αr15 rna αr15 rna αr15 is a family of bacterial small non-coding rnas with representatives in a broad group of α-proteobacteria from the order rhizobiales. the first members of this family (smr15c1 and smrc15c2) were found tandemly arranged in the same intergenic region (igr) of the sinorhizobium meliloti 1021 chromosome (c). further homology and structure conservation analysis have identified full-length smr15c1 and smr15c2 homologs in several nitrogen-fixing symbiotic rhizobia (i.e. r. leguminosarum bv. viciae, r. leguminosarum bv. trifolii, r. etli, and several mesorhizobium species), in the plant pathogens belonging to agrobacterium species (i.e. a. tumefaciens, a. vitis, a. radiobacter, and agrobacterium h13) as well as in a broad spectrum of brucella species (b. ovis, b. canis, b. abortus and b. microtis, and several biovars of b. melitensis). the smr15c1 (115 nt) and smr15c2 (121 nt) homologs are also encoded in tandem within the same igr region of rhizobium and agrobacterium species, whereas in brucella species the αr15c loci are spread in the igrs of chromosome i. moreover, this analysis also identified a third αr15 loci in extrachromosomal replicons of the mentioned nitrogen-fixing α-proteobacteria and in the chromosome ii of brucella species. αr15 rna species are 99-121 nt long (table 1) and share a well defined common secondary structure consisting of three stem loops (figure 1). the transcripts of the αr15 family can be catalogued as trans-acting srnas encoded by independent transcription units with recognizable promoter and transcription termination signatures within intergenic regions (igrs) of the α-proteobacterial genomes (figure 5). discovery and structure smr15c1 y smr15c2 srnas were described by del val et al., as a result of a computational comparative genomic approach in the intergenic regions (igrs) of the reference s. meliloti 1021 strain (http://iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi). although the primary nucleotide sequence of smr15c1 y smr15c2 showed high similarity (84% identity), specific probes for each srna could be designed which detected transcripts of different size and expression profiles. tap-based 5’-race experiments mapped the smr15c1 and smr15c2 transcription start sites (tss) in the s. meliloti 1021 genome (http://iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi). the smr15c1 tss was mapped to the chromosomal position 1698731 nt and the tss of smr15c2 to the nt 1698937. the 3’-ends were assumed to be located at the 1698617 nt and 1698817 nt respectively, matching the last residue of the consecutive us stretch of a bona fide rho-independent terminator (figure 5). parallel and later studies, in which smr15c1 and smr15c2 transcripts are referred to as two copies of sra41 or sm3/sm3', independently confirmed the expression of these srnas in s. melilloti and in its closely related strain 2011. recent deep sequencing-based characterization of the small rna fraction (50-350 nt) of s. meliloti 2011 also revealed the expression of smr15c1 and smr15c2, here referred to as smelc411 and smelc412 respectively, mapping the 5’- and 3´-ends of the full-length transcripts to essentially the same positions as del val et al. in the s. meliloti 1021 chromosome. however, this study identified an additional tss for smr15c2 at position 1698948. the nucleotide sequences of smr15c1 and smr15c2 were initially used as query to search against the rfam database (version 10.0; http://rfam.xfam.org). this search revealed partial homology of both transcripts, restricted to the second hairpin and the rho-independent terminator, to the rf00519 family of rnas known as suhb (http://rfam.xfam.org/family/rf00519). however, no structural homologs of the full-length srnas were found in this database. both s.melilloti αr15 srnas were also blasted with default parameters