Prediction of Sinorhizobium meliloti sRNA genes and experimental detection in strain 2011

doi:10.1186/1471-2164-9-416

BMC Genomics
Volume 9

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Valverde C
Livny J
Schlüter JP
Reinkensmeier J
Becker A
Parisi G

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Research article

Prediction of Sinorhizobium meliloti sRNA genes and experimental detection in strain 2011

Claudio Valverde¹ , Jonathan Livny² , Jan-Philip Schlüter³^,4 , Jan Reinkensmeier⁵ , Anke Becker³^,4 and Gustavo Parisi⁶

¹Programa Interacciones Biológicas, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, Bernal, Buenos Aires, B1876BXD, Argentina

²Channing Laboratories, Brigham and Women's Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA

³Institute for Genome Research and Systems Biology, Center for Biotechnology (CeBiTec), Bielefeld University, 33594 Bielefeld, Germany

⁴Institute of Biology III, Faculty of Biology, University of Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany

⁵Faculty of Technology, Bielefeld University, 33615 Bielefeld, Germany

⁶Grupo de Bioinformática Estructural, Centro de Estudios e Investigaciones, Universidad Nacional de Quilmes, Roque Saénz Peña 352, Bernal, Buenos Aires, B1876BXD, Argentina

author email corresponding author email

BMC Genomics 2008, 9:416doi:10.1186/1471-2164-9-416


Published:	16 September 2008

Additional files

Additional file 1:

Crude sRNApredictHT predictions on S. meliloti 1021 genome. Direct output of the sRNAPredictHT algorithm applied to the complete set of S. meliloti 1021 chromosomal IgRs.

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Additional file 2:

Oligonucleotides used for synthesis of Northern blot probes. Oligonucleotides used to PCR amplify the IgR sequences encompassing sRNA candidates.

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Additional file 3:

Denaturing PAGE fractionation of S. meliloti 2011 total RNA. Electrophoretic pattern of S. meliloti 2011 total RNA in a denaturing polyacrylamide gel (8.3 M urea, 8% acrylamide and 0.2% bisacrylamide in 1× TBE buffer; 25 cm-long). Approximately 20–60 μg of total RNA, corresponding to all cells present in 20 ml of RDM cultures, were loaded in each lane. The gel was stained with ethidium bromide and visualized on an UV transilluminator. Under this conditions, effective fractionation of RNAs < 600 nt was achieved. RNA bands of varying intensity in different samples are indicated with arrowheads. Stat, stationary phase cells; log, exponential phase cells; NaCl 0.3 M, saline stress, H₂O₂, oxidative stress; pH 4.0, acid stress; SDS and EtOH, membrane stress; -P, phosphate starvation; 0°C, cold shock; 45°C, heat shock.

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Additional file 4:

Curated sRNApredictHT predictions. The sRNAPredictHT output listed in Additional file 1 was curated upon elimination of IgRs containing annotated and non annotated repeats.

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Additional file 5:

Expression of putative sRNAs in IgR#4 and IgR#14. Northern blot analysis of putative sRNAs encoded in IgR#4 and IgR#14. See legend to Figure 1 for details.

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Additional file 6:

Novel candidate sRNA gene sm8 in IgR#2. Conservation of the novel candidate sRNA gene sm8 (IgR#2) in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), Sinorhizobium medicae WSM419 (Smed), Agrobacterium tumenfaciens C58 (At), Rhizobium etli CFN42 (Retli) and Rhizobium leguminosarum bv viciae 3841 (Rleg). The Rho-independent terminator was predicted for S. meliloti 1021 (see text) and confirmed from conserved positions in the alignment. The putative sigma 70-dependent promoter (-10 and -35 hexamers) and transcription start site (+1) were deduced from conserved positions in the alignment. The secondary structure presented for S. meliloti Sm8 RNA was calculated with the Mfold server [75] and corresponds to the predicted structure with lower free energy.

