Prediction, microarray and Northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida
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ABSTRACT:
ORGANISM(S): Aliivibrio salmonicida LFI1238
SUBMITTER: Erik Hjerde
PROVIDER: E-GEOD-25558 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Prediction, microarray and Northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida (wild type vs. LitR mutant)
| PRJNA142439 | ENA
Project description: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.
2010-12-01 | E-GEOD-25546 | biostudies-arrayexpress
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.
2011-07-01 | E-GEOD-28087 | biostudies-arrayexpress
Project description:Prediction, microarray and Northern blot analyses identify new intergenic small RNAs in Aliivibrio salmonicida (LB medium vs. iron-limited condition)
| PRJNA142437 | ENA
Project description: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, cells grown in LB medium (control samples) vs. cells grown under iron-limited conditions (2,2`-dipyridyl) (stimulated samples). Samples collected from three different timepoints (15, 30 and 60 min). Technical replicates for each timepoint: 3 control, 3 stimulated, independently grown and harvested. One replicate per array.
2010-12-01 | E-GEOD-25545 | biostudies-arrayexpress
Project description:Aliivibrio salmonicida causes âcold-water vibriosisâ (or âHitra diseaseâ) in fish, including marine-reared Atlantic salmon. During development of the disease the bacterium will encounter macrophages with antibacterial activities such as production of damaging reactive oxygen species (ROS). To defend itself the bacterium will presumably start producing detoxifying enzymes, reducing agents, and proteins involved in DNA and protein repair systems. Even though responses to oxidative stress are well studied for a few model bacteria, little work has been done in general to explain how important groups of pathogens, like members of the Vibrionaceae family, can survive at high levels of ROS. We have used bioinformatic tools and an âomics approach to study how A. salmonicida responds to hydrogen peroxide (H2O2). First, we used the recently published genome sequence to predict potential binding sites for OxyR (H2O2 response regulator). The computer-based search identified OxyR sites associated with 20 single genes and 8 operons, and these predictions were compared to experimental data from Northern blot analysis, microarray analysis and 2D gel electrophoresis. In general, OxyR binding site predictions and experimental results are in agreement. Up- and down-regulated genes are distributed among all functional gene categories, but a striking number of â¥2 fold up-regulated genes encode proteins involved in detoxification or DNA protection and repair, are part of reduction systems, or are involved in carbon metabolism and regeneration of NADH/NADPH. Our predictions and âomics data corroborates well with findings from other model bacteria, but also suggest species-specific gene regulation. Two-condition experiment, cells grown in LB medium (control samples) vs. cells grown under oxidative stress (H2O2) (stimulated samples). Samples collected from three different timepoints (15, 30 and 60 min). Technical replicates for each timepoint: 3 control, 3 stimulated, independently grown and harvested. One replicate per array.
2010-05-06 | E-GEOD-20082 | biostudies-arrayexpress
Project description:Quorum sensing (QS) is a cell density regulated communication system that bacteria use to coordinate activities, including biofilm formation, involved in colonization and pathogenesis. We have previously shown that inactivation of the QS master regulator LitR attenuates the Vibrio (Allivibrio) salmonicida strain LFI1238 in a fish model. In this work we show that LFI1238 as well as a panel of naturally occurring V. salmonicidia strains are poor biofilm producers. Inactivation of litR strongly enhances medium and temperature dependent adhesion, rugose colony morphology and biofilm formation. Chemical treatment and scanning electron microscopy of the biofilm identified an extracellular matrix consisting mainly of protein filaments and polysaccharides. Further, microarray analysis of planktonic and biofilm cells identified a number of genes regulated by LitR, and among these were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. Disruption of syp alleviated the different phenotypes regulated by LitR in V. salmonicida. Hence, LitR is a repressor of syp expression that is necessary for rugose colony morphology, adhesion and biofilm formation, three phenotypes of the DlitR mutant that are expressed at temperatures below 12M-BM-:C. The DlitR mutant mimics low cell density behavior suggesting that these phenotypes are important during the initial steps of colonization. Although the syp operon in V. salmonicida shows identical gene synteny to the one in the squid symbiont V. fischeri, its regulation is probably more related to vibrio polysaccharide (vps) expression in the human pathogenic Vibrio cholera which is controlled by the LitR homologue HapR. V. salmonicida wild type strain LFI1238 (control) and the isogenic DlitR mutant were grown as statical biofilm in SWT medium, at 4M-BM-0C and harvested after 72 hours. Biological replicates for each sample: 4 wild type, 4 DlitR mutant (including one dye swap), independently grown and harvested. One replicate per array.
2012-12-19 | E-GEOD-42995 | biostudies-arrayexpress
Project description:Quorum sensing (QS) is a cell density regulated communication system that bacteria use to coordinate activities, including biofilm formation, involved in colonization and pathogenesis. We have previously shown that inactivation of the QS master regulator LitR attenuates the Vibrio (Allivibrio) salmonicida strain LFI1238 in a fish model. In this work we show that LFI1238 as well as a panel of naturally occurring V. salmonicidia strains are poor biofilm producers. Inactivation of litR strongly enhances medium and temperature dependent adhesion, rugose colony morphology and biofilm formation. Chemical treatment and scanning electron microscopy of the biofilm identified an extracellular matrix consisting mainly of protein filaments and polysaccharides. Further, microarray analysis of planktonic and biofilm cells identified a number of genes regulated by LitR, and among these were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. Disruption of syp alleviated the different phenotypes regulated by LitR in V. salmonicida. Hence, LitR is a repressor of syp expression that is necessary for rugose colony morphology, adhesion and biofilm formation, three phenotypes of the DlitR mutant that are expressed at temperatures below 12M-BM-:C. The DlitR mutant mimics low cell density behavior suggesting that these phenotypes are important during the initial steps of colonization. Although the syp operon in V. salmonicida shows identical gene synteny to the one in the squid symbiont V. fischeri, its regulation is probably more related to vibrio polysaccharide (vps) expression in the human pathogenic Vibrio cholera which is controlled by the LitR homologue HapR. V. salmonicida wild type strain LFI1238 (control) and the isogenic DlitR deletion mutant were grown in suspension in SWT medium at 8M-BM-0C with 200 rpm and harvested at OD=0.8. Biological replicates for each sample: 4 wild type, 4 DlitR mutant (including one dye swap), independently grown and harvested. One replicate per array.
2012-12-19 | E-GEOD-42994 | biostudies-arrayexpress
Project description:Quorum sensing (QS) is a cell density regulated communication system that bacteria use to coordinate activities, including biofilm formation, involved in colonization and pathogenesis. We have previously shown that inactivation of the QS master regulator LitR attenuates the Vibrio (Allivibrio) salmonicida strain LFI1238 in a fish model. In this work we show that LFI1238 as well as a panel of naturally occurring V. salmonicidia strains are poor biofilm producers. Inactivation of litR strongly enhances medium and temperature dependent adhesion, rugose colony morphology and biofilm formation. Chemical treatment and scanning electron microscopy of the biofilm identified an extracellular matrix consisting mainly of protein filaments and polysaccharides. Further, microarray analysis of planktonic and biofilm cells identified a number of genes regulated by LitR, and among these were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. Disruption of syp alleviated the different phenotypes regulated by LitR in V. salmonicida. Hence, LitR is a repressor of syp expression that is necessary for rugose colony morphology, adhesion and biofilm formation, three phenotypes of the DlitR mutant that are expressed at temperatures below 12ºC. The DlitR mutant mimics low cell density behavior suggesting that these phenotypes are important during the initial steps of colonization. Although the syp operon in V. salmonicida shows identical gene synteny to the one in the squid symbiont V. fischeri, its regulation is probably more related to vibrio polysaccharide (vps) expression in the human pathogenic Vibrio cholera which is controlled by the LitR homologue HapR.
2012-12-19 | GSE42994 | GEO