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A 6.0-kbSal I DNA fragment containing an entire rRNA operon (rrnB ) was cloned from a cosmid gene bank of the phytopathogenic strainRhodococcus fascians D188. The nucleotide sequence of the 6-kb fragment was determined and had the organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without tRNA-encoding genes in the spacer regions. The 5′ and 3′ ends of the mature 16S, 23S, and 5S rRNAs were determined by alignment with therrn operons ofBacillus subtilis and other gram-positive bacteria. Four copies of therrn operons were identified by hybridization with anrrnB probe inR. fascians type strain ATCC 12974 and in the virulent strainR. fascians D188. However, another isolate, CECT 3001 (= NRRL B15096), also classified asR. fascians , produced fiverrn -hybridizing bands. An integrative vector containing a 2.5-kb DNA fragment internal torrnB was constructed for targeted integration of exogenous genes at therrn loci. Transformants carrying the exogenous chloramphenicol resistance gene (cmr ) integrated in differentrrn operons were obtained. These transformants had normal growth rates in complex medium and minimal medium and were fully stable for the integrated marker.

applied and environmental microbiology

Characterization of the rrnB Operon of the Plant Pathogen Rhodococcus fascians and Targeted Integrations of Exogenous Genes at rrn Loci