BMC Genomics 2009, 10:101 http://www.biomedcentral.com/1471-2164/10/101 goFDefiignatgehurseraewmgei5mtnheasctiocnrseeprrveesdenRtNatAionstroufcltipuorepsrolitnekine_d1toantdheC3R'AeSnPd-s1 Diagrammatic representation of lipoprotein_1 and CRASP-1 genes with conserved RNA structures linked to the 3' ends of the genes. [9,12,13,1,17], other small nucleotide sequence repeats [11,47] and small non-coding RNA genes [48,3-7]. Here we analyzed intergenic plasmid regions from three species of Borrelia and have detected intergenic sequences that can fold into conserved RNA secondary structures. Compel- ling evidence for evolutionary conservation comes from comparisons of homologous sequences, where numerous base-pair changes are found to maintain stem loop struc- tures. These stem loops are specific to plasmid sequences, and none have been detected in Borrelia chromosomes or in sequences from other bacterial species. Two RNA-motifs associated with super families of protein genes (lipoprotein_1 and CRASP-1) show a high conser- vation of secondary structure between homologs, yet these gene families show extensive amino acid substitu- tions and deletions/insertions. Perhaps the cell maintains these RNA motifs as reservoirs and as potential functional units in the formation of new variant proteins. A major focus in future work should be to determine if variant CRASP-1 and lipoprotein-1 loci are translated. Sequence #2 contains inverted repeats and is located less than 35 bp downstream of putative lipoprotein_1 genes, and in one case overlaps the terminal codon sequences. This is very similar to the location of several miniature inverted repeats, the MITEs that are present in other bac- terial species. These inverted repeats are also found down- stream of genes, and in some cases are found to overlap C- terminal codons [13,14,16,17,49]. In Yersinia, genes situ- ated upstream of MITEs appear to be regulated by these inverted repeat elements, which are transcribed into RNA [50]. Although Sequence #2 differs from bacterial MITEs in not having a large nucleotide segment between inverted repeats, the proximity of this sequence to C-terminal cod- ing ends of genes is similar to that of several MITEs. Borrelia contains transposase genes that are found in other bacterial species [20]. Some plasmids show a high percent- age of transposase-specific nucleotide sequences which may not be evident from gene annotations, e.g., the first ~1400 bp of the left side of B. afzelii PKo plasmid lp28 starting at nucle- otide position 1 consists entirely of transposase-related sequences (unpublished results). There may also be non- autonomous transposable elements present in Borrelia that are moved and replicated by transposases. As many other bacteria contain these elements [1], it would not be surpris- ing if Borrelia had its own set of non-autonomous small transposable elements, possibly with their own specific sig- natures. Repeat Sequence #2 described above should be fur- ther analyzed for a possible relationship to bacterial MITEs. Stem loops that are proximal to protein genes have been reported before. Dunn et al [51] described two inverted repeat sequences in tandem with perfect base paired stems in B. burgdorferi in circular plasmid cp8.3. The hairpins are adjacent to putative -35 promoter sequences of an open reading frame. Also, an inverted repeat sequence is found in the 5' flanking region of the bba64 (P35) gene in B. burgdorferi [52]. However the above sequences, which are upstream of genes in promoter regions, are unrelated to those reported here. Stem loop 2, from Sequence #3 is downstream of the CRASP-1-related genes and appears to have classic Rho-inde- pendent termination signatures in terms of size and oligo U tail. The adjacent stem loop 1 may be part of a putative 3' UTR of CRASP-1 and CRASP-1-related proteins. Functions can not presently be assigned, but it should be noted that some small RNAs in E. coli represent 3' UTR transcripts which show different expression levels from associated mRNAs and may have independent functions [8]. Sequences #1, #4, and #5 appear to have typical RNA signatures with long stem loops and bulged/looped positions. Without further charac- terization, functional roles cannot be assigned. But of partic- ular interest is the conservation of the bulged U at position 23 of the Sequence #1 stem loop. Many RNA secondary structures display conserved bulged positions and these have functional roles in RNA/RNA interactions [53,54]. Sequence #1 does not appear to be linked to any protein genes and is present in nine different plasmids. This poses the question of how it was transferred and why the sequence is duplicated. Interestingly, Sequence #4 is found in three different species, B. burgdorferi str. B31, B. afzelii PKo and B. garinii PB but in only one copy number. Thus this RNA motif may provide an essential function in Borrelia, as it is found in all three species. Once complete genome sequences of other Borrelia species are determined, it would be of interest to see if Sequence #4 and/or its characteristic secondary structural model is also present in these species. Only a limited number of plasmids have been analyzed for repeat sequences that fold into RNA motifs, but a more Page 9 of 12 (page number not for citation purposes)