Distinguished Professor and Pernot Endowed Chair, Microbiology
KEYWORDS: Ribosomal RNA ; Molecular Phylogeny; Microbial Ecology
Oceanography. Microorganisms in the ocean surface layer are important because they play an integral role in the exchange of carbon between the atmosphere and the ocean. The global dissolved organic carbon (DOC) pool is estimated to be approximately 700 Pg C, a value comparable to the mass of inorganic C in the atmosphere. Thus, perturbations in the metabolism of DOC by microorganisms have the potential to impact the balance between oceanic and atmospheric CO2. Our attention is currently focused on understanding why DOC stocks accumulate in surface waters during summer periods, but are depleted in the upper mesopelagic following convective mixing (Carlson et al. 2004). Recently we have shown that blooms of several specific microbial groups (SAR11b, OCS116 and the marine actinobacteria) are correlated with DOC drawdown (Morris 2005) Relatively simple explanations for these patterns, such as macronutrient depletion, have been ruled out (Carlson, 2002). These studies are carried out at the Sargasso Sea Oceanic Microbial Observatory, with support from the Microbial Observatories Program of NSF. Our close collaborator on this project is Dr. Craig Carlson of UCSB, one of the world’s leading experts on dissolved organic carbon. The study site, BATS (Bermuda Atlantic Time-series Study site), is located in the northwestern Sargasso Sea approximately 80 km southeast of the island of Bermuda. This regionis an oligotrophic, subtropical gyre characterized by wintertime convective overturn, spring phytoplankton blooms, and regular patterns of DOC cycling. At BATS we study the major bacterioplankton groups - SAR11, SAR86, SAR202 and SAR116, marine actinobacteria, SAR324, and SAR406. One of our core activities at BATS is the monthly sampling of microbial distributions. This microbial observatory has yielded a wealth of data about patterns in microbial community structure and their correlation with hydrographic and geochemical variables.
High Throughput Microbial Cultivation. We are working to culture microorganisms that are important to the oceanic DOC cycle so that these strains can be studied in controlled settings that model oceanographic conditions. Weintroduced high throughput culturing (HTC) procedures that have led to a dramatic increase in the number of important bacterioplankton groups that are being cultivated and studied in a laboratory setting (Connon and Giovannoni, 2002). This approach is based on Button’s method for isolating cells by extinction culturing in natural seawater. To achieve high throughput rates we developed cell array methods for screening cultures. Part of our current effort is devoted to developing denser cell arrays and faster methods for scoring arrays. Approximately 2500 cultures of pelagic marine bacteria were isolated over the course of 3 years and 14 separate samplings. Up to 14% of cells from coastal seawater were cultured using this method, a number that is 1400 to140-fold higher than obtained by traditional microbiological culturing techniques. Among the cultured organisms are many unique cell lineages that have been named as new phyla, families, and genera (see Cho et al., 2003, 2004). Ninety percent of the cells recovered by the HTC project do not replicate in Petri dishes of agar media. A majority of the isolates obtained are obligate oligotrophs that display logarithmic growth curves in seawater, reaching stationary phase cell densities of 104 - 106 cells ml-1. It is apparent that some abundant bacterioplankton groups do not replicate in the current generation of HTC media, and will require further innovation before isolation can be achieved.
Bioinformatics. Twenty-two genomes from OSU HTC lab isolates are now being sequenced by the J. Craig Venter Institute with support from the Gordon and Betty Moore Foundation. Genome sequences for Erythrobacter longus, Janibacter sp. (HTCC2649), Oceanicola batsensis (HTCC2597) Croceibacter atlanticus (HTCC2559), Oceanicaulis alexandrii (HTCC2633), Rhodobacterales bacterium (HTCC2654), Parvularcula bermudensis (HTCC2502) have been deposited in Genbank. To annotate and interpret these genomes, and also to support our proteomics effort, we formed a bioinformatics group. This group's main objectives are to identify common adaptive themes and explain how species partition nutrient resources. For this purpose they created GenDB FactFinder. Multiple genomes can be loaded into GenDB FactFinder's large MySQL data warehouse, which is patterned after the GenDB database. The whole dataset can then be mined using a simple keyword search that acts as a front end into a three-tier, Web-enabled data retrieval and presentation tool programmed in Perl and Mason by the Giovannoni lab and the Center for Genome Research and Biocomputing at Oregon State University. The data warehouse includes BLAST hits to the Swissprot, PIR, and NCBI protein databases as well as Interpro protein domains and the results of Hidden Markov model searches against pfam. Links to sequence information in the sponsoring databases are stored for each hit and from here linkouts to literature and curation data are immediately available for research. Gene context for the genome of interest is displayed in a genome browser, and a linkout is provided to Lawrence Livermore’s Microbes On-line database for comparisons of gene synteny across user-defined phylogenetic groups. Metabolic comparisons of any two genomes in the database are possible with color coded KEGG diagrams incorporated into FactFinder with KEGG standard APIs. If available, proteomics confirmation data is also available for mining. Finally, links are provided for powerful tools such as the Transport Classification Database Tool at UCSD for doing specialized research on gene subsets.
Proteomics by Mass Spectrometry. A major thrust of our current research is the application of mass spectrometry methods to understand the regulatory responses of Pelagibacter to environmental variables, and to explore the proteome state of Pelagibacter cells in the oceans, so that they can be used as proxies to report the biological state of the system (Staples et al., 2004). This work is being undertaken in collaboration with OSU Professor Doug Barofsky, co-inventor of the tandem time-of-flight mass spectrometer, and Martha Staples of Waters Corp., formerly a postdoc on the project. We are presently testing the new Waters NanoAquity system and iTRACK methods for quantitative comparisons of proteome composition.
Our specific project objectives are: 1) to measure proteome changes by LC/MS/MS in response to natural variation in environmental variables (e.g. response to light or N, P, or Fe limitation); 2) the identification of indicator proteins that report metabolic status; and 3) in situ measurements of indicator proteins in a framework of oceanographic time-series measurements. To supplement LC/MS/MS data we plan to make limited use of microarrays, mainly to obtain baseline data about global changes in gene expression associated with significant environmental factors in physiological experiments with pure cultures. We predict that mass spectrometry will be more effective than microarrays for studying microbial populations in nature because of the high synonymous substitution rate associated with very large bacterioplankton populations. We have already demonstrated that this approach can be effective by detecting Pelagibacter peptides in coastal seawater (Giovannoni et. al, 2005a).
Pelagibacter Genome Biology. The complete genome sequence of Pelagibacter was assembled, closed and annotated by my laboratory in collaboration with Diversa Corporation (Giovannoni et al., 2005b). The genome is 1,308,506 bp, and encodes the smallest number of predicted ORFs (1373) known for an independently replicating organism. In contrast to parasitic bacteria and archaea with small genomes, P. ubique has complete biosynthetic pathways for all twenty amino acids, and all but a few cofactors. P. ubique has no pseudogenes, introns, transposons, extrachromosomal elements or inteins, few paralogs, and the shortest intergenic spacers yet observed for any cell. The pattern of genome reduction observed in P. ubique is consistent with the hypothesis of genome streamlining. During annotation we discovered that Pelagibacter is the first cultured bacterium that possesses light-dependent retinylidene ion pump, and we are now studying the relationship of this photochemical mechanism to the bioenergetic budget of the cell. Also, in collaboration with Richard McIntosh and Daniella Nicastro, we are defining Pelagibacter cytoarchitecture by electron tomography, and planning is in progress to develop in silico models with the objective of understanding how these cells optimize their growth in the oceans.