Wednesday, August 5, 2009: 4:00 PM
San Miguel, Albuquerque Convention Center
Background/Question/Methods Mineralization of recalcitrant dissolved organic phosphorus (DOP) by heterotrophic prokaryotes is dependent on exogenous supplies of carbon, nitrogen and phosphorus (P). Twenty years of research in the oligotrophic north pacific subtropical gyre (NPSG) has documented a significant decrease in P availability as compared to nitrogen, suggesting microbial activity in this region may frequently be P limited. Extended P starvation could result in a change in community composition, selecting for prokaryotes with specialized metabolic pathways that can acquire P from sources other than the dissolved inorganic pool to temporarily alleviate limitation. One common refractory compound is phosphonate, approximated to be 25% of the ocean’s high molecular weight DOP. Utilization of methylphosponate (MPn), one form of these common compounds, could not only provide bacteria needed P, but at the same time release methane into the surrounding water, contributing to the documented supersaturation in methane concentrations in many aerobic ocean surface waters (the methane paradox). Using a bioinformatic and experimental approach, we asked two questions: (1) Which heterotrophic Bacterial species may be responsible for the utilization of MPn in the NPSG? (2) Is their evidence that MPn utilization could be a significant method by which aerobic organisms obtain P and produce methane?
Results/Conclusions Bioinformatic analysis of existing marine gene databases found an abundance of alpha proteobacterial species carrying genes for the C-P lyase pathway used for MPn degradation (gene operon phnCDEFGHIJKLMNOP), including genes found in samples from station ALOHA where this work was completed. We conducted an enrichment experiment using gas-tight bags to identify key heterotrophic species responsible for MPn utilization. We measured significant methane production linked to MPn degradation, then extracted DNA and RNA from the same water for analysis of community composition. T-RFLP fingerprinting and clone library construction using 16S rRNA genes documented a small but significant shift in Bacterial community structure during the 4-day experiment. The majority of the sequences unique to the MPn treatment had closest matches to the gamma proteobacterial genus, Vibrio. Of the fully-sequenced Vibrio genomes to date, 4 have the phn operon. Primers were designed to target gamma proteobacterial phnM genes using the genomes of Vibrio angustum, Vibrionales bacterium, Vibrio shilonii, and Photobacterium profundum. Quantitative PCR was used to evaluate the extent to which proteobacterial expression of the C-P lyase pathway genes was linked to methane production in our experiment, as well as the percentage of the Bacterial community actively expressing these genes.
Results/Conclusions Bioinformatic analysis of existing marine gene databases found an abundance of alpha proteobacterial species carrying genes for the C-P lyase pathway used for MPn degradation (gene operon phnCDEFGHIJKLMNOP), including genes found in samples from station ALOHA where this work was completed. We conducted an enrichment experiment using gas-tight bags to identify key heterotrophic species responsible for MPn utilization. We measured significant methane production linked to MPn degradation, then extracted DNA and RNA from the same water for analysis of community composition. T-RFLP fingerprinting and clone library construction using 16S rRNA genes documented a small but significant shift in Bacterial community structure during the 4-day experiment. The majority of the sequences unique to the MPn treatment had closest matches to the gamma proteobacterial genus, Vibrio. Of the fully-sequenced Vibrio genomes to date, 4 have the phn operon. Primers were designed to target gamma proteobacterial phnM genes using the genomes of Vibrio angustum, Vibrionales bacterium, Vibrio shilonii, and Photobacterium profundum. Quantitative PCR was used to evaluate the extent to which proteobacterial expression of the C-P lyase pathway genes was linked to methane production in our experiment, as well as the percentage of the Bacterial community actively expressing these genes.
