Construction of a combined physical and genetic map of the chromosome of Lactobacillus acidophilus ATCC 4356 and characterization of the rRNA operons

Abstract

The combination of PFGE and hybridization approaches was used to study the genome ofLactobacillus acidophilusneotype strain ATCC 4356. PFGE analysis of chromosomal DNA after digestion with each of the rare-cutting restriction enzymes I-CeuI,NotI,CspI,SmaI,ApaI andSgrAI allowed the size of the circular chromosome ofL. acidophilusto be estimated at 2·061 Mbp. The physical map contained 86 restriction sites for the six enzymes employed, with intervals between the sites varying from 1 to 88 kbp (∼0·05–4·3 % of the chromosome). Based on the physical map, a genetic map was constructed via Southern blot analyses ofL. acidophilusDNA using specific gene probes. A total of 73 probes representing key genes, including 12 rRNA (rrn) genes, were positioned on the latter map. Mapping analysis also indicated the presence of fourrrnoperons (rrnA–D) on the chromosome, each containing a single copy of each of the threerrngenes 16S (rrl), 23S (rrs) and 5S (rrf). OperonrrnDwas inverted in orientation with respect to the others and contained a long 16S–23S intergenic spacer region with tRNAIleand tRNAAlagenes, whereas the other operons contained a short spacer lacking any tRNA genes. The high-resolution physical/genetic map constructed in this study provides a platform for genomic and genetic studies ofLactobacillusspecies and for improving industrial and probiotic strains.