Transmission strategies in a chemosynthetic symbiosis: detection and quantification of symbionts in host tissues and their environment.

Transmission of bacteria vertically through host tissues ensures offspring acquire symbionts; however, horizontal transmission is an effective strategy for many associations and plays a role in some vertically transmitted symbioses. The bivalve Solemya velum and its gammaproteobacterial chemosynthetic symbionts exhibit evolutionary evidence of both transmission modes, but the dominant strategy on an ecological time scale is unknown. To address this, a specific primer set was developed and validated for the S. velum symbiont using a novel workflow called specific marker design (SMD). Symbionts were quantified in spawned eggs and sediment and seawater samples from S. velum habitats with qPCR. Each egg was estimated to contain 50-100 symbiont genomes. By contrast, symbiont DNA was found at low abundance/occurrence in sediment and seawater, often co-occurring with host mitochondrial DNA, obscuring its origin. To ascertain when eggs become infected, histological sections of S. velum tissues were labelled for symbiont 16S rRNA via in situ hybridization. This revealed symbionts in the ovary walls and mature oocytes, suggesting association in late oogenesis. These data support the hypothesis that S. velum symbionts are vertically transmitted every host generation, thus genetic signatures of horizontal transmission are driven by ecologically infrequent events. This knowledge furthers our understanding of vertical and horizontal mode integration and provides insights across animal-bacterial chemosynthetic symbioses.

Prokaryotic Cells in the Hydrothermal Vent Tube Worm Riftia pachyptila Jones: Possible Chemoautotrophic Symbionts.

The existence of a symbiotic association between vestimentiferan tube worms from deep-sea hydrothermal vents and chemoautotrophic sulfur-oxidizing prokaryotes, based on histological and enzymatic evidence, is suggested.

Biogeography and ecological setting of Indian Ocean hydrothermal vents.

Within the endemic invertebrate faunas of hydrothermal vents, five biogeographic provinces are recognized. Invertebrates at two Indian Ocean vent fields (Kairei and Edmond) belong to a sixth province, despite ecological settings and invertebrate-bacterial symbioses similar to those of both western Pacific and Atlantic vents. Most organisms found at these Indian Ocean vent fields have evolutionary affinities with western Pacific vent faunas, but a shrimp that ecologically dominates Indian Ocean vents closely resembles its Mid-Atlantic counterpart. These findings contribute to a global assessment of the biogeography of chemosynthetic faunas and indicate that the Indian Ocean vent community follows asymmetric assembly rules biased toward Pacific evolutionary alliances.

Isotopic discrimination and kinetic parameters of RubisCO from the marine bloom-forming diatom, Skeletonema costatum.

The cosmopolitan, bloom-forming diatom, Skeletonema costatum, is a prominent primary producer in coastal oceans, fixing CO2 with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) that is phylogenetically distinct from terrestrial plant RubisCO. RubisCOs are subdivided into groups based on sequence similarity of their large subunits (IA-ID, II, and III). ID is present in several major oceanic primary producers, including diatoms such as S. costatum, coccolithophores, and some dinoflagellates, and differs substantially in amino acid sequence from the well-studied IB enzymes present in most cyanobacteria and in green algae and plants. Despite this sequence divergence, and differences in isotopic discrimination apparent in other RubisCO enzymes, stable carbon isotope compositions of diatoms and other marine phytoplankton are generally interpreted assuming enzymatic isotopic discrimination similar to spinach RubisCO (IB). To interpret phytoplankton δ(13) C values, S. costatum RubisCO was characterized via sequence analysis, and measurement of its KCO2 and Vmax , and degree of isotopic discrimination. The sequence of this enzyme placed it among other diatom ID RubisCOs. Michaelis-Menten parameters were similar to other ID enzymes (KCO2 = 48.9 ± 2.8 µm; Vmax = 165.1 ± 6.3 nmol min(-1 ) mg(-1) ). However, isotopic discrimination (ε = [(12) k/(13) k - 1] × 1000) was low (18.5‰; 17.0-19.9, 95% CI) when compared to IA and IB RubisCOs (22-29‰), though not as low as ID from coccolithophore, Emiliania huxleyi (11.1‰). Variability in ε-values among RubisCOs from primary producers is likely reflected in δ(13) C values of oceanic biomass. Currently, δ(13) C variability is ascribed to physical or chemical factors (e.g. illumination, nutrient availability) and physiological responses to these factors (e.g. carbon-concentrating mechanisms). Estimating the importance of these factors from δ(13) C measurements requires an accurate ε-value, and a mass-balance model using the ε-value for S. costatum RubisCO is presented. Clearly, appropriate ε-values must be included in interpreting δ(13) C values of environmental samples.

Population Dynamics of Two Toluene Degrading Bacterial Species in a Contaminated Stream.

Toluene uptake by a benthic biofilm community was previously shown to vary seasonally from 0.03 m hr?1 in winter to 0.2 m hr?1 in summer in a solvent-contaminated stream of the Aberjona watershed. We used quantitative PCR to estimate the population dynamics of previously isolated species of toluene-degrading Xanthobacter autotrophicus and Mycobacterium sp. in both toluene-contaminated and uncontaminated reaches of the stream, and to estimate their relative roles in overall biodegradation rate. Quantification using specific 16S rDNA primers forX. autotrophicus and Mycobacterium sp. showed that populations of both species were much larger in the toluene-contaminated than the toluene-free reach, in agreement with earlier culture-based investigations. A relatively brief bloom of X. autotrophicus occurred in the contaminated reach in the summer, while Mycobacterium sp. populations occurred at elevated densities for more than 5 months. Calculations showed that Mycobacterium, previously thought to be less important than Xanthobacter in annual toluene degradation based on single time-point CFU estimates, appears actually more important because of this longer persistence.

