Use of Salmonella typhimurium TA 98, YG 1024 and YG 1021 and deconjugating enzymes for evaluating the mutagenicity from smokers'' urine

Four smokers were chosen for their different smoking habits, and their declared cigarette consumption confirmed by urinary measurement of nicotine and its metabolites. The promutagenicity of their urine was evaluated by the Ames test, modified according to Kado et al. (Mutation Res., 31 (1983) 25–32) after extraction on XAD2 Amberlite resin. The different Salmonella typhimurium strains TA 98, YG 1021 and YG 1024 were compared to determine the presence of amino aromatic compounds in the urine of smokers of blond and black tobacco. The strain YG 1024 shows higher mutagenicity than TA 98 for extracts from the smoker's urine and more particularly from black tobacco smokers. In addition, the pretreatment of urine by external enzymatic systems (β-glucuronidase or arylsulfatase) reveals the presence in the urine of glucurono- and sulfoconjugated forms of promutagens, including amino aromatic compounds.     

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.     

Plasmid-determined alterations of Salmonella typhimurium lipopolysaccharides

Summary Salmonella typhimurium Rc902 infected with derepressed ColIb mutants gave rise to changes in the composition of bacterial lipopolysaccharides (LPS). Bacteria carrying ColIbdrd7, derepressed in transfer, exhibited a marked decrease in the content of all 0-side-chain sugars of LPS. Similar effects were found upon the introduction of R64-11, also derepressed in transfer. In LPS of S. typhimurium containing ColIbdrd2, derepressed in colicin synthesis, a decrease of abequose content associated with an increase of glucose level was observed. Bacteria carrying the wild-type ColIb, the revertant of a drd mutant to the wild type, or the non colicinogenic strain resulting from the elimination of ColIbdrd2, showed no changes in the sugar composition of LPS.     

Envelope mutation promoting autolysis in Salmonella typhimurium

Summary Two strains independently isolated in Salmonella typhimurium display abnormal autolytic activity when nutrient broth becomes alkaline. They also show increased sensitivity to deoxycholate, EDTA, and sodium dodecyl sulfate. Response to acridine orange remains normal. In both strains a single stable mutation is responsible for all the changes. The same gene, called envD, appears to be involved in both mutant strains. envD has been located at minute 33 of the Salmonella genetic map, between markers sucA and nadA, very close to the latter. envD also affects morphological characteristics of the cells. Many mutant cells are shorter than wild type bacteria, and appear frequently associated in short chains of 4 to 10 cells. Furthermore, envD mutants display division by septation under conditions that preclude its observation in wild type strains.Career Investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina     

Salmonella typhimurium LT2 metF operator mutations

Summary Using an Escherichia coli lac deletion strain lysogenized with lambda phage carrying a metF-lacZ gene fusion (Flac), in which -galactosidase levels are dependent on metF gene expression, cis-acting mutations were isolated that affect regulation of the Salmonella typhimurium metF gene. The mutations were located in a region previously defined as the metF operator by its similarity to the E. coli metF operator sequence. Regulation of the metF gene was examined by measuring -galactosidase levels in E. coli strains lysogenized with the wild-type Flac phage and mutant Flac phage. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product, but not the vitamin B₁₂ control system mediated by the metH gene product. The results also demonstrate that negative control of the metF gene by the metH gene product and vitamin B₁₂ is dependent on a functional metJ gene product.Abbreviations Ap ampicillin - dNTP deoxyribonucleoside triphosphates - GM glucose minimal - Km kanamycin - L-agar Luria agar - LM lactose minimal - SAM s-adenosyl-L-methionine - TPEG phenylethyl -D-thiogalactoside - X-gal 5-bromo-4-chloro-3-indolyl -D-galactopyranoside - [] designates plasmid-carrier state - :: novel joint     

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.     

Salmonella typhimurium mutants affecting establishment of lysogeny

Summary Salmonella typhimurium mutants have been isolated in which phage P22 fails to establish lysogeny. These appear to be defective in cAMP metabolism. A phage mutation overcoming the bacterial defect has been mapped between gene c ₁ and gene 12.     

Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains

Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<2,2′-dimethyl-5,5′-dinitroazoxybenzene (2,2′-DM-5,5′-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB), and aminonitrotoluenes (2A4NT, 4A2NT)<2,4-DNT<4HA2NT4,4′-dimethyl-3,3′-dinitroazoxybenzene (4,4′-DM-3,3′-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2,6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2′-dimethyl-3,3′-dinitroazoxybenzene (2,2′-DM-3,3′-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.     

A comparison of aflatoxin B1-induced cytotoxicity,mutagenicity and prophage induction in Salmonella typhimurium mutagen tester strains TA1535, TA1538, TA98 and TA100

Treatment of Ames mutagen tester strains with aflatoxin B1 (AFB1) and S9 mix results not only in the production of a poten mutagen, but induces a pathway that leads to the induction of prophages present in all Ames tester strains.Characterization of the prophage induction and mutagenic response following AFB1 treatment showed that plasmid pKM101 dramatically enhances mutagenesis, but suppressed prophage induction. Spontaneous release of phage by TA98 and TA100 was also lower than in TA1535 and TA1538.In addition to mutagenesis and prophage induction, survival of all 4 tester strains was quantitated after AFB1 treatment. The data show that the frameshift tester strains (TA1538 and TA98) are more sensitive to the bactericidal action of AFB1 than the base-pair tester strains (TA1535 and TA100), survival being significantly affected above 100 ng. One of several hypotheses examined was the difference in the number and types of prophages present in base-pair tester strains that are not detectable in the frame-shift tester strains.These data suggest that prophage induction can detect DNA damage that is non-mutagenic; and that it is important to characterize the lysogenic nature of the Ames strains since it may influence the observed histidine revertant rate and the survival of the tester strain.     

