ciat 652 (nc_010994), reciatr15c1 = rhizobium etli ciat 652 (nc_010994), reciatr15pc = rhizobium etli ciat 652 plasmid pc (nc_010997), reciatr15b = rhizobium etli ciat 652 plasmid pb (nc_010996), arr15ci2 = agrobacterium radiobacter k84 chromosome 1 (nc_011985), arr15ci1 = agrobacterium radiobacter k84 chromosome 1 (nc_011985), arr15cii = agrobacterium radiobacter k84 chromosome 2 (nc_011983), rlt2304r15c2 = rhizobium leguminosarum bv. trifolii wsm2304 chromosome (nc_011369), rlt2304r15c1 = rhizobium leguminosarum bv. trifolii wsm2304 chromosome (nc_011369), avr15c2 = agrobacterium vitis s4 chromosome 1 (nc_011989), avr15c1 = agrobacterium vitis s4 chromosome 2 (nc_011989), avr15patc = agrobacterium vitis s4 plasmid pats4c (nc_011984), avr15ate = agrobacterium vitis s4 plasmid pats4e (nc_011981), avr15ti = agrobacterium vitis s4 plasmid ptis4 (nc_011982), rlvr15c2 = rhizobium leguminosarum bv. viciae 3841 (nc_008380), rlvr15c1 = rhizobium leguminosarum bv. viciae 3841 (nc_008380), rlvr15p10 = rhizobium leguminosarum bv. viciae 3841 plasmid prl10 (nc_008381), rlvr15p11 = rhizobium leguminosarum bv. viciae 3841 plasmid prl11 (nc_008384), rlt1325r15c2 = rhizobium leguminosarum bv. trifolii wsm1325 (nc_012850), rlt1325r15c1 = rhizobium leguminosarum bv. trifolii wsm1325 (nc_012850), rlt1325r15p02 = rhizobium leguminosarum bv. trifolii wsm1325 plasmid pr132502 (nc_012858), recfnr15c2 = rhizobium etli cfn 42 (nc_007761), recfnr15c1 = rhizobium etli cfn 42 (nc_007761), recfnr15d = rhizobium etli cfn 42 symbiotic plasmid p42d (nc_004041), recfnr15a = rhizobium etli cfn 42 plasmid p42a (nc_007762), mlr15a = mesorhizobium loti maff303099 plasmid pmla (nc_002679), bcr15cii = brucella canis atcc 23365 chromosome ii (nc_010104), bcr15ci2 = brucella canis atcc 23365 chromosome i (nc_010103), bcr15ci1 = brucella canis atcc 23365 chromosome i (nc_010103), bs23445r15ci2 = brucella suis atcc 23445 chromosome i (nc_010169), bs23445r15ci1 = brucella suis atcc 23445 chromosome i (nc_010169), bs23445r15cii = brucella suis atcc 23445 chromosome ii (nc_010167), bm16mci = brucella melitensis bv. 1 str. 16m chromosome i (nc_003317), bm16mr15cii = brucella melitensis bv. 1 str. 16m chromosome ii (nc_003318), bas19r15cii = brucella abortus s19 chromosome 2 (nc_010740), bas19r15ci2 = brucella abortus s19 chromosome 1 (nc_010742), bas19r15ci1 = brucella abortus s19 chromosome 1 (nc_010742), bm23457r15cii = brucella melitensis atcc 23457 chromosome ii (nc_012442), bm23457r15ci = brucella melitensis atcc 23457 chromosome i (nc_012441), bs1330r15cii = brucella suis 1330 chromosome ii (nc_004311), bs1330r15ci2 = brucella suis 1330 chromosome i (nc_004310), bs1330r15ci1 = brucella suis 1330 chromosomei (nc_004310), ba19941r15ci2 = brucella abortus bv. 1 str. 9-941 chromosome i (nc_006932), ba19941r15ci1 = brucella abortus bv. 1 str. 9-941 chromosome i (nc_006932), ba19941r15cii = brucella abortus bv. 1 str. 9-941 chromosome ii (nc_006933), bmar15cii = brucella melitensis biovar abortus 2308 chromosome ii (nc_007624), bmar15ci2 = brucella melitensis biovar abortus 2308 chromosome i (nc_007618), bmar15ci1 = brucella melitensis biovar abortus 2308 chromosome i (nc_007618), bor15ci2 = brucella ovis atcc 25840 chromosome i (nc_009505), bor15ci1 = brucella ovis atcc 25840 chromosome i (nc_009505), bor15cii = brucella ovis atcc 25840 chromosome ii (nc_009504), bmir15cii = brucella microti ccm 4915 chromosome 2 (nc_013118), bmr15ci2 = brucella microti ccm 4915 chromosome 1 (nc_013119), bmir15ci1 = brucella microti ccm 4915 chromosome 1 (nc_013119), oar15ci = brucella anthropi atcc 49188 chromosome 1 (nc_009667), oar15cii = brucella anthropi atcc 49188 chromosome 2 (nc_009668). expression and functional information several studies have assessed smr15c1 