BioProject

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. More...

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. Overall design: 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 manufacturer’s 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. Less...