αr7 is a family of bacterial small non-coding RNAs with representatives in a broad group of Alphaproteobacterial species from the order Hyphomicrobiales. The first member of this family (Smr7C 150nt) was found in a Sinorhizobium meliloti 1021 locus located in the chromosome (C). Further homology and structure conservation analysis identified full-length 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 biobars of B. melitensis). αr7 RNA species are 134-159 nucleotides (nt) long (Table 1) and share a well defined common secondary structure (Figure 1, Figure 2). αr7 transcripts can be catalogued as trans-acting sRNAs expressed from well-defined promoter regions of independent transcription units within intergenic regions (IGRs) of the Alphaproteobacterial genomes (Figure 4).
Discovery and structure Smr7C sRNA was described by del Val et al. in the intergenic regions (IGRs) of the reference S. meliloti 1021 strain (Sinorhizobium meliloti 1021). Northern hybridization experiments detected two RNA species expressed from the smr7C locus, which accumulated differentially in free-living and endosymbiotic bacteria. TAP-based 5'-RACE experiments mapped the transcription start site (TSS) to two close G residues. From the six independent sequences obtained, five mapped to residue 201,681 in the chromosome and one to the upstream residue 201,679. Furthermore, an additional 5'-RACE product could be obtained from this transcript in both TAP- and mock-treated RNA samples. The sequence of this second RACE product mapped the processing site of Smr7C to residue 201,723 nt in the S. meliloti 1021 genome. The 3'-end was assumed to be located at the 201,828 nt position matching the last residue of the consecutive stretch of Us of a bona fide Rho-independent terminator. Parallel and later studies in which Smr7C transcript is referred to as sra03 or Sm13, independently confirmed the expression this sRNA 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 2021 further also confirmed the expression of Smr7C (here referred to as SmelC023), and mapped the 5'- end of the full-length transcript to the same position in the S. meliloti 1021 genome, and the 3' end to position 201,825.
The nucleotide sequence of Smr7C was initially used as query to search against the Rfam database (version 10.0;). This homology search rendered no matches to known bacterial sRNA in this database. Smr7C was next BLASTed with default parameters against all the currently available bacterial genomes (1,615 sequences at 20 April 2011;). 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). This CM was used in a further search for new members of the αr7 family in the existing bacterial genomic databases (Table 1) and to create the final CM model (Figure 1).
+ Table 1: αr7 homologs in other symbionts and pathogens CM model Name GI accession number begin end strand %GC length Organism αr7 Smr7C gi15963753refNC_003047.1 201679 201828 + 64 150 Sinorhizobium meliloti 1021 αr7 Smedr7C gi150395228refNC_009636.1 3585152 3585302 + 60 151 Sinorhizobium medicae WSM419 chromosome αr7 Sfr7C gi227820587refNC_012587.1 3727302 3727450 - 66 149 Sinorhizobium fredii NGR234 chromosome αr7 Atr7C gi159184118refNC_003062.2 112535 112678 - 65 144 Agrobacterium tumefaciens str. C58 chromosome circular αr7 AH13r7C gi325291453refNC_015183.1 112523 112667 - 63 145 Agrobacterium sp. H13-3 chromosome αr7 ReCIATr7C gi190889639refNC_010994.1 205678 205811 - 61 134 Rhizobium etli CIAT 652 αr7 Arr7CI gi222084201refNC_011985.1 243240 243377 - 62 138 Agrobacterium radiobacter K84 chromosome 1 αr7 Rlt2304r7C gi209547612refNC_011369.1 4280520 4280653 - 63 134 Rhizobium leguminosarum bv. trifolii WSM2304 chromosome αr7 Avr7CI gi222147015refNC_011989.1 255869 256019 + 60 151 Agrobacterium vitis S4 chromosome 1 αr7 Rlvr7C gi116249766refNC_008380.1 192620 192753 - 62 134 Rhizobium leguminosarum bv. viciae 3841 αr7 Rlt1325r7C gi241202755refNC_012850.1 4553564 4553697 - 62 134 Rhizobium leguminosarum bv. trifolii WSM1325 αr7 ReCFNr7C gi86355669refNC_007761.1 163762 163895 - 60 134 Rhizobium etli CFN 42 αr7 Mlr7C gi57165207refNC_002678.2 2648243 2648390 - 65 148 Mesorhizobium loti MAFF303099 chromosome αr7 MsBNCr7C gi110632362refNC_008254.1 4251581 4251722 + 60 142 Mesorhizobium sp. BNC1 αr7 Mcr7C gi319779749refNC_014923.1 