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

Abstract

The bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila play a key role in providing their host with fixed carbon. Results of prior research suggest that the symbionts are selected from an environmental bacterial population, although a free-living form has been neither cultured from nor identified in the hydrothermal vent environment. To begin to assess the free-living potential of the symbiont, we cloned and characterized a flagellin gene from a symbiont fosmid library. The symbiont fliC gene has a high degree of homology with other bacterial flagellin genes in the amino- and carboxy-terminal regions, while the central region was found to be nonconserved. A sequence that was homologous to that of a consensus sigma28 RNA polymerase recognition site lay upstream of the proposed translational start site. The symbiont protein was expressed in Escherichia coli, and flagella were observed by electron microscopy. A 30,000-Mr protein subunit was identified in whole-cell extracts by Western blot analysis. These results provide the first direct evidence of a motile free-living stage of a chemoautotrophic symbiont and support the hypothesis that the symbiont of R. pachyptila is acquired with each new host generation.

FIG. 1

Restriction map of the symbiont fliC gene region from the fosmid clone pFOS1O9. The size and transcriptional direction of the rbfC, fliC, and flaG genes are shown by arrows. The fliC fragments cloned for the expression study are indicated below the appropriate sequence.

FIG. 2

Electron microscopy of motility mutant E. coli JA11 expressing the flagellin gene from the R. pachyptila symbiont. JA11 cells contained the following plasmids: pBluescript (vector control) (A); pDH90, containing the R. pachyptila symbiont fliC gene (FliC_RS) (B); pDH95 (FliC_RS) (C); and pMS1, containing the Salmonella H2 flagellin gene (FliC_ST) (D). Cells at the mid-exponential phase were prepared for electron microscopy by negative staining with 0.5% uranyl acetate. Bar, 500 nm.

FIG. 3

Immunoblots showing reactivity of the anti-FliC_E antibody (15D8) with purified Salmonella H2 flagellin (lane 1) and E. coli JA11 containing pMS1 (lane 2), pBluescript (lane 3), pDH90 (lane 4), or pDH95 (lane 5).