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Additional file 7:

Novel candidate sRNA gene sm137 in IgR#4. Conservation of the novel candidate sRNA gene sm137 (IgR#4) in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), S. medicae WSM419 (Smed), R. etli CFN42 (Retli) and R. leguminosarum bv viciae 3841 (Rleg). The Rho-independent terminator was predicted for S. meliloti 1021 (see text) and confirmed from conserved positions in the alignment, but there was no prediction of a promoter in this IgR.

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Additional file 8:

Novel candidate sRNA gene smIgR#6. Conservation of the novel candidate sRNA gene smIgR#6 in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), R. etli CFN42 (Retli) and R. leguminosarum bv viciae 3841 (Rleg). Two putative sigma 70-dependent promoters (-10 and -35 hexamers), transcription start sites (+1) and a single Rho-independent terminator were predicted for S. meliloti 1021 (see text). The secondary structure presented for both possible S. meliloti sRNAs from IgR#6 were calculated with the Mfold server [75] and correspond to the predicted structures with lower free energy.

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Additional file 9:

Novel candidate sRNA gene sm26 in IgR#7. Conservation of the novel candidate sRNA gene sm26 (IgR#7) in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), S. medicae WSM419 (Smed), R. etli CFN42 (Retli) and R. leguminosarum bv viciae 3841 (Rleg). The putative sigma 70-dependent promoter (-10 and -35 hexamers), transcription start site (+1) and Rho-independent terminator were predicted for S. meliloti 1021 (see text). The secondary structure presented for S. meliloti Sm26 RNA was calculated with the Mfold server [75] and corresponds to the predicted structure with lower free energy.

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Additional file 10:

Candidate sRNA gene sm64 (sra25) in IgR#10. Conservation of the candidate sRNA gene sm64 (IgR#10; sra25, [27]) in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), S. medicae WSM419 (Smed), R. etli CFN42 (Retli) and R. leguminosarum bv viciae 3841 (Rleg). The putative sigma 70-dependent promoter (-10 and -35 hexamers), transcription start site (+1) and Rho-independent terminator were predicted for S. meliloti 1021 (see text) and confirmed from conserved positions in the alignment. The secondary structure presented for S. meliloti Sm64 RNA was calculated with the Mfold server [75] and corresponds to the predicted structure with lower free energy.

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Additional file 11:

Novel candidate sRNA gene sm145 in IgR#11. Conservation of the novel candidate sRNA gene sm145 (IgR#11) in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), S. medicae WSM419 (Smed), A. tumenfaciens C58 (At), R. etli CFN42 (Retli) and R. leguminosarum bv viciae 3841 (Rleg). The putative sigma 70-dependent promoter (-10 and -35 hexamers), transcription start site (+1) and Rho-independent terminator were predicted for S. meliloti 1021 (see text) and confirmed from conserved positions in the alignment. The secondary structure presented for S. meliloti Sm145 RNA was calculated with the Mfold server [75] and corresponds to the predicted structure with lower free energy.

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Additional file 12:

Novel candidate sRNA gene sm76 in IgR#14. Conservation of the novel candidate sRNA gene sm76 (IgR#14) in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), S. medicae WSM419 (Smed), A. tumenfaciens C58 (At), R. etli CFN42 (Retli), R. leguminosarum bv viciae 3841 (Rleg), Mesorhizobium loti MAFF303099 (Mloti), Ochrobactrum anthropi ATCC49188 (Oa) and Brucella ovis ATCC25840 (Bo). The putative sigma 70-dependent promoter (-10 and -35 hexamers), transcription start site (+1) and Rho-independent terminator were predicted for S. meliloti 1021 (see text) and confirmed from conserved positions in the alignment. A second putative promoter was predicted for S. meliloti upstream then conserved one, but it seems to be specific for Sinorhizobium. The alternative secondary structures presented for S. meliloti Sm76 RNA were calculated with the Mfold server [75] and corresponds to the predicted structure with lower free energy for the two possible transcripts.