against all the currently available bacterial genomes (1,615 sequences at 20 april 2011; https://www.ncbi.nlm.nih.gov). the regions exhibiting significant homology to the query sequence (78-89% similarity) were extracted to create a covariance model (cm) from a seed alignment using infernal (version1.0) (figure 2). this cm was used in a further search for new members of the αr15 family in the existing bacterial genomic databases. figure 2: covariance model in stockholm format showing the consensus structure for the αr15 family. each of the stems represented by the structure line #=gc ss_cons is in a different color, corresponding the red one to the rho independent terminator stem. covariance model in stockholm format can be downloaded here. table 1: smr15c1 and smr15c2 homologs in other symbionts and pathogens cm modelnamegi accession numberbeginendstrand%gclengthorganism αr15 smr15c1 gi|15963753|ref|nc_003047.1| 1698617 1698731 - 54 115 sinorhizobium meliloti 1021 αr15 smr15c2 gi|15963753|ref|nc_003047.1| 1698817 1698937 - 50 121 sinorhizobium meliloti 1021 αr15 smr15a gi|16262453|ref|nc_003037.1| 552873 552984 + 51 112 sinorhizobium meliloti 1021 plasmid psyma αr15 smedr15c1 gi|150395228|ref|nc_009636.1| 1337011 1337126 - 53 116 sinorhizobium medicae wsm419 chromosome αr15 smedr15c2 gi|150395228|ref|nc_009636.1| 1337212 1337331 - 50 120 sinorhizobium medicae wsm419 chromosome αr15 smedr15p03 gi|150378263|ref|nc_009622.1| 40054 40165 - 52 112 sinorhizobium medicae wsm419 plasmid psmed03 αr15 sfr15c1 gi|227820587|ref|nc_012587.1| 1612511 1612626 - 59 116 sinorhizobium fredii ngr234 chromosome αr15 sfr15c2 gi|227820587|ref|nc_012587.1| 1612711 1612830 - 51 120 sinorhizobium fredii ngr234 chromosome αr15 sfr15b gi|227818258|ref|nc_012586.1| 134078 134190 - 55 113 sinorhizobium fredii ngr234 plasmid pngr234b αr15 atr15c1 gi|159184118|ref|nc_003062.2| 2163254 2163370 + 53 117 agrobacterium tumefaciens str. c58 chromosome circular αr15 atr15c2 gi|159184118|ref|nc_003062.2| 2163454 2163554 + 57 101 agrobacterium tumefaciens str. c58 chromosome circular αr15 ah13r15c1 gi|325291453|ref|nc_015183.1| 2112823 2112939 + 52 117 agrobacterium sp. h13-3 chromosome αr15 ah13r15c2 gi|325291453|ref|nc_015183.1| 2113023 2113121 + 54 99 agrobacterium sp. h13-3 chromosome αr15 ah13r15a gi|325168279|ref|nc_015184.1| 211698 211807 - 53 110 agrobacterium sp. h13-3 plasmid pasph13-3a αr15 reciatr15c1 gi|190889639|ref|nc_010994.1| 3155217 3155332 + 51 116 rhizobium etli ciat 652 αr15 reciatr15c2 gi|190889639|ref|nc_010994.1| 3155440 3155555 + 48 116 rhizobium etli ciat 652 αr15 reciatr15pc gi|190894340|ref|nc_010997.1| 941345 941452 + 53 108 rhizobium etli ciat 652 plasmid pc αr15 reciatr15b gi|190893983|ref|nc_010996.1| 187927 188041 - 50 115 rhizobium etli ciat 652 plasmid pb αr15 arr15ci1 gi|222084201|ref|nc_011985.1| 2506215 2506331 + 52 117 agrobacterium radiobacter k84 chromosome 1 αr15 arr15ci2 gi|222084201|ref|nc_011985.1| 2506418 2506534 + 54 117 agrobacterium radiobacter k84 chromosome 1 αr15 arr15cii gi|222080781|ref|nc_011983.1| 1011511 1011624 - 57 114 agrobacterium radiobacter k84 chromosome 2 αr15 rlt2304r15c1 gi|209547612|ref|nc_011369.1| 2770612 2770727 + 50 116 rhizobium leguminosarum bv. trifolii wsm2304 chromosome αr15 rlt2304r15c2 gi|209547612|ref|nc_011369.1| 2770835 2770949 + 50 115 rhizobium leguminosarum bv. trifolii wsm2304 chromosome αr15 avr15ci1 gi|222147015|ref|nc_011989.1| 2608532 2608647 + 54 116 agrobacterium vitis s4 chromosome 1 αr15 avr15ci2 gi|222147015|ref|nc_011989.1| 2608739 2608839 + 46 101 agrobacterium vitis s4 chromosome 1 αr15 avr15atc gi|222083145|ref|nc_011984.1| 122624 122736 - 50 113 agrobacterium vitis s4 plasmid pats4c αr15 avr15ate gi|222102412|ref|nc_011981.1| 198928 199039 + 51 112 agrobacterium vitis s4 plasmid pats4e αr15 avr15ti gi|222080117|ref|nc_011982.1| 52286 