Kinetics of cobalamin repression of the cob operon in Salmonella typhimurium

Abstract The cob operon in Salmonella typhimurium encodes 25 proteins involved in the biosynthesis of cobalamin. Expression of the cob operon is negatively feedback regulated by cobalamin via a translational control mechanism. The concentration of cobalamin required to repress cob expression to half-maximal was determined in vivo and in vitro to 0.4 μM and 0.6 μM, respectively. These results suggest that cob expression in wild-type cells is partially repressed by de novo synthesized cobalamin.     

Biosynthesis of cobalamin in Salmonella typhimurium: transformation of riboflavin into the 5,6-dimethylbenzimidazole moiety

In order to elucidate the biosynthesis of the base moiety of cobalamin in Salmonella typhimurium LT2, this organism was grown in the presence of [1′-¹⁴C]riboflavin. The vitamin B₁₂ isolated was ¹⁴C-labeled. It was shown by chemical degradation that the ¹⁴C-label was exclusively localized in carbon atom 2 of the 5,6-dimethylbenzimidazole moiety. This demonstrated the precursor function of riboflavin in the biosynthesis of 5,6-dimethylbenzimidazole in S. typhimurium. Received: 25 August 1998 / Accepted: 27 October 1998     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

Abstract The susceptibility of Salmonella typhimurium LT2 and of S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae . The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Galactose metabolism in gal mutants of Salmonella typhimurium and Escherichia coli

Abstract We report a new pathway for galactose metabolism in Escherichia coli and Salmonella typhimurium . Growth of gal mutants on galactose is restored by the addition of pyrrolo-quinoline quinone (PQQ) to the medium. In such strains galactose is oxidized to galactonate by a PQQ-dependent, membrane-bound dehydrogenase. A pathway for galactonate metabolism in these organisms has already been described.     

Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model

Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed. One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon. The virulence of these strains has been determined by inoculating BALB/c or Swiss mice. The 50% lethal dose of both strains is identical to that obtained for the wild-type. Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections. The frequency of Nal(R) mutants recovered from animals inoculated with either wild-type or MutS(-) cells was not affected by the presence of pRW30. These results indicate that the DNA damage which S. typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.     

Genetic variability among archival cultures of Salmonella typhimurium

The existence in our laboratory of over 10000 Salmonella typhimurium LT2 cultures sealed in agar stab vials for 33-46 years offers an opportunity for evolutionary and mutational studies. In each of 77 vials examined, 10(3)-10(5) colony forming units per vial were recovered (less than 0.01% of the original population) even after decades of undisturbed storage. Considerable genetic variability was observed in these populations. Three genetic variables, chromosome fragment size as determined by pulsed-field gel electrophoresis, extensive mutational reversions from nutritional auxotrophy to prototrophy, and differences in protein content as assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were measured.     

The outer membrane permeability mutation of the virulence-associated plasmid of Salmonella typhimurium is located in a traT-like gene

Abstract The SS-A mutation carried by the virulence-as-associated plasmid of Salmonella typhimurium results in increased outer membrane permeability to hydrophobic compounds. A 7.8-kilobase pair Bam HI- Sal I fragment containing the SS-A mutation was cloned from the virulence-associated plasmid into the cloning vector pACYC184. The cloned DNA segment hybridized with a radioactive probed prepared from the traT gene of R6-5. A similar DNA fragment, cloned from the wild-type virulence-associated plasmid, complemented the SS-A mutant phenotype. Both clones produced a protein that immunologically resembled the R6-5 TraT protein; however, the protein produced by the SS-A containing clone appeared truncated by approximately M _r 1000 indicating an alteration in the primary structure or processing of the protein. We conclude that the mutation producing the SS-A phenotype has occured in a traT -like gene of the Salmonella plasmid.     

Soluble plasma antigen in experimental Salmonella typhimurium infection in mice

Abstract To detect and characterize Salmonella antigen in blood, outbred CF-1 female mice were inoculated intraperitoneally with S. typhimurium LT-2 and blood was assayed by ELISA for Salmonella common structural antigen. Plasma antigen was detectable early in the course of infection and increased in quantity later in the course of illness when animals showed high grade bacteremia and high counts of splenic bacteria. Antigen was associated with a cell-free plasma fraction of blood, passed through filters with cut-offs of 0.2 μ and molecular mass of 1000 kDa, and was enhanced in detectability after heating to 100°C for 15 min. Antigen was concentrated by diluting plasma 1:4 in 0.1 M EDTA, heating to 100°C, and concentrating the supernate with an ultrafiltration membrane with a molecular mass cut-off of 15 kDa. By gel filtration, antigen was associated with a peak at about molecular mass 300 kDa in heated plasma and a peak at about 380 kDa in unheated plasma. These results indicate that murine typhoid infection results in circulating soluble plasma antigen, which is heat-stable with a molecular mass of approximately 300 kDa.     

Purification and characterization of distinct type of mannose-sensitive fimbriae from Salmonella typhimurium

Abstract The fimbriae (E₄) of a virulent strain of Salmonella typhimurium were purified by ion exchange chromatography in an FPLC system. They had a channelled appearance under transmission electron microscope and showed a major structural subunit of 17-kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified fimbriae were found to agglutinate guinea pig erythrocytes, but this effect was inhibited in presence of D-mannose. Immune sera raised against the Mono-Q purified fimbriae (E₄) showed cross-reactivity with the type-1 fimbriae (F₁) composed of 21-kDa fimbrin subunit, purified by a different method from the same strain.     

