Subsurface Microbiology
As participants in the Department of Energy's (DOE) Subsurface Science Program (SSP), we have been interested in developing molecular approaches to examine the microbial community structure of subsurface sediments. We have developed a simple in situ hybridization procedure using 16S rRNA targeted phylogenetic group- and species- specific probes to identify unknown organisms. This procedure can be used on actively growing isolates or directly on microbial communities in sediments.
Additionally, we are extracting total community DNA from subsurface sediments and using the Polymerase Chain Reaction (PCR) to amplify the 16S rRNA genes of all the bacteria present in a given sediment sample. Using this approach, we are attempting to classify the nonculturable bacteria that reside in these sediment types. The focus of this project are sediment samples from the integrated Geological, Microbiological and Hydrological (GEMHEX) experiment. Geological evidence indicate that it is unlikely that transport of bacteria has occurred into or out of these sediments, thus bacteria present would be descendants of the ancestral bacteria that were deposited when these sediments were formed, approximately 6 to 8 million years ago.
Because some of these bacteria could be remnants of ancient
communities, we are also in the process of examining these bacterial
isolates for the presence of intervening sequences, or introns. There
are reports in the literature that some bacteria possess a group I
self-splicing intron in a tRNA gene. Using dot blot hybridization,
Restriction Fragment Length Polymorphism (RFLP) analysis and PCR,
we are attempting to identify those bacteria that may contain
these sequences.
Plant Symbiotic Associations
The aquatic fern Azolla grows in a symbiotic association with nitrogen fixing cyanobacteria (cyanobionts) and other eubacteria (bactobionts). In many Asian counties, Azolla has been used as a natural biofertilizer for rice fields. Additionally, this fern is being used to make green manure for other crops, as a weed suppressant, to purify water, in aquaculture, and as an animal fodder. Obviously, this is an agriculturally significant plant. We are attempting to understand the interactions between the three members of this association.
Traditional culturing techniques have been used to classify the endosymbionts, with little success. Evidence from our lab suggests that there are multiple endosymbionts present in the leaf cavities of Azolla. Using an approach similar to that for subsurface sediments, we are identifying the endosymbionts based on 16S rRNA gene sequence. Once we know the bacterial species present, it may be possible to better understand this association.
Assimilation of fixed nitrogen by the host Azolla occurs via the enzyme glutamine synthetase (GS). In other symbiotic associations, it has been shown that there may be symbiotic regulation of the expression of GS. Preliminary evidence using immunochemical techniques suggests that this may also be the case in Azolla. We are in the process of cloning and sequencing the members of the GS gene family (the gln genes) in an effort localize the expression of these genes within the host plant.
Expression of the nitrogen fixing genes (nif genes) occurs in some cyanobacteria in a specialized cell called a heterocyst. As these cells differentiate, there is a irreversible genetic rearrangement of the genome to allow for expression of the nif genes. The enzyme responsible for this is an excise (xis gene). We are currently attempting to identify and localize expression of this gene in the Azolla endosymbionts.
Freshwater Ecology
Recently, we have begun to expand into utilizing molecular techniques in freshwater systems. Currently we are working on two projects in this area.The zebra mussel is an exotic aquatic pest that was transported to the United States in the late 1980's. Since that time, it has spread rapidly thoughout many of the major waterbodies to the east of the Mississippi. This pest can cause dramatic changes to the aquatic environment, and is considered a major nuisance for industry who use water for such things as cooling. The zebra mussel has a free-swimming veliger stage. We are in the process of developing a genetic probe to be able to rapidly and precisely identify these veligers in water samples.
The second project we have begun is an examination of phytoplankton community structure. We are using as our system Lake George (site of the D-FWI), which is a lake being impacted by human development.
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