Isolation and mapping of a uracil-sensitive mutant of Salmonella typhimurium

Summary A uracil-sensitive mutant of Salmonella typhimurium was isolated by diethyl sulfate mutagenesis and penicillin counterselection. This mutation identifies a new Salmonella gene that is well separated from the structural genes for arginine and pyrimidine biosynthesis. The use-1 mutation was located between the ilv gene cluster (isoleucine-valine operon) and hisR (structural gene for histidine tRNA) at 83 map units.     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

Formate dehydrogenase mutants of Salmonella typhimurium: A new medium for their isolation and new mutant classes

Summary We have designed a new medium for the differentiation of mutants of Salmonella typhirmurium defective in the ability to reduce nitrate with formate, and have characterized 24 formate dehydrogenase (FDH) mutants isolated on this medium. The mutants were assayed for the ability to use formate to reduce benzyl viologen and phenazine methosulfate, and were mapped by means of conjugation and P22-mediated transduction. Mutants lacking the ability to reduce either dye were found to map at three distinct sites: at a site co-transducible with xyl (presumably fdhA), at a site or sites between 13U and 33U, but not co-transducible with aroA, bio, purB, pyrC, or pyrD (near, but not identical with fdhB), and at a site 10–20% co-transducible with pyrE, for which we suggest the designation fdhC. Six mutant isolates reduced benzyl viologen, but not phenazine methosulfate. They retained the ability to produce nitrite during growth with nitrate. They mapped between 83U and 89U, but no co-transduction was found with metE, glnA, metB, or argH. The combined biochemical and genetic data suggest the existence of a gene in this area which is essential for the reduction of nitrate with formate, but not for formate hydrogenlyase activity or for nitrate reductase activity.     

Molecular cloning of genes involved in purine biosynthetic and salvage pathways of Salmonella typhimurium

Summary Genes have been cloned from Salmonella typhimurium which when present on the multicopy plasmid pBR322 in the E. coli strain NT31 confer a Gua⁺ phenotype on this strain. NT31 is a purE gpt double mutant and it was expected that a Gua⁺ phenotype could be conferred on it by the cloning of either gpt or purE. It was, however found that in addition to these two loci the molecular cloning of another gene, which has been identified as hpt, in pBR322 confers a Gua⁺ phenotype on NT31. This result is explained by the overproduction of the hpt gene product, hypoxanthine phosphoribosyl transferase, which compensates for the lack of the gpt product guanine-xanthine phosphoribosyl transferase. Restriction analysis of the three loci, gpt, hpt and purE is also presented.Abbreviations Kb kilobase pairs - Tc tetracyline - m.o.i multiplicity of infection - 8AG 8-azaguanine     

Growth of coliphage BF23 on rough strains of Salmonella typhimurium: the bfe locus

Summary Coliphage BF23 develops in Salmonella typhimurium rough strains. The phage is neither restricted nor modified by S. typhimurium. The growth patterns of the phage were slightly different in S. typhimurium than in Escherichia coli, although phage propagated on S. typhimurium is identical to the phage propagated in E. coli by several criteria used. Mutants of S. typhimurium resistant to BF23 were isolated and found to map (by P22-and Plmediated transduction) in the same position as bfe mutants of E. coli. The order of genes was: metB-argC-bfe-rif-purD-metA.Phage BF23 does not form plaques on smooth S. typhimurium strains, since the phage fails to adsorb irreversibly to smooth cells. Nevertheless, on solid agar, the phage prevents growth of many (but not all) smooth strains. Moreover, UV-and alkali-inactivated phage BF23, although unable to form plaques on sensitive hosts, retains the ability to prevent growth of the host on solid medium. This ability is sensitive to protease and resistant to DNAse and RNase. Heat treatment of the phage causes rapid loss of the cell-growth-preventing-ability whereas the ability to form plaques is lost much more slowly. These results lead to a proposal that phage BF23 virions carry a colicin-like factor that kills sensitive cells.     

