SALMONELLA-TYPHIMURIUM LT2 POSSESSES 3 DISTINCT 23S RIBOSOMAL-RNA INTERVENING SEQUENCES

The rrl genes for 23S rRNA of Salmonella typhimurium LT2 are known to carry intervening sequences (IVSs) at two sites, helix-25 and helix-45 , which are excised by RNase Zn: during rRNA maturation, resulting in rRNA which is fragmented but nevertheless functional. We isolated DNA fragments containing the seven rrl genes from BlnI, I-CeuI, and SpeI g enomic digests following pulsed-field gel electrophoresis and used the se DNA fragments as templates for PCRs utilizing primers upstream and downstream of helix-25 and helix-45. Variance in amplicon length and c ycle sequencing indicated that rrlG and rrlH have IVSs in helix-25 of similar to 110 bp which are only 56% identical, rrnA, rrnB, rrnC, rrnD , rrnE, and rrnH have IVSs of similar to 90 bp in helix-45, and all ha ve the same nucleotide sequence. Twenty-one independent wild-type stra ins of S. typhimurium from Salmonella Reference Collection A were anal yzed for IVSs by using PCRs with genomic DNAs and by denaturing agaros e electrophoresis of RNAs, Many strains resemble LT2, but some have no IVSs in helix-25 and others have IVSs in helix-45 in all seven rrl ge nes. However, the IVSs in individual wild-type lines are relatively st able, for several LT2 isolates separated over many years by many singl e-colony isolations are indistinguishable from one another, with the e xception of line LB5010, which differs by one helix-25 IVS, We postula te that IVSs have entered strain LT2 by three independent lateral-tran sfer events and that the IVS in helix-45 was dispersed to and maintain ed in the same sequence in six of the seven rrl genes by the mechanism of gene conversion.