K-047. Genetic Identification of the Bacteriohopanepolyol C-2 Methyltransferase in Rhodopseudomonas palustris TIE-1

P. V. Welander1, A. L. Sessions2, D. K. Newman1;
1Massachusetts Inst. of Technology, Cambridge, MA, 2California Inst. of Technology, Pasadena, CA.

Sedimentary 2-methylhopanes can be recognized as the molecular fossils of 2-methylbacteriohopanpolyols (2-MeBHP), methylated bacteriohopanepolyols (BHPs) which have been found primarily in cyanobacteria. Because cyanobacteria are the only bacteria that utilize oxygenic photosynthesis, 2-methylhopanes discovered in the sedimentary record have been used as biomarker proxies for both cyanobacteria and oxygenic photosynthesis itself. However, significant 2-MeBHP production has been recently discovered in the anoxic phototroph Rhodopseudomonas palustris, indicating that cyanobacteria are not the only significant source of methylated BHPs. To better determine the distribution of 2-MeBHPs among bacteria and to better understand the functional role of methylated BHPs, we set out to identify and characterize the gene required for C-2 methylation in R. palustris TIE-1. A key step in the biosynthesis of BHPs in bacteria is the cyclization of squalene by the squalene-hopene cylcase (Shc) to form the hopane skeleton. Close examination of the DNA sequence surrounding the shc gene in TIE-1 revealed a cluster of ORFs similar to a putative hopanoid biosynthetic cluster. Downstream of this gene cluster a hypothetical protein belonging to the radical S-adenosylmethionine (SAM) protein family was discovered (ORF2917). Because TIE-1 has been shown to incorporate the C-2 methyl group from methionine, most likely through a SAM intermediate, ORF2917 is an excellent C-2 methyltransferase candidate. We are currently attempting to delete this gene in TIE-1 and will determine whether the resulting deletion strain is able to produce methylated BHPs. Furthermore, the production of methylated BHPs in TIE-1 has been shown to vary with different growth conditions, indicating that methylation of BHPs may be regulated. Quantitative RT-PCR will be used to assess the expression levels of ORF2917, as well as other putative radical SAM proteins, under heterotrophic growth (low 2-MeBHP production) and photoautotrophic growth (high 2-MeBHP production).