52397 - 57 112 agrobacterium vitis s4 plasmid ptis4 αr15 rlvr15c1 gi|116249766|ref|nc_008380.1| 3605490 3605605 + 51 116 rhizobium leguminosarum bv. viciae 3841 αr15 rlvr15c2 gi|116249766|ref|nc_008380.1| 3605714 3605829 + 48 116 rhizobium leguminosarum bv. viciae 3841 αr15 rlvr15p10 gi|116254467|ref|nc_008381.1| 138799 138912 + 56 114 rhizobium leguminosarum bv. viciae 3841 plasmid prl10 αr15 rlvr15p11 gi|116255200|ref|nc_008384.1| 567053 567166 - 53 114 rhizobium leguminosarum bv. viciae 3841 plasmid prl11 αr15 rlt1325r15c1 gi|241202755|ref|nc_012850.1| 2981275 2981390 + 50 116 rhizobium leguminosarum bv. trifolii wsm1325 αr15 rlt1325r15c2 gi|241202755|ref|nc_012850.1| 2981499 2981613 + 50 115 rhizobium leguminosarum bv. trifolii wsm1325 αr15 rlt1325r15p02 gi|241666492|ref|nc_012858.1| 36176 36289 + 51 114 rhizobium leguminosarum bv. trifolii wsm1325 plasmid pr132502 αr15 recfnr15c1 gi|86355669|ref|nc_007761.1| 3117667 3117782 + 50 116 rhizobium etli cfn 42 αr15 recfnr15c2 gi|86355669|ref|nc_007761.1| 3117890 3118004 + 50 115 rhizobium etli cfn 42 αr15 recfnr15d gi|89255298|ref|nc_004041.2| 172760 172874 - 50 115 rhizobium etli cfn 42 symbiotic plasmid p42d αr15 recfnr15a gi|86359705|ref|nc_007762.1| 157296 157409 + 56 114 rhizobium etli cfn 42 plasmid p42a αr15 mlr15a gi|13488050|ref|nc_002679.1| 65044 65154 - 51 111 mesorhizobium loti maff303099 plasmid pmla αr15 bcr15cii gi|161620094|ref|nc_010104.1| 707465 707572 + 56 108 brucella canis atcc 23365 chromosome ii αr15 bcr15ci1 gi|161617991|ref|nc_010103.1| 1379297 1379398 - 51 102 brucella canis atcc 23365 chromosome i αr15 bcr15ci2 gi|161617991|ref|nc_010103.1| 1451969 1452087 - 50 119 brucella canis atcc 23365 chromosome i αr15 bs23445r15ci1 gi|163842277|ref|nc_010169.1| 1401085 1401186 - 51 102 brucella suis atcc 23445 chromosome i αr15 bs23445r15ci2 gi|163842277|ref|nc_010169.1| 1473791 1473909 - 50 119 brucella suis atcc 23445 chromosome i αr15 bs23445r15cii gi|163844199|ref|nc_010167.1| 696081 696188 + 56 108 brucella suis atcc 23445 chromosome ii αr15 bm16mr15ci gi|17986284|ref|nc_003317.1| 607684 607785 + 51 102 brucella melitensis bv. 1 str. 16m chromosome i αr15 bm16mr15cii gi|17988344|ref|nc_003318.1| 589501 589608 - 56 108 brucella melitensis bv. 1 str. 16m chromosome ii αr15 bas19r15cii gi|189022234|ref|nc_010740.1| 508055 508162 - 56 108 brucella abortus s19 chromosome 2 αr15 bas19r15ci1 gi|189023268|ref|nc_010742.1| 1396794 1396895 - 51 102 brucella abortus s19 chromosome 1 αr15 bas19r15ci2 gi|189023268|ref|nc_010742.1| 1469407 1469525 - 50 119 brucella abortus s19 chromosome 1 αr15 bm23457r15cii gi|225685871|ref|nc_012442.1| 687290 687397 + 56 108 brucella melitensis atcc 23457 chromosome ii αr15 bm23457r15ci gi|225851546|ref|nc_012441.1| 1400641 1400742 - 51 102 brucella melitensis atcc 23457 chromosome i αr15 bs1330r15cii gi|56968493|ref|nc_004311.2| 708185 708292 + 56 108 brucella suis 1330 chromosome ii αr15 bs1330r15ci1 gi|56968325|ref|nc_004310.3| 1380381 1380482 - 51 102 brucella suis 1330 chromosome i αr15 bs1330r15ci2 gi|56968325|ref|nc_004310.3| 1453011 1453129 - 50 119 brucella suis 1330 chromosome i αr15 ba19941r15ci1 gi|62288991|ref|nc_006932.1| 1398464 1398565 - 51 102 brucella abortus bv. 1 str. 9-941 chromosome i αr15 ba19941r15ci2 gi|62288991|ref|nc_006932.1| 1471073 1471191 - 50 119 brucella abortus bv. 1 str. 9-941 chromosome i αr15 ba19941r15cii gi|62316961|ref|nc_006933.1| 508851 508958 - 56 108 brucella abortus bv. 1 str. 9-941 chromosome ii αr15 bmar15cii gi|83268957|ref|nc_007624.1| 508839 508946 - 56 108 brucella melitensis biovar abortus 2308 chromosome ii αr15 bmar15ci1 gi|82698932|ref|nc_007618.1| 1395614 1395715 - 51 102 brucella melitensis biovar abortus 2308 chromosome i αr15 bmar15ci2 gi|82698932|ref|nc_007618.1| 1468227 1468345 - 50 119 brucella melitensis biovar abortus 2308 chromosome i