Proline over-production results in enhanced osmotolerance in Salmonella typhimurium

Summary Mutants of S. typhimurium with enhanced osmotolerance were isolated. These mutants were obtained as strains which over-produced proline due to regulatory mutations affecting proline biosynthesis. The mutations are located on FproBA and upon transfer to other S. typhimurium strains, they confer enhanced osmotolerance on the recipients. The osmotolerant mutants not only have higher intracellular proline levels than the osmosensitive parental strain, but the proline levels in the osmotolerant mutants are regulated such that they increase in response to osmotic stress. Possible reasons why elevated proline levels lead to enhanced osmotolerance are discussed.     

The role of cAMP in flagellation of Salmonella typhimurium

Summary A mutational alteration either in adenylate cyclase (cya ^-) or in cyclic-35-AMP (cAMP) receptor protein (crp ^-) rendered Salmonella typhimurium incapable of producing flagella. The amount of mRNA specific for flagellin in these mutants was almost negligible when assayed in an in vitro protein synthesizing system. A secondary mutation, cfs, partially suppressing the cya ^- mutation, was identified among the revertants of cya ^-. A mutation in the same cistron as cfs resulted in a non-flagellate phenotype either by itself or in combination with cfs. The cistron, which was given the gene symbol flaT, was located between flaE and flaL. It was suggested that cAMP receptor protein together with cAMP modulates the gene flaT, which in turn acts as a positive effector on the synthesis of active mRNA specific for flagellin.     

Regulation of L-arabinose transport in Salmonella typhimurium LT2

Summary The inducible L-arabinose transport system was characterized in Salmonella typhimurium LT2. Only one L-arabinose transport system with a Km of 2x10^-4 M was identified. The results suggested that araE may be the only gene which codes for L-arabinose transport activity under the conditions tested. An araE-lac fusion strain was used to study the induction of the araE gene. No araE expression was detected when the L-arabinose concentration was lower than 1 mM. The expression of araE reached a maximum in the presence of 50 mM L-arabinose, and was significantly reduced in the presence of D-glucose. Expression of the araBAD and araE genes was coordinately regulated. The concentration of L-arabinose that allowed maximum araBAD gene expression was 50-fold lower in an araE ⁺ strain compared to an araE strain.     

Pentachlorophenol-mediated mutagenic synergy with aromatic amines in Salmonella typhimurium

Pentachlorophenol (PCP), a widely used pesticide, enhanced the mutagenic potency of plant- or mammalian-activated 2-aminofluorene (2AF) as well as the direct-acting mutagen 2-acetoxyacetylaminofluorene (2AAAF) when assayed with specific Salmonella typhimurium strains. With 2AF the mutagenic synergy was observed in strains YG1024, TA1538, and MP153. With 2AAAF the PCP-mediated synergy was observed with these strains and with strain TA98/1,8-DNP₆. The synergy was dependent upon the presence of an activated N-acetoxy functional group and was only expressed at the hisD3052 allele and not at the hisG46 allele. Spectrophotometric analysis demonstrated that the rate of degradation of 2AAAF was reduced in the presence of PCP in phosphate buffer or with S. typhimurium cytosol and thus PCP may be affecting the stability of the N-acetoxy group of activated aromatic amines.     

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.     

Mutagenic activities of selected nitrofluoranthene derivatives in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6

The mutagenic activities of novel nitrofluoranthene derivatives in Salmonella strains TA98, TA98NR and TA98/1,8-DNP6 (with and without S9 addition) are given. These derivatives were produced from the reactions of fluoranthene (FL) and its directly mutagenic 2- and 3-nitro derivatives with covalent dinitrogen pentoxide (N2O5) in CCl4 solution at ambient temperature. The influence of the addition of a nitro group on the observed activity of the resulting di- and tri-nitrofluoranthenes is discussed.     

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...     

S-adenosylmethionine synthetase in methionine regulatory mutants of Salmonella typhimurium

Summary In wild-type bacteria, S-adenosylmethionine (SAM) synthetase activity was repressed by growth in methionine. MetJ regulatory mutants had elevated activities which did not show this repression. Two metK mutants with normal regulation of the methionine biosynthetic enzymes had elevated Km's (methionine) for SAM synthetase while five metK mutants with constitutive methionine enzymes showed no measurable SAM synthetase activity. One mutant, metK ^X 721, similar in phenotype to these five had a wild-type level of SAM synthetase in conditions where SAM decarboxylase activity was blocked. By using an F-factor carrying the metK region of the genome, this mutant was shown to complement six other metK mutants.These results indicate that SAM or a derivative of it, rather than methionine itself, is the co-repressor of the methionine system, regulatory abnormalities resulting from the absence or reduction of the amount of SAM formed by the SAM synthetase reaction. As SAM is essential for bacteria it is likely that there is some alternative biosynthetic route for SAM.