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Additional file 13:

Novel candidate sRNA gene sm84 in IgR#15. Conservation of the novel candidate sRNA gene sm84 (IgR# 15) in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), S. medicae WSM419 (Smed), A. tumenfaciens C58 (At), R. etli CFN42 (Retli) and R. leguminosarum bv viciae 3841 (Rleg). The putative sigma 70-dependent promoter (-10 and -35 hexamers), transcription start site (+1) and Rho-independent terminator were predicted for S. meliloti 1021 (see text) and confirmed from conserved positions in the alignment. The secondary structure presented for S. meliloti Sm84 RNA was calculated with the Mfold server [75] and corresponds to the predicted structure with lower free energy.

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Additional file 14:

Novel candidate sRNA gene sm270 in IgR#16. Conservation of the novel candidate sRNA gene sm270 (IgR#16) in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), S. medicae WSM419 (Smed), A. tumenfaciens C58 (At), R. etli CFN42 (Retli) and R. leguminosarum bv viciae 3841 (Rleg). The putative sigma 70-dependent promoter (-10 and -35 hexamers), transcription start site (+1) and the putative Rho-independent terminator were predicted for S. meliloti 1021 (see text) and confirmed from conserved positions in the alignment. The secondary structure presented for S. meliloti Sm270 RNA was calculated with the Mfold server [75] and corresponds to the predicted structure with lower free energy.

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Additional file 15:

Novel candidate sRNA gene sm5 in IgR#17. Conservation of the novel candidate sRNA gene sm5 (IgR#17) in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), R. etli CFN42 (Retli) and R. leguminosarum bv viciae 3841 (Rleg). The putative sigma 70-dependent promoter (-10 and -35 hexamers), transcription start site (+1) and the putative Rho-independent terminator were predicted for S. meliloti 1021 (see text) and confirmed from conserved positions in the alignment. The secondary structure presented for S. meliloti Sm5 RNA was calculated with the Mfold server [75] and corresponds to the predicted structure with lower free energy.

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Additional file 16:

Novel candidate sRNA gene sm190 in IgR#18. Conservation of the novel candidate sRNA gene sm190 (IgR#18) in α-proteobacteria. Sequence alignment generated with ClustalW for the corresponding IgRs of S. meliloti 1021 (1021), R. etli CFN42 (Retli) and R. leguminosarum bv viciae 3841 (Rleg). The putative Rho-independent terminator was predicted for S. meliloti 1021 (see text) and confirmed from conserved positions in the alignment. The secondary structure presented for S. meliloti Sm190 RNA was calculated with the Mfold server [75] and corresponds to the predicted structure with lower free energy assuming that the sRNA extends along the conserved sequence upstream the terminator and includes the terminator itself.

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Additional file 17:

List of S. meliloti sRNA gene predictions based on the GS method. Complete list of S. meliloti 1021 chromosomal IgRs predicted to encode sRNAs according to the global scoring procedure (summarized in Figure 3).

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Additional file 18:

Predicted transcriptional units in S. meliloti 1021 chromosomal IgRs. sRNA candidates identified as putative transcriptional units in intergenic regions of S. meliloti 1021 chromosome.

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Additional file 19:

Homologs of sRNA genes smrC14-smrC15 and smrB35 are present in pSymA and pSymB IgRs. Identification of extra copies of sRNA genes smrC14-smrC15 and smrB35 in pSymA and pSymB IgRs. Genetic surroundings and sequence alignments generated with ClustalW for the sRNA genes of S. meliloti 1021 smrC14, smrC15 [26] and the corresponding identified homolog in pSymA (smA4b; Table 4), and for the sRNA gene smrB35 [26] and the corresponding identified homolog in pSymB (smB5b; Table 4). The putative sigma 70-dependent promoters, transcription start sites (+1) and Rho-independent terminators were predicted for S. meliloti 1021 pSymA and pSymB (Table 4) and confirmed from conserved positions in the alignment.

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