Monterey Bay Microbial Observatory
- Edward DeLong
Massachusetts Institute of Technology
End Date: March 01, 2008
In the Monterey Bay, a typical milliliter of surface water contains on the order of a million microbial cells. These small, single-celled planktonic microbes represent the most abundant organisms in the world’s oceans. Some of these microbes contribute significantly to primary production in the sea, while others are responsible for consuming a large proportion of marine primary productivity. Planktonic microbial species are key players in the central chemical transformations of carbon, nitrogen, and sulfur, which help maintain the oceanic biosphere. However, despite their importance to the functioning of the Monterey Bay ecosystem, and as a model for understanding marine microbial communities in general, we are still only beginning to describe the microbes of the Bay.
Previous efforts by the DeLong Lab have generated large-insert genomic libraries from genomic fragments of Monterey Bay microbes. These libraries have been characterized extensively, both phylogenetically through surveys of rDNA genes and functionally through surveys of genes known to be involved in, for example, nutrient cycling. In this Monterey Bay Microbial Observatory project funded by NSF, the genomic information in the libraries is now being used to develop microarrays targeting specific microbes within the Bay, to allow a high-throughput means for studying their ecology in the Bay across space and time. The targets of these microarrays will be DNA, extracted from the water, and they will be used to for identification and relative quantification of specific microbes (targeting RNA for expression studies is not currently within the scope of this project). DNA samples from several sites in Monterey Bay are collected and archived monthly by the DeLong Lab group, in conjunction with the MBARI Biological Oceanography Group’s regular CTD cruises. The microarrays will be used to query these time-series DNA samples.
Among the questions to be addressed in this project are: What are the seasonal microbial population changes in Monterey Bay? How do these vary with depth? How do microbial populations vary and ‘bloom’ on shorter time scales, in correlation with nutrient fields, hydrodynamics, and other biotic and abiotic factors? What are the community transitions along the coastal to offshore transect? The prototype array has been made and is currently being tested, and the next generation array, which is due to be printed in early 2005, will allow us to begin answering these questions.
Summary to DateThe prototype array is constructed and is in the early testing stages. The probes successfully hybridize to their target DNA and can be visualized using standard techniques. The prototype also has the ability to distinguish between two closely-related genome fragments. The two genome fragments share 98% identity at the rDNA level and are syntenic, but there is very little cross-hybridization of the probes for one with the target DNA of the other. See Project Images for a picture of the prototype array. This project findings section will be updated frequently as we validate the prototype, print the experimental array, and begin to generate ecological data.
One of the overall goals of this Monterey Bay microbial microarray work is to correlate the presence and absence of particular groups with other physical, chemical and biotic variables. The DeLong Lab’s microbial DNA collections have been and will continue to be parallel with the CTD efforts of the MBARI Biological Oceanography Group (BOG). A list of the oceanographic variables measured by BOG can be found at http://www.mbari.org/bog/Projects/centralcal/summary/ts_summary.htm. In addition to looking for relationships between microbial species or groups and BOG-collected oceanographic variables, we will also investigate positive and negative correlations of individual microarray-targeted picoplankton groups with one another, testing for potential symbiotic and/or synergistic interactions, as well as competitive or antagonistic interactions between individual functional or phylogenetic groups.
- Habitat association
- metabolic potential
Study MethodsThe DeLong Lab’s large-insert genomic libraries are the key reagent in this project. They have been created from genome fragments from Monterey Bay microbes from several depths (0m, 80m, 100m and 750m), using large-insert vectors (bacterial artificial chromosomes and fosmids). For the methods used to construct the bacterial artificial chromosome (BAC) libraries, please see:
Beja, O; Suzuki, MT; Koonin, EV; Aravind, L; Hadd, A; Nguyen, LP; Villacorta, R; Amjadi, M; Garrigues, C; Jovanovich, SB; Feldman, RA; DeLong, EF. 2000. Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage. ENVIRONMENTAL MICROBIOLOGY; v.2, no.5, p.516-529
These BAC libraries have been screened and characterized, and a number of inserts have been fully-sequenced. These represent the core initial sequence targets for the design of the DNA microarray. At least 40 BAC-encoded genome sequences from predominant picoplankton are currently available (ftp://ftp.tigr.org/pub/data/MBMO/). Each of these encodes 40 to > 100 genes, that can serve as multiple targets for microarray probes. The DeLong Lab has annotated these genome sequences and they are currently in the process of being published and submitted to GenBank. Currently, all the ORF-calling has been done using publicly available software, as well as automated annotation software developed by Softberry (http://www.softberry.com). Together, these sequences cover a broad array of some of the most abundant phylogenetic groups, as well as functionally important ones (planktonic heterotrophs, anoxygenic and oxygenic phototrophs, proteorhodopsin phototrophs, autotrophs, ammonia and nitrite oxidizing chemlithoautotrophs). For each BAC insert, we are designing microarray probes targeting 20 to 40 open reading frames (ORFs), which provides multiple, redundant targets for each group – this should also help control for genetic microheterogeneity than may exist in these populations. Besides genome sequences determined from Monterey Bay BAC clones, we will also include ORF targets (20 to 40 ORFs as ‘genome-proxies’) from relevant whole genome picoplankton sequences, as they become available. Genomes in progress or now finished include Synechococcus spp., Prochlorococcus spp, Silicibacter pomeroy (Roseobacter group), and Pelagibacter ubique (SAR11). The prototype array currently being tested targets fourteen of the BAC clones and Prochlorococcus MED4.
The sequenced genome fragments are used to select appropriate oligonucleotide probes targeting those genomes, using the program ArrayOligoSelector, written by Jingchun Zhu, Zbynek Bozdech, and Joe DeRisi, of the DeRisi Lab at UCSF (http://arrayoligosel.sourceforge.net/). This program uses a sliding window to evaluate the calculated hybridization properties of each possible oligonucleotide probe (of desired length) along an input nucleotide sequence. We then select among the possible candidate probes to select those with consistent hybridization properties, and purchase the synthesized oligonucleotides of the desired sequences (from Illumina). The oligonucleotides are spotted onto poly-L-lysine-coated slides, and processed according to standard microarray procedures (http://derisilab.ucsf.edu/microarray/index.html).
Figures and Images
MBARI's R/V Pt. Lobos. Photo: MBARI.
CTD cast. Photo: MBARI.
A comparison of the signal generated from two sets of probes designed to two closely-related (98% identity at the rDNA level) syntenic SAR-86 genome fragments, when queried with the DNA from the genome represented by the probes on the left. There is very little cross-hyrbidization (it is within that seen for the other non-related probe sets in this experiment).