Αr45 RNA


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 was found in a Sinorhizobium meliloti 1021 locus located in the chromosome. Further homology and structure conservation analysis identified homologs in several nitrogen-fixing symbiotic rhizobia, in the plant pathogens belonging to Agrobacterium species as well as in a broad spectrum of Brucella species, in Bartonella species, in several members of the Xanthobactereacea family, and in some representatives of the Beijerinckiaceae family. αr45C RNA species are 147-153 nt long and share a well defined common secondary structure. 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 of the α-proteobacterial genomes.

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 of the reference S. meliloti 1021 strain . 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 of the full-length Smr45C transcript to the 3,105,445 nt position in the S. meliloti 1021 genome 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 of a putative, but low-rated, Rho-independent terminator. Recent deep sequencing-based characterization of the small RNA fraction of S. meliloti 2011 further confirmed the expression of Smr45C, and mapped the full-length transcript to the same 5' end and to the 3' end position 3,105,298.
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The nucleotide sequence of Smr45C was initially used as query to search against the Rfam database. 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. The regions exhibiting significant homology to the query sequence were extracted to create a Covariance Model from a seed alignment using Infernal . This CM was used in a further search for new members of the αr45 family in the existing bacterial genomic databases.
CM modelNameGI accession numberbeginendstrand%GClengthOrganism
αr45Smr45Cgi15963753refNC_003047.131052653105445-59148Sinorhizobium meliloti 1021
αr45Smedr45Cgi150395228refNC_009636.128927292892909-59148Sinorhizobium medicae WSM419 chromosome
αr45Sfr45Cgi227820587refNC_012587.130690953069275-59148Sinorhizobium fredii NGR234 chromosome
αr45Atr45Cgi159185562refNC_003063.2205601205782-57148Agrobacterium tumefaciens str. C58 chromosome linear
αr45ReCIATr45Cgi190889639refNC_010994.139537563953936-59148Rhizobium etli CIAT 652
αr45Arr45CIgi222084201refNC_011985.131242633124442-57147Agrobacterium radiobacter K84 chromosome 1
αr45Rlt2304r45Cgi209547612refNC_011369.135042093504389-59148Rhizobium leguminosarum bv. trifolii WSM2304
αr45Avr45CIgi222147015refNC_011989.131669673167147-59148Agrobacterium vitis S4 chromosome 1
αr45Rlvr45Cgi116249766refNC_008380.144052104405390-59148Rhizobium leguminosarum bv. viciae 3841
αr45Rlt1325r45Cgi241202755refNC_012850.137393713739551-59148Rhizobium leguminosarum bv. trifolii WSM1325
αr45ReCFNr45Cgi86355669refNC_007761.138409783841158-59148Rhizobium etli CFN 42
αr45Mlr45Cgi57165207refNC_002678.223906872390867-62147Mesorhizobium loti MAFF303099 chromosome
αr45Mcr45Cgi319779749refNC_014923.119991101999290+63147Mesorhizobium ciceri biovar biserrulae WSM1271 chromosome
αr45Bcr45CIIgi161620094refNC_010104.19714897326+60143Brucella canis ATCC 23365 chromosome II
αr45Bs23445r45CIIgi163844199refNC_010167.19725497432+60146Brucella suis ATCC 23445 chromosome II
αr45Bm16Mr45CIIgi17988344refNC_003318.111726511172829-60146Brucella melitensis bv. 1 str. 16M chromosome II
αr45BaS19r45CIIgi189022234refNC_010740.19694897126+60146Brucella abortus S19 chromosome 2
αr45Bm23457r45CIIgi225685871refNC_012442.19712997307+60146Brucella melitensis ATCC 23457 chromosome II
