Unknown,Transcriptomics,Genomics,Proteomics

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Prediction, microarray and Northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida (wild type vs. LitR mutant)

ABSTRACT: Bacterial small RNAs (sRNAs) are trans-encoded regulatory RNAs that typically bind mRNAs by short sequence complementarities and change the expression of the corresponding proteins. Some of the well characterized sRNAs serve critical steps in the regulation of important cellular processes, such as quorum sensing (Qrr), iron homeostasis (RyhB), oxidative stress (OxyS) and carbon metabolism (Spot42). However, many sRNAs remains to be identified, and the functional roles of sRNAs are known for only a small fraction. For example, of the hundreds of candidate sRNAs from members of the bacterial family Vibrionaceae the function is known for only nine. We have in this study significantly contributed to the discovery and verification of new sRNAs in a representative of Vibrioneceae, i.e., the A. salmonicida which causes severe disease in farmed Atlantic salmon and other fishes. A computational search for intergenic non-coding (nc) RNAs in the 4.6 Mb genome identified a total of 233 potential sRNAs and 19 other types of ncRNAs with homologs in Rfam. Depending on a set threshold value our microarray approach identified 50-80 sRNAs that are expressed under different growth conditions, twelve of which were verified by Northern blot analysis. In total our data identify nine previously unrecognized sRNAs. Two-condition experiment, wild type cells (control samples) vs. LitR mutants grown in LB medium (stimulated samples). Samples collected from three different optical densities (OD600nm 0.15, OD600nm 0.5 and OD600nm 0.8). Technical replicates for each sample point: 3 control, 3 stimulated, independently grown and harvested. One replicate per array.

ORGANISM(S): Aliivibrio salmonicida LFI1238

SUBMITTER: Erik Hjerde

PROVIDER: E-GEOD-25546 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role remained obscure until it was shown to cause discoordinate expression of the galactose operon (gal operon). Recently Spot 42 has also been implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria, which includes a number of serious pathogens of humans and animals, including the infamous Vibrio cholerae. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84 % identity to E. coli Spot 42 (spf). We generated a A. salmonicida spf deletion mutant. We then and used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose we found a surprisingly large number of ≥2× differentially expressed genes, and several major cellular processes were affected, such as carbohydrate metabolism and transport, motility and chemotaxis, iron homeostasis and quorum sensing. A gene encoding a pirin-like protein (VSAL_I1200) showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, in a global search we discovered a sRNA, named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is highly dependent on glucose. Our hypothesis is that this novel sRNA works in concert with Spot 42 to regulate carbohydrate metabolism and uptake. Two-condition experiment, wild type cells (control samples) vs. spf mutants grown in A. salmonicida-specific minimal media (ASMM) (stimulated samples). Samples were prepared by culturing the A. salmonicida wild type and a spf deletion mutant strains at 12 °C in ASMM without any added carbon source to OD 600nm 0.4. At this density glucose to 44.4 mM was added. Samples were collected 15 min after stimulation. Biological replicates for each sample: 3 wild type, 3 spf mutant, independently grown and harvested. One replicate per array.

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Prediction, microarray and Northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida (wild type vs. LitR mutant)

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