and smr15c2 expression in s. meliloti 1021 under different biological conditions; i.e. bacterial growth in ty, minimal medium (mm) and luteolin-mm broth and endosymbiotic bacteria (i.e. mature symbiotic alfalfa nodules), high salt stress, oxidative stress and cold and hot shock stresses. the results showed different expression profiles for both srnas, which is consistent with their organization in independent and differentially regulated transcription units within the same igr (figure 4 and figure 5). the expression of smr15c1 and smr15c2 in free-living bacteria was found to be growth-dependent but in an opposite manner. while smr15c1 is accumulated in the stationary phase smr15c2 is the expression of smr15c1 and smr15c2 in free-living bacteria was found to be growth-dependent but in an opposite manner. while smr15c1 is accumulated in the stationary phase, smr15c2 is preferentially expressed in log bacterial cultures. additionally, schlüter et al. recently described the up-regulation of smr15c2 under cold shock stress, while no effects of a temperature downshift were observed in the expression of smr15c1. the growth-dependent opposite expression profiles of smr15c1 and smr15c2, have not been observed in their agrobacterium tumefaciens counterparts referred to as abcr1 and abcr2, respectively, by wilms et al. (atr15c1 and atr15c2 in this work). abcr1 and abcr2 are induced simultaneously and both accumulate in stationary phase. this behavior agrees with the fact that abcr1 and abcr2 have identical promoter-like sequences, being these very similar to the one of smr15c2, but not to the promoter sequence of smr15c1 (see promoter analysis). furthermore, a first approach to the function of the abcr genes revealed that these srnas silence the gaba uptake system through the down-regulation of the corresponding abc transporter genes in an hfq-dependent manner. gaba is one of the plants signals recognized by rhizobacteria in some plant-bacteria interactions. thus, these results, point to the shutting off synthesis of the gaba uptake system as a way used by a. tumefaciens to subvert the plant defense mechanism. recent co-immunoprecipitation experiment showed that both, smr15c1 and smr15c2, do bind the s. meliloti rna chaperone hfq, supporting also a role for these transcripts in this bacterium as trans-acting antisense riboregulators.they were also shown to fine-tune nutrient uptake. promoter analysis all αr15 loci have recognizable σ70-dependent promoters showing a -35/-10 consensus motif cttgac-n17-ctatat, which has been previously shown to be widely conserved among several other genera in the α-subgroup of proteobacteria. a multiple sequence alignment of these promoter regions revealed a conserved sequence stretch extending up to 80 bp upstream of the transcription start site in all the αr15 loci with the only exceptions of the s. meliloti, s.fredii and s. medicae αr15c1 promoters. to identify binding sites for other known transcription factors we used the fasta sequences provided by regpredict(http://regpredict.lbl.gov/regpredict/help.html), and used those position weight matrices (pswm) provided by regulondb (http://regulondb.ccg.unam.mx). we built pswm for each transcription factor from the regpredict sequences using the consensus/patser program, choosing the best final matrix for motif lengths between 14 and 30 bps a threshold average e-value < 10e-10 for each matrix was established, (see "thresholded consensus" in http://gps-tools2.its.yale.edu). moreover, we searched for conserved unknown motifs using meme (http://meme.sdsc.edu/meme4_6_1/intro.html) and used relaxed regular expressions (i.e. pattern matching) over all αr15 homologs promoters. these studies revealed a difference in regulation between