Project description:The endosymbiont population of the hydrothermal vent tube worm Riftia pachyptila consists of a single 16S phylotype of sulfur-oxidizing gammaproteobacteria. The intracellular symbiont exhibits remarkable morphological heterogeneity, from small rod-shaped or coccoid cells to large cocci, which were suggested to be part of a common cell cycle. To assess whether these morphological differences are accompanied by distinct metabolic profiles, we physically enriched individual symbiont cells sizes by density gradient centrifugation and subjected these enrichments to metaproteomic analysis and statistical evaluation using clustering and random forests. Unlike previous molecular studies, which examined the metabolism of the symbiont population as whole, we were thus able to unravel comprehensive protein abundance patterns of individual symbiont subpopulations. Supported by microscopic analyses, our metaproteomic results show that Riftia symbiont cells of different sizes are stages of a physiological differentiation process: Small symbionts actively divide and may establish cellular symbiont-host interaction, as indicated by highest abundance of the cell division key protein FtsZ and highly abundant chaperones and porins in this initial phase. We furthermore present evidence that large symbionts, on the other hand, do not divide, but still replicate DNA, leading to DNA endoreduplication. Highest abundance of enzymes for CO2 fixation, carbon storage and biosynthesis indicates that in its late differentiation stage, the symbiont’s metabolism is efficiently geared on the production of organic material. We propose that this symbiont aging process enhances the productivity of the symbiosis as a whole.
Project description: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.
Project description:The uncultivated bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila play a central role in providing their host with fixed carbon. While this intimate association between host and symbiont indicates tight integration and coordination of function via cellular communication mechanisms, no such systems have been identified. To elucidate potential signal transduction pathways in symbionts that may mediate symbiont-host communication, we cloned and characterized a gene encoding a histidine protein kinase homolog isolated from a symbiont fosmid library. The gene, designated rssA (for Riftia symbiont signal kinase), resembles known sensor kinases and encodes a protein capable of phosphorylating response regulators in Escherichia coli. A second open reading frame, rssB (for Riftia symbiont signal regulator), encodes a protein similar to known response regulators. These results suggest that the symbionts utilize a phosphotransfer signal transduction mechanism to communicate external signals that may mediate recognition of or survival within the host. The specific signals eliciting a response by the signal transduction proteins of the symbiont remain to be elucidated.
Project description:Biological carbon fixation is foundational to the biosphere. Most autotrophs are thought to possess one carbon fixation pathway. The hydrothermal vent tubeworm Riftia pachyptila’s chemoautotrophic symbionts, however, possess two functional pathways: the Calvin Benson-Bassham (CBB) and the reductive tricarboxylic acid (rTCA) cycles. Little is known about how Riftia’s symbionts and related organisms coordinate the functioning of these two pathways. Here we investigated net carbon fixation rates, transcriptional/metabolic responses, and transcriptional co-expression patterns of Riftia pachyptila’s endosymbionts by incubating tubeworms at environmental pressures, temperature, and geochemistry. Results showed that rTCA and CBB transcriptional patterns varied in response to different geochemical regimes and that each pathway is allied to specific metabolic processes, suggesting distinctive yet complementary roles in metabolic function. Net carbon fixation rates were also exemplary, and accordingly we propose that co-activity of CBB and rTCA may be an adaptation for maintaining high carbon fixation rates, conferring a fitness advantage in dynamic vent environments.