Application of phage lambda technology to Salmonella typhimurium. Construction of a lambda-sensitive Salmonella strain

Summary We have previously constructed a novel strain of S. typhimurium carrying the E. coli lamB gene and shown that this strain adsorbs phage lambda and, for example, can be used for transposon mutagenesis with lambda vectors. In this study, we show that this strain can support the lytic growth of phage lambda nin derivatives, but not growth of wild-type lambda. However, lysogenization with lambda nin does not occur. Using this strain as starting material we took the construction one step further by introducing the E. coli nusA gene in a multicopy plasmid to this strain. We could show that this new Salmonella derivative can support both the lytic and lysogenic mode of growth of several different lambda derivatives. Using the same approach it should be possible to construct lambdasensitive derivatives of other enteric bacteria thus rendering them more amenable to in vivo genetic manipulation.     

Cosmid cloning and transposon mutagenesis in Salmonella typhimurium using phage lambda vehicles

Summary We have constructed a strain of Salmonella typhimurium which contains the malB region from Escherichia coli and carries the bacteriophage receptor protein in its outer membrane. Phage adsorbs to this strain but cannot grow, thus providing a very useful system for transposon mutagenesis of S. typhimurium using vehicles carrying transposons. This system can also be used for cosmid cloning.     

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.     

Salmonella serotype typhimurium phage typing. A critical evaluation

Of 12,930 Salmonella serotype typhimurium strains, phage typed during 1985-1988, 45.68% were "nontypable" by Anderson's set; the percent of typable strains decreased from 54.17 in 1986 to 30.54 in 1988. Of 90 phage patterns of sensitivity, 22 were currently encountered. Phage types 1, 18 and 104 were most frequent to strain of both human and non-human origin. In food generating S. typhimurium outbreaks, phage types 1 and 36 were prevalent. Except lysotypes 198 and 95, isolated from "single cases" in man only, all other phage types were common in man and animals, too. Introducing other typing methods to serotype typhimurium "nontypable" strains (by Anderson's set) was considered necessary for epidemiological purposes.     

Use of lambda vehicles to isolate ompC-lacZ gene fusions in Salmonella typhimurium LT2

Summary A novel plasmid vector, pAMH70 carrying both the lamB and nusA genes of Escherichia coli K12 was constructed. Introduction of this plasmid into Salmonella typhimurium LT2 renders this bacterium both sensitive to adsorption and able to sustain growth and lysogenization by . Using this strain as a recipient, stable gene fusions to the gene encoding a major outer membrane porin protein OmpC, were constructed with a vehicle placMu. To confirm the actual site of fusions they were genetically mapped and transducing phages carrying the ompC-lacZ fusion were isolated and relysogenized. The fusions were also shown to be to ompC by their regulatory properties.     

Molecular markers with potential to replace phage typing for Salmonella enterica serovar typhimurium

Using Amplified Fragment Length Polymorphism (AFLP) analysis of isolates from 23 phage types, we isolated 11 molecular markers that are potentially useful for molecular typing of Salmonella enterica serovar typhimurium. We tested these and 11 previously studied markers for their ability to discriminate among isolates and for correlation of their distribution with phage types. The Simpson's index of discriminatory power for the molecular markers is 0.96. One hundred and twenty one isolates from 33 phage types tested were divided into 51 types which are further grouped into 24 patterns. Eight patterns can unambiguously identify 8 phage types and a further 12 correlated with phage type distribution, showing the usefulness of these markers for molecular phage typing.     

Integration of transposon Tn10 into phage L (Salmonella typhimurium)

Transposon Tn10 was transposed into phage L (Salmonella typhimurium) from F'ts114lac+zzf::Tn10 plasmid of strain TT629 (Chumely et al. 1979). Phage L with the insertion Tn10 (L::Tn10-8) was isolated in the form of a prophage in the lysogenic strain S. typhimurium LT2-18 (L::Tn10-8), in which it can be induced with UV light. The phage induced in this way is defective; however, it forms plaques at a multiplicity of infection (moi) greater than one and transduces the tetracycline-resistance determinant to tetracycline-sensitive cells. Analysis of its DNA by restriction endodeoxyribonucleases revealed insertion of the intact transposon Tn10 of 9300 bp in the E fragment, formed during the action of EcoRI, at a distance of 16,800 bp from the pac site.     

Effect of the transfer of the R plasmids of resistant Salmonella typhimurium strains of different origins on the phage sensitivity of Salmonella typhimurium

It has been shown that after the transfer of R-plasmids from S. typhimurium strains of different origin to S typhimurium strains sensitive to antibiotics these latter strains, as a rule, cannot be typed according to the scheme of Felix and Kellow These strains retain sensitivity to phages from I. G. Chiarkadze's collection, but the spectrum of their phage sensitivity is narrower. In one case the recipient was noted to change its phagovar to that of the donor. S. typhimurium donor strains, differing in the degree of their influence on the virulence of recipients, do not differ in their capacity for changing their sensitivity to the phages from the international collection of Felix and Kellow and from I. G. Chirakadze's collection.     

Characterization of SE1, a new general transducing phage of Salmonella typhimurium

A transducing phage, SE1, which is able to infect Salmonella typhimurium was isolated from a Salmonella enteritidis strain. SE1 is a temperate phage which is heteroimmune with respect to phages P22, L, KB1 and ES18. It is similar in morphology and size to phages P22, L and KB1 and is serologically related to phages P22 and L but not to KB1. Efficiencies of generalized transduction effected by phage SE1 are similar to those for P22HT (int7), a mutant which mediates a high frequency of chromosomal gene transduction. The lengths of chromosomal DNA transduced by SE1 and P22HT (int7) are similar. Furthermore, the SE1 prophage does not exclude the transducing particles from cells it has lysogenized; consequently it is possible to use both SE1 lysogens and non-lysogenic strains as recipients in SE1-mediated transduction experiments, and obtain similar transduction efficiencies. However, the SE1 prophage gives rise to a lysogenic conversion that decreases the rate of adsorption of SE1 and L phages by about 50%, but does not affect adsorption of P22. Altogether these results suggest that phage SE1 may be a useful tool in the genetic manipulation of S. typhimurium.     

Isolation of Salmonella typhimurium mutants affecting the plaque morphology of phage P22

Summary Salmonella typhimurium mutants affecting the plaque morphology of P22 and other phages have been isolated. Using one such bacterial mutant phage mutants making turbid plaques have been isolated.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

The susceptibility of Salmonella typhimurium LT2 and 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.     

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.     

Superinfection exclusion and changes in cellular transport processes in phage infected Salmonella typhimurium.

Summary The changes induced by bacteriophage P22 in the cellular transport process(es) of the host Salmonella typhimurium (Taneja et al., 1975; Khandekar et al., 1975; Bandyopadhyay and Chakravorty, 1976) involve interactions between the superinfection exclusion system of the resident prophage and the C immunity region of the superinfecting phage. The sieA gene of the prophage interferes with the changes in the cellular transport process induced by the superinfecting phage. However, if the superinfecting phage carries active C ₁ and C ₂ genes of the superinfecting phage seem to be expressed in the sie A⁺ lysogen.