αr45 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 (Smr45C) was found in a Sinorhizobium meliloti 1021 locus located in the chromosome (C). Further homology and structure conservation analysis identified homologs in several nitrogen-fixing symbiotic rhizobia (i.e. S. medicae, S. fredii, Rhizobium 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), in Bartonella species (i.e. B. henselae, B. clarridgeiae, B. tribocorum, B. quintana, B. bacilliformis, B. grahamii), in several members of the Xanthobactereacea family (i.e. Azorhizobium caulinodans, Starkey novella, Xhantobacter autotrophicus), and in some representatives of the Beijerinckiaceae family (i.e. Methylocella silvestris, Beijerinckia indica subsp. indica). αr45C RNA species are 147-153 nt long (Table 1) and share a well defined common secondary structure (Figure 1). All of the αr45 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 Smr45C sRNA was 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) . Northern hybridization experiments confirmed that the predicted smr45C locus did express a single transcript of 130-179 nt length, which accumulated differentially in free-living and endosymbiotic bacteria. TAP-based 5'-RACE experiments mapped the transcription start site (TSS) of the full-length Smr45C transcript to the 3,105,445 nt position in the S. meliloti 1021 genome (http://iant.toulouse.inra.fr/bacteria/annotation/cgi/rhime.cgi) whereas the 3'-end was initially assumed to be located at the 3,105,265 nt position matching the last residue of a short stretch of Us (Figure 2) of a putative, but low-rated, Rho-independent terminator. Recent deep sequencing-based characterization of the small RNA fraction (50-350 nt) of S. meliloti 2011 further confirmed the expression of Smr45C (here referred to as SmelC706), and mapped the full-length transcript to the same 5' end and to the 3' end position 3,105,298.

The nucleotide sequence of Smr45C 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. Smr45C 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 αr45 family in the existing bacterial genomic databases.

+ Table 1: αr45 homologs in other symbionts and pathogens CM model Name GI accession number begin end strand %GC length Organism αr45 Smr45C gi15963753refNC_003047.1 3105265 3105445 - 59 148 Sinorhizobium meliloti 1021 αr45 Smedr45C gi150395228refNC_009636.1 2892729 2892909 - 59 148 Sinorhizobium medicae WSM419 chromosome αr45 Sfr45C gi227820587refNC_012587.1 3069095 3069275 - 59 148 Sinorhizobium fredii NGR234 chromosome αr45 Atr45C gi159185562refNC_003063.2 205601 205782 - 57 148 Agrobacterium tumefaciens str. C58 chromosome linear αr45 ReCIATr45C gi190889639refNC_010994.1 3953756 3953936 - 59 148 Rhizobium etli CIAT 652 αr45 Arr45CI gi222084201refNC_011985.1 3124263 3124442 - 57 147 Agrobacterium radiobacter K84 chromosome 1 αr45 Rlt2304r45C gi209547612refNC_011369.1 3504209 3504389 - 59 148 Rhizobium leguminosarum bv. trifolii WSM2304 αr45 Avr45CI gi222147015refNC_011989.1 3166967 3167147 - 59 148 Agrobacterium vitis S4 chromosome 1 αr45 Rlvr45C gi116249766refNC_008380.1 4405210 4405390 - 59 148 Rhizobium leguminosarum bv. viciae 3841 αr45 Rlt1325r45C gi241202755refNC_012850.1 3739371 3739551 - 59 148 Rhizobium leguminosarum bv. trifolii WSM1325 αr45 ReCFNr45C gi86355669refNC_007761.1 3840978 3841158 - 59 148 Rhizobium etli CFN 42 αr45 Mlr45C gi57165207refNC_002678.2 2390687 2390867 - 62 147 Mesorhizobium loti MAFF303099 chromosome αr45 Mcr45C gi319779749refNC_014923.1 1999110 1999290 + 63 147 Mesorhizobium ciceri biovar biserrulae WSM1271 chromosome αr45 Bcr45CII gi161620094refNC_010104.1 97148 97326 + 60 143 Brucella canis ATCC 23365 chromosome II αr45 Bs23445r45CII gi163844199refNC_010167.1 97254 97432 + 60 146 Brucella suis ATCC 23445 chromosome II αr45 Bm16Mr45CII gi17988344refNC_003318.1 1172651 1172829 - 60 146 Brucella melitensis bv. 1 str. 16M chromosome II αr45 BaS19r45CII gi189022234refNC_010740.1 96948 97126 + 60 146 Brucella abortus S19 chromosome 2 αr45 Bm23457r45CII gi225685871refNC_012442.1 97129 97307 + 60 146 Brucella melitensis ATCC 23457 chromosome II αr45 Bs1330r45CII gi56968493refNC_004311.2 97128 97306 + 60 146 Brucella suis 1330 chromosome II αr45 Ba19941r45CII gi62316961refNC_006933.1 96885 97063 + 60 146 Brucella abortus bv. 1 str. 9-941 chromosome II αr45 Bmar45CII gi83268957refNC_007624.1 96950 97128 + 60 146 Brucella melitensis biovar Abortus 2308 chromosome II αr45 Bor45CII gi148557829refNC_009504.1 96814 96992 + 60 146 Brucella ovis ATCC 25840 chromosome II αr45 Bmir45CII gi256014795refNC_013118.1 97102 97280 + 60 146 Brucella microti CCM 4915 chromosome 2 αr45 Oar45CII gi153010078refNC_009668.1 1673505 1673683 - 60 146 Brucella anthropi ATCC 49188 chromosome 2 αr45 MsBNCr45C gi110632362refNC_008254.1 2952752 2952930 + 60 146 Mesorhizobium sp. BNC1 αr45 Bahr45C gi49474831refNC_005956.1 1421835 1422014 - 54 147 Bartonella henselae str. Houston-1 αr45 Bacr45C gi319898193refNC_014932.1 1277567 1277776 - 54 147 Bartonella clarridgeiae 73 αr45 Batr45C gi163867306refNC_010161.1 1865843 1866022 - 54 147 Bartonella tribocorum CIP 105476 αr45 Baqr45C gi49473688refNC_005955.1 1185140 1185319 - 53 147 Bartonella quintana str. Toulouse αr45 Babr45C gi121601635refNC_008783.1 1120052 1120229 - 56 147 Bartonella bacilliformis KC583 αr45 Bagr45C gi240849682refNC_012846.1 1761390 1761567 - 53 147 Bartonella grahamii as4aup αr45 Acr45C gi158421624refNC_009937.1 385443 385629 - 64 154 Azorhizobium caulinodans ORS 571 αr45 Stnr45C gi298290017refNC_014217.1 4175745 4175703 + 65 156 Starkeya novella DSM 506 chromosome αr45 Xar45C gi154243958refNC_009720.1 2404335 2404523 - 63 156 Xanthobacter autotrophicus Py2 chromosome αr45 Mesr45C gi217976200refNC_011666.1 1244610 1244795 - 64 155 Methylocella silvestris BL2 chromosome αr45 Beir45C gi182677002refNC_010581.1 1241316 1241498 - 61 153 Beijerinckia indica subsp. indica ATCC 9039 chromosome

