Insights into vitamin B12 production, acquisition, and use by marine microbes

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2012The distribution and magnitude of marine primary production helps determine the ocean's role in global carbon cycling. Constraining factors that impact this productivity and elucidating selective pressures that drive the composition of marine microbial communities are thus essential aspects of marine biogeochemistry. Vitamin B12, also known as cobalamin, is a cobalt containing organometallic micronutrient produced by some bacteria and archaea and required by many eukaryotic phytoplankton for methionine biosynthesis and regeneration. Although the potential for vitamin B12 availability to impact primary production and phytoplankton species composition has long been recognized, the lack of molecular-level tools for studying B12 production, use and acquisition has limited inquiry into the role of the vitamin in marine biogeochemical processes. This thesis describes the development of such tools and implements them for the study of B12 dynamics in an Antarctic shelf ecosystem. Nucleic acid probes for B12 biosynthesis genes were designed and used to identify a potentially dominant group of B12 producers in the Ross Sea. The activity of this group was then verified by mass spectrometry-based peptide measurements. Then, possible interconnections between iron and B12 dynamics in this region were identified using field-based bottle incubation experiments and vitamin uptake measurements, showing that iron availability may impact both B12 production and consumption. Changes in diatom proteomes induced by low B12 and low iron availability were then examined and used to identify a novel B12 acquisition protein, CBA 1, in diatoms. This represents the first identification of a B12 acquisition protein in eukaryotic phytoplankton. Transcripts encoding CBAl were detected in natural phytoplankton communities, confirming that B12 acquisition is an important part of phytoplankton molecular physiology. Selected reaction monitoring mass spectrometry was used to measure the abundance of CBA 1 and methionine synthase proteins in diatoms cultures, revealing distinct protein abundance patterns as a function ofB12 availability. These peptide measurements were implemented to quantify methionine synthase proteins in McMurdo Sound, revealing that there is both B12 utilization and starvation in natural diatom communities and that these peptide measurements hold promise for revealing the metabolic status of marine ecosystems with respect to vitamin B12.The work described in this thesis was supported by a National Science Foundation (NSF) Graduate Research Fellowship (2007037200) and an Environmental Protection Agency STAR Fellowship (F6E20324), the WHOI Ocean Life Institute, the WHOI Ocean Ventures Fund, the MIT Houghton Fund, NSF awards ANT 0732665, OCE 0752291, OPP 0440840, OCE 0327225, OCE 0452883, OCE 0723667, OCE 1031271, and OCE 0928414, the Center for Environmental Bioinorganic Chemistry at Princeton, the Center for Microbial Oceanography: Research and Education (CMORE), the Australian Research Council and the Gordon and Betty Moore Foundation