DNA sequence of a Salmonella-specific DNA fragment and the use of oligonucleotide probes for Salmonella detection

Summary Hybridization specificity of a 1.8-kb HindIII DNA fragment isolated from Salmonella typhimurium by a molecular cloning technique was confirmed by colony hybridization with 327 Salmonella isolates of various serotypes and 56 non-Salmonella isolates including Enterobacteriaceae closely related to Salmonella, such as Escherichia coli, Klebsiella, Citrobacter and Shigella. It was found that this 1.8-kb DNA fragment was highly specific for all the Salmonella isolates tested. The DNA sequence of this 1.8-kb fragment was then determined by the dideoxynucleotide chain termination method. According to this DNA sequence, six oligonucleotide fragments ranging from 17- to 26-mer were then chemically synthesized and tested for their hybridization specificities. Results show that three of the six oligonucleotide fragments are highly specific for all 327 Salmonella strains tested and can be used as probes for the specific detection of Salmonella in foods or other samples. Offprint requests to: H.-Y. Tsen     

Characterization of the Citrobacter freundii phoE gene and development of C. freundii-specific oligonucleotides

The phoE gene of Citrobacter freundii, encoding a pore-forming outer membrane protein, was cloned and its nucleotide sequence was determined. The homologies in terms of identical amino acids between the C. freundii PhoE protein and those of Escherichia coli, E. cloacae and Klebsiella pneumoniae were 90%, 86% and 84%, respectively. Two synthetic oligonucleotides, corresponding to hypervariable, cell surface-exposed regions of the protein, were tested for their specificity in polymerase chain reactions. They were specific for the species C. freundii, i.e., no reaction was detected with 35 non-C. freundii strains tested, including 17 Salmonella, two C. amalonaticus and three C. diversus strains, whereas all five C. freundii strains tested were correctly recognized.     

Characterization of a HindIII-generated DNA fragment carrying the glutamine synthetase gene of Salmonella typhimurium

The glnA gene, encoding glutamine synthetase in Salmonella typhimurium, has been cloned into the plasmid pBR322. One hybrid plasmid, pJB1, containing an 8.5 kb insert generated by a HindIII digest, was analyzed using eleven different restriction enzymes. Evidence that the region controlling glutamine synthetase expression remained on the insert was obtained by showing that the regulation is normal in cells carrying plasmids with the insert in the original and reversed orientation. Several new plasmids derived from pJB1 following SalI and EcoRI digestions were examined for their ability to complement a glnA202 mutation in order to locate the DNA segment needed for glutamine synthetase expression. The results show that cells containing plasmid pJB8, which has a 21 kb deletion, produce and regulate glutamine synthetase normally, whereas cells with a plasmid (pJB11) similar to pJB8, but lacking a 0.25 kb EcoRI fragment, do not exhibit glutamine synthetase activity. The analysis of proteins produced in minicells containing pJB8 and pJB11 show that they both produce a protein that migrates with the glutamine synthetase subunit. Because pJB11 makes an inactive protein of similar size to the glutamine synthetase subunit, the 0.25 kb deletion may encode only the C-terminus of this protein. Consistent with this finding is the presence of a strong RNA polymerase-binding site on pJB8 to the right of the 0.25 kb EcoRI that could correspond to a promoter near the N-terminus of the glnA gene.     

Landscape phage probes for Salmonella typhimurium

We selected from landscape phage library probes that bind preferentially Salmonella typhimurium cells compared with other Enterobacteriaceae. The specificity of the phage probes for S. typhimurium was analyzed by the phage-capture test, the enzyme-linked immunosorbent assay (ELISA), and the precipitation test. Interaction of representative probes with S. typhimurium was characterized by fluorescence-activated cell sorting (FACS), and fluorescent, optical and electron microscopy. The results show that the landscape phage library is a rich source of specific and robust probes for S. typhimurium suitable for long-term use in continuous monitoring devices and biosorbents.     

Oligonucleotide probes specific for the genus Salmonella and for Salm. typhimurium

The Crystal Enteric/Nonfermenter (E/NF) identification kit (Becton Dickinson Microbiology Systems, USA) was evaluated using water-derived bacterial isolates and results compared to those obtained by the API 20E system (BioMérieux, UK). Both the E/NF and 20E systems correctly identified 93% of the Enterobacteriaceae reference cultures. Both systems agreed in the identification of 64·9% of environmental isolates. The E/NF system gave a positive identification to 88·0% of isolates and the 20E to 79·5% of isolates. The principal tests which gave differing reactions between the two systems were arginine dihydrolase, lysine decarboxylase, urease and citrate utilization.     