αr15 bor15ci1 gi|148558820|ref|nc_009505.1| 1387928 1388029 - 50 102 brucella ovis atcc 25840 chromosome i αr15 bor15ci2 gi|148558820|ref|nc_009505.1| 1460506 1460624 - 50 119 brucella ovis atcc 25840 chromosome i αr15 bor15cii gi|148557829|ref|nc_009504.1| 709415 709524 + 54 110 brucella ovis atcc 25840 chromosome ii αr15 bmir15cii gi|256014795|ref|nc_013118.1| 709102 709209 + 56 108 brucella microti ccm 4915 chromosome 2 αr15 bmir15ci1 gi|256368465|ref|nc_013119.1| 1387776 1387877 - 51 102 brucella microti ccm 4915 chromosome 1 αr15 bmir15ci2 gi|256368465|ref|nc_013119.1| 1461298 1461416 - 50 119 brucella microti ccm 4915 chromosome 1 αr15 oar15ci gi|153007346|ref|nc_009667.1| 1751482 1751598 + 49 117 brucella anthropi atcc 49188 chromosome 1 αr15 oar15cii gi|153010078|ref|nc_009668.1| 1270083 1270191 + 56 109 brucella anthropi atcc 49188 chromosome 2 αr15 oar15p02 gi|153011934|ref|nc_009670.1| 22208 22320 + 54 113 brucella anthropi atcc 49188 plasmid poant02 the results were manually inspected to deduce a consensus secondary structure for the family (figure 1 and figure 2). the consensus structure was also independently predicted with the program locarnate comparing the obtained predictions. the manual inspection of the sequences found with the cm using infernal allowed finding 38 true homologues in phylogenetically related α-proteobacterial genomes. the 26 closest αr15 family members were found as tandem in the same chromosomal igrs for the following species besides s. melilloti: sinorhizobium species: s. medicae and s. fredii rhizobium species: two r. leguminosarum trifolii strains (wsm304 and wsm35), two r. etli strains cfn 42 and ciat 652, the reference r. leguminosarum bv. viciae 3841 strain agrobacterium species: a. vitis,a. tumefaciens, a. radiobacter and a. h13 all these sequences showed significant bit scores and infernal e-values (1.71e-28 - 2.03e-20). however, the plasmidic copies of all mentioned α-proteobacterial genomes and those αr15 members encoded by brucella species (b. ovis, b. canis, b. abortus, b. microtis, and several biovars of b. melitensis), brucella anthropi and mesorhizobium lotishowed high e-values between (1e-19 and 8e-03) but very low bit-scores. figure 1: consensus secondary structure of smr15c1, smr15c2 and the αr15 family predicted by rna and rnaalifold. smr15c1 and smr15c2 are colored with a base probability scheme. the coloring of the αr15 family structure follows a base pairs conservation scheme: red: base pair occurring in all sequences used to generate the consensus; yellow: two types of base pairing occur; green: three types of base pairing occur. the shading of base pairs represents: saturated, no inconsistent sequences; pale, one inconsistent sequence; very pale, two inconsistent sequences. figure 3: phylogenetic distribution of known and predicted αr9 genes. gene numbers are based on computational analysis using the program infernal. legend: smr15c1 = sinorhizobium meliloti 1021 (nc_003047), smr15c2 = sinorhizobium meliloti 1021 (nc_003047), smr15a = sinorhizobium meliloti 1021 plasmid psyma (nc_003037), smedr15c1 = sinorhizobium medicae wsm419 chromosome (nc_009636), smedr15c2 = sinorhizobium medicae wsm419chromosome (nc_009636), smedr15p03 = sinorhizobium medicae wsm419 plasmid psmed03 (nc_009622), sfr15c1 = sinorhizobium fredii ngr234 chromosome (nc_012587), sfr15c2 = sinorhizobium fredii ngr234 chromosome (nc_012587), sfr15b = sinorhizobium fredii ngr234 plasmid pngr234b (nc_012586), atr15c2 = agrobacterium tumefaciens str. c58 chromosome circular (nc_003062), atr15c1 = agrobacterium tumefaciens str. c58 chromosome circular (nc_003062), ah13r15c2 = agrobacterium sp. h13-3 chromosome (nc_015183), ah13r15c1 = agrobacterium sp. h13-3 chromosome (nc_015183), ah13r15a = agrobacterium sp. h13-3 plasmid pasph13-3a (nc_015184), reciatr15c2 = rhizobium etli