Αr15 RNA

Smr15C1 y Smr15C2 sRNAs were described by del Val et al.,^[1] 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.^[1]

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,^[2][3] 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.^[4]

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)^[5] (Figure 2). This CM was used in a further search for new members of the αr15 family in the existing bacterial genomic databases.

Table 1: Smr15C1 and Smr15C2 homologs in other symbionts and pathogens

CM model	Name	GI accession number	begin	end	strand	%GC	length	Organism
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^[6] 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.

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),^[1] high salt stress, oxidative stress and cold and hot shock stresses.^[3] The results showed different expression profiles for both sRNAs,^[1] 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.^[1] Additionally, Schlüter et al.^[4] 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.^[9] 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.^[9] 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^[10] 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.^[11]