Unknown,Transcriptomics,Genomics,Proteomics

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Streptococcus sanguinis gene expression at late-log growth phase

ABSTRACT: We found that many of the genes for which mutants could not be obtained using promoterless aphA-3 gene replacement were annotated as acquired via horizontal gene transfer or as encoding hypothetical proteins. We were curious about the explanation of this finding. The expression of these genes was therefore examined by microarray analysis. The microarray data showed many had undetectable expression. This led us to suspect that many of the unrecoverable mutants might have resulted from insufficient expression of the promoterless aphA-3 gene. S. sanguinis cultures at late log growth phase at 37 oC under microaerobic condition were used for microarray analysis. RNA from each of three independent samples was isolated by RNeasy mini kit (Qiagen, Valencia, CA). Spotted microarray slides were obtained from the Pathogen Functional Genomics Resource Center at JCVI. Each gene had four repeats in one spotted microarray slides. The microarray was performed according to the manufacturerM-bM-^@M-^Ys protocol. Each sample was divided into two parts and labeled by Cy5 and Cy3 dyes respectively for microarray. The microarray data were analyzed by programs TIGR Spotfinder and Midas to obtain the ratio of one dye to another dye for gene relative expression. Additionally, the ratio of dye intensity to background in a microarray slide was obtained after Spotfinder analysis. Absolute expression of each gene was represented by the average ratio of dye intensity to background for each slide.

ORGANISM(S): Streptococcus sanguinis

SUBMITTER: Xiuchun Ge

PROVIDER: E-GEOD-25340 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The model prokaryote Escherichia coli can exist as a either a commensal or a pathogen in the gut of diverse mammalian hosts. These associations, coupled with its ease of cultivation and genetic variability, have made E. coli a popular indicator organism for tracking the origin of fecal water contamination. Source tracking accuracy is predicated on the assumption that E. coli isolates recovered from contaminated water present a genetic signature characteristic of the host from which they originated. In this study, we compared the accuracy with which E. coli isolated from humans, bear, cattle and deer could be identified by standard fingerprinting methods used for library-based microbial source tracking (repetitive element PCR and pulsed-field gel electrophoresis) in relation to microarray-based analysis of genome content. Our results show that patterns of gene presence or absence were more useful for distinguishing E. coli isolates from different sources than traditional fingerprinting methods, particularly in the case of human strains. Host-associated differences in genome composition included the presence or absence of mobile IS1 elements as well as genes encoding the ferric dicitrate iron transporter (fec), E. coli common pilus (ECP), type 1 fimbriae and the CRISPR associated cas proteins. Many of these differences occurred in regions of the E. coli chromosome previously shown to be M-bM-^@M-^\hot spotsM-bM-^@M-^] for the integration of horizontally-acquired DNA. PCR primers designed to amplify the IS1 and fec loci confirmed array results and demonstrated the ease with which gene presence/absence data can be converted into a diagnostic assay. The data presented here suggest that, despite the high level of genetic diversity observed among isolates by PFGE, human-derived strains may constitute a distinct ecotype distinguished by multiple potential library-independent source tracking markers. Twelve isolates of E. coli ( 3 from bear, 3 from cattle, 3 from deer and 3 from humans) were isolated from feces and/or raw sewage. Genome content for each strain was assessed in duplicate using comparative genome hybridization with E. coli K12 MG1655 as the reference for a total of 24 arrays.

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Streptococcus sanguinis gene expression at late-log growth phase

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