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 of Lactobacillus acidophilus neotype strain ATCC 4356. PFGE analysis of chromosomal DNA after digestion with each of the rare-cutting restriction enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI allowed the size of the circular chromosome of L. acidophilus to 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 (approximately 0.05-4.3 % of the chromosome). Based on the physical map, a genetic map was constructed via Southern blot analyses of L. acidophilus DNA 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 four rrn operons (rrnA-D) on the chromosome, each containing a single copy of each of the three rrn genes 16S (rrl), 23S (rrs) and 5S (rrf). Operon rrnD was inverted in orientation with respect to the others and contained a long 16S-23S intergenic spacer region with tRNAIle and tRNAAla genes, 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 of Lactobacillus species and for improving industrial and probiotic strains.