αr45Bs1330r45CIIgi56968493refNC_004311.29712897306+60146Brucella suis 1330 chromosome II
αr45Ba19941r45CIIgi62316961refNC_006933.19688597063+60146Brucella abortus bv. 1 str. 9-941 chromosome II
αr45Bmar45CIIgi83268957refNC_007624.19695097128+60146Brucella melitensis biovar Abortus 2308 chromosome II
αr45Bor45CIIgi148557829refNC_009504.19681496992+60146Brucella ovis ATCC 25840 chromosome II
αr45Bmir45CIIgi256014795refNC_013118.19710297280+60146Brucella microti CCM 4915 chromosome 2
αr45Oar45CIIgi153010078refNC_009668.116735051673683-60146Brucella anthropi ATCC 49188 chromosome 2
αr45MsBNCr45Cgi110632362refNC_008254.129527522952930+60146Mesorhizobium sp. BNC1
αr45Bahr45Cgi49474831refNC_005956.114218351422014-54147Bartonella henselae str. Houston-1
αr45Bacr45Cgi319898193refNC_014932.112775671277776-54147Bartonella clarridgeiae 73
αr45Batr45Cgi163867306refNC_010161.118658431866022-54147Bartonella tribocorum CIP 105476
αr45Baqr45Cgi49473688refNC_005955.111851401185319-53147Bartonella quintana str. Toulouse
αr45Babr45Cgi121601635refNC_008783.111200521120229-56147Bartonella bacilliformis KC583
αr45Bagr45Cgi240849682refNC_012846.117613901761567-53147Bartonella grahamii as4aup
αr45Acr45Cgi158421624refNC_009937.1385443385629-64154Azorhizobium caulinodans ORS 571
αr45Stnr45Cgi298290017refNC_014217.141757454175703+65156Starkeya novella DSM 506 chromosome
αr45Xar45Cgi154243958refNC_009720.124043352404523-63156Xanthobacter autotrophicus Py2 chromosome
αr45Mesr45Cgi217976200refNC_011666.112446101244795-64155Methylocella silvestris BL2 chromosome
αr45Beir45Cgi182677002refNC_010581.112413161241498-61153Beijerinckia indica subsp. indica ATCC 9039 chromosome

The results were manually inspected to deduce a consensus secondary structure for the family. 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, 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, Brucella anthropi, the Mesorhizobium species loti, M. ciceri and M. BNC., Bartonella species. All these sequences showed significant Infernal E-values and bit-scores. The rest of the sequences found with the model showed high E-values between but lower bit-scores and are encoded by several members of the Xanthobactereacea family, 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 and luteolin-MM broth and endosymbiotic bacteria. 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 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, and used those position weight matrices 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,. Moreover, we searched for conserved unknown motifs using MEME and used relaxed regular expressions 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, 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. 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

All members of the αr45 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 genomic regions of the αr45 sRNAs from Sinorhizobium, Rhizobium, A. vitis and A. radiobacter exhibited a great degree of conservation including the upstream and downstream genes which have been predicted to code for a LysR family transcriptional regulator and an ornithine descarboxilase, respectively. Partial synteny of the αr45 genomic regions was observed in the Mesorhizobium and Brucella species where instead of aLysR family transcriptional regulator' gene an amidase coding gene was found. In Bartonella species the αr45 upstream gene was always found to code for a protein containing a rhodanase domain. In the genomic regions of the αr45 homologs in more distantly related α-proteobacteria synteny was restricted to the downstream ornithine descarboxylase gene.
