Gene expression analysis using a modified HiCEP method applicable to prokaryotes: a study of the response of Rhodococcus to isoniazid and ethambutol.
We established a novel method to analyze the gene expression of prokaryotes by modifying and optimizing the HiCEP (high coverage gene expression analysis) method, which was originally developed for eukaryotic gene expression profiling. Following the removal of abundant rRNA, the mRNA of prokaryotic cells was enzymatically polyadenylated and subjected to HiCEP analysis. This method was highly reproducible due to selective PCR that was performed by using adaptor specific primers. We confirmed induction of tipA and induction or suppression of cspA, which are genes that are obtained from distinctive actinomycetes and responded to thiostrepton and temperature stress, respectively. Further, we applied this method to explore the gene expression profile of Rhodococcus erythropolis in response to drugs that inhibit cell wall synthetic pathways, and we were able to identify 35 upregulated genes. Among these genes, we confirmed the upregulation of 22 genes by using RT-PCR (reverse transcriptase-polymerase chain reaction). Some of these genes are involved in the synthesis of mycolic acid and arabinogalactan, suggesting a cell response to drug treatment by regulation of the genes involved in cell wall synthesis. This method could prove to be a useful technique for gene expression analysis of prokaryotes, particularly nonmodel strains with unknown genome sequences.
Mitani
Yasuo
Y
Proteolysis and Protein Turnover Research Group, Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Sapporo 062-8517, Japan.
Suzuki
Katsuhisa
K
Kondo
Kazuhiro
K
Okumura
Koji
K
Tamura
Tomohiro
T
D000995:Antitubercular Agents / Q000008:administration & dosage; D001426:Bacterial Proteins / Q000378:metabolism*; D016172:DNA Fingerprinting / Q000379:methods*; D004977:Ethambutol / Q000008:administration & dosage*; D015870:Gene Expression / Q000502:physiology; D020869:Gene Expression Profiling / Q000379:methods*; D007538:Isoniazid / Q000008:administration & dosage*; D016133:Polymerase Chain Reaction / Q000379:methods; D019105:Random Amplified Polymorphic DNA Technique / Q000379:methods*; D012240:Rhodococcus / Q000187:drug effects / Q000378:metabolism*
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004977:Ethambutol; D007538:Isoniazid