s. melilloti, s.fredii and s. medicae αr15c1 srnas and the rest of the αr15 family members independently of their group of origin (rhizobium or agrobacterium ) and genomic location (αr15c1, αr15c2, αr15plasmid) (figure 4). the rest of the family members presented a very well conserved 30 bp long region between positions -36 and -75. this conserved region, was used to query databases of known transcription factor motifs with tomtom, the best matching motif was smb20667_rhizobiales, motif belonging to regtransbase (http://regtransbase.lbl.gov/cgi-bin/regtransbase?page=main). the smb20667 correspond to the binding site for a dicarboxylate metabolism regulator in rhizobiales that belongs to the laci family. this motif was identified clustering genes of tartrate dehydrogenase, succinate semialdehyde dehydrogenase, 3-hydroxyisobutyrate dehydrogenase and hydroxypyruvate isomerase in s. meliloti, and several rhizobiums and it is marked in the promoter alignment figure (figure 5) with an orange box. moreover, another conserved sequence was found using meme in the promoter region of the sinorhizobium species αr15c1. this conserved region was used to query databases of known transcription factor motifs with tomtom, and the best matching motif was smb20537_rhizobiales (figure 5 in red), that identifies the binding site for a sugar utilization regulator in rhizobiales from the laci family as well. figure 5: graphic representation of the 15c seed members' promoter region. all members presented putative σ70 promoters with -30 and -10 boxes marked in green and red respectively. conserved motifs are marked with orange and red color boxes. genomic context most of the αr15 family members are trans-encoded srnas transcribed from independent promoters in chromosomal igrs. many of the αr15 members neighboring genes were not annotated, and thus they were further manually curated. as a result, we could classify the members of the family in four subgroups according to their genomic context. in the first group are the tandem αr15c1 and αr15c2 loci of the rhizobium, sinorhizobium and agrobacterium species. they exhibited a great degree of conservation in the up and downstream genes, which have been predicted to code for a lysr transcriptional regulator and an asr transcriptional regulator protein respectively (figure 5). the only exception in this group was found for s. fredii that presented a very different genomic context. the second group includes the αr15ci1 loci in the brucella species (additional file 2), which presented a very well conserved genomic context (aspartate amino transferase and lysr/unknown transcriptional regulator) with partial synteny to the first group. a very different genomic context, not even partially conserved in most cases, was present in all plasmid-borne αr15 loci (additional file 1), which integrates the defined group three, where the flanking genes corresponded to abc transporter proteins, excisonase or transposase among others. the αr15ci2 loci in the brucella species (additional file 3) conform the group four and presented an up and downstream conserved genomic context, coding all regions for udp-3-o-hydroxymyristoyl n-acetylglucosamine deacetylase and gtpase cell division protein ftsz. to the last group correspond the αr15cii loci in the brucella group (additional file 4) where only one of the genes could be annotated always as a glycine deshidrogenase, the other srna flanking position was mostly coped by a hypothetical protein conserved in the brucella group to which no domain, motif or go functional annotation could be assigned. figure 4: genomic context scheme of smr15 and its closest homologues in other organisms. the αr15 rna genes are represented by red arrows and the flanking orfs by arrows on