Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in Sinorhizobium meliloti strain 1021

<p>Abstract</p> <p>Background</p> <p>Small untranslated RNAs (sRNAs) seem to be far more abundant than previously believed. The number of sRNAs confirmed in <it>E. coli </it>through various approaches is above 70, with several hundred more sRNA candidate genes under biological validation. Although the total number of sRNAs in any one species is still unclear, their importance in cellular processes has been established. However, unlike protein genes, no simple feature enables the prediction of the location of the corresponding sequences in genomes. Several approaches, of variable usefulness, to identify genomic sequences encoding sRNA have been described in recent years.</p> <p>Results</p> <p>We used a combination of <it>in silico </it>comparative genomics and microarray-based transcriptional profiling. This approach to screening identified ~60 intergenic regions conserved between <it>Sinorhizobium meliloti </it>and related members of the alpha-proteobacteria sub-group 2. Of these, 14 appear to correspond to novel non-coding sRNAs and three are putative peptide-coding or 5' UTR RNAs (ORF smaller than 100 aa). The expression of each of these new small RNA genes was confirmed by Northern blot hybridization.</p> <p>Conclusion</p> <p>Small non coding RNA (<it>sra</it>) genes can be found in the intergenic regions of alpha-proteobacteria genomes. Some of these <it>sra </it>genes are only present in <it>S. meliloti</it>, sometimes in genomic islands; homologues of others are present in related genomes including those of the pathogens <it>Brucella </it>and <it>Agrobacterium</it>.</p

Extraction of RNA from Cheese without Prior Separation of Microbial Cells▿

In situ gene expression studies are promising approaches for improving our understanding of the cheese microbial flora. This requires efficient RNA extraction methods, but studies of cheeses are scarce. The objective of the present study was to determine whether RNA samples compatible with quantitative mRNA transcript analyses can be obtained without separating the cells from the cheese matrix. In the method that we describe, the cellular processes are stopped at the very beginning of the procedure. When cheeses were produced with Lactococcus lactis LD61 as the only starter microorganism, the integrity of the purified RNA was good, even for 2-week-old cheeses that had been incubated at 30°C. In addition, the RNA samples did not contain any traces of RNases, and the amount of genomic DNA was negligible. A good level of reproducibility could also be achieved. When real-time reverse transcription-PCR analyses were normalized to the total RNA concentration, the amounts of 16S and 23S rRNA transcripts were constant during the 2-week incubation period, whereas the amount of tuf mRNA transcripts decreased substantially. RNA samples obtained using the method described in this study were compared to samples obtained using the method described by Ulvé et al. (J. Appl. Microbiol., in press), which is based on separation of the cells from the cheese matrix. For most of the 29 genes investigated, the transcript abundance was the same for both types of samples. Differences were observed mainly for genes whose expression has previously been shown to be modified by heat, acid, or osmotic stresses, such as busAA and glnQ

Characterization and expression patterns of Sinorhizobium meliloti tmRNA (ssrA).

International audiencetmRNA (ssrA) in Sinorhizobium meliloti is a small RNA annotated by homology with the Bradyrhizobium japonicum sra molecule. Here, this molecule is described in Sinorhizobium meliloti as a model for such molecules in Alphaproteobacteria subgroup-2. Northern blot analysis and mapping of both 5' and 3' ends of this tmRNA allow the identification of two pieces: a 214 nt mRNA-like domain and an 82 nt tRNA-like domain, both highly stable, whereas the premature form is unstable. Transcriptional studies reveal that Sinorhizobium meliloti tmRNA is mainly expressed during growth resumption, replication initiation and various stress responses

Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021-0

<p><b>Copyright information:</b></p><p>Taken from "Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021"</p><p>http://www.biomedcentral.com/1471-2164/8/467</p><p>BMC Genomics 2007;8():467-467.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2245857.</p><p></p>f total RNA from strain 1021 was analyzed by Northern blotting hybridization with specific oligonucleotides [see Additional file ]; the molecular sizes were calculated in nucleotides (nt): > rnpB (372 nt), > tmRNA 3' end (204 nt), > 5S (120 nt), > 4.5S (95 nt), and > tmRNA 5' end (82 nt) [39]. Band > was not hybridized but its size is consistent with it being a tRNA. Exposure times were optimized for each panel and signal intensity does not indicate relative abundance of ncRNAs

Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021-4

<p><b>Copyright information:</b></p><p>Taken from "Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021"</p><p>http://www.biomedcentral.com/1471-2164/8/467</p><p>BMC Genomics 2007;8():467-467.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2245857.</p><p></p> of 1000 replicates. B. In northern blots, a signal for the predicted was detected corresponding to lengths of 144 and 106 nucleotides in total RNA from 1021 and to 140 and 132 nt in that from and , respectively

Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021-1

<p><b>Copyright information:</b></p><p>Taken from "Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021"</p><p>http://www.biomedcentral.com/1471-2164/8/467</p><p>BMC Genomics 2007;8():467-467.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2245857.</p><p></p>e minimal, maximal and mean values of the percentage of GC

Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021-5

<p><b>Copyright information:</b></p><p>Taken from "Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021"</p><p>http://www.biomedcentral.com/1471-2164/8/467</p><p>BMC Genomics 2007;8():467-467.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2245857.</p><p></p>f total RNA from strain 1021 was analyzed by Northern blotting hybridization with specific oligonucleotides [see Additional file ]; the molecular sizes were calculated in nucleotides (nt): > rnpB (372 nt), > tmRNA 3' end (204 nt), > 5S (120 nt), > 4.5S (95 nt), and > tmRNA 5' end (82 nt) [39]. Band > was not hybridized but its size is consistent with it being a tRNA. Exposure times were optimized for each panel and signal intensity does not indicate relative abundance of ncRNAs

Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021-2

<p><b>Copyright information:</b></p><p>Taken from "Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021"</p><p>http://www.biomedcentral.com/1471-2164/8/467</p><p>BMC Genomics 2007;8():467-467.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2245857.</p><p></p> indicated as signal-to-noise SNR values (log2 scale), represented by a red panel. The position of each gene in the heat map is determined by its intensity. For the complementary Northern dot analysis, 10 μg of RNA, isolated from the most favourable expression condition, was spotted (in duplicate) onto a nylon membrane and hybridized with a radiolabelled specific probe [see Additional file ]

Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021-3

<p><b>Copyright information:</b></p><p>Taken from "Identification of chromosomal alpha-proteobacterial small RNAs by comparative genome analysis and detection in strain 1021"</p><p>http://www.biomedcentral.com/1471-2164/8/467</p><p>BMC Genomics 2007;8():467-467.</p><p>Published online 19 Dec 2007</p><p>PMCID:PMC2245857.</p><p></p>ted DNA oligonucleotide probes [see Additional file ] and exposed for various times (therefore the intensities of the signals do not correspond to the relative abundance of each sRNA). The positions of RNA size standards are shown on the left. B. Distribution of the genes along the chromosome. The origin of replication (= sra01) and positions of , and are also indicated. Blue and red arrows represent genes on the reverse or forward strands, respectively. Genomic islands are indicated with grey boxes (Sme21T, Sme19T and Sme80S)