BMC Genomics 2009, 10:101 http://www.biomedcentral.com/1471-2164/10/101 sRFeNiqguAuersneecc2eosnfrdoarmy as)trBubctlupr1e7mb)odBealslpo2f5Saenqduecn) cBeb#lp12n8t-4 RNA secondary structure models of Sequence #1 nt sequences from a) Bb lp17 b) Ba lp25 and c) Bb lp28- 4. The mfold (version 3.2) program by Zuker and Turner [28,29] was used for secondary structure modeling. Figure 2c shows base substitutions (marked with red arrows) that are found in all positions in the nine plasmid sequences. tated upstream of the stem loop repeat element in Bb lp56. This upstream region has high nucleotide and amino acid sequence identities to lipoprotein_1, but the translated lipoprotein_1 amino acid sequence contains several stop codons (Figure 3). With plasmid Bg lp54, the stem loop repeat element is located 93 bp from the left end (5' end) of the plasmid. Thus, most of the upstream sequence of the lipoprotein_1 gene would have been lost upon a putative translocation of lipoprotein_1. Neverthe- less, a C-terminal 14 aa fragment is found that is highly similar to lipoprotein_1 C-terminal sequences (Figure 3). The Bg lp54 34 nt stem loop is 26 bp downstream of the 3' end of the sequence encoding the 14 aa C-terminal frag- ment. On the other hand, there is an 18 bp overlap of the stem loop sequence with the 3' end sequence of locus BB_H32 encoding a lipoprotein_1 gene in Bb lp28-3, yet the stem loop structure is conserved with multiple base- pair compensatory changes (e.g., compare figures a and d, Additional file 3). Thus in cases where the stem loop has been detected, there is phylogenetic conservation of sec- ondary structure and conservation of its location down- stream of or partially overlapping lipoprotein_1 genes/ lipoprotein_1 gene fragments. The high conservation of repeat element secondary structure suggests there is evolu- tionary pressure to maintain a subset of lipoprotein_1 genes/gene fragments with downstream sequences that can fold into a stem loop structure. However, not all lipoprotein_1 genes have the conserved hairpin and the stem loop may be specific to the subset of lipoprotein-1 genes outlined above. For example, locus BAPKO_4514 in Ba lp28 encodes a putative 261aa lipoprotein_1, which does not have the characteristic stem loop. Sequence #3 Borrelia sp. encode the virulence factor termed complement regulator-acquiring surface protein 1 (CRASP-1) [34-36]. This protein binds factor H, resulting in inhibition of com- plement activation in mammals. CRASP-1 proteins from B. afzelii lp54 and other Borrelia species plasmids have been isolated and their properties characterized [36]. Multiple copies of sequences analogous to CRASP-1 genes have been detected in Borrelia plasmids. These include sequences in loci BAPKO_2065- BAPKO_2070 from B. afzelii lp60 [see Additional file 4]. A comparison shows that the translated aa sequence from Ba lp60 locus BAPKO_2068 and the aa sequence derived experimentally from the Ba lp54 CRASP-1 protein (whose gene locus is termed Ba_lp54 mmsa71) are almost identical and show only 5 aa changes out of 241 aa. In addition, the factor H binding motif, 232KDLDSFNP239 is present in locus BAPKO_2068 and the Ba lp54 CRASP-1 mmsa71 gene [see Additional file 4]. BAPKO_2068 and Ba lp54 mmsa71 (CRASP-1) probably are paralogous genes and the BAPKO_2065-2070 superfamily are also paralogs result- ing from gene duplication. However an amino acid sequence alignment of this family of loci shows several major insertions/deletions and amino acid substitutions [see Additional file 4]. The bottom figure in Additional file 4 shows a phylogram of this gene family. Alignment of nucleotide sequences immediately down- stream of open reading frame stop codons from BAPKO_2065 to BAPKO_2070 and CRASP-1 genes from Bg lp54 zqa68 and Ba lp54 mmsa71 (reference position, TAG726 BAPKO_2068) shows that these sequences are highly conserved (Figure 4). Secondary structure mode- ling of putative transcripts of downstream sequences Page 5 of 12 (page number not for citation purposes)