鼠伤寒沙门氏菌维生素B_2营养缺陷型的分离和初步遗传定位

通过在筛选培养基中补加超高浓度维生素B_2(Riboflavin)的方法(简称B_2),首次成功的诱变分离到21株独立的沙门氏菌'B_2营养缺陷型。B_2基因的转导分析结果表明,21株营养缺陷型可分为彼此不连锁的4个类群,这意味着B_2生物合成至少涉及4个结构基因。初步的遗传定位指出,2个基因位于沙门氏菌染色体遗传图的7'—22'区域,另2个分别位于22'—34'和61.5'—69'区域。     

Isolation of a cytotoxin from L-form Salmonella typhimurium

Abstract A cytotoxin protein was isolated from the sodium dodecyl sulphate (SDS)-solubilized extract of the stable L forms of Salmonella typhimurium by ion-retardation chromatography, ion-exchange chromatography, isoelectric focusing and gel filtration. The purified toxin, with a molecular mass of 32 kDa and with isoelectric point of 6.4, was thermolabile and trypsin-sensitive. Against mouse macrophages, its cytolytic effect was detectable in vitro at concentrations higher than 0.7 μg/ml, with a complete lysis obtained at 5 μg/ml. In contrast, it stimulated C3H/HeJ macrophages in the dose range of 0.1–0.5 μg/ml to allow the cell to respond to endotoxin, resulting in the significant production of tumor necrosis factor α. By Northern blot analysis, this effect was detectable at a dose as low as 0.01 μg/ml. These findings suggest that the transformation of bacillary S. typhimurium into L forms in vivo may induce alterations in host resistance against murine typhoid.     

Salmonella enteritidis temperature-sensitive mutants protect mice against challenge with virulent Salmonella strains of different serotypes

The protection conferred by temperature-sensitive mutants of Salmonella enteritidis against different wild-type Salmonella serotypes was investigated. Oral immunization with the single temperature-sensitive mutant E/1/3 or with a temperature-sensitive thymine-requiring double mutant (E/1/3T) conferred: (i) significant protection against the homologous wild-type Salmonella strains; (ii) significant cross-protection toward high challenge doses of S. typhimurium. Significant antibody levels against homologous lipopolysaccharide and against homologous and heterologous protein antigens were detected in sera from immunized mice. Moreover, a wide range of protein antigens from different Salmonella O serotypes were recognized by sera from immunized animals. Besides, primed lymphocytes from E/1/3 immunized mice recognized Salmonella antigens from different serotypes. Taken together, these results indicate that temperature-sensitive mutants of S. enteritidis are good candidates for the construction of live vaccines against Salmonella.     

A temperature-sensitive DNA adenine methyltransferase mutant of Salmonella typhimurium

A temperature-sensitive mutant of Salmonella typhimurium was isolated earlier after transposon mutagenesis with Tn10d Tet. The mutant D220 grows well at 28 °C but has a lower growth rate and forms filaments at 37 °C. Transposon-flanking fragments of mutant D220 DNA were cloned and sequenced. The transposon was inserted in the dam gene between positions 803 and 804 (assigned allele number: dam-231 : : Tn10d Tet) and resulted in a predicted ten-amino-acid-shorter Dam protein. The insertion created a stop codon that led to a truncated Dam protein with a temperature-sensitive phenotype. The insertion dam-231 : : Tn10d Tet resulted in a dam“leaky” phenotype since methylated and unmethylated adenines in GATC sequences were present. In addition, the dam-231 : : Tn10d Tet insertion rendered dam mutants temperature-sensitive for growth depending upon the genetic background of the S. typhimurium strain. The wild-type dam gene of S. typhimurium exhibited 82% identity with the Escherichia coli dam gene.     

Quercetin but not luteolin suppresses the induction of lethal shock upon infection of mice with Salmonella typhimurium

Tumor necrosis factor-alpha (TNF-alpha) is important for the induction of systemic inflammatory responses that lead to lethal shock. Quercetin and luteolin, which differ by one hydroxyl group, are known to suppress the lipopolysaccharide-induced production of TNF-alpha in vitro. We show differing inhibitory effects of quercetin and luteolin on the induction of lethal shock in Salmonella typhimurium aroA-infected mice. In a time- and dose-dependent manner, quercetin reduced the plasma levels of TNF-alpha, lowered bacterial titers in livers, prevented liver damage and prolonged survival, while luteolin had little or no effect. Compared with luteolin, quercetin increased the infiltration of Gr-1(+)CD69(+) neutrophils into the peritoneal cavity and lowered heat shock protein 70 expression. Obviously, the additional hydroxyl group in quercetin is important for suppressing infection-induced lethal shock in mice.     

鼠伤寒沙门氏菌asd基因的克隆及序列分析

以鼠伤寒沙门氏茵标准株基因组DNA作为模板,用PCR的方法扩增鼠伤寒沙门氏菌的asd基因并克隆入质粒pUCl9,并对其进行测序,序列与献报道一致。同时将质粒pYA248上的链球菌asd基因进行了置换,观察了分别含有链球菌asd基因与鼠伤寒沙门氏菌asd基因的质粒在减毒鼠伤寒沙门氏菌X4072中的生长情况,结果表明含有鼠伤寒沙门氏菌的asd基因的高拷贝质粒pUCl9的菌株生长情况更好。为完善染色体／质粒平衡致死系统,构建减毒鼠伤寒沙门氏活菌疫苗奠定了基础。