BMC Genomics 2009, 10:101 http://www.biomedcentral.com/1471-2164/10/101 FAilgigunrmee1nt of plasmid nucleotide sequences related to B. afzelii PKo lp25 Sequence #1 Alignment of plasmid nucleotide sequences related to B. afzelii PKo lp25 Sequence #1. Adenosine residues are colored red, all other residues are green. Color scheme is for ease of viewing only. The EMBL-EBI CLUSTALW 2.0.8 multiple sequence alignment program [31]http://www.ebi.ac.uk/Tools/clustalw2/index.html[32] was used for alignment. Numbers adja- cent to plasmid names refer to nt positions in plasmid sequences. A star (*) denoted invariant positions. structures which contain 11–13 base pairs. Additional file 3 shows representative secondary structures and depicts several base-pair compensatory changes in the stem, e.g., A8-U27 pairing in B. afzelii lp34 changes to C8-G29 in B. burgdorferi lp25 and A5-U30→U5-A29 in B. burgdorferi lp28- 3. Again, these are examples of pyrimidine→purine and purine→pyrimidine mutational transversions, and these are found between orthologs. Additional base substitu- tions in other plasmid sequences result in G-U non- canonical pairing (data not shown). The presence of non- canonical pairs implies that the conserved structure may function at the RNA level as opposed to the DNA level. The loop structure sustained base substitutions and inser- tions/deletions, which resulted in major differences in loop sequences [see Additional file 3]. The stem length varies, but the invariant A4-U31 pair is always at the termi- nal end of the stem and is straddled by invariant A3 and A32 (numbering position relative to the Ba lp34 sequence [see Additional file 3]. This arrangement is found in all eleven of the stem loop structures (data not shown). By bioinformatics methods, random mutations were introduced in Sequence #2 to ascertain the probability of compensatory base pair changes arising by random base changes. For example, after adding 3 mutations to the 34 nt Sequence #2 and initiating 30 trials of random muta- genesis, the stem was found disrupted (with mispairs) in >90% of trials, and all 30 trials showed a resultant decrease (towards [+] side) in delta G, and in some cases there was a decrease by a factor of 10 in the delta G value. The configuration of the stem was drastically altered in 10 of the 30 trials (data not shown). Single base compensa- tory changes in the stem occurred in about 10% of trials, but at the same time the accompanying mutations (again, 3 mutations/34 nt were induced) caused a partial disrup- tion of the stem. Double compensatory mutations, such as U-A → C-G and the less probable transversion, U-A → G-C, did not appear. These trials show a trend towards dis- ruption of an ordered structure by addition of random mutations. In sharp contrast, biological mutations within 11 homologous sequences (23 positions showing muta- tions out of ~34 nt of Sequence #2) display numerous base-pair compensatory changes, including transversions, show no mispairing, no stem alterations (such as forma- tion of a bulged or looped positions), and several inser- tions/deletions that were closely confined to the unpaired looped region where they do not induce changes in the stem loop configuration. Strong evolutionary pressures appear to maintain the secondary structure motif of Sequence #2. Borrelia plasmids contain the superfamily of protein genes that encode Borrelia_lipoprotein_1 [20,33,23]. Signifi- cantly, the eleven stem loop sequences are found prima- rily between 14 and 33 bp downstream of a family of lipoprotein_1 genes, as well sequences that encode frag- ments of lipoprotein_1. The stem loop-associated lipoprotein_1/lipoprotein_1 fragment amino acid sequences are shown in Figure 3. The stem loop sequence in Ba lp60 is 24 bp downstream of locus BAPKO_2001, a putative lipoprotein_1 gene encoding a 237 aa protein. On the other hand, there is no lipoprotein_1 gene anno- Page 4 of 12 (page number not for citation purposes)