Genetic Mapping of Is 200 Copies in Salmonella Typhzmurim Strain

The wild-type Saliiaonella typhiinnurium strain LT2 contains six copies of the insertion sequence element IS200 which is unique to Salmonella. We have determined the chromosomal locations of all six copies of IS200 in strain LT2. This was done by mapping the positions of TnlO elements inserted near each copy of IS200. Such TnlO insertions were detected by Southern hybridization as IS200-containing restriction fragments with altered electrophoretic mobility. The copies are located at quite evenly spaced sites in the chromosome. Some are found in regions with many known genes; others are in regions with few known functions. There is no indication of a possible function for ISZOO. The method described here should be applicable to the mapping of IS elements in general. NSERTION sequences (IS) are genetic elements that can insert copies of I themselves into different sites in a genome. These elements can also mediate various chromosomal rearrangements (including inversion, deletion and fusion of DNA segments) and alter the expression of adjacent genes (STARLINGER 1980; CALOS and MILLER 1980). Although IS elements appear to be present in most bacterial chromosomes, no functional role has been found for them. A new insertion sequence element, IS200, has recently been identified in Salmonella typhimurium (LAM and ROTH 1983). The wild-type S. typhimurium strain LT2 contains six copies of IS200. A survey of the distribution of IS200 in enteric bacteria suggests that the element may be limited to the genus Salmonella. Similar surveys carried out for IS], IS2, IS4 and IS5 (NISEN, PURUCKER and SHAPIRO 1979; NYMAN et al. 1981; LAM and ROTH 1983) show that these elements may also be confined to relatively few host bacteria. These findings raise the possibility that IS elements may serve some functional role in their respective hosts such that they are selectively maintained in a particular group of organisms and do not persist in other hosts. In hopes of finding some indication of possible function, we have determined the chromosomal locations of all six copies of IS200 in LT2. In this report we describe a method that permits mapping of these copies despite the fact that no mutants are available and no known phenotype is associated with IS200. Present address: Department of Plant Pathology, Montana State University, Bozeman, Montana 59717. Genetics 105: 801-81 1 December, 1983. 802 S. LAM AND J. R. ROTH MATERIALS AND METHODS Bacterial strains and nomenclature: The strains used in this study are listed in Table 1. The nomenclature for TnlO insertions outside of known genes has been described, by CHUMLEY, MENZEL and ROTH (1979). TnlO insertions for which cotransducible markers were found were designated by a three-letter symbol indicating the map positions of the cotransduced markers. For the two TnlO insertions for which no cotransducible markers could be found, the last letter of the three-letter symbol was designated z to indicate the uncertainty of the map position within the 10min segment. Media: Difco nutrient broth containing 0.5% NaCl was used as rich medium. Minimal medium was the E medium of VOCEL and BONNER (1956). Solid medium contained 1.5% Difco agar. To select for growth on various sugars as sole carbon sources, the NCE medium of BERKOWITZ et al. (1968) was used, supplemented with 0.2% final concentration of the appropriate sugar. When required, tetracycline was added to a final concentration of 25 pg/ml in rich medium or 10 rg/ ml in minimal medium. Kanamycin sulfate was added to a final concentration of 50 rg/ml in rich medium or 100 pg/ml in minimal medium. Histidine was used at a final concentration of 0.1 mM, and histidinol was used at a final concentration of 1 mM in minimal medium. Phage growth and transductional methods: Phage P22 (HT 105/2) which transduces with high frequency (SCHMIEGER 1971) and carries an intmutation (isolated by G. ROBERTS) was used. Phage was grown according to HOPPE et al. (1979). In transductional crosses, phage and bacteria were mixed directly on selective media. When selection involved tetracycline, phage and bacteria were mixed and incubated for 20 min in nonselective liquid medium before plating. When selection involved kanamycin, phage and bacteria were plated on nonselective rich medium, allowed to incubate overnight and then printed onto selective medium. Transductants were purified and made free of phage by streaking alternately on selective and rich media. Hfr construction and conjugational crosses: Methods for the construction of Hfr’s using homology provided by TnlO sequences have been described by CHUMLEY, MENZEL and ROTH (1979). In conjugational crosses, Hfr’s constructed using TnlO homology were used as donors, and various strains carrying single auxotrophic markers with known map positions were used as recipients. The selection was for prototrophic recombinants. In crosses in which the Hfr donor carried the his644 deletion as a counterselective marker, the selective medium used was the minimal (E) medium of VOCEL and BONNER (1956). In crosses in which the Hfr donor was prototrophic, the selection medium used was E medium containing 2 mg/ml of streptomycin to select against the donor; all auxotrophic recipient strains used in these crosses are streptomycin resistant. For conjugation, donor and recipient strains (0.1 ml of each) were mixed and plated directly on selective medium without prior incubation. Prototrophic recombinants were counted after incubation at 37” for 2 days. Resfriction fragment hybridization: The probe used in the Southern hybridization experiments was radioactively labeled replicative-form DNA of the M 13 phage derivative M13Hol76-hisD984. This recombinant phage contains an IS200 element inserted into S. typhimurium his operon sequences. The construction of M13Hol76-his984 will be described elsewhere (S. LAM and J. R. ROTH, unpublished results). The probe DNA was radioactively labeled by nick translation as described by DAVIS, BOTSTEIN and ROTH (1980). [a-”P]dATP was obtained from Amersham. DNA polymerase I was from New England Biolabs, Inc. DNA was isolated from 2-ml cultures by the method described by DAVIS, BOTSTEIN and ROTH (1980). All restriction enzymes were obtained from New England Biolabs, Inc. and used according to the supplier’s instructions. DNA fragments were separated by horizontal gel electrophoresis in 0.7-1.2% agarose (MCB Chemicals) gels as described by DAVIS, BOTSTEIN and ROTH (1980). Procedures for the transfer of DNA from agarose gel onto nitrocellulose filter (SCHLEICHER and SCHUELL BA85) and hybridization to radioactive probe were as discussed by DAVIS, BOTSTEIN and ROTH (1980). Filters were routinely prehybridized for 30 min in hybridization buffer before addition of radioactive probe. Hybridization buffer was SXSSPE, 0.3% SDS, 100 pg/ml of denatured sonicated salmon sperm DNA; lXSSPE was 0.18 M NaCI, 10 mM (Nal.5)P04, 1 mM NaZEDTA, pH 7.0 (see DAVIS, BOTSTEIN and ROTH 1980). Hybridization was at 65” without formamide for 12-18 hr. After hybridization, blots were washed four times for approximately 15