Importance of Xanthobacter autotrophicus in toluene biodegradation within a contaminated stream.

Toluene-degrading strains T101 and T102 were isolated from rock surface biomass in a toluene-contaminated freshwater stream. These organisms were present at a density of 5.5 x 10(6) cells/g of rock surface biomass. Both are aerobic, rod-shaped, Gram-negative, non-motile, catalase-positive, oxidase-positive, with yellow pigments, and can grow on benzene. Phylogenetic analyses show that strains T101 and T102 have 16S rDNA sequences identical to Xanthobacter autotrophicus. Fatty acid analyses indicate that they are different strains of the same species Xanthobacter autotrophicus, and that they have high levels of cis-11-octadecenoic acid and cis-9-hexadecenoic acid; 3-hydroxyhexadecanoic acid is the major hydroxy fatty acid present. Strains T101 and T102 had maximal velocities (Vmax) for toluene biodegradation of 3.8 +/- 0.5 and 28.3 +/- 2.2 mumoles toluene/mgprotein-hr, and half-saturation constants (Ks) of 0.8 +/- 0.5 and 11.5 +/- 2.4 microM, respectively. Strain T102 has a higher capacity than strain T101 to degrade toluene, and kinetic calculations suggest that strain T102 may be a major contributor to toluene biodegradation in the stream.

Vertical transmission of chemoautotrophic symbionts in the bivalve Solemya velum (Bivalvia: Protobranchia).

Adults of the bivalve species Solemya velum live in symbiosis with chemoautotrophic bacteria in specialized gill bacteriocytes. The bacteria play an essential nutritional role in the mature association, fixing CO2 via the Calvin cycle with energy obtained through the oxidation of reduced sulfur compounds. To understand how the continuity of this partnership is maintained between host generations, we investigated the mode of symbiont transfer in S. velum. A diagnostic assay using the polymerase chain reaction and primers specific for the S. velum symbiont ribulose-1,5-bisphosphate carboxylase (RubisCO) gene consistently detected bacterial sequence in female gonad tissue, suggesting the presence of symbiont cells in host ovaries and a vertical mode of symbiont transmission from mother to offspring. Furthermore, intracellular bacteria were present in the developing gills of juveniles that had not yet hatched from the gelatinous capsule in which larval development occurs (11 days after fertilization). By 64 days postfertilization, the typical adult gill ultrastructure of alternating bacteriocytes and symbiont-free-intercalary cells was apparent. Knowledge about the mode of symbiont transfer in S. velum allows further study into the dynamics of host-symbiont interactions in chemoautotrophic associations.

The Calyptogena magnifica chemoautotrophic symbiont genome.

Chemoautotrophic endosymbionts are the metabolic cornerstone of hydrothermal vent communities, providing invertebrate hosts with nearly all of their nutrition. The Calyptogena magnifica (Bivalvia: Vesicomyidae) symbiont, Candidatus Ruthia magnifica, is the first intracellular sulfur-oxidizing endosymbiont to have its genome sequenced, revealing a suite of metabolic capabilities. The genome encodes major chemoautotrophic pathways as well as pathways for biosynthesis of vitamins, cofactors, and all 20 amino acids required by the clam.

Dominance of one bacterial phylotype at a Mid-Atlantic Ridge hydrothermal vent site.

Microbial community structure in natural environments has remained largely unexplored yet is generally considered to be complex. It is shown here that in a Mid-Atlantic Ridge hydrothermal vent habitat, where food webs depend on prokaryotic primary production, the surface microbial community consists largely of only one bacterial phylogenetic type (phylotype) as indicated by the dominance of a single 16S rRNA sequence. The main part of its population occurs as an ectosymbiont on the dominant animals, the shrimp Rimicaris exoculata, where it grows as a monoculture within the carapace and on the extremities. However, the same bacteria are also the major microbial component of the free-living substrate community. Phylogenetically, this type forms a distinct branch within the epsilon-Proteobacteria. This is different from all previously studied chemoautotrophic endo- and ectosymbioses from hydrothermal vents and other sulfidic habitats in which all the bacterial members cluster within the gamma-Proteobacteria.

Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes.

The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized. Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy. The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp. By using sequence data from 16S rRNA, the identity and evolutionary origins of the S. terraeregina and S. pusilla symbionts were also determined. Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species. In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills. Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S. terraeregina and S. pusilla symbionts as members of the gamma subdivision of the Proteobacteria. In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S. terraeregina and S. pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S. velum, S. occidentalis, and S. reidi). Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria. While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins.

Bias in template-to-product ratios in multitemplate PCR.

Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.

Independent phylogenetic origins of methanotrophic and chemoautotrophic bacterial endosymbioses in marine bivalves.

The discovery of bacterium-bivalve symbioses capable of utilizing methane as a carbon and energy source indicates that the endosymbionts of hydrothermal vent and cold seep bivalves are not restricted to sulfur-oxidizing chemoautotrophic bacteria but also include methanotrophic bacteria. The phylogenetic origin of methanotrophic endosymbionts and their relationship to known symbiotic and free-living bacteria, however, have remained unexplored. In situ localization and phylogenetic analysis of a symbiont 16S rRNA gene cