The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis

Abstract

Endonuclease digestion of the 4,800-kb chromosome of SabloneUa lyphimurium LT2 yielded 24 XbaI fragments, 12 BIn! fragments, and 7 CeuI fragments, which were separated by pulsed-field gel electrophoresis.The 90-kb plasmid pSLT has oneXbaI site and one BinI site. The locations of the fragments around the circular chromosome and of the digestion sites of the different endonucleases with respect to each other were determined by excision of agarose blocks containing fragments from single digestion, redigestion with a second enzyme, end labelling with 32P by using T7 DNA polymerase, reelectrophoresis, and autoradiography. Forty-three cleavage sites were established on the chromosome, and the fragments and cleavage sites were designated in alphabetical order and numerical order, respectively, around the chromosome. One hundred nine independent TnlO insertions previously mapped by genetic means were located by pulsed-field gel electrophoresis on the basis of the presence of XbaI and Bin1 sites in TnlO. The genomic cleavage map was divided into 100 units called centisomes; the endonuclease cleavage sites and the genes defined by the positions of Tn1O insertions were located by centisome around the map. There is very good agreement between the genomic cleavage map, defined in centisomes, and the linkage map, defined in minutes. All seven rRNA genes were located on the map; all have the Ceu! digestion site, all four with the tRNA gene for glutamate have theXbaI and the BinI sites, but only four of the seven have the Bin! site in the 16S rRNA (rrs) gene. Their inferred orientation of transcription is the same as in Escherichia coli. A rearrangement of the rrnB and rrnD genes with respect to the arrangement in E. coli, observed earlier by others, has been confirmed. The sites for all three enzymes in the rrn genes are strongly conserved compared with those in E. coli, but the XbaI and BinI sites outside the mrn genes show very little conservation.

Bacterial copper‐ and zinc‐cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis

“…Chromosomal mapping of sodC A physical map has been constructed for S. typhimurium LT2 (Liu et al, 1993). Chromosomal DNA from LT2 was digested with the rare-cutting restriction enzymes XbaI and BlnI, and analysed by pulsed-field gel electrophoresis (PFGE).…”

Muller's ratchet decreases fitness of a DNA-based microbe.

“…From these data, we can estimate an average genomic mutation rate which is based on the largest genetic target studied so far and which does not rely on the extrapolation of a mutation rate from one specific target gene or region-i.e., the lacI or his operon genes (13). In the S. typhimurium genome of 4800 kbp (14), "200 genes are involved in the biosynthetic pathways for amino acids, purines, pyrimidines, vitamins, etc. (15).…”

Highly plastic chromosomal organization in Salmonella typhi.

“…In the second group, which includes Bacillus cereus and Leptospira interrogans, a high level of intraspecies heterogeneity is found. We assume that genomic rearrangements have occurred commonly during divergence of E. coli and S. typhimurium, but that constraints on gene order, due to genomic balance or gene dosage or some other cause, have prevented survival of strains with rearrangements [except for an inversion over the terminus (3)]. However, these constraints appear to have been partially relaxed during evolution of S. typhi.…”

“…The genetic and physical genome maps of Salmonella typhimurium LT2 (3)(4)(5) (Fig. 1), Escherichia coli K-12 (9, 10), Salmonella enteritidis (11), and Salmonella paratyphi B (12) are very similar in the order of genes, although they sometimes differ by an inversion in the TER region (for termination of replication), by insertions of nonhomologous DNA ("loops"), and by the presence or absence of a plasmid.…”

“…PFGE Methods. Preparation of high-molecular weight genomic DNA, endonuclease cleaving of DNA in agarose blocks, separation of the DNA fragments by PFGE, and double-digestion techniques were as reported (3,4); analysis with I-CeuI was as described (13). The analysis by PFGE was done with the Bio-Rad DRII, the Bio-Rad Mapper, or the Hoefer Hula electrophoresis systems.…”