FamilyFeatureNameStrandBeginEndProtein nameAnnotationOrganism
αr45geneSMc02983R31039123105051NP_386981.1Ornithine or Arginine decarboxylaseSinorhizobium meliloti 1021
αr45sRNASmr45CR31052653105445--Sinorhizobium meliloti 1021
αr45geneSMc02984D31056383106531NP_386982.1LysR family transcriptional regulatorSinorhizobium meliloti 1021
αr45geneSmed_2772R28913762892509YP_001328437.1Ornithine decarboxylaseSinorhizobium medicae WSM419 chromosome
αr45sRNASmedr45CR28927292892909--Sinorhizobium medicae WSM419 chromosome
αr45geneSmed_2773D28930452893959YP_001328438.1LysR family transcriptional regulatorSinorhizobium medicae WSM419 chromosome
αr45geneNGR_c29260R30677403068873YP_002827423.1Ornithine decarboxylaseSinorhizobium fredii NGR234 chromosome
αr45sRNASfr45CR30690953069275--Sinorhizobium fredii NGR234 chromosome
αr45geneNGR_c29280D30694813070374YP_002827424.1LysR family transcriptional regulatorSinorhizobium fredii NGR234 chromosome
αr45geneRL4156R44038504404983YP_769731.1Lysine-Ornithine decarboxylaseRhizobium leguminosarum bv viciae 3841
αr45sRNARlvr45CR44052104405390--Rhizobium leguminosarum bv viciae 3841
αr45geneRL4157D44055124406405YP_769732.1LysR family transcriptional regulatorRhizobium leguminosarum bv viciae 3841
αr45geneRHECIAT_CH0003899R39523963953529YP_001980014.1Ornithine decarboxylaseRhizobium etli CIAT 652
αr45sRNAReCIATr45CR39537563953936--Rhizobium etli CIAT 652
αr45geneRHECIAT_CH0003900D39540593954952YP_001980015.1LysR family transcriptional regulatorRhizobium etli CIAT 652
αr45geneRleg2_3390R35028503503983YP_002282883.1Ornithine decarboxylaseRhizobium leguminosarum bv trifolii WSM2304
αr45sRNARlt2304r45CR35042093504389--Rhizobium leguminosarum bv trifolii WSM2304
αr45geneRleg2_3391D35045123505405YP_002282884.1LysR family transcriptional regulatorRhizobium leguminosarum bv trifolii WSM2304
αr45geneRleg_3689R37380113739144YP_002977471.1Ornithine decarboxylaseRhizobium leguminosarum bv trifolii WSM1325
αr45sRNARlt1325r45CR37393713739551--Rhizobium leguminosarum bv trifolii WSM1325
αr45geneRleg_3690D37396713740564YP_002977472.1LysR family transcriptional regulatorRhizobium leguminosarum bv trifolii WSM1325
αr45geneRHE_CH03628R38389313839521YP_471110.1acetyltransferaseRhizobium etli CFN 42
αr45sRNAReCFNr45CR38409783841158--Rhizobium etli CFN 42
αr45geneRHE_CH03631D38423033842944YP_471113.1DUF1007Rhizobium etli CFN 42
αr45geneAvi_3806R31656003166733YP_002550752.1Ornithine decarboxylaseAgrobacterium vitis S4 chromosome 1
αr45sRNAAvr45CIR31669673167147--Agrobacterium vitis S4 chromosome 1
αr45geneAvi_3808D31672823168211YP_002550753.1LysR family transcriptional regulatorAgrobacterium vitis S4 chromosome 1
αr45geneArad_3911R31229033124036YP_002545680.1Ornithine decarboxylaseAgrobacterium radiobacter K84 chromosome 1
αr45sRNAArr45CIR31242633124442--Agrobacterium radiobacter K84 chromosome 1
αr45geneArad_3912D31245693125462YP_002545681.1LysR family transcriptional regulatorAgrobacterium radiobacter K84 chromosome 1
αr45geneAtu3196R204249205382NP_357406.2Ornithine decarboxylaseAgrobacterium tumefaciens str. C58 chromosome linear
αr45sRNAAtr45CR205601205782--Agrobacterium tumefaciens str. C58 chromosome linear
αr45geneAtu3197D205919206812NP_357405.2LysR family transcriptional regulatorAgrobacterium tumefaciens str. C58 chromosome linear
αr45geneMeso_2714R29514552952642YP_675256.1amidaseMesorhizobium sp. BNC1
αr45sRNAMsBNCr45CD29527522952930--Mesorhizobium sp. BNC1
