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

Abstract

The 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, CBA1, in diatoms. This represents the first identification of a B₁₂ acquisition protein in eukaryotic phytoplankton. Transcripts encoding CBA 1 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 CBA1 and methionine synthase proteins in diatoms cultures, revealing distinct protein abundance patterns as a function of B₁₂ 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.