1781903 1782047 + 68 145 Mesorhizobium ciceri biovar biserrulae WSM1271 chromosome αr7 Bcr7CI gi161617991refNC_010103.1 134573 134715 - 62 143 Brucella canis ATCC 23365 chromosome I αr7 Bs23445r7CI gi163842277refNC_010169.1 134908 135050 - 62 143 Brucella suis ATCC 23445 chromosome I αr7 Bm16Mr7CI gi17986284refNC_003317.1 1868345 1868487 + 62 143 Brucella melitensis bv. 1 str. 16M chromosome I αr7 BaS19r7CI gi189023268refNC_010742.1 134335 134477 - 62 143 Brucella abortus S19 chromosome 1 αr7 Bm23457r7CI gi225851546refNC_012441.1 134555 134697 - 62 143 Brucella melitensis ATCC 23457 chromosome I αr7 Bs1330r7CI gi56968325refNC_004310.3 134590 134732 - 62 143 Brucella suis 1330 chromosome I αr7 Ba19941r7CI gi62288991refNC_006932.1 135949 136091 - 62 143 Brucella abortus bv. 1 str. 9-941 chromosome I αr7 Bmar7CI gi82698932refNC_007618.1 132316 132458 - 62 143 Brucella melitensis biovar Abortus 2308 chromosome I αr7 Bor7CI gi148558820refNC_009505.1 134888 135029 - 63 142 Brucella ovis ATCC 25840 chromosome I αr7 Bmir7CI gi256368465refNC_013119.1 136167 136309 - 62 143 Brucella microti CCM 4915 chromosome 1 αr7 Oar7CI gi153007346refNC_009667.1 149814 149972 - 58 159 Brucella anthropi ATCC 49188 chromosome 1
The results were manually inspected to deduce a consensus secondary structure for the family (Figure1 and Figure2). 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 Alphaproteobacterial genomes. The rhizobial species encoding the closer homologs to Smr7C were: S. medicae and S. fredii, two R. leguminosarum trifolii strains (WSM2304 and WSM1325), two R. etli strains CFN 42 and CIAT 652, the reference R. leguminosarum bv. viciae 3841 strain, and the Agrobacterium species A. vitis, A. tumefaciens, A. radiobacter and A. H13 and the Mesorhizobum species loti, M. ciceri and M. BNC. All these sequences showed significant Infernal E-values (3.66E-49 – 2.93E-12) and bit-scores. The rest of the sequences found with the model showed high E-values between (1.58E-10 and 2.88E-09) but lower bit-scores and are encoded by Brucella species (B. ovis, B. canis, B. abortus, B. microtis, several biobars of B. melitensis, and B. anthropi).
Expression information Parallel studies assessed Smr7C 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). Expression of Smr7C in free-living bacteria was found to be growth-dependent, being the gene strongly down-regulated when bacteria entered the stationary phase. Expression of SmrC7 increased ~13-fold in nodules when compared with free-living bacteria (log phase TY or MM cultures), suggesting the induction of this sRNAs during bacterial infection and/or bacteroid differentiation. SmrC7 expression has also been proved in parallel studies.
Promoter analysis All the promoter regions of the αr7 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 closest homologous 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 Alphaproteobacteria. To identify binding sites for other known transcription factors we used the fasta sequences provided by RegPredict, and used those position weight matrices (PSWM) provided by RegulonDB. 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 gps-tools2.its.yale.edu). Moreover, we searched for conserved unknown motifs using MEME and used relaxed regular expressions (i.e. pattern matching) over all promoters of the Smr7C homologs promoters.
This studies revealed a 20 bp conserved motif in ll promoter regions, marked in orange as MEME conserved motif, in (Figure 4), but no significant similarity to known transcription factor binding sites matrices could be established.
Genomic context All identified members of the αr7 family are trans-encoded sRNAs transcribed from independent promoters in chromosomal IGRs. Most of the neighboring genes of the seed alignment's members were not annotated and thus were further manually curated.
The αr7 sRNAs' genomic regions of the Sinorhizobium, Rhizobium and Agrobacterium group members exhibited a great degree of conservation with the upstream and downstream genes coding for a DNA polymerase, and a MarR family transcriptional regulator, respectively. Partial synteny of the αr7 genomic regions was observed in the Mesorhizobium and Brucella group, where instead of the MarR family transcriptional regulator gene, a peptidase encoding gene was found.