Characterization of the fliL gene in the flagellar regulon of Escherichia coli and Salmonella typhimurium.

filL is a small gene of unknown function that lies within the beginning of a large flagellar operon of Salmonella typhimurium and Escherichia coli. A spontaneous fliL mutant of S. typhimurium, containing a frameshift mutation about 40% from the 3' end of the gene, was moderately motile but swarmed poorly, suggesting that FliL might be a component of the flagellar motor or switch. However, in-frame deletions of the E. coli gene, including an essentially total deletion, had little or no effect on motility or chemotaxis. Thus, FliL does not appear to have a major role in flagellar structure or function and is therefore unlikely to be a component of the motor or switch; the effect on motility caused by truncation of the gene is probably an indirect one.     

DNA damage-inducible loci in Salmonella typhimurium.

lac operon fusions to DNA damage-inducible (din) loci were generated in Salmonella typhimurium LT2. Many of these din fusions were efficiently repressed by cloned Escherichia coli LexA, while others were not; all required RecA for induction. Several din fusions exhibited strong inducibility and will be useful in developing an SOS induction assay in S. typhimurium to detect genotoxins.     

Regulation of the metR gene of Salmonella typhimurium.

Regulation of the Salmonella typhimurium metR gene was studied by measuring beta-galactosidase levels in Escherichia coli strains lysogenic for a lambda bacteriophage carrying a metR-lacZ fusion. The results indicate that the metR gene is negatively regulated by its own gene product and that this autoregulation involves homocysteine as a corepressor. In addition, the results indicate that the metR gene is negatively regulated by the metJ gene product over a 70- to 80-fold range.     

Molecular characterization of the Salmonella typhimurium parE gene.

The DNA sequence of the wild type S. typhimurium parE gene was determined. The predicted protein has 96.7% amino acid identity with the ParE protein of E.coli, but is 29 amino acids longer, due to an additional basepair in the 3' end of the S. typhimurium gene. Subclones of the S. typhimurium parE gene localized the sites of four heat sensitive mutations within parE. The parE206 and parE374 mutations are identical (Val67-Met) and lie in a highly conserved region corresponding to the ATP binding pocket of GyrB. Two additional heat sensitive mutations were sequenced and predict the following amino acid substitutions: parE377 (Gly399-Ser) and parE493 (Thr583-Pro). All of the heat sensitive mutations lie in regions with strong amino acid homology to GyrB.     

Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair.

Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, a umu-like function of S. typhimurium has been found; the phenotype of this function is weaker than that of its E. coli counterpart, which is consistent with the weak mutagenic response of S. typhimurium to UV compared with the response in E. coli.     

Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6

The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity. The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides. GTP and CTP are both required for the initiation of oligoribonucleotide synthesis. In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5'-end of oligoribonucleotides and CMP at the second position. On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5'-GCA-3'. This trinucleotide sequence, containing a cryptic dA at the 3'-end, differs from other known bacterial and phage primase recognition sites. SP6 primase shares some properties with the well-characterized E. colibacteriophage T7 primase. The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis. However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E. coli- or the T7-encoded ssDNA binding protein. An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys(3)His zinc finger motif that may be involved in DNA binding.     

Characterization of amber and ochre suppressors in Salmonella typhimurium.

Amber and ochre suppressor mutations in Salmonella typhimurium were selected. The amino acid insertions directed by the suppressors were inferred from suppression patterns of Escherichia coli lacI amber mutations. These amber mutations only respond to nonsense suppressors that direct the insertion of particular amino acids. Four Salmonella amber suppressors characterized insert serine, glutamine, tyrosine, and (probably) leucine. Of the three ochre suppressors characterized, two direct the insertion of tyrosine and one directs that of lysine. Of the three amber and two ochre suppressors which have been mapped by phage P22 cotransduction, all are located in the same relative position on the Salmonella map as the analogous E. coli suppressors are on the E. coli map.     

Physical map of Salmonella typhimurium LT2 DNA in the vicinity of the proA gene.

More than 55 kilobases of chromosomal DNA of Salmonella typhimurium LT2, including the gpt, proA, ataA, and newD genes, were cloned in plasmid vector pULB113. The locations of the genes and selected restriction endonuclease cleavage sites were established, and some of the restriction enzyme fragments were subcloned in plasmid vector pBR322.