Salmonella typhimurium LT2 possesses three distinct 23S rRNA intervening sequences

Abstract

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 III 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 genomic digests following pulsed-field gel electrophoresis and used these DNA fragments as templates for PCRs utilizing primers upstream and downstream of helix-25 and helix-45. Variance in amplicon length and cycle sequencing indicated that rrlG and rrlH have IVSs in helix-25 of ϳ110 bp which are only 56% identical. rrnA, rrnB, rrnC, rrnD, rrnE, and rrnH have IVSs of ϳ90 bp in helix-45, and all have the same nucleotide sequence. Twenty-one independent wild-type strains of S. typhimurium from Salmonella Reference Collection A were analyzed for IVSs by using PCRs with genomic DNAs and by denaturing agarose 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 genes. However, the IVSs in individual wild-type lines are relatively stable, for several LT2 isolates separated over many years by many single-colony isolations are indistinguishable from one another, with the exception of line LB5010, which differs by one helix-25 IVS. We postulate that IVSs have entered strain LT2 by three independent lateral-transfer events and that the IVS in helix-45 was dispersed to and maintained in the same sequence in six of the seven rrl genes by the mechanism of gene conversion.The prokaryotic 50S ribosomal subunit contains contiguous 23S and 5S rRNAs; in certain genera of bacteria, such as Campylobacter (14,19,32), Leptospira (11,25,26), Rhodobacter (15, 17), Salmonella (4,12,13,29,30,33), and Yersinia (29), the 23S rRNA can be fragmented into two or more pieces. In Salmonella typhimurium LT2, 23S rRNA fragmentation is caused by RNase III excision (without repair) of novel intervening sequences (IVSs) of ϳ90 to 110 bp (4). Most IVSs do not contain open reading frames or terminal consensus sequences to facilitate any other type of excision or mobilization. For this reason, IVSs are not true introns but are related elements that disrupt the normal continuity of a gene without affecting its function. The 23S rRNA fragments maintain functionality, presumably through secondary structure and ribosomal protein interactions in the 50S subunit.S. typhimurium possesses two distinct types of IVSs, on the basis of rRNA fragment stoichiometry (4), one at about bp 550 and another at about bp 1170 in the rrl gene (for the 23S rRNA) (Escherichia coli gene numbering [23]). These positions correspond to helix-25 and helix-45 in the postulated secondary structure of the 23S rRNA of E. coli; both of these helices represent small tetraloops. IVSs partly replace these small helices with an extended helix and loop. Sequencing of two helix-45 IVSs isolated from two independent strains of S. typhimurium, ATCC 23566 (4) and 13311 (...

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