Department of Biological and Environmental Sciences
Graduate Project, Annika Castaneda
Fall 2006
Mentor: Ruth A. Gyure
EPILITHIC BACTERIAL BIOFILM COMMUNITY ASSOCIATED WITH RIVERWEED (PODOSTEMACEAE) ATTACHMENT TO ROCKS IN WATERFALLS
INTRODUCTION:
Plants have long been known to harbor associated bacterial communities, often in structures that may be referred to as aggregates or biofilms. Such structures may form on leaves or roots, or on the surfaces of plant-attached material such as rocks. This microbial life on surfaces allows interaction with plants that would not be possible without them, and these partnerships have evolutionary significance. Biofilms associated with plants and their intracellular spaces affect the ecology of bacteria involved and the plant-bacterial relationship. It is also suggested that an understanding of plant-associated bacteria may be necessary for developing new strategies for disease control and maintaining the safety of the food supply (11).
These epilithic (rock or particle-associated) communities are different from free-living bacteria, and may be up to 100x more likely to account for metabolic activity in water. Thus, these attached microbial communities in rivers play a particularly important role in global nutrient cycles due to the rapid turnover, capture and release that they mediate are of great interest in the study of aquatic ecosystems (2, 3).
In addition to their overall role in ecosystem nutrient flux, these communities serve as substrates for emergent higher plant colonization through their ability to mediate attachment (1). The matrix of the biofilm provides a carbon and energy reserve and also possible protection from hazards like pollutants (5).
In spite of their importance, there have been few studies done to identify and enumerate the culturable or unculturable bacteria of these freshwater biofilms. Aside from the more visible and noticeable Cyanobacteria, another group of interest in these niches is the Cytophaga-Flavobacterium group. These bacteria are ubiquitous in diverse natural moist or freshwater environments from Antarctica (9) to ordinary terrestrial soils (6). So, they are likely candidates for also playing a role in epilithic environments and indeed have been found to be important by O’Sullivan et al. (12). In their study, the groups Cytophaga, Flexibacter, and Bacteroides represented 25% of the clones obtained from the epilithon of the River Taff in South Wales.
In particular, the plant body of the riverweed Podostemaceae fastens to rocks in fast-running water by means of sticky biofilms mainly comprised of Cyanobacteria. These biofilms are poorly understood and not fully characterized. Jäger-Zürn et al suspected that the Cyanobaterial members of the community play a major role in the secretion of adhesive compounds that allow the plants to attach to the rocks and maintain that attachment during changing water levels and other environmental conditions. It has been commonly accepted that bacterial bioflims exist under the plant body of Podostemaceae. However, it was also believed that projections from the thallus of the plant may be responsible for secretion of the polysaccharide glue that maintained the plant/rock attachment
In collecting plant material, the biofilm-plant threadlike connections were typically destroyed and not until a careful study done by Jäger-Zürn in 1999 (7) was it first demonstrated that adhesive hairs of the plant were never directly attached to bare rock, but instead always attached to a sticky bacterial layer in between the plant hair and rock.
In 2000, with careful microscopic investigation, it was demonstrated that the bacterial threads of Cyanobateria were mainly involved in secretion of adhesive and attachment, and these findings were replicated in Podostemaceae sample collected from Kenya, Madagascar, Sri Lanka and India (8). To date, these Cyanobacteria, though described from microscopic analysis, have not been isolated or identified to species.
Questions:
1. What types of bacteria comprise the biofilm community at the epillithic interface between Podostemacea and their substrate?
2. Can these biofilms be coaxed into growth away from the natural environment of waterfall and actively growing plant body?
3. Can the Cyanobacteria associated with the biofilm be isolated and identified?
Hypotheses:
A. Novel species of Cyanobacteria play a major role in the attachment of Podostemaceae to their rocky substrates.
B. Cyanobacterial species sequence differences will reflect geographic, geologic and environmental differences as well as Podostemaceae phylogenetic variation.
C. Aside from the major role of Cyanobacteria in adhesion, the Cytophaga-Flavobacterial group will comprise a significant proportion of the bacterial species present in the biofilm/mat.
Methods:
A. Collection:
Podostemaceae samples collected from at least 3 locations in Brazil and 2 sites in the CT/New York area. Samples will be preserved by two methods in the field: immersion in 100% ethanol (rock samples with attached plant material) and drying (samples thoroughly air-dried for transport and storage until return to the laboratory. Samples of bacteria from river water will also be collected to serve as a control in distinguishing biofilm species from the planktonic aquatic community flowing over and around the attached plants. These samples are to be collected by filtration (0.45um) and the filters will either be alcohol preserved as above, or dired for transport.
B. Enrichment for isolation of Cyanobacteria:
Dried material will be immediately placed into enrichment media and conditions upon return to the laboratory. Plant attached rock or pieces of filter will be placed on Chu’s agar or Algal basal media and incubated in the light (18h light6h dark) cycle at 25C. Evidence of Cyanobaterial growth (blue green films and colonies) will be transfered, photographed and cultured for isolation and identification (4).
C. DNA extraction:
DNA will be extracted from biofilm samples (dried and ethanol preserved) after it is carefully scraped from the surface of the rock. DNA will also be extracted from any slimy biofilm growth obtained in the enrichments. Two types of extraction kits from MoBio will be used to obtain this DNA.
D. DNA analysis:
Community DNA obtained in this way will be analyzed by PCR amplification of 16s rDNA gene as well as other genes, using group specific primers for domain Bacteria, group Cyanobacteria, and group Cytophaga-Flavobacteria.
The following groups will also be amplified: domain Archaea, Pseudomonads, Actinomycetes, Green and purple photosynthetic Bacteria, Ammonia oxidizers and genus Nitrobacter. Presence or absence of groups will denoted by degree of amplification success (presence or absence of bands) , compared to positive known controls and spiked samples. Amplicons will also be digested with restriction enzymes to generate restriction patters that can be compared among samples.
Terminal Restriction Fragment Length Polymorphism analysis will also be done with community DNA to compare community profiles among samples and identify key species in the communities.
If funds allow: 16s rDNA amplicon will be cloned into a library using TOPO-TA cloning vector system and 50 clones will be generated and identified by DNA sequencing. One such set will be generated from 1 biofilm sample, and another from the river microbial community control.
E. Identification:
Isolates of Cyanobacteria obtained in the biofilm/Cyanobacterial enrichments will be identified using standard microscopy (acridine orange staining, direct observation, and sequencing of the 16S gene). Assistance in identification will be sought from a laboratory that works with these bacteria routinely.
References
1. Allan J. D. 1995. Stream ecology structure and function of running waters. Chapman and Hall, London, UK.
2. Crump, B. C. and J. A. Baross. 1996. Particle-attached bacteria and heterotrophic plankton in the Columbia River estuary. Mar. Ecol. Prog. Ser. 138: 265-273.
3. Crump B. C., E. V. Armbrust, and J. A. Baross. 1999. Phylogenetic analysis of particle-attached and free-living bacterial communities in the Columbia River, its estuary, and the adjacent coastal ocean. Appl. Env. Microbiol. 65: 3192-3204.
4. Ferris, M, and C.F. Hirsch. 1991. Method for isolation and purification of Cyanobacteria. Appl Env. Microbiol. 57(5):1448-1452.
5. Freeman, C. and M. A. Lock. 1995. The biofilm polysaccharide matrix: a buffer against changing organic substrate supply? Limnol Oceanogr. 40: 273-278.
6. Hengstmann, U., K. J. Chin, P. H. Janssen, and W. Liesack. 1999. Comparative phylogenetic assignment of environmental sequences of genes encoding 16s rRNA and numerically abundant culturable bacteria from anoxic rice paddy soil. Appl. Env. Microbiol. 65: 5050-5058.
7. Jäger-Zürn I 1999. The superglue of Podostemaceae is a bacterial slime. In H. Manitz, FH Hellwig, eds. Symposium Biodiversität und Evolutionbiologie. 14. Inst. Spez. Bot. Friedrich Schuller Universitäat, Jena.
8. Jäger-Zürn I and M. Grubert. 2000. Podostemaceae depend on sticky biofilms with respect to attachment on rocks in waterfalls. Int. J. Plant Sci. 161(4): 599-607.
9. McCammon, S. A., and J. P. Bowman. 2000. Taxonomy of Antarctic Flavobacterium species: description of Flavobacterium gillisiae sp. nov., Flavobacterium tegetincola sp. nov. and Flavobacterium xantham sp. nov. , nom. Rev. and reclassification of [Flavobacteirum] salengens as Salegentibacter salegens gen. nov. , comb. Nov. Int. J. Syst, Bacteriol. 50:1055-1063.
10. McMurdy, H. and W. Hodgson 1973. Method for the selective enumeration of blue green bacteria in water, Appl and Env. Microbiol. 26(5) 682-686.
11. Morris, C. and J. Monier, 2003. The ecological significance of biofilm formation by plant-associated bacteria. Annual Review of Phytopathology, 41: 429-453.
12. O’Sullivan, L., Weightman, A. and J. Fry. 2002. New degenerate Cytophaga-Flexibacter-Bacteroides-Specific 16S ribosomal DNA-targeted oligonucleotide probes reveal high bacterial diversity in river Taff epilithon, Appl Environ Microbiol. 68(4): 2093.
13. Taton, A., S. Grubisic, E. Brambilla, R. De Witt, and A. Wilmotte. 2003. Cyanobacterial diversity in natural and artificial microbial mats of Lake Fryxell (McMurdo Dry Valleys, Antarctica): a morphological and molecular approach.