｙｒ的酪素平板上筛选到分泌可溶性黑色素的转化子,所含重组质粒ｐＷＳＹ约７００ｂｐ的外源ＤＮＡ片段上携有ｍｅｌ基因,该片段无ＢａｍＨＩ、ＨｉｎｄＩＩＩ、ＥｃｏＲＩ、ＢｃｌＩ等酶的识别位点。Ｓｏｕｔｈｅｒｎ杂交证实此片段确实     

Cloning and DNA sequence analysis of the wild-type and mutant cyclic AMP receptor protein genes from Salmonella typhimurium.

The crp gene from Salmonella typhimurium, as well as two mutant adenylate cyclase regulation genes designated crpacr-3 and crpacr-4, were cloned into the EcoRI site of plasmid pUC8. Initially cloned on 5.6-kilobase fragments isolated from EcoRI digests of chromosomal DNA, these genes were further subcloned into the BamHI-EcoRI site of plasmid pBR322. When tested, Escherichia coli crp deletion strains harboring the clones regained their ability to pleiotropically ferment catabolite-repressible sugars. Also, the crpacr-containing strains displayed sensitivity to exogenous cyclic AMP (cAMP) when grown on eosin-methylene blue medium with xylose as the carbon source. The proteins encoded by the S. typhimurium wild-type and mutant crp genes were found to have similar molecular weights when compared with the wild-type cAMP receptor protein (CRP) from E. coli. DNA sequence analysis of the wild-type crp gene showed only a three-nucleotide difference from the E. coli sequence, suggesting little divergence of the crp gene between these organisms. The crpacr sequences, however, each contained single nucleotide changes resulting in amino acid substitutions at position 130 of the CRP. Based on the site at which these substitutions occur, the crpacr mutations are believed to affect CRP-cAMP interactions.     

猪丹毒丝菌C43065株表面保护性抗原AN端保护区在大肠杆菌中的表达

目的：在大肠杆菌中表达猪丹毒丝菌C43065株表面保护性抗原A（SpaA）N端保护区（SpaA-N）,并检测其抗原性。方法：利用PCR方法从猪丹毒丝菌C43065株基因组中扩增出spaA基因片段,构建pMD18-spaA重组质粒并对插入片段进行测序;以pMD18-spaA重组质粒为模板,PCR扩增得到spaA-N基因片段,构建重组表达质粒pGEX-spaA-N,经序列测定证实正确后转化大肠杆菌BL21（DE3）,再经IPTG诱导表达GST-SpaA-N融合蛋白并纯化。结果：扩增得到的spaA基因长1881bp,编码由626个氨基酸残基构成的多肽;SDS-PAGE和Western印迹检测结果表明,诱导表达获得相对分子质量约64000的GST-SpaA-N融合蛋白,该融合蛋白能与相应抗体发生特异性反应。结论：获得了在大肠杆菌中可溶性表达的GST-SpaA-N融合蛋白,为进一步研究猪丹毒丝菌免疫保护性抗原奠定了基础。     

Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase

The Escherichia coli cca gene which encodes the enzyme tRNA nucleotidyltransferase has been cloned by taking advantage of its proximity to the previously cloned dnaG locus. A series of recombinant bacteriophages, spanning the chromosomal region between the dnaG and cca genes at 66 min on the E. coli linkage map, were isolated from a lambda Charon 28 partial Sau3A E. coli DNA library using recombinant plasmids containing regions between dnaG and cca as probes. Two of the recombinant phage isolates, lambda c1 and lambda c4, contained the cca gene. A BamHI fragment from lambda c1 was subcloned into pBR328, and cells containing this recombinant plasmid, pRH9, expressed tRNA nucleotidyltransferase activity at about 10-fold higher level than the wild type control. The cca gene was further localized to a 1.4-kilobase stretch of DNA by Bal31 deletion analysis. The nucleotide sequence of the cca gene was determined by the dideoxy method, and revealed an open reading frame extending for a total of 412 codons from an initiator GTG codon that would encode a protein of about 47,000 daltons. Southern analysis using genomic blots demonstrated that the cca gene is present as a single copy on the E. coli chromosome and that there is no homology on the DNA level between the E. coli cca gene, and the corresponding gene in the Bacillus subtilis, Saccharomyces cerevisiae, Petunia hybrida, or Homo sapiens genomes. Homology was found only with DNA from the closely related species, Salmonella typhimurium. These studies have also allowed exact placement of the cca gene on the E. coli genetic map, and have shown that it is transcribed in a clockwise direction.     

