αr9 is a family of bacterial small non-coding RNAs with representatives in a broad group of α-proteobacteria from the order Hyphomicrobiales. The first member of this family (Smr9C) was found in a Sinorhizobium meliloti 1021 locus located in the chromosome (C). Further homology and structure conservation analysis have identified full-length Smr9C 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). αr9C RNA species are 144-158 nt long (Table 1) and share a well defined common secondary structure consisting of seven conserved regions (Figure 1). Most of the αr9 transcripts can be catalogued as trans-acting sRNAs expressed from well-defined promoter regions of independent transcription units within intergenic regions (IGRs) of the α-proteobacterial genomes (Figure 5).
Discovery and structure
Smr9C sRNA was described by del Val et al. in the intergenic regions (IGRs) of the reference S. meliloti 1021 strain (http://iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi). Northern hybridization experiments confirmed that the predicted smr9C locus did express a single transcript of the expected size, which accumulated differentially in free-living and endosymbiotic bacteria. TAP-based 5'-RACE experiments mapped the transcription start site (TSS) of the full-length Smr9C transcript to the 1,398,425 nt position in the S. meliloti 1021 genome (http://iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi) whereas the 3'-end was assumed to be located at the 1,398,277 nt position matching the last residue of the consecutive stretch of Us of a bona fide Rho-independent terminator (Figure 5). Parallel and later studies in which Smr9C transcript is referred to as Igr#3 or sra32 independently confirmed the expression this sRNA in S. meliloti and in its closely related strain 2011. Recent deep sequencing-based characterization of the small RNA fraction (50-350 nt) of S. meliloti 2011 further confirmed the expression of Smr9C (here referred to as SmelC289), and mapped the 5'- and 3'-ends of the full-length transcript to positions 1,398,423 and 1,398,279, respectively, in the S. meliloti 1021 genome.
The nucleotide sequence of Smr9C was initially used as query to search against the Rfam database (version 10.0; http://www.sanger.ac.uk/Software/Rfam). This homology search rendered no matches to known bacterial sRNA in this database. Smr9C was next 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 αr9 family in the existing bacterial genomic databases.
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 with very similar predictions. The manual inspection of the sequences found with the CM using Infernal allowed finding 26 true homolog sequences, all of them present as single chromosomal copies in the α-proteobacterial genomes. The rhizobial species encoding the 12 closer homologs to Smr9C were: two R. leguminosarum trifolii strains (WSM2304 and WSM1235), two R. etli strains CFN 42 and CIAT 652, the reference R. leguminosarum bv. viciae 3841 strain, Rhizobium NGR234, and the Agrobacterium species A. vitis,A. tumefaciens, A. radiobacter and A. H13. All these sequences showed significant Infernal E-values (1.50e-39 – 14.02e-21) and bit-scores. The rest of the sequences found with the model showed high E-values between (3.40e-12 and 2.62e-04) but lower bit-scores and are encoded by Brucella species (B. ovis, B. canis, B. abortus, B. microtis, and several biobars of B. melitensis), Brucella anthropi and the Mesorhizobum species M. loti, M. ciceri and M. BNC.
+ Table 1: Smr9C homologs in other symbionts and pathogens CM model Name GI accession number begin end strand %GC length Organism αr9 Smr9C gi15963753refNC_003047.1 1398277 1398425 - 46 149 Sinorhizobium meliloti 1021 αr9 Smedr9C gi150395228refNC_009636.1 979747 979902 - 48 150 Sinorhizobium medicae WSM419 chromosome αr9 Sfr9C gi227820587refNC_012587.1 1130001 1130150 - 47 150 Sinorhizobium fredii NGR234 chromosome αr9 Atr9C gi159184118refNC_003062.2 1275297 1275443 - 47 147 Agrobacterium tumefaciens str. C58 chromosome circular αr9 AH13r9C gi325291453refNC_015183.1 1215580 1215726 - 48 147 Agrobacterium sp. H13-3 chromosome αr9 ReCIATr9C gi190889639refNC_010994.1 1684929 1685072 - 48 144 Rhizobium etli CIAT 652 αr9 Arr9CI gi222084201refNC_011985.1 1481975 1482119 - 49 145 Agrobacterium radiobacter K84 chromosome 1 αr9 Rlt2304r9C gi209547612refNC_011369.1 1296506 1296650 - 48 145 Rhizobium leguminosarum bv. trifolii WSM2304 chromosome αr9 Avr9CI gi222147015refNC_011989.1 1448510 1448659 - 45 150 Agrobacterium vitis S4 chromosome 1 αr9 Rlvr9C gi116249766refNC_008380.1 1802936 1803079 - 49 144 Rhizobium leguminosarum bv. viciae 3841 αr9 Rlt1325r9C gi241202755refNC_012850.1 1354593 1354736 - 47 144 Rhizobium leguminosarum