We have developed a DNA-DNA hybridization test for the presence of Salmonella spp. in foods. This test requires an initial pre-enrichment of food samples in nutrient broth but does not require selective enrichment. Samples of food cultures are collected on membrane filters and assayed by molecular hybridization to labeled probes. The probes consist of DNA sequences which are unique to the genus Salmonella and are widely distributed in the genus. A diverse panel of foods was assayed successfully by this methodology.

We have developed a rapid and sensitive fluorimetric method, based on the formation of a fluorescent product from nitrosation of 2,3-diaminonaphthalene, for measuring the ability of bacteria to catalyze nitrosation of amines. We The concept that bacteria might participate in nitrosation (i.e., formation of an NNO bond to yield the N-nitroso derivative of amines) has been considered to some extent in most hypotheses concerning the possible role of endogenous nitrosation in cancer etiology (2,4,5,16,20,22,29). As recently as 1981, it was felt that the major contribution of bacteria to nitrosation was the reduction of nitrate to nitrite, resulting in an increase in the concentrations of nitrosating species (21). More recently, however, several groups have shown that bacteria can in fact participate directly in the nitrosation of amines, and this area has consequently gained renewed interest (4,5,16,17,18).A number of bacterial genera, including Neisseria, Pseudomonas, Escherichia, Klebsiella, Proteus, Alcaligenes, and Bacillus, have been investigated from several experimental perspectives and reported to have some ability to catalyze nitrosation (4,5,8,16,17,19,20,29). This catalysis is generally believed to be an anaerobic process which occurs in intact resting cells. It should probably be noted that, at this stage of research, it is not clear that all investigators have been examining the same catalytic activity.Typically, these studies have been concerned with the identification of bacterial strains (mostly clinical isolates) capable of nitrosation, rather than characterization of the reaction itself. We have thus begun a detailed investigation of this activity in Escherichia coli, which is well characterized from both biochemical and genetic perspectives. To facilitate the study of a number of bacterial strains under a wide range of reaction conditions, we have developed a new assay. This assay is based on the formation of a fluorescent product upon nitrosation of 2,3-diaminonaphthalene (31) as an alternative to the gas chromatographic-thermal energy analysis method (11) for following nitrosation of secondary amines.We have attempted to characterize the bacterial catalysis of nitrosation from a biochemical point of view by examining the effects of nitrite, nitrate, and electron donors on this process.

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Six independent secretion-defective mutations were found that result in failure to release protein from the membrane into the periplasmic space of Salmonella typhimurium after removal of the signal peptide. The mutant protein is found in a membrane-bound form accessible to trypsin added to intact spheroplasts. The phenotype of these mutations supports the existence in general of an intermediate in bacterial secretion. All six mutations changed one or the other of the two cysteine residues in the mature protein to tyrosine, suggesting that these residues are involved in the release of protein into the periplasmic space, most likely by affecting protein folding.

The target site for bacteriophage Mu integration in a lytic cycle of infection was investigated. DNA synthesis in five Hfr strains of Escherichia coli K-12 (3,9,10). Mu can also promote the integration of plasmids and direct the transposition of chromosomal markers (11-13). As is the case with some insertion elements and transposons, Mu prophages in stable lysogens are flanked by a direct repeat of five base pairs of host DNA (14,15). No form of Mu that is free of host DNA has yet been observed.One possible explanation for the ability of this phage to integrate randomly utilizes host chromosomal replication forks as target sites for Mu insertion. In a population of cells replicating asynchronously, the chromosomal replication forks must be distributed over all parts of the bacterial genome; integration of Mu at host replication forks would therefore result in an apparent lack of site specificity for insertion. Integration at host replication forks has been associated with Mu insertion events leading to the formation of stable lysogens (16,17).In this study we asked if the integration events that occur in a lytic cycle of infection involve host chromosomal replication forks. We constructed a system consisting of five Escherichia coli K-12 Fig. 1. Nonsuppressing (Su-) derivatives of the Hfr stocks were isolated by using the procedure of Templin et al. (22). The his-4 allele from strain AB1157 (23) was introduced into each Hfr strain by phage P1 transduction. Strain AT3757 was constructed by phage P1 cotransduction of the pdxAl::Mu -and ara + alleles from AT796 into a spontaneous arabinose-negative mutant of strain AT3753.Bacteriophages. Mucts62SamlO04 was obtained by heat induction of strain MH1671. Mucts62AamlO93 and Mucts62Bam7234 were obtained by heat induction of strains MH219 and MH2808, respectively. A phage strains JC1929 (XN7N53cI26) and JC1930 (h80iXN7N53cI26), used for the construction of Su-strains, were the gift of A. J. Clark.Media. Bacteria were cultured in L broth or in M9 minimal medium (24). Dilutions and washings were done in 0.85% NaCI, except where otherwise noted. Agar plates contained 1% agar (Difco) in L broth plus 5 mM MgSO4 and nalidixic acid (Sigma) at 30 ,ug/ml; agar overlays contained 0.3% agar in L broth plus nalidixic acid at 15 ,ug/ml. Amino Acid Starvation and Nalidixic Acid Treatment.Overnight cultures of Hfr strains in M9 minimal medium supplemented with histidine at 40 ug/ml were diluted 1:100 in 40 ml of the same medium and grown at 37°C for 2 hr with vigorous shaking. After this period of incubation, the cells were collected by filtration (Amicon, 0.45 ,m), washed, resuspended in 40 ml of M9 without histidine, and incubated at 37°C for 200 min to allow existing rounds of replication to terminate (25)(26)(27). We have previously determined by thymidine incorporation assays that DNA synthesis ceases in these his-4 strains after 180 min of starvation (data not shown). Starved cells were collected by filtration and resuspended in 10 ml of L broth Abbreviations: cfu, ...