Repair of cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium and Escherichia coli.

The role of nucleotide excision repair and 3-methyladenine DNA glycosylases in removing cytotoxic lesions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium and Escherichia coli cells was examined. Compared to the E. coli wild-type strain, the S. typhimurium wild-type strain was more sensitive to the same dose of MNNG. Nucleotide excision repair in both bacterial species does not contribute significantly to the survival after MNNG treatment, indicating that the observed differences in survival between S. typhimurium and E. coli should be attributed to DNA-repair systems other than nucleotide excision repair. The survival of the E. coli alkA mutant strain is seriously affected by the lack of 3-methyladenine DNA glycosylase II, accentuating the importance of this DNA-repair enzyme in protecting E. coli cells against the lethal effects of methylating agents. Following indications from our experiments, the existence of an alkA gene analogue in S. typhimurium has been questioned. Dot-blot hybridisation, using the E. coli alkA gene as a probe, was performed, and such a nucleotide sequence was not detected on S. typhimurium genomic DNA. The existence of constitutive 3-methyladenine DNA glycosylase, analogous to the E. coli Tag gene product in S. typhimurium cells, suggested by the results is discussed.     

A novel sensor-regulator protein that belongs to the homologous family of signal-transduction proteins involved in adaptive responses in Escherichia coli

Expression of the Escherichia coli outer membrane porins, OmpC and OmpF, is regulated in response to changes in the medium osmolarity through the functions of the regulatory factors, EnvZ and OmpR. A 3.0 kilobase pair DNA fragment cloned from E. coli is able phenotypically to suppress the defect in ompC and ompF expression caused by an envZ deletion mutation, provided that a certain gene located in this fragment is expressed on a high copy-number plasmid. Nucleotide sequencing revealed that the putative gene encodes a protein of 102,452 Da. The deduced amino acid sequence of the protein shows a high degree of homology to those of both EnvZ and OmpR, i.e. it contains both a 'sensory kinase domain' and a 'response regulator domain' in its primary amino acid sequence. The protein identified in this study is probably a novel member of the homologous family of proteins involved in bacterial adaptive responses. Hence, the gene encoding this novel sensor-regulator protein was designated as barA (bacterial adaptive responses) and mapped at 60 min on the E. coli genetic map. The BarA protein in isolated membranes was demonstrated in vitro to undergo phosphorylation in the presence of ATP.     

Cloning and characterization of gdhA, the structural gene for glutamate dehydrogenase of Salmonella typhimurium.

Glutamic acid is synthesized in enteric bacteria by either glutamate dehydrogenase or by the coupled activities of glutamate synthase and glutamine synthetase. A hybrid plasmid containing a fragment of the Salmonella typhimurium chromosome cloned into pBR328 restores growth of glutamate auxotrophs of S. typhimurium and Escherichia coli strains which have mutations in the genes for glutamate dehydrogenase and glutamate synthase. A 2.2-kilobase pair region was shown by complementation analysis, enzyme activity measurements, and the maxicell protein synthesizing system to carry the entire glutamate dehydrogenase structural gene, gdhA. Glutamate dehydrogenase encoded by gdhA carried on recombinant plasmids was elevated 5- to over 100-fold in S. typhimurium or E. coli cells and was regulated in both organisms. The gdhA promoter was located by recombination studies and by the in vitro fusion to, and activation of, a promoter-deficient galK gene. Additionally, S. typhimurium gdhA DNA was shown to hybridize to single restriction fragments of chromosomes from other enteric bacteria and from Saccharomyces cerevisiae.