Week 5 - Screening for Rubisco and 3-HPO bicycle

The weeks are really flying by. I am already at the halfway point! I have already progressed so far in my project and have learned so many valuable skills. I have spent the week working to quantify and characterize the phylogenetic placement of Form I Rubisco large subunit (cbbL) genes in the Warren Cave metagenome. Rubisco is the enzyme used by plants, algae and bacteria to fix atmospheric carbon dioxide into biomass via the CBB cycle (Badger and Bek 2008). Because there is no photosynthetic activity in Warren Cave, this carbon fixation pathway is a primary source of biomass production and energy conservation. After screening the metagenome for Rubisco, I assembled the potential cbbL genes and used the high quality contigs to build phylogenetic trees with RAxML and MEGA.

I then searched for the 3-hydroxypropionate bicycle, an alternative carbon fixation pathway that is primarily found in Chloroflexus species. Chloroflexus is predominant in many of the Antarctic ice caves, so it was a logical pathway to screen for. The 3-HP bicycle is composed of at least 14 enzymes, many of which are present in other metabolic activities (Klatt et al. 2007). So it was essential we identified enzymes that were specific to 3-HP. After reading through the literature, it was apparent that malonyl-CoA reductase, propionyl-CoA synthase, and malyl-CoA/beta-methylmalyl-CoA/citramalyl-CoA lyase (MMC) are the novel enzymes in the 3-HP bicycle. I identified genes encoding these enzymes on the IMG/JGI database and screened the metagenome for these genes. After extracting the potential propionyl-CoA synthase genes from the metagenome and assembling them in Geneious, the resulting contigs had poor coverage and very short nucleotide sequence length. So, I extracted the consensus sequences from the contigs and used the NCBI BLASTx search tool to identify homologous sequences. The BLAST search resulted in hits to enzymes not involved in the 3-HP pathway. After screening for genes encoding MMC and finding similar results, I concluded that the 3-HP bicycle is not a key metabolic pathway in the community because the essential enzymes in the pathway are not present.

Friday, I used the FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) to trim the transposon (CTGTCTCTTATA) from the entire metagenome. Once the transposon is trimmed I am going to attempt to assemble the sequences into genomes in order to learn more about the microbial diversity in the caves. It will still be interesting to see if it is practical to assemble a metagenome of this size and if there is enough sequence coverage to yield high quality contigs. 

Badger MR, Bek EJ (2008) Multiple Rubisco forms in proteobacteria: their functional significance in relation to CO₂ acquisition by the CBB cycle. Journal of Experimental Botany 59: 1525-1541
Klatt CG, Bryant DA, Ward DM (2007) Comparitive genomics provides evidence for the 3-hydroxypropionate autotrophic pathway in filamentous anoxygenic phototrophic bacteria and in hot spring microbial mats. Environmental Microbiology 9: 2067-2078