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 39 closer homolog sequences, all of them present as single chromosomal copies in the α-proteobacterial genomes. The rhizobial species encoding these homologs to Smr45C were: S. medicae and S. fredii, 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, and the Agrobacterium species A. vitis,A. tumefaciens, A. radiobacter and A. H13, Brucella species (B. ovis, B. canis, B. abortus, B. microtis, and several biobars of B. melitensis), Brucella anthropi, the Mesorhizobium species loti, M. ciceri and M. BNC., Bartonella species (i.e. B. henselae, B. clarridgeiae, B. tribocorum, B. quintana, B. bacilliformis, B. grahamii). All these sequences showed significant Infernal E-values (8.93E-40 – 6.12E-36) and bit-scores. The rest of the sequences found with the model showed high E-values between (3.28E-06 and 4.56E-04) but lower bit-scores and are encoded by several members of the Xanthobactereacea family (i.e. A. caulinodans, Sa. novella, X. autotrophicus), Me. silvestris, and Be. indica subsp. indica.

Expression information The expression of Smr45C in S. meliloti 1021 was assessed 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). The expression of Smr45C in free-living bacteria was found to be growth-dependent, being the gene strongly down-regulated when bacteria entered the stationary phase. However, luteolin moderately stimulated the expression of Smr45C (2-fold) but the gene was not detectable in endosymbiotic bacteria.

Recent co-inmuno precipitation experiments corroborate that Smr45C, does bind the bacterial protein Hfq for efficient target binding.

Promoter Analysis All the promoter regions of the αr45 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 homolog 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 Smr45C homologs promoters.

This study predicts differences in the regulation of the expression of the αr45 representatives in the different α-proteobacterial species. The Sinorhizobium, Rhizobium, and Agrobacterium groups presented a very well conserved motif that matches the consensus sequence recognized by the maltose repressor Mall. Furthermore, the promoters of the αr45 members of the . Furthermore, the promoters of the αr45 members of the Sinorhizobium group presented an additional conserved region between positions -60 and -85 (boxed in orange in Figure 4), with significant similarity to the matrix SMb21598_Rhizobiales from Reg_Predict. This binding site corresponds to a transcriptional regulator of the LacI family. The Rhizobium group, presented also a well conserved motif in this region for which no significant similarity could be found (marked in green in Figure 4). This analysis also revealed an extended conserved sequence stretch among the promoters of the Brucella and Bartonella αr45 sRNA loci, but no known transcription factor binding sites were recognizable in these motifs.

Genomic Context