BMC Genomics 2009, 10:101 http://www.biomedcentral.com/1471-2164/10/101 show two stem loop structures [see Additional file 5]. Stem loop #2 is highly conserved with numerous base pair compensatory changes, but it also has a 3' terminal oli- gouridine. It probably represents a Rho-independent tran- scription termination site for CRASP-1 and related putative gene transcripts. Stem loop 1 is present in all repeat sequences but shows variations in secondary struc- ture (e.g., compare figures a and b in Additional file 5). The significance of this stem loop is unknown, but it may reside within a putative 3'UTR region. The high conserva- tion of the stem loop 2 secondary structure contrasts with the variability in overall amino acid sequence, differences in factor H binding site sequence (232KDLDSFNP239) and peptide chain length of associated protein genes [see Additional file 4]. Figure 5 shows a diagrammatic representation of viru- lence protein genes lipoprotein_1 and CRASP-1 with their associated RNA motifs at the 3' ends. The amino acids sequences of both genes vary between homologous sequences and some gene copies are degenerate. However, the associated RNA secondary structures are evolutionar- ily highly conserved. Sequence #4 Sequence #4 (Table 1) is 122 nt and has an inverted repeat segment (positions 26245–26290 in Ba lp60). This sequence is not located near any protein genes. It is 233 bp down- stream of locus BAPKO_2033, which encodes an oligopep- tide ABC transporter, and 171 bp upstream of BAPKO_2034, a putative lipoprotein gene. Sequence #4 from Ba lp60 is highly conserved in Bg lp54, but only a part of the sequence is conserved in Bb lp54 [see Additional file 6]. Comparison of positions 52–122 show less than 40% similarity between sequences of Bb lp54 and Ba lp60. A comparison and analy- sis of Sequence #4 and flanking regions in plasmid Bb lp54 shows that the 122 bp sequence has been recombined in the opposite orientation (data not shown). This resulted in a major change in sequence between Bb p54 and Ba lp60 and Bg lp54 downstream of position 53 [see Additional file 6]. The approximate 5' half of the sequences are very similar due to the inverted repeat, which provides a similar sequence in the opposite orientation. Secondary structure models of the 122 nt sequence reveal that a structural motif is conserved between the three sequences [see Additional file 7]. The three models dis- play two stem loops and one small stem (stem 3). Stem loop 1 consists of the inverted repeat and has 21 contigu- ous Watson-Crick base pairs (positions 7–52 in Ba lp60). The sequence in Bb 54 is not a perfect inverted repeat but there are base compensatory changes that maintain the 21 Watson-Crick base pairs, i.e., there are two G-C pairs in Bb lp54 in place of two non canonical G-U pairs in Ba lp 60 and Bg lp54. The presence of stem loop 2 is of major sig- nificance in that it is in a region with very low nt sequence identity, yet a similar stem loop is maintained but with extensive differences in base pairing (compare stem loop 2, figures 1a and 1b, see Additional file 7). This type of phylogenetically conserved motif is characteristic of sec- ondary structural properties of some small non-coding RNAs, where secondary structure and not necessarily sequence is conserved, e.g., see [37]. This conservation implies a functional importance for this 122 nt sequence, which may represent a non-coding RNA. Sequences #5 Sequence #5 is 150 nt in length and is found in plasmids of three Borrelia species, Ba lp60 from B. afzelii PKo, Bb lp54 from B. burgdorferi str. B31 and Bg lp54 in B. garinii PB. This sequence is highly conserved with nt sequence identities > 95% between the three sequences. The sequence at positions 14913–14964 from B. burgdorferi B31 plasmid lp54 represent a perfect inverted repeat. Sec- ondary structure modeling shows the presence of three stem loops in all three plasmid sequences. The structure from Bb lp54 is shown in Additional file 8. It is highly sta- ble thermodynamically with a delta G of -53.3 kcal/mol. A comparison of different plasmid structures shows three base substitutions in stem 3 that maintain the base-pair- ing, but two other substitutions produce A-A and A-G non-canonical pairs (data not shown). Stem 3 is not destabilized, but there are small decreases in the delta G value to -43.3 kcal/mol (Bg lp54) and -43.6 kcal/mol (Ba lp60). In the context of surrounding base pairs, non- canonical pairs such as A-A and A-G are present in RNA double helices [38] and have been found to contribute to RNA stem double helical conformations [39,40]. An analysis of Sequence #5 by the RNAz bioinformatics program shows a predicted evolutionarily conserved RNA structure with a mean z-value of -6.04 [see Additional file 9]. Sequence #5 is immediately downstream of loci BAPKO_2021, BGA_19 and BB_A21. These loci represent highly conserved proteins that have been annotated as hypothetical proteins. However they have amino acid sequence and putative protein domain similarities to a plasmid partition protein (PF-49 encoded on plasmid cp32-11 in B. burgdorferi) when analyzed by Pfam [41-44]. The aa sequence identity to PF-49 is 55% as determined by the ExPASy Proteomics Server [45,46]. Although the Sequence #5 stem loops reveal an interesting highly stable structure that is linked to a conserved protein, additional sequences homologous to Sequence #5 would be needed to further support a proposed phylogenetic conservation of secondary structure. Discussion In genomes of many bacterial species, intergenic regions are found to be rich in repeat elements such as MITEs Page 6 of 12 (page number not for citation purposes)