SGER: Culture of Gigantic Bacteria: Prerequisite for Advanced Studies

Northern Arizona University, Flagstaff AZ

Investigators

Abstract

The objective of this research is to develop pure cultures of giant Eubacteria (to at least 600 x 80 micrometers) which occupy the central intestines of tropical reef-dwelling surgeonfishes ("tangs" of the aquarium trade, Fam. Acanthuridae). Only one species of these bacteria has been named (Epulopiscium fishelsoni from the Red Sea) although approximately 10 structural "morphs" (here termed "epulos") are distinguishable in a light microscope. Epulos represent a distinct phylogenetic lineage related to Clostridium and Metabacterium, and exhibit a daily life cycle of cell growth and reproduction that tracks the feeding and resting behavior of the host fish. Several characteristics emphasize the unique nature of these cells: epulos possess a distinct nucleoid separated from the cytoplasm by an unidentified, thin, membranous structure; condensed DNA is arranged in elongate, chromosome-like structures with cross-striations similar to those seen in dinoflagellates, so-called "mesokaryotes;" the cytoplasm is packed with an intricately-folded system of what appear to be membranes; cells are highly motile, driven by a covering of bacterial flagella. Cells are large enough to allow use of light microscopy at moderate magnification (100-400 x in most previous studies), manipulative and invasive techniques (e.g., microprobe, microinjection), and other methods normally tractable only with large cells. Such techniques would complement standard physiological, biochemical and molecular microbiological methods used to study the "inner workings" of bacterial cells, as well as to understand: factors which limit cell size and morphology in prokaryotes; vertebrate host-symbiont interactions in an essentially open system (gut vs. specialized culture organ); and bacterial dispersal and evolution in widespread but patchily distributed habitats (surgeonfishes). Major advances on any front, however, are hampered by the inability to maintain them in pure culture. All prior work on epulos has been done on fixed cells or on live cells within about 1 hour of their collection at a tropical field laboratory. Furthermore, all collections have required a diving ichthyologist to identify, capture and dissect the host fish and collect the bacteria. In a preliminary attempt (Summer 1999) to overcome this "culture barrier," epulos remained alive, bud did not grow or reproduce. The goal of this research is to build from the first step to (1) define complete culture conditions and (2) begin to identify gut environmental factors which cue cyclical daily changes in bacterial physiology and activity