Project description:The Manila clam (Ruditapes philippinarum) is a cultured bivalve species with high worldwide commercial importance. Nevertheless, diseases can cause high economical losses. For this reason, the study of immune genes in bivalve mollusks has increased in the last years. The present work describes the construction of the first R. philippinarum microarray containing immune-related hemocyte sequences and its application for the study of the gene transcription profiles of hemocytes from clams challenged with Vibrio alginolyticus through a time course.
Project description:Although many members of the genus Vibrio are known to inhabit the marine photic zone, an understanding of the influence of light on the molecular physiology of Vibrio spp. has largely been neglected. To begin to characterize the photophysiology of one such Vibrio sp. (Vibrio campbellii ATCC strain BAA-1116) we used microarray-based expression profiling to compare the transcriptomes of illuminated versus dark cell cultures. Specficially, we compared the transcriptomes of wild type V. campbellii (STR) cells that were cultured in M9 minimal salts medium plus glucose under two conditions: (i) after 24 hours of continuous dark and (ii) after a 12 hour dark:12 hour light cycle (white light illumination at 54 µmol photons s-1 m-2). The results revealed a large photostimulon (differential expression of ~20% of the V. campbellii genome; adjusted p value < 0.0001) that surprisingly included ~75% of the type III secretion system (T3SS) genes which were found to be 1.6 – 5.4X more abundant in illuminated cultures. These findings, which were confirmed by quantitative reverse transcription PCR and quantitative membrane proteomics, strongly suggest that the photostimulon of strain BAA-1116 includes the T3SS.
Project description:Although many members of the genus Vibrio are known to inhabit the marine photic zone, an understanding of the influence of light on the molecular physiology of Vibrio spp. has largely been neglected. To begin to characterize the photophysiology of one such Vibrio sp. (Vibrio campbellii ATCC strain BAA-1116) we used microarray-based expression profiling to compare the transcriptomes of illuminated versus dark cell cultures. Specficially, we compared the transcriptomes of wild type V. campbellii (STR) cells that were cultured in M9 minimal salts medium plus glucose under two conditions: (i) after 24 hours of continuous dark and (ii) after a 12 hour dark:12 hour light cycle (white light illumination at 54 M-BM-5mol photons s-1 m-2). The results revealed a large photostimulon (differential expression of ~20% of the V. campbellii genome; adjusted p value < 0.0001) that surprisingly included ~75% of the type III secretion system (T3SS) genes which were found to be 1.6 M-bM-^@M-^S 5.4X more abundant in illuminated cultures. These findings, which were confirmed by quantitative reverse transcription PCR and quantitative membrane proteomics, strongly suggest that the photostimulon of strain BAA-1116 includes the T3SS. Five biological replicates of V. campbellii BAA-1116 (STR) were grown to log phase (200 rpm, 30M-BM-0C, 25 mL M9 minimal salts medium plus glucose in 125 mL baffled Erlenmeyer flasks) under continuous dark for 24 hours or under a 12 hour dark:12 hour light cycle (white light illumination at 54 M-BM-5mol photons s-1 m-2) and total RNA was extracted from 1.0E+9 cells. Messenger RNA was isolated from the total RNA extracts treated with DNase, labeled with biotin, fragmented and hybridized to V. campbellii BAA-1116 whole genome microarrays (520694F, Affymetrix).
Project description:VarS/A is one of the global factors regulating diverse aspects of metabolism and virulence of bacteria including pathogenic Vibrio spp. An experiment to identify VarS/A-regulon in V. vulnificus revealed that a putative LuxR-type transcriptional regulator was down-regulated in ΔvarA mutant. To investigate the roles of this regulatory cascade from VarS/A to a LuxR-type regulator in V. vulnificus, the target gene regulated by a LuxR-regulator was identified and its expression was characterized.
Project description:Objectives: determination of transcription start sites in Vibrio harveyi genome and discovery of new transcripts Methods: we performed differential seqencing of total RNA isolated from o.n. control Vibrio harveyi cultures. Sample treatment with Terminator EXonuclease (TEX) allowed differenciation of primary and secondary transcripts, helping in the definition of transcription start sites (TSS) Results: by data-mining RNA-seq data and performing some Northern Blot experiments we were able to detect new putative small-RNAs, along with these results, a more deep analisys of our RNA-seq data will give futher insight into genetic organization of Vibrio harveyi genome to help in its investigation