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 2012

The work described in this thesis was supported by a National Science Foundation (NSF)Graduate Research Fellowship (2007037200) and an Environmental Protection Agency STARFellowship (F6E20324), the WHOI Ocean Life Institute, the WHOI Ocean Ventures Fund, theMIT Houghton Fund, NSF awards ANT 0732665, OCE 0752291, OPP 0440840, OCE 0327225,OCE 0452883, OCE 0723667, OCE 1031271, and OCE 0928414, the Center for EnvironmentalBioinorganic Chemistry at Princeton, the Center for Microbial Oceanography: Research andEducation (CMORE), the Australian Research Council and the Gordon and Betty MooreFoundation.

The distribution and magnitude of marine primary production helps determine theocean's role in global carbon cycling. Constraining factors that impact this productivity andelucidating selective pressures that drive the composition of marine microbial communities arethus essential aspects of marine biogeochemistry. Vitamin B12, also known as cobalamin, is acobalt containing organometallic micronutrient produced by some bacteria and archaea andrequired by many eukaryotic phytoplankton for methionine biosynthesis and regeneration.Although the potential for vitamin B12 availability to impact primary production andphytoplankton species composition has long been recognized, the lack of molecular-level tools forstudying B12 production, use and acquisition has limited inquiry into the role of the vitamin inmarine biogeochemical processes. This thesis describes the development of such tools andimplements 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 apotentially dominant group of B12 producers in the Ross Sea. The activity of this group was thenverified by mass spectrometry-based peptide measurements. Then, possible interconnectionsbetween iron and B12 dynamics in this region were identified using field-based bottle incubationexperiments and vitamin uptake measurements, showing that iron availability may impact bothB12 production and consumption. Changes in diatom proteomes induced by low B12 and low ironavailability were then examined and used to identify a novel B12 acquisition protein, CBA 1, indiatoms. This represents the first identification of a B12 acquisition protein in eukaryoticphytoplankton. Transcripts encoding CBAl were detected in natural phytoplanktoncommunities, confirming that B12 acquisition is an important part of phytoplankton molecularphysiology. Selected reaction monitoring mass spectrometry was used to measure the abundanceof CBA 1 and methionine synthase proteins in diatoms cultures, revealing distinct proteinabundance patterns as a function ofB12 availability. These peptide measurements wereimplemented to quantify methionine synthase proteins in McMurdo Sound, revealing that thereis both B12 utilization and starvation in natural diatom communities and that these peptidemeasurements hold promise for revealing the metabolic status of marine ecosystems with respectto vitamin B12.