Genetic Separation of Hypoxanthine and Guanine-Xanthine Phosphoribosyltransferase Activities by Deletion Mutations in Salmonella typhimurium

Certain proAB deletion mutants of Salmonella typhimurium were found to be simultaneously deleted in a gene required for the utilization of guanine and xanthine (designated gxu). These mutants were resistant to 8-azaguanine and when carrying an additional pur mutation were unable to use guanine or xanthine as a purine source. The defect was correlated with deficiencies in the uptake and phosphoribosyltransferase activities for guanine and xanthine. Hypoxanthine and adenine activities were unaltered. The deficiency was restored to normal by transduction to pro(+) and in F' merodiploids.     

Complex typing of Salmonella typhimurium hospital strains

A salmonellosis outbreak, caused by S. typhimurium, was investigated with the use of some microbiological and molecular-biological methods of typing. This investigation revealed that the outbreak was caused by the "outbreak" electrotype of the multi-resistant variant of the infective agent, found to have several plasmidovars. The possibilities and limitations of typing by sensitivity to antibiotics and plasmid DNA profile were shown. These methods of intraspecific typing were regarded as methods making it possible to establish the heterogeneity of S. typhimurium with the use of intraclonal markers.     

Genetic mapping of the Salmonella typhimurium pepB locus.

Transposon technology has been used to map the pepB locus of Salmonella typhimurium. This locus is cotransducible by phage P22 with glyA and strB at min 56 on the Salmonella genetic map. The gene order is strB pepB glyA.     

Genetic mapping of the Salmonella typhimurium pncB locus.

The nicotinic acid phosphoribosyltransferase locus pncB was located on the Salmonella typhimurium linkage map counterclockwise relative to pyrC. P22 and P1 transductional analyses revealed linkage of pncB with aroA and pyrD, indicating a pncB map position of approximately 20 map units. The results of these cotransduction experiments also indicated that the genetic map distance between gal and pyrD is greater than the published 2.2 map units.     

Transformation of Salmonella typhimurium by Plasmid Deoxyribonucleic Acid

A modified transformation procedure that is effective for the introduction of plasmid deoxyribonucleic acid at high frequency into Salmonella typhimurium, as well as into Escherichia coli, is described. Transformed bacteria acquire a circular deoxyribonucleic acid species having the genetic and molecular characteristics of the transforming plasmid.     

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.     

Genetic analysis of thr mutations in Salmonella typhimurium

Previous workers divided threonine-requiring (Thr(-)) strains of Salmonella into three phenotypes with mutations in four complementation groups. The mutations were deemed to define four genes in the order thrD-C-A-B at minute zero on the Salmonella linkage map. In the present study 12 of these mutants were reexamined together with eight new Thr(-) strains. The three phenotypes were: homoserine-requiring (Hom(-)); Thr(-), feeders of Hom(-) strains; Thr(-), nonfeeders. Exact correlation between these phenotypic groups and three complementation groups was confirmed by abortive transduction. No evidence was found for intergenic complementation between mutations in Hom(-) strains. It is proposed that thr mutations define three genes rather than four and that these be renamed thrA (Hom(-)), thrB (Thr(-) feeders), and thrC (Thr(-) nonfeeders) to correspond with the sequence of reactions in threonine biosynthesis. Double mutant trpRthr strains were used in reciprocal three-point transduction tests to establish the order of thr mutation sites. Although revisions were made in the classification or location of several mutations, there was an overall correlation of complementation group, phenotype, and map position. The present data provide a basis for further correlation of threonine genes and biosynthetic enzymes, and analysis of cross regulation in aspartate amino acid biosynthesis in Salmonella.     

Deoxyribonucleic Acid Breaks in Heated Salmonella typhimurium LT-2 After Exposure to Nutritionally Complex Media

Minimal medium recovery of heat-treated Salmonella typhimurium LT-2 has been expressed as the reduced viability on trypticase soy agar supplemented with 0.5% yeast extract (TSY) relative to a glucose-salts (M-9) agar. Incubation of S. typhimurium LT-2 in water at 50 C for 15 min did not change the sedimentation patterns of deoxyribonucleic acid (DNA) in alkaline sucrose gradients. The same pattern was obtained if the heated cells were further incubated for 15 min at 37 C in M-9 broth. However, a marked increase in DNA single-strand breakage accompanied by a loss of viability was observed after a similar incubation of heated bacteria in TSY broth. If heated bacteria were incubated in M-9 broth before TSY broth, there was a decrease in the single-strand breakage occurring in the TSY broth. This decrease is believed to be the result of repair of heat-induced damage. We conclude that minimal medium recovery after heat treatment is due to DNA damage caused by sequential exposure to heat and TSY medium, such damage not occurring after sequential exposure to heat and M-9 medium.     