αr45geneMeso_2715D29531762954309YP_675257.1Ornithine decarboxylaseMesorhizobium sp. BNC1
αr45geneOant_4245R16720551673188YP_001372774.1Ornithine decarboxylaseBrucella anthropi ATCC 49188 chromosome 2
αr45sRNAOar45CIIR16735051673683--Brucella anthropi ATCC 49188 chromosome 2
αr45geneOant_4246D16738661675053YP_001372775.1amidaseBrucella anthropi ATCC 49188 chromosome 2
αr45geneBOV_A0091R9452995420A5VTL3amidaseBrucella ovis ATCC 25840 chromosome II
αr45sRNABor45CIID9681496992--Brucella ovis ATCC 25840 chromosome II
αr45geneBOV_A0092D9721498347YP_001257169.1Ornithine decarboxylaseBrucella ovis ATCC 25840 chromosome II
αr45sRNABcr45CIID9714897326--Brucella canis ATCC 23365 chromosome II
αr45geneBCAN_B0102R9575896963YP_001594073.1amidaseBrucella canis ATCC 23365 chromosome II
αr45geneBCAN_B0104D9754898681YP_001594074.1Lysine-Ornithine decarboxylaseBrucella canis ATCC 23365 chromosome II
αr45IsRNABs23445r45CIID9725497432--Brucella suis ATCC 23445 chromosome II
αr45geneBSUIS_B0104R9586497069YP_001621952.1amidaseBrucella suis ATCC 23445 chromosome II
αr45geneBSUIS_B0106D9765498787YP_001621953.1Lysine-Ornithine decarboxylaseBrucella suis ATCC 23445 chromosome II
αr45sRNABm16Mr45CIIR11726511172829--Brucella melitensis bv. 1 str. 16M chromosome II
αr45geneBMEII1133R11712961172429NP_542111.1Ornithine decarboxylaseBrucella melitensis bv. 1 str. 16M chromosome II
αr45geneBMEII1134D11730141174219NP_542112.1amidaseBrucella melitensis bv. 1 str. 16M chromosome II
αr45sRNABaS19r45CIID9694897126--Brucella abortus S19 chromosome 2
αr45geneBAbS19_II00900R9555996764YP_001932063.1amidaseBrucella abortus S19 chromosome 2
αr45geneBAbS19_II00910D9734898481YP_001932064.1Ornithine decarboxylaseBrucella abortus S19 chromosome 2
αr45sRNABm23457r45CIID9712997307--Brucella melitensis ATCC 23457 chromosome II
αr45geneBMEA_B0101R9573996944YP_002733935.1amidaseBrucella melitensis ATCC 23457 chromosome II
αr45geneBMEA_B0103D9752998662YP_002733936.1Lysine-Ornithine decarboxylaseBrucella melitensis ATCC 23457 chromosome II
αr45sRNABmir45CIID9710297280--Brucella microti CCM 4915 chromosome 2
αr45geneBMI_II99R9571396918YP_003104900.1amidaseBrucella microti CCM 4915 chromosome 2
αr45geneBMI_II100D9750298635YP_003104901.1Ornithine decarboxylaseBrucella microti CCM 4915 chromosome 2
αr45sRNABs1330r45CIID9712897306--Brucella suis 1330 chromosome II
αr45geneBRA0099R9573896943NP_699304.1amidaseBrucella suis 1330 chromosome II
αr45geneBRA0100D9752898661NP_699305.1Ornithine Arginine decarboxylaseBrucella suis 1330 chromosome II
αr45sRNABa19941r45CIID9688597063--Brucella abortus bv. 1 str. 9-941 chromosome II
αr45geneBruAb2_0098R9549596700YP_222910.1amidaseBrucella abortus bv. 1 str. 9-941 chromosome II
αr45geneBruAb2_0099D9728598418YP_222911.1Ornithine Arginine decarboxylaseBrucella abortus bv. 1 str. 9-941 chromosome II
αr45sRNABmar45CIID9695097128--Brucella melitensis biovar Abortus 2308 chromosome II
αr45geneBAB2_0097R9556096765YP_418341.1amidaseBrucella melitensis biovar Abortus 2308 chromosome II
αr45geneBAB2_0098D9735098483YP_418342.1Ornithine Arginine decarboxylaseBrucella melitensis biovar Abortus 2308 chromosome II
αr45geneMesci_1912R19978041999000YP_004141115.1amidaseMesorhizobium ciceri biovar biserrulae WSM1271 chromosome
αr45sRNAMcr45CD19991101999290--Mesorhizobium ciceri biovar biserrulae WSM1271 chromosome
αr45geneMesci_1913D19995412000674YP_004141116.1Ornithine decarboxylaseMesorhizobium ciceri biovar biserrulae WSM1271 chromosome
