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RT-PCR FOR DETECTING EXPRESSION OF THE DISSIMILATORY BISULFITE REDUCTASE DSR GENE IN THE SEAGRASS RHIZOSPHERE
Citation:
Devereux, R D., S S. Wilkinson, AND D F. Yates. RT-PCR FOR DETECTING EXPRESSION OF THE DISSIMILATORY BISULFITE REDUCTASE DSR GENE IN THE SEAGRASS RHIZOSPHERE. Presented at American Society for Microbiology, 102nd General Meeting, Salt Lake City, UT, May 19-23, 2002.
Description:
Sulfate-reducing bacteria (SRB) colonize the epidermis and cortex layers in roots of the seagrass, Halodule wrightii. The purpose of this study is to determine whether SRB reduces sulfate to sulfide within these roots. Summer lac-1 is a lactate-utilizing SRB isolated from the roots of H. wrightii. Summer lac-1 is affiliated with the Desulfovibrionaceae, but shares only 90% 16S rRNA sequence similarity with members of this family. The sequence of the dsr gene in Summer lac-1 coding for dissimilatory bisulfite reductase, a key enzyme in the reduction of sulfate to sulfide, was determined and used to develop primers for PCR and RT-PCR. Reaction
conditions with these primers were optimized for annealing temperature and magnesium concentration. RT-PCR with RNA obtained from Summer lac-1 grown with sulfate and pyruvate yielded a band of the expected size. RT-PCR with RNA from Summer lac-1 grown in the absence of sulfate did not yield a band. These results indicate that RT-PCR may be used to determine whether SRB are actively reducing sulfate in situ. Total RNA and total DNA were separately extracted from H. wrightii roots that had been incubated overnight in anoxic
medium containing pyruvate with and without sulfate. Inhibition of PCR using 16S rDNA primers was apparent with some of the DNA samples. RT-PCR with dsr primers yielded a band of the expected size with one of three RNA samples extracted from roots incubated with sulfate and none of three RNA samples obtained from roots incubated in the absence of sulfate. These results indicate sulfate reduction may be associated with seagrass roots but do not provide information about in situ activities under natural conditions. RT-PCR with dsr primers has been extended to the examination of intact cells and can differentiate between Summer lac-1 cells grown with and without sulfate. Work is presently underway to further develop RT-PCR for direct observation of SRB activity in seagrass roots. The SRB distribution and activity in seagrass roots could be useful for understanding anaerobic stress and sulfide toxicity that affect the viability of these plants.