Genetic analysis of carbohydrate transport-deficient mutants of Salmonella typhimurium

Mutants (car) isolated from Salmonella typhimurium were unable to utilize or ferment the following carbohydrates (all d-configuration): glucose, fructose, mannose, N-acetylglucosamine, sorbitol, mannitol, maltose, melibiose, and glycerol. The mutants did utilize galactose, glucose 6-phosphate, gluconic acid, glucuronic acid, pyruvate, and l-lactate. Biochemical analysis showed that there were two classes of mutants, each lacking one component of a phosphotransferase system. CarA mutants were deficient in enzyme I; carB lacked the phosphate carrier protein, HPr. Mapping experiments showed that the carA gene was located near pro; the carB gene mapped near purC.     

Genetic map of Salmonella typhimurium, edition VIII.

We present edition VIII of the genetic map of Salmonella typhimurium LT2. We list a total of 1,159 genes, 1,080 of which have been located on the circular chromosome and 29 of which are on pSLT, the 90-kb plasmid usually found in LT2 lines. The remaining 50 genes are not yet mapped. The coordinate system used in this edition is neither minutes of transfer time in conjugation crosses nor units representing "phage lengths" of DNA of the transducing phage P22, as used in earlier editions, but centisomes and kilobases based on physical analysis of the lengths of DNA segments between genes. Some of these lengths have been determined by digestion of DNA by rare-cutting endonucleases and separation of fragments by pulsed-field gel electrophoresis. Other lengths have been determined by analysis of DNA sequences in GenBank. We have constructed StySeq1, which incorporates all Salmonella DNA sequence data known to us. StySeq1 comprises over 548 kb of nonredundant chromosomal genomic sequences, representing 11.4% of the chromosome, which is estimated to be just over 4,800 kb in length. Most of these sequences were assigned locations on the chromosome, in some cases by analogy with mapped Escherichia coli sequences.     

Replication of Deoxyribonucleic Acid During the Division Cycle of Salmonella typhimurium

The rate of thymidine incorporation into cells of Salmonella typhimurium growing in different media has been measured. In glucose-minimal medium, deoxyribonucleic acid (DNA) replication occurs during the first two-thirds of the division cycle; the final one-third of the division cycle was devoid of DNA replication. The measured doubling time of S. typhimurium in this medium is approximately 48 min, indicating that C (the time for a round of replication) and D (the time between termination and cell division) are approximately 32 and 16 min, respectively. At slower growth rates the pattern of replication is the same as glucose minimal medium. At faster growth rates the "gap" in DNA synthesis disappears. At rapid growth rates evidence for multiple forks is obtained.     

Heterogeneity of the Stability of Messenger Ribonucleic Acid in Salmonella typhimurium

Cells of Salmonella typhimurium strain SL 282, deflagellated by mechanical shear, regenerated their flagella in the absence of tryptophan, an amino acid required for growth but not found in flagellin. Ribonucleic acid (RNA) synthesis was severely inhibited by tryptophan starvation. These findings suggested that the messenger RNA (mRNA) for flagellin might be stable. Actinomycin D was used to inhibit RNA synthesis in ethylenediaminetetraacetate-treated bacteria. The introduction of an F(lac) episome into strain SL 282 permitted the simultaneous study of the synthesis of flagellin, beta-galactosidase, and total protein. In the actinomycin-treated bacteria protein and beta-galactosidase syntheses were inhibited by 90%, whereas flagellin synthesis was unaffected. We conclude that the mRNA for flagellin synthesis is stable and that species of mRNA vary with respect to metabolic stability in S. typhimurium.     

Deoxyribonucleic Acid Adenine and Cytosine Methylation in Salmonella typhimurium and Salmonella typhi

The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence - [Formula: see text] - were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC- (DpnI and MboI) and - [Formula: see text] - (EcoRII). The restriction patterns obtained for this plasmid isolated from transformed S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at - [Formula: see text] - are met hylated by enzymes coded for by the genes dam and dem, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.     

Genetic analysis of the histidine operon control region of Salmonella typhimurium

Mutants of the histidine operon control region (hisO) include two classes: (1) those completely unable to express the operon (His auxotrophs), and (2) prototrophs that are unable to achieve fully induced levels of operon expression (still His⁺ but sensitive to the drug amino-triazole). Using new, as well as previously existing hisO mutants, we constructed a fine-structure deletion map of hisO. Mutations that presumably alter the his promoter map at one end of hisO; mutations that alter the his attenuator map at the other end of hisO. Between the promoter and the attenuator lie a number of mutations that affect either the translation of the his leader peptide gene, or the formation and stability of his leader messenger RNA structures. All of the point mutations mapping in this central region revert to His⁺ at a very high frequency (10^?5 to 10^?6); this frequency is increased by both base substitution and frameshift-inducing mutagens. Many of the His^? mutants are suppressed by informational suppressors; all three types of nonsense mutations have been identified, demonstrating that translation of a region of hisO between the promoter and attenuator is essential for his operon expression. All of the hisO mutations tested are cis-dominant.     