Induction and cleavage of Salmonella typhimurium UmuD protein

Summary Two different chromosomal locations of major genes controlling extreme resistance to potato virus X (PVX) were found by restriction fragment length polymorphism (RFLP) analysis of two populations segregating for the resistance. The resistance geneRx1 mapped to the distal end of chromosome XII, whereasRx2 was located at an intermediate position on linkage group V in a region where reduced recombination and segregation distortion have also been observed. These linkage anomalies were due to abnormal behaviour of the chromosome contributed by the resistant parent P34. The results presented were obtained using two different strategies for mapping genes of unknown location. One approach was the use of probes revealing polymorphic loci spread throughout the genome and resulted in the mapping ofRx1. The second approach was based on the assumption of possible linkage between the resistance gene and clone-specific DNA fragments introduced from a wild potato species.Rx2 was mapped by adopting this strategy.     

A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803

Summary Synechocystis 6803 contains at least four cryptic plasmids of 2.27 kb (pUS1, pUS2 and pUS3) and 5.20 kb (pUS4). The 1.70 kb HpaI fragments of the related plasmids pUS2 and pUS3 were cloned into the Ap^r gene of the E. coli plasmid pACYC177, yielding the Km^r hybrid plasmids pUF12 and pUF3 respectively. pUF3 recombines in Synechocystis 6803 with a 2.27 kb plasmid giving the Km^r shuttle vector pUF311. The 1.35 kb HaeII fragment containing the Cm² gene of the E. coli plasmid pACYC184 was cloned in pUF311 generating the Cm^r Km^r shuttle vector pFCLV7. Wild-type cells of Synechocystis 6803 are transformed, albeit poorly, by the plasmids pUF3, pUF12 and pFCLV7. pFCLV7 very efficiently transforms the SUF311 strain of Synechocystis 6803 containing pUF311 as a resident plasmid. This is due to recombination between the homologous parts of pFCLV7 and pUF311. For the same reason the strain SUF311 is also efficiently transformable by E. coli plasmids, as shown for pLF8, provided that they have some homology with the E. coli part of pUF311.The combined use of Synechocystis 6803 strain SUF311 and of plasmids pFCLV7 and pLF8 generates an efficient host-vector system for gene cloning in this facultatively heterotrophic cyanobacterium.     

Inhibition of growth and aspartokinase activity of Salmonella typhimurium by thialysine

Thialysine (S-2-aminoethyl cysteine) is an analog of lysine and has been reported to inhibit the lysyl-tRNA synthetase activity of Escherichia coli. This analog inhibits the growth of Salmonella typhimurium when added to glucose minimal medium at concentrations of 1.25 mM or greater. The addition of lysine with thialysine restores the normal growth rate, whereas, methionine, valine, or leucine each enhances the growth inhibition caused by thialysine. Enzyme assays demonstrate that thialysine inhibits not only the lysyl-tRNA synthetase from S. typhimurium, but also the aspartokinase activity. Lysine and thialysine appear to inhibit the same 40% of the total aspartokinase because simultaneous addition of the two compounds to the reaction mixture does not increase the inhibition caused by either alone. Furthermore, the slow growth of cells in the presence of 2.5 mM thialysine decreases the level of aspartokinase activity, suggesting that thialysine causes repression of enzymes synthesis as well as inhibition of activity.     

A mutation affecting expression of the gene coding for serine transacetylase in Salmonella typhimurium

Summary A 1,2,4-triazole resistant mutant of S. typhimurium has been isolated, in which serine transacetylase activity is seven times higher than in wild type. Partially purified serine transacetylase from a strain carrying the trz-312 mutation has kinetic properties which are virtually identical to those of the wild type enzyme and binds to O-acetylserine sulfhydrylase A to form a cysteine synthetase complex which is also indistinguishable from that found in wild type. Thus the increased activity of serine transacetylase associated with trz-312 appears to result from increased quantities of a kinetically normal, enzyme protein. Resistance to 1,2,4-triazole is probably due to the ability of trz-312 strains to synthesize O-acetyl-l-serine at a rapid enough rate to compensate for that utilized by the O-acetylserine triazolylase reaction.Genetic mapping experiments, using P1-mediated transduction, show that trz-312 is 91–99% linked to cysE, the structural gene for serine transacetylase. The results of three point crosses indicate that this mutation is located at one extreme end of the cysE locus, as would be expected for a promotor mutation.     