αr45genemll2974R23893012390434NP_104188.1Ornithine decarboxylaseMesorhizobium loti MAFF303099 chromosome
αr45sRNAMlr45CR23906872390867--Mesorhizobium loti MAFF303099 chromosome
αr45genemlr2975D23909772392173NP_104189.1amidaseMesorhizobium loti MAFF303099 chromosome
αr45geneBH12730R14204571421590YP_034014.1Ornithine decarboxylaseBartonella henselae str. Houston-1
αr45sRNABahr45CR14218351422014--Bartonella henselae str. Houston-1
αr45geneBH12750D14224711423014PseudogenBartonella henselae str. Houston-1
αr45geneBARCL_1139R12762161277577YP_004159385.1Lysine-Ornithine decarboxylaseBartonella clarridgeiae 73
αr45sRNABacr45CR12775971277776--Bartonella clarridgeiae 73
αr45geneBARCL_1140R12780011278918YP_004159386.1rhodanese domain.Bartonella clarridgeiae 73
αr45geneBtr_1749R18644671865600YP_001610035.1Lysine-Ornithine decarboxylaseBartonella tribocorum CIP 105476
αr45sRNABatr45CR18658431866022--Bartonella tribocorum CIP 105476
αr45geneBtr_1751R18679231868852YP_001610036.1rhodanese domain.Bartonella tribocorum CIP 105476
αr45geneBQ10050R11837591184892YP_032587.1Ornithine decarboxylaseBartonella quintana str. Toulouse
αr45sRNABaqr45CR11851401185319--Bartonella quintana str. Toulouse
αr45geneBQ10070R11860011186915YP_032589.1rhodanese domain.Bartonella quintana str. Toulouse
αr45sRNABabr45CR11200521120229--Bartonella bacilliformis KC583
αr45geneBARBAKC583_1090R11186761119809YP_989352.1pyridoxal-dependent decarboxylasBartonella bacilliformis KC583
αr45geneBARBAKC583_1091R11204631121380YP_989353.1rhodanese domain.Bartonella bacilliformis KC583
αr45sRNABagr45CR17613901761567--Bartonella grahamii as4aup
αr45geneBgr_15560R17600161761149YP_002972413.1Ornithine decarboxylaseBartonella grahamii as4aup
αr45geneBgr_15590D17638851764550YP_002972415.12-dehydro-3-deoxyphosphogluconate aldolaseBartonella grahamii as4aup
αr45geneAZC_0333R384008385138YP_001523249.1Ornithine decarboxylaseAzorhizobium caulinodans ORS 571
αr45sRNAAcr45CR385443385629--Azorhizobium caulinodans ORS 571
αr45geneAZC_0334D386047387324YP_001523250.15-aminolevulinate synthaseAzorhizobium caulinodans ORS 571
αr45geneSnov_3938D41750114175250YP_003695827.1hypothetical proteinStarkeya novella DSM 506 chromosome
αr45sRNAStnr45CD41755154175703--Starkeya novella DSM 506 chromosome
αr45geneSnov_3939D41759604177090YP_003695828.1Ornithine decarboxylaseStarkeya novella DSM 506 chromosome
αr45geneXaut_2140R24029022404032YP_001417041.1Ornithine decarboxylaseXanthobacter autotrophicus Py2 chromosome
αr45sRNAXar45CR24043352404523--Xanthobacter autotrophicus Py2 chromosome
αr45geneXaut_2141D24049532405258YP_001417042.1hypothetical proteinXanthobacter autotrophicus Py2 chromosome
αr45geneMsil_1157R12431781244308YP_002361488.1Ornithine decarboxylaseMethylocella silvestris BL2 chromosome
αr45sRNAMesr45CR12446101244795--Methylocella silvestris BL2 chromosome
αr45geneMsil_1158D12451601246743YP_002361489.1glucan biosynthesis proteinMethylocella silvestris BL2 chromosome
αr45geneBind_1080R12398831241013YP_001832212.1Ornithine decarboxylaseBeijerinckia indica subsp. indica ATCC 9039 chromosome
αr45sRNABeir45CR12413161241498--Beijerinckia indica subsp. indica ATCC 9039 chromosome
αr45geneBind_1081D12423731243974YP_001832213.1glucan biosynthesis proteinBeijerinckia indica subsp. indica ATCC 9039 chromosome
αr45geneRPE_4802R53805825381724YP_783701.1Ornithine decarboxylaseRhodopseudomonas palustris BisA53 chromosome
αr45sRNARhpr45CR53819125382109--Rhodopseudomonas palustris BisA53 chromosome
αr45geneRPE_4803R53824365382696YP_783702.1hypothetical proteinRhodopseudomonas palustris BisA53 chromosome