Isolation and identification of an epibiotic bacterium associated with heterocystous Anabaena cells.

Heterotrophic bacteria are commonly found in close associations with photosynthetic cyanobacteria in aquatic ecosystems. Some of these associations can be speciesspecific and mutualistic, resulting in optimal growth and nitrogen-fixing potential for the cyanobacteria. A two-membered culture, consisting of a heterotrophic, epibiotic bacterium attached to an Anabaena sp. was studied in the work reported here. The epibiotic bacterium was grown in pure culture, and both organisms were identified on the basis of their 16S rRNA gene sequence. The specificity of the epibiont for the Anabaena sp. heterocysts was confirmed by re-association experiments. The epibiont is a member of the Alphaproteobacteria in the order Rhizobiales, with close relatives that include a group of aerobic anoxygenic photosynthetic marine isolates commonly associated with dinoflagellate phytoplankton. The close association of the epibiotic bacterium with its Anabaena host, and its phylogenic affiliation allude to the evolutionary history of association with photosynthetic organisms for a group of Rhizobia and warrant further investigation. In diverse aquatic environments, bacteria routinely form close associations with photosynthetic cyanobacteria (1). These cyanobacteria represent a favorable habitat for heterotrophic bacteria because of their buoyancy, production of a mucilaginous sheath, and excretion of copious organic carbon and nitrogen compounds (2–4). Some epibiotic bacteria exhibit specificity for their cyanobacterial hosts through chemotaxis and specificity for the site of their attachment (5–7). These relationships are mutualistic: for example, epibiotic cells incorporate fixed organic carbon and nitrogen from the cyanobacterium Anabaena within an hour under optimal conditions (8). Aerobic respiration of the epibiont results in beneficial localized removal of O2 and perhaps CO2 recycling, which in turn leads to higher photosynthetic growth and N2-fixing potential for the Anabaena (7, 9). The work reported here focuses on a two-membered association between an epibiotic bacterium and the heterocystous cells of a species of Anabaena. This two-membered assemblage was isolated from a brackish marsh located in Woods Hole, Massachusetts, by capturing a single Anabaena filament with attached epibiotic cells, serially washing the filament in sterile autotrophic medium (25% SO) to remove any contaminating bacteria, and using the washed filament to inoculate an axenic culture of the Anabaena sp. (10). The resulting two-membered culture has been studied for at least 10 years as part of the curriculum of the Microbial Diversity summer course at the Marine Biological Laboratory in Woods Hole. The epibiotic bacterium was cultivated in the absence of its Anabaena host. Both bacteria that comprise this assemblage were characterized on the basis of the molecular phylogeny of their 16S rRNA gene sequences. In cultures of the two-membered assemblage, 10 to 50 epibiotic cells were attached to each heterocyst (Fig. 1; panels A and B). Individual heterocysts severed from senescent Anabaena filaments were also observed to have numerous epibiont cells attached. The epibiont cells were 2 to 5 m long, 0.2m wide gram-negative rods, narrower at one end. The thinner end of the cell was the site of attachment to the Anabaena heterocysts. Although the observation was not quantified, the epibiont cells attached preferentially to the polar ends of the heterocysts near the junction with the adjacent vegetative cells, as noted in studies of similar epibiont-Anabaena associations (7). The epibiotic bacterium was successfully cultivated in the absence of the Anabaena basibiont in a brackish, heterotroReceived 8 November 2005; accepted 28 January 2006. * To whom correspondence should be addressed. E-mail: bradley.stevenson@ ou.edu Reference: Biol. Bull. 210: 73–77. (April 2006) © 2006 Marine Biological Laboratory