Cleavage of the O antigen 4,5,12 of Salmonella typhimurium by hydrofluoric acid

The effect of hydrofluoric acid (aqueous 48% HF) upon different lipopolysaccharides (LPS) was studied, employing conditions (48 h at + 4°C) that are commonly used to dephosphorylate LPS. From the LPS of Salmonella typhimurium having the O antigen 4,5,12 almost all of the O-antigenic sugars (Abe, Gal, Glc, Man, Rha) were liberated in dialysable form, whereas the saccharide chains of Salmonella LPS with O antigen 6,7 (Man, Glc, GlcNAc) were resistant to HF. The lability towards HF was shown to be due to the presence of the deoxysugar L-rhamnose in the saccharide backbone of the O antigen 4,5,12, since only Rha was found as the terminal sugar in the corresponding dialysable material. Hydrofluoric acid can thus be used to specifically cleave Rha-containing polysaccharides.     

Cloning of the regulatory locus ompB of Salmonella typhimurium LT-2

Summary We have cloned the complete functional ompB locus of Salmonella typhimurium LT-2 into Escherichia coli K-12 using a cosmid vector and in vitro packaging into . The ompB locus of Salmonella was found to complement both envZ and ompR mutations in E. coli as well as an ompR mutation of Salmonella. The ompR part of the ompB locus was further subcloned into the multicopy plasmid pKN410 as a 1.3 kb fragment. This fragment coded for a single 28.5 kd protein corresponding to about 820 bp in length. Furthermore, the OmpR proteins of S. typhimurium and E. coli were shown to be structurally and functionally highly similar.Abbreviations SDS sedium dodecyl sulfate - kb kilobase pairs - bp base pairs - kd kilodaltons     

Cytochrome-c reductases from wild-type and mutant strains of Aspergillus nidulans

Summary The sedimentation coefficients of the NADPH: cytochrome-c oxidoreductase enzymes from wild-type and mutant strains of Aspergillus nidulans have been estimated by sucrose density gradient centrifugation. In the wild-type, two species of cytochrome-c reductase were found, with sedimentation coefficients of 13.7s and 7.6s respectively. The 13.7s species did not appear to be associated with the enzymes of nitrate reduction, whereas the 7.6s species was closely associated with NADPH: nitrate oxidoreductase. In mutant strains lacking nitrate reductase, a thir species of cytochrome-c reductase with a sedimentation coefficient of 4.5s was found. There is some evidence that this 4.5s cytochrome-c reductase is a subunit or breakdown product of nitrate reductase and a model is presented for the role of this 4.5s cytocnorome-c reductase in the assembly of the intact nitrate reductase molecule.     

Regulation of the nitrate-reducing system enzymes in wild-type and mutant strains of Chlamydomonas reinhardii

Summary Six mutant strains (301, 102, 203, 104, 305, and 307) affected in their nitrate assimilation capability and their corresponding parental wild-type strains (6145c and 21gr) from Chlamydomonas reinhardii have been studied on different nitrogen sources with respect to NAD(P)H-nitrate reductase and its associated activities (NAD(P)H-cytochrome c reductase and reduced benzyl viologen-nitrate reductase) and to nitrite reductase activity. The mutant strains lack NAD(P)H-nitrate reductase activity in all the nitrogen sources. Mutants 301, 102, 104, and 307 have only NAD(P)H-cytochrome c reductase activity whereas mutant 305 solely has reduced benzyl viologen-nitrate reductase activity. Both activities are repressible by ammonia but, in contrast to the nitrate reductase complex of wild-type strains, require neither nitrate nor nitrite for their induction. Moreover, the enzyme from mutant 305 is always obtained in active form whereas nitrate reductase from wild-types needs to be reactivated previously with ferricyanide to be fully detected. Wild-type strains and mutants 301, 102, 104, and 307, when properly induced, exhibit an NAD(P)H-cytochrome c reductase distinguishable electrophoretically from contitutive diaphorases as a rapidly migrating band. Nitrite reductase from wild-type and mutant strains is also repressible by ammonia and does not require nitrate or nitrite for its synthesis. These facts are explained in terms of a regulation of nitrate reductase synthesis by the enzyme itself.     

通过易错PCR提高鼠伤寒沙门氏菌丙氨酸消旋酶催化活性

[目的] 通过易错PCR技术提高鼠伤寒沙门氏菌中丙氨酸消旋酶的催化活性。[方法] 利用易错PCR技术构建丙氨酸消旋酶基因alr_St的突变体文库,采用缺陷菌株UT5028筛选突变体基因,以D-氨基酸氧化酶偶联法检测各突变蛋白的活性,通过凝胶过滤层析法分析酶蛋白寡聚化状态,并采用HPLC检测酶蛋白的动力学参数。[结果] 经过易错PCR及定点突变技术最终获得了3个催化活性有所提高的突变体A3V、Y343H和A3VY343H,酶学特性分析发现,与野生型蛋白StAlr相比,突变体Y343H仅对底物L/D-丝氨酸的催化效率略有提高,k_cat/K_m值分别是StAlr的2.01和3.68倍;而突变体A3V则对底物L/D-丙氨酸或L/D-丝氨酸的K_m、k_cat和k_cat/K_m值均有较大幅度的改变,其k_cat/K_m值分别是StAlr的105.51、97.36、4.63和10.73倍。凝胶过滤层析结果显示,突变体A3V在蛋白含量极低时就呈现出单体和二聚体共存状态,且随着蛋白含量的增加,其向二聚体状态迁移的速率最为明显。[结论] 丙氨酸消旋酶StAlr的第3位点是影响其催化活性和低聚合状态的关键位点。     

Functional homology of fla genes between Salmonella typhimurium and Escherichia coli

Summary Genetic studies have shown the presence of more than 20 fla genes indispensable for the formation of flagella in Salmonella typhimurium and Escherichia coli. Functional homology of the fla genes in these two bacterial species was examined through intergeneric complementation tests by bacteriophage Pl-mediated transduction from E. coli donors to S. typhimurium recipients. It was found that most of the fla gene products in these two bacterial species were interchangeable and the following correspondence was established (S. typhimurium genes vs. E. coli genes): flaFIV to flaV; flaFV to flaK; flaFVII to flaL; flaFIX to flaM; flaC to flaH; flaM to flaG; flaE to flaI; flaAI to flaN; flaAII·1 to flaB; flaAIII to flaC; flaS to flaO; flaR to flaE; flaQ to flaA; and flaB to flaR. These results suggest that the chromosomal alignment of the functionally homologous genes is very similar in these two bacterial species. Furthermore, five additional fla genes were inferred to exist in E. coli in addition to the fla genes already identified. They were termed flaU, flaX, flaY, flaZ, and flbB (flb is equivalent to fla), which corresponded to flaFI, flaFVI, flaFVIII, flaFX, and flaK of Salmonella in this order. The flaK mutants of E. coli showed no complementation with any of the flaFV, flaFVI, flaFVII, flaFVIII, or flaFIX mutants of Salmonella.     

鼠伤寒沙门氏菌baeR过表达株的构建及其耐药性

[背景]鼠伤寒沙门氏菌（Salmonella typhimurium）是一种重要的人兽共患病原菌,其多重耐药性问题日益严重,双组分系统可调控鼠伤寒沙门氏菌的耐药性。[目的]通过构建鼠伤寒沙门氏菌baeR过表达株及回补株探究BaeSR双组分系统对鼠伤寒沙门氏菌耐药性的影响。[方法]在BaeSR双组分系统和AcrB外排泵双缺失株（CRΔbaeSRΔacrB）的基础上构建baeR过表达株（CRpbaeRΔbaeSRΔacrB）及baeR回补株（CRcbaeRΔbaeSRΔacrB）,测定双缺失株、回补株和过表达株的最小抑菌浓度（minimum inhibitory concentration,MIC）,并对其生长特性、生物膜形成能力及运动性进行分析。采用转录组学技术筛选与耐药相关的差异表达基因,RT-qPCR验证耐药相关基因。[结果]构建了鼠伤寒沙门氏菌baeR过表达株和baeR回补株。与双缺失株相比,过表达株对氧氟沙星、恩诺沙星、氟苯尼考、乙酰甲喹、头孢他啶、头孢噻呋、阿莫西林和氨苄西林的MIC分别升高2-256倍,对大观霉素、安普霉素的MIC下降了50%;与双缺失株相比,回补株对头孢他啶...