different colors depending on their product function (legend). numbers indicate the αr15 rna gene's and flanking orfs coordinates in each organism genome database. the gene strand is represented with the file direction. on the left of the figure identification names are used which correspond to a certain organism: legend: αr15_smr15c = sinorhizobium meliloti 1021 (nc_003047), αr15_smedr15c = sinorhizobium medicae wsm419 chromosome (nc_009636), αr15_sfr15c = sinorhizobium fredii ngr234 chromosome (nc_012587), αr15_atr15c = agrobacterium tumefaciens str. c58 chromosome circular (nc_003062), αr15_ah13r15c = agrobacterium sp. h13-3 chromosome (nc_015183), αr15_reciatr15c = rhizobium etli ciat 652 (nc_010994), αr15_arr15ci = agrobacterium radiobacter k84 chromosome 1 (nc_011985), αr15_rlt2304r15c = rhizobium leguminosarum bv. trifolii wsm2304 chromosome (nc_011369), αr15_avr15c = agrobacterium vitis s4 chromosome 1 (nc_011989), αr15_rlvr15c = rhizobium leguminosarum bv. viciae 3841 (nc_008380), αr15_rlt1325r15c = rhizobium leguminosarum bv. trifolii wsm1325 (nc_012850), αr15_recfnr15c = rhizobium etli cfn 42 (nc_007761). additional files: genomic context graph of the αr15 family plasmid copies image:smbr15 1.png genomic context graph of the αr15ci1 loci in the brucella group image:smbr15 2.png genomic context graph of the αr15ci2 loci in the brucella group image:smbr15 3.png genomic context graph of the αr15cii loci in the brucella group image:smbr15 4.png table 2: detailed genomic context information of the αr15 srna seed members. familyfeature typefeature namestrandbeginendprotein nameannotationorganism αr15_smr15c gene smc01226 r 1698201 1698491 np_385671.1 transcription regulator protein sinorhizobium meliloti 1021 (nc_003047) αr15_smr15c srna smr15c1 r 1698617 1698731 - srna sinorhizobium meliloti 1021 (nc_003047) αr15_smr15c srna smr15c2 r 1698817 1698937 - srna sinorhizobium meliloti 1021 (nc_003047) αr15_smr15c gene smc01225 r 1699144 1700052 np_385672.1 transcription regulator protein sinorhizobium meliloti 1021 (nc_003047) αr15_smed15c gene smed_1246 r 1336608 1336898 yp_001326931.1 arsr family transcriptional regulator sinorhizobium medicae wsm419 chromosome (nc_009636) αr15_smed15c srna smedr15c1 r 1337011 1337126 - srna sinorhizobium medicae wsm419 chromosome (nc_009636) αr15_smed15c srna smedr15c2 r 1337212 1337331 - srna sinorhizobium medicae wsm419 chromosome (nc_009636) αr15_smed15c gene smed_1247 r 1337537 1338433 yp_001326932.1 lysr family transcriptional regulator sinorhizobium medicae wsm419 chromosome (nc_009636) αr15_rlt2304r15c gene rleg2_2722 d 2769491 2770402 yp_002282219.1 lysr family transcriptional regulator rhizobium leguminosarum bv. trifolii wsm2304 chromosome (nc_011369) αr15_rlt2304r15c srna rlt2304r15c1 d 2770612 2770727 - srna rhizobium leguminosarum bv. trifolii wsm2304 chromosome (nc_011369) αr15_rlt2304r15c srna rlt2304r15c2 d 2770835 2770949 - srna rhizobium leguminosarum bv. trifolii wsm2304 chromosome (nc_011369) αr15_rlt2304r15c gene rleg2_2723 d 2771064 2771357 yp_002282220.1 arsr family transcriptional regulator rhizobium leguminosarum bv. trifolii wsm2304 chromosome (nc_011369) αr15_rlt1325r15c gene rleg_2983 d 2980263 2981174 yp_002976781.1 lysr family transcriptional regulator rhizobium leguminosarum bv. trifolii wsm1325 (nc_012850) αr15_rlt1325r15c srna rlt1325r15c1 d 2981275 2981390 - srna rhizobium leguminosarum bv. trifolii wsm1325 (nc_012850) αr15_rlt1325r15c srna rlt1325r15c2 d 2981499 2981613 - srna rhizobium leguminosarum bv. trifolii wsm1325 (nc_012850) αr15_rlt1325r15c gene rleg_2984 d 2981728 2982021 yp_002976782.1 arsr family transcriptional regulator rhizobium leguminosarum bv. trifolii wsm1325 (nc_012850) αr15_recfnr15c gene rhe_ch02976 d 3116670 3117566