bv. trifolii WSM1325 αr9 ReCFNr9C gi86355669refNC_007761.1 1703203 1703345 - 47 143 Rhizobium etli CFN 42 αr9 Mlr9C gi57165207refNC_002678.2 1127217 1127363 + 54 145 Mesorhizobium loti MAFF303099 chromosome αr9 Mcr9C gi319779749refNC_014923.1 3449639 3449786 - 52 148 Mesorhizobium ciceri biovar biserrulae WSM1271 chromosome αr9 Bcr9CI gi161617991refNC_010103.1 796281 796428 - 49 148 Brucella canis ATCC 23365 chromosome I αr9 Bs23445r9CI gi163842277refNC_010169.1 817762 817909 - 49 148 Brucella suis ATCC 23445 chromosome I αr9 Bm16Mr9CI gi17986284refNC_003317.1 1187596 1187743 + 49 146 Brucella melitensis bv. 1 str. 16M chromosome I αr9 BaS19r9CI gi189023268refNC_010742.1 817898 818045 - 49 148 Brucella abortus S19 chromosome 1 αr9 Bm23457r9CI gi225851546refNC_012441.1 819377 819523 - 50 147 Brucella melitensis ATCC 23457 chromosome I αr9 Bs1330r9CI gi56968325refNC_004310.3 797917 798064 - 49 148 Brucella suis 1330 chromosome I αr9 Ba19941r9CI gi62288991refNC_006932.1 819597 819744 - 49 148 Brucella abortus bv. 1 str. 9-941 chromosome I αr9 Bmar9CI gi82698932refNC_007618.1 815875 816022 - 49 148 Brucella melitensis biovar Abortus 2308 chromosome I αr9 Bor9CI gi148558820refNC_009505.1 822712 822859 - 49 148 Brucella ovis ATCC 25840 chromosome I αr9 Bmir9CI gi256368465refNC_013119.1 802078 802225 - 49 148 Brucella microti CCM 4915 chromosome 1 αr9 Oar9CI gi153007346refNC_009667.1 2529108 2529255 + 48 146 Brucella anthropi ATCC 49188 chromosome 1 αr9 MsBCNr9C gi110632362refNC_008254.1 1141776 1141933 - 50 158 Mesorhizobium sp. BNC1
Expression information
Parallel studies assessed Smr9C 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) and high salt stress, oxidative stress and cold and hot shock stresses. Expression of Smr9C in free-living bacteria was found to be growth-dependent, being the gene strongly down-regulated when bacteria entered the stationary phase, whereas no expression was detected in endosymbiotic bacteria. Recent deep sequencing data further revealed up-regulation of Smr9C upon salt, acidic, cold-shock and heat shock stresses. Recent co-inmuno precipitation experiments corroborate that Smr9C, does bind the bacterial protein Hfq for efficient target binding.
Promoter analysis
All the promoter regions of the αr9 family members examined so far are very conserved in a sequence stretch extending up to 80 bp upstream of the transcription start site of the sRNA. All loci have recognizable σ70-dependent promoters showing a -35/-10 consensus motif CTTAGAC-n17-CTATAT, which has been previously shown to be widely conserved among several other genera in the α-subgroup of proteobacteria. 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 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 Smr9C homologs promoters. This studies revealed the presence of 30 bp long region very conserved between positions -40 and -75, conserved MEME motif, (Figure 5). This sequence was present in all but one of the smrC9 homologs found, but no significant similarity to known transcription factor binding sites matrices could be established.
Genomic context
Most of the members of the αr9 family are trans-encoded sRNAs transcribed from independent promoters in chromosomal IGRs. Exceptions are the cis-encoded antisense Smr9C homologs of A. tumefaciens and B. microti, which are located in the opposite strand of annotated genes, partially overlapping ORFs. Most of the neighboring genes of the seed alignment's members were not annotated and thus were further manually curated. The predicted protein products of these overlapping ORFs could not be assigned to any functional category on the basis of the amino acid sequence homology. However, the genomic regions of almost all αr9 sRNAs exhibited a great degree of conservation including the sRNA-coding sequence and the upstream and downstream genes which have been predicted to code for a prolyl-tRNA syntethase (proS) and a transmembrane protein, respectively. Partial synteny of the αr9 genomic regions was observed in a few cases such as, S. medicae where instead of a proS gene an FAD-dependent pyridine nucleotide-disulfide oxidoreductase encoding gene was found upstream of the αr9 locus, and Mesorhizobium loti where no transmembrane coding gene was recognizable downstream of the sRNA gene. A special case is the Brucella group, where primary automatic annotation over their genomes identified ORFs smaller than 30 aa overlapping with the predicted αr9 sRNA in the same strand. These predicted ORFs, neither show any similarity with database entries nor any motif or signatures when searched against family and motif databases such as Interpro, PFAM or Smart, and thus, are considered here as missannotations not registered in the genomic context graph.