Acid adaptation sensitizes Salmonella typhimurium to hypochlorous acid.

Acid adaptation of Salmonella typhimurium at a pH of 5.0 to 5.8 for one to two cell doublings resulted in marked sensitization of the pathogen to halogen-based sanitizers including chlorine (hypochlorous acid) and iodine. Acid-adapted S. typhimurium was more resistant to an anionic acid sanitizer than was its nonadapted counterpart. A nonselective plating medium of tryptose phosphate agar plus 1% pyruvate was used throughout the study to help recover chemically stressed cells. Mechanisms of HOCl-mediated inactivation of acid-adapted and nonadapted salmonellae were investigated. Hypochlorous acid oxidized a higher percentage of cell surface sulfhydryl groups in acid-adapted cells than in nonadapted cells, and sulfhydryl oxidation was correlated with cell inactivation. HOCl caused severe metabolic disruptions in acid-adapted and nonadapted S. typhimurium, such as respiratory loss and inability to restore the adenylate energy charge from a nutrient-starved state. Sensitization of S. typhimurium to hypochlorous acid by acid adaptation also involved increased permeability of the cell surface because nonadapted cells treated with EDTA became sensitized. The results of this study establish that acid-adapted S. typhimurium cells are highly sensitized to HOCl oxidation and that inactivation by HOCl involves changes in membrane permeability, inability to maintain or restore energy charge, and probably oxidation of essential cellular components. This study provides a basis for improved practical technologies to inactivate Salmonella and implies that acid pretreatment of food plant environments may increase the efficacy of halogen sanitizers.     

鼠伤寒沙门菌spvB基因缺陷突变株的构建

高度保守的spvB基因,其编码产物具有ADP核糖基转移酶活性,是导致受染后细胞发生凋亡的重要毒力因子.为进一步研究该基因致病机制,构建了鼠伤寒沙门菌spvB基因缺陷突变株.根据GenBank鼠伤寒沙门菌spvB基因序列,用Primer Premier 5.0设计PCR特异性引物,获得spvB基因缺陷性核苷酸片段后连接至自杀质粒PGMB151.将构建的自杀载体导入含有spvB基因的鼠伤寒沙门菌标准株SR-11中进行同源重组,PCR筛选缺陷突变株.经PCR和DNA序列测定,成功获得了spvB基因缺陷的鼠伤寒沙门菌突变株.     

Genetic characterization of pyridine nucleotide uptake mutants of Salmonella typhimurium

Two classes of pyridine nucleotide uptake mutants isolated previously in a strain of Salmonella typhimurium defective in both de novo NAD biosynthesis (nad) and pyridine nucleotide recycling (pncA) were analysed in terms of their genetic relationship to each other and their roles in the transport of nicotinamide mononucleotide as a precursor to NAD. The first class of uptake mutants, pnuA (99 units), failed to grow on nicotinamide mononucleotide (NMN) as a precursor for NAD. The second class, pnuB, grew on lower than normal levels of NMN and suppressed pnuA mutations. A third class of uptake mutant, pnuC, isolated in a nadB pncA pnuB background, also failed to grow on NMN. Transport studies and enzyme analyses confirmed these strains as defective in NMN uptake. A fourth locus, designated pnuD, was found to diminish NMN utilization in a nad pncA+ background. Tn10 insertions near pnuA, pnuC and pnuD were isolated and utilized in mapping studies. pnuA was found to map between thr and serB near trpR. The pnuC locus was cotransducible with nadA at 17 units while pnuD mapped at approximately 60 units. The biochemical and genetic data suggest that the pnuA and pnuC gene products cooperate in the utilization of NMN under normal conditions. A pnuB mutant, however, does not require the pnuA gene product for NMN uptake but does rely on the pnuC product. Fusion studies indicate that pnuC is regulated by internal NAD concentrations.     

Host Specificity of Salmonella typhimurium Deoxyribonucleic Acid Restriction and Modification

The restriction and modification genes of Salmonella typhimurium which lie near the thr locus were transferred to a restrictionless mutant of Escherichia coli. These genes were found to be allelic to the E. coli K, B, and A restriction and modification genes. E. coli recombinants with the restriction and modification host specificity of S. typhimurium restricted phage λ that had been modified by each of the seven known host specificities of E. coli at efficiency of plating levels of about 10⁻². Phage λ modified with the S. typhimurium host specificity was restricted by six of the seven E. coli host specificities but not by the RII (fi⁻ R-factor controlled) host specificity. It is proposed that the restriction and modification enzymes of this S. typhimurium host specificity have two substrates, one of which is a substrate for the RII host specificity enzymes.