- The combined activities of diverse heterotrophic marine microorganismssignificantly shape global biogeochemical cycles, but models of these activities arecurrently limited to aggregate microbial community processes, and it remains unclearhow community structure and the functional roles of specific microbial taxa should beintegrated into these models. Therefore, understanding the contributions of specificmicrobial populations toward net community processes remains a critical step fordetermining the appropriate taxonomic resolution that should be employed in morecomplex models of ecosystem processes. The application of ‘omics’ methods, such asmetagenomics and metaproteomics, has revealed the phylogenetic diversity of naturalmicrobial communities and the functional potential of discrete populations. From theintegration of these observations, an understanding of microbial community dynamicshas emerged in which niche processes influence general patterns of community structureat broad taxonomic scales across spatial and temporal resource gradients. Within thesedynamic microbial systems, the partitioning of specific resources is governed by resourcepreferences and competitive interactions that cannot be ascertained with ‘omics’approaches alone. In order to link phylogenetic identity with ecological function, andcharacterize resource partitioning in coastal marine microbial communities, we appliednovel mass spectrometry techniques to stable isotope probing (SIP) experimentsconducted on microbes sampled from coastal North Pacific surface waters.Chapter 2 presents the first application of proteomic stable isotope probing(proteomic SIP) to track 13C-labeled substrates into the proteomes of planktonic marinemicrobial communities. We developed two metrics for describing observations of labelincorporation into peptides. Label frequency is a measure of protein synthesis activitythat is calculated as the ratio of labeled to total detected peptide mass spectra for adefined set of proteins. Average enrichment is a measure of substrate specialization, andis calculated from the average percent of stable isotope content measured for a set oflabeled peptides. Using these metrics, we compared the assimilation of 13C-labeled aminoacids by abundant microbial taxa over two time-points, 15 and 32 hours. Thecommunities sampled from Newport, OR and Monterey Bay, CA exhibited similarbehaviors. Alteromonadales and Rhodobacterales proteomes had significantly high labelfrequency at the first time-point but had diverging trajectories in the second time-pointindicating that Rhodobacterales held a competitive advantage as the amended substratebecame depleted.In Chapter 3, we examined the assimilation of six 13C-labeld substrates bymicrobial taxa sampled from Monterey Bay, CA. Comparisons between relativeabundance shifts and substrate assimilation were inconsistent, emphasizing the need forcaution when interpreting relative abundances shifts in microbial communityexperiments. Specialization patterns were significantly conserved among abundantpopulations within class level divisions, suggesting that resource preferences have deepevolutionary origins, but variation in the activities of individual species or strains withinthese lineages may be driven by other environmental factors, such as resourceconcentrations or temperature, or pressure from grazing and viral lysis. Although activitymeasures were also conserved among these classes, measures of activity divided theseclades into higher or lower activity, suggesting different strategies for responding toincreased nutrient availability.Finally, Chapter 4 explores physiological bases for observations of diverginglevels of activity among microbial taxonomic lineages. We found that substrate additionsresulted in reproducible taxonomic and functional changes in the whole communitymetaproteome, and that relative abundances of specific protein functional assignmentsdifferentiated abundant taxa. Comparisons of the protein functional profiles (i.e., therelative abundances of mass spectra assigned to functional categories) for specific taxa,between time-points and also between treatments, generally revealed minimal changes inthe expressed proteins, which suggests some inherent overall stability in the functions ofthese taxa, despite environmental changes. However, there were significantly differentamounts of variation observed in the proteomes of abundant taxa; higher levels of whichcorrelated with higher observed label frequency, greater numbers of detected ribosomes,and larger nitrogen requirements encoded in their genomes. Taken together, theseobservations suggest that the capacity of organisms to respond rapidly to increasednutrient availability relies on the ability to transition into states of increased proteinsynthesis, and that this strategy does not select for reductions in nitrogen requirements.However, these adaptations for exploiting abundant resources were not correlated withother physiological features, such as the abundance of transporters, motility proteins, andgene regulatory mechanisms.The outcome of these experiments, enabled by the concurrent use of ‘omics’ andnovel mass spectrometry methods, was a deeper understanding of how resourcepreferences of individual microbial taxa impact carbon cycling processes within complexmarine microbial communities. Although SIP approaches can only access assimilatoryprocesses, they complement established ‘omics’ techniques by revealing interactions suchas competition for and partitioning of resources that can only be examined bysimultaneous measuring of whole community dynamics and the relative contributions ofindividual populations.
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