Sulfur-oxidizing symbionts have not co-evolved with their hydrothermal vent tube worm hosts: an RFLP analysis.

A fine-scale phylogenetic comparison was made among the symbionts of different genera of hydrothermal vent tube worms. These included Riftia pachyptila and Tevnia jerichonona, which inhabit sites along the east Pacific Rise, and Ridgeia piscesae from the Juan de Fuca Ridge. An analysis of restriction fragment length polymorphism (RFLP) was employed using three symbiont-specific gene probes: eubacterial 16S rRNA, RuBPC/O Form II, and ATP sulfurylase (recently cloned from the Riftia symbiont). Results indicated that all of the symbionts from the three different hosts were conspecific and the Riftia and Tevnia symbionts were indistinguishable over and 1800-km range. Significantly, this indicates that the symbionts have not co-evolved with their respective hosts, which are known to belong to separate families. This study strongly supports the conclusion that the symbionts are acquired de novo by each generation of juvenile tube worms from a common source in the surrounding sea water.

Using a phase-locked mutant of Myxococcus xanthus to study the role of phase variation in development.

The bacterium Myxococcus xanthus undergoes a primitive developmental cycle in response to nutrient deprivation. The cells aggregate to form fruiting bodies in which a portion of the cells differentiate into environmentally resistant myxospores. During the growth portion of the M. xanthus life cycle, the organism also undergoes a phase variation, in which cells alternate between yellow and tan colony-forming variants. Phase variation occurs in our laboratory strain (M102, a derivative of DK1622) at a frequency high enough that a single colony of either the yellow or the tan phase already contains cells of the alternate phase. In this study we demonstrate that tan cells within a predominantly yellow population of phase variation-proficient cells are preferentially recovered as heat- and sonication-resistant spores. To further investigate the possibility of a differential role of tan and yellow cells during development, a tan-phase-locked mutant was used to compare the developmental phenotypes of a pure tan population with a predominantly yellow, phase variation-proficient population. Pure tan-phase populations did not produce fruiting bodies or mature spores under conditions in which predominantly yellow wild-type populations did so efficiently. Pure populations of tan-phase cells responded to developmental induction by changing from vegetative rod-shaped cells to round forms but were unable to complete the maturation to heat- and sonication-resistant, refractile spores. The developmental defect of a tan-phase-locked mutant was rescued by the addition of phase variation-proficient cells from a predominantly yellow culture. In such mixtures the tan-phase-locked mutant not only completed the process of forming spores but also was again preferentially represented among the viable spores. These findings suggest the intriguing possibility that the tan-phase cells within the vegetative population entering development are the progenitors of spores and implicate a requirement for yellow-phase cells in spore maturation.

Use of a phase variation-specific promoter of Myxococcus xanthus in a strategy for isolating a phase-locked mutant.

The bacterium Myxococcus xanthus alternates between two colony types distinguished by colony morphology and pigmentation. Because the two phases are interconvertible, this phenomenon has been termed phase variation. In one phase, the colonies are bright yellow, rough, and swarming. In the alternate phase, the colonies are tan and mucoid with smooth edges. During exponential vegetative growth, the populations within a colony reach an equilibrium of approximately 99% yellow and 1% tan cells. Neither the biological function nor the genetic mechanism of phase variation is currently understood. To investigate phase variation, a yellow-phase-specific promoter was identified by Tn5lac mutagenesis. A tan-phase-locked mutant was isolated by a strategy, described in this study, which involved introducing a selectable marker expressed under phase-regulated expression. This was accomplished by a fusion of the cloned yellow-phase-specific promoter to a promoterless kanamycin resistance gene. The defect in the phase-locked mutant, given the designation var-683, caused the rate of switching from the tan to yellow phase to be reduced by at least 10(3)-fold below the wild-type rate of switching. This strain will provide a stable tan population for genetic and biological analysis. Evidence is presented for the existence of a transcriptional regulator which controls the expression of phase-regulated promoters.

Characterization of the gene encoding the autotrophic ATP sulfurylase from the bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

ATP sulfurylase is a key enzyme in the energy-generating sulfur oxidation pathways of many chemoautotrophic bacteria. The utilization of reduced sulfur compounds to fuel CO2 fixation by the still-uncultured bacterial endosymbionts provides the basis of nutrition in invertebrates, such as the tubeworm Riftia pachyptila, found at deep-sea hydrothermal vents. The symbiont-containing trophosome tissue contains high levels of ATP sulfurylase activity, facilitating the recent purification of the enzyme. The gene encoding the ATP sulfurylase from the Riftia symbiont (sopT) has now been cloned and sequenced by using the partial amino acid sequence of the purified protein. Characterization of the sopT gene has unequivocally shown its bacterial origin. This is the first ATP sulfurylase gene to be cloned and sequenced from a sulfur-oxidizing bacterium. The deduced amino acid sequence was compared to those of ATP sulfurylases reported from organisms which assimilate sulfate, resulting in the discovery that there is substantial homology with the Saccharomyces cerevisiae MET3 gene product but none with the products of the cysDN genes from Escherichia coli nor with the nodP and nodQ genes from Rhizobium meliloti. This and emerging evidence from other sources suggests that E. coli may be atypical, even among prokaryotic sulfate assimilators, in the enzyme it employs for adenosine 5'-phosphosulfate formation. The sopT gene probe also was shown to specifically identify chemoautotrophic bacteria which utilize ATP sulfurylase to oxidize sulfur compounds.