BMC Genomics 2009, 10:101 http://www.biomedcentral.com/1471-2164/10/101 related species, B. afzelii PKo and Borrelia garinii PB, have been sequenced, as well as many of the associated plas- mids [20-23]. These organisms possess multiple plasmids. For example, B.bugrdorferi strain B31 has 12 linear plas- mids and 9 circular plasmids [20,21]. Borrelia chromo- somes are small relative to many bacterial genomes, e.g., the genome of Borrelia burgdorferi str. B31 is ~0.9 Mb and Yersinia pestis str. Co92 genome is 4.6 Mb. Borrelia chromo- somes represent a tight packing of protein genes where there is little intergenic space. On the other hand, plas- mids contain a much larger amount of intergenic space. These regions are known to have sequences that translate to repeat units of small peptides. In addition, they contain a high percentage of fragmented genes, including those from transposase genes, and interesting fusions of protein motifs as well as [21]. This shows a rapid evolutionary trend in these regions and perhaps plasmid intergenic regions are where new protein and RNA genes and other functional units may evolve. A small number of Borrelia non-coding RNA genes have been detected [24,25]. It has been assumed that Borrelia has few small RNA genes, based on comparative genomic searches for similarities to known bacterial small RNA sequences [24]. However many regulatory RNA gene sequences diverge between species, e.g., micC, micF and ryhB (see Rfam website [26,27]), and analogous genes in other species can be missed, especially between distantly related species. In some cases, such as the regulatory RNA DsrA, nucleotide sequences from different species show few similarities [25]. Intergenic regions have not been fur- ther analyzed for evolutionarily conserved RNA secondary structure motifs. These motifs can signal the presence of functional units. In a bioinformatics study, we show that several repeat sequences in plasmid intergenic spaces and/or sequences immediately downstream of coding regions sustained multiple mutations, yet these sequences fold into highly conserved RNA-type stem loop structures. Evolutionary conservation indicates an essential role for these struc- tures in the cell. In contrast, super family protein genes associated with some conserved RNA-type structures dis- play marked amino acid and peptide chain length differ- ences and appear to be in a process of change and/or decay. This raises interesting questions concerning how these peptide-RNA linked elements will evolve with time. Results Repeat sequences of intergenic nucleotide sequences of Borrelia plasmids were analyzed for secondary structure motifs using the Zuker m-fold program [28,29]. In addi- tion, the RNAz program was used to confirm thermody- namically stable and evolutionarily conserved RNA secondary structures [30]. Intergenic sequences from plas- mids lp60 and lp28 of B. afzelii Pko were completely scanned manually for repeat sequences and RNA motifs. In addition, selected regions that contain relatively large intergenic regions from B. burgdorferi B31 and Borrelia garinii PB plasmids were also scanned. Most regions did not yield conserved stem loop structures, however five intergenic nucleotide sequences were found to display evolutionary conserved stem loop structures (Table 1). Sequence #1 A 60 nt intergenic sequence (Sequence #1, Table 1) was found in nine plasmids from B. afzelii Pko and B. burgdor- feri B31. Alignment of these sequences reveals a major conserved region that is approximately at the center of the polynucleotide nucleotide chain (Figure 1). The EMBL- EBI CLUSTALW 2.0.8 multiple sequence alignment pro- gram [31,32] was used for alignment. Twenty out of 60 nucleotide positions show base substitutions. A compari- son of sequences shows a 77–100% sequence identity between the nine plasmid sequences. Sequences homolo- gous to Sequence #1 have not been detected in B. garinii PBi plasmids or Borrelia chromosomal sequences. RNA secondary structure modeling of the nine sequences shows a high conservation of secondary structure with multiple base substitutions that maintain base pairing. In addition, a bulged U at position 23 is found invariant in all nine sequences. Figure 2a–c depicts RNA secondary structure models from three of the nine plasmid sequences. Base substitutions at individual positions are depicted in Figure 2c. Mutations at six base-paired posi- tions in the upper portion of the stem loop show compen- satory changes that conserve the stem structure (Figure 2c). Prominent are the base pair changes at positions C14- G47 that result in A14-U47 pairing in the sequence of plas- mid Bb pl17 (Fig. 2a) and U14-A47 pairing in two other plasmid sequences (Figure 2c). Base pair positions 14 and 47 appear to be "hot spots" for mutations, but neverthe- less, Watson-Crick base pairing is maintained. The C14- G47→A14-U47 substitution is highly significant in that it shows the double mutation, pyrimidine→purine, purine→pyrimidine. This is a transversion and has a lower probability of occurring than purine→purine and pyrimi- dine→pyrimidine transitions. The C-G→U-G transition at positions 19,20 and 41,42 (Figure 2c) are between orthol- ogous genes in Bb lp28-4 and Ba lp60-2. The base pairing at the terminal end of the stem differs between several plasmid structures (e.g., compare Figure 2a and 2b with 2c). Although the three A-U base pairs at the base of the stem (positions A6–8 and U53–55) are conserved in all plas- mid sequences, in plasmids Bb lp28-2 and Bb lp36, a G56→A56 substitution appears to partially destabilize the base stem structure (data not shown). The cut off at the 60 nt length for Sequence #1 was made because sequences extended from the 5' and 3' ends do not yield additional conserved secondary structure motifs. This however does not preclude that the 60 nt stem loop is part of a larger Page 2 of 12 (page number not for citation purposes)