GENE ORGANIZATION, PRIMARY STRUCTURE AND RNA PROCESSING ANALYSIS OF A RIBOSOMAL-RNA OPERON IN LACTOCOCCUS-LACTIS

Southern blot analysis of genomic DNA ofthe mesophilic lactic bacterium Lactococcus lactis subsp. lactis strain IL1403, illuminated six rRNA gene clusters. Each cluster contains one copyeach of three rRNA genes, displaying the typical eubacterial organization of physically linked 16 S, 23 S and 5 S rRNA genes. Five of the six rRNAclusters were cloned into plasmid pBR322. One recombinant plasmid, pSLCM6, containing a 6500 base-pair genomic DNA fragment, was characterized by physical mapping and the sequences encoding rRNAsand tRNAs were localized by Southern hybridization. This fragment contains a single operon composed of one promoter, a leader sequence, a 16 S rRNA gene, a tRNA(Ala) gene, a 23 S rRNA gene, a 5 SrRNA gene and a tRNA(Asn) gene. S1 nuclease mapping and primer extension analysis of in vivo transcripts localized one transcriptional initiation site 150 base-pairs upstream from the start of the 16 S rRNA gene. These procedures also suggest that this transcript is processed by anRNase III-like activity similar to Bacillus subtilis; i.e. the L. lactis nuclease might be sequence-specific. The chronology of specific cleavagesoccurring during the maturation process of the precursor transcriptis described. One interesting observation is that the regions flanking the 16 S and 23 S rRNAs containing the primary processing sites are identical and contain sequences that could be involved in transcriptional antitermination. S1 mapping of the 3′ ends ofin vivo transcripts indicate that a terminator-like sequence a few base-pairs downstream from the distal tRNAAsn gene is inefficient in arresting transcription. © 1993 Academic Press Limited.