Identification and characterization of a flagellin gene from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila

The bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyp tila play a key role in providing their host with fixed carbon. Results of prior research suggest that the symbionts are selected from an environmenta l bacterial population, although a free-living form has been neither cultur ed from nor identified in the hydrothermal vent environment. To begin to as sess the free-living potential of the symbiont, we cloned and characterized a flagellin gene from a symbiont fosmid library. The symbiont fliC gene ha s a high degree of homology with other bacterial flagellin genes in the ami no- and carboxy-terminal regions, while the central region was found to be nonconserved. A sequence that was homologous to that of a consensus sigma(2 8) RNA polymerase recognition site lay upstream of the proposed translation al start site. The symbiont protein was expressed in Escherichia coli, and flagella were observed by electron microscopy. A 30,000-M-r protein subunit was identified in whole-cell extracts by Western blot analysis. These resu lts provide the first direct evidence of a motile free-living stage of a ch emoautotrophic symbiont and support the hypothesis that the symbiont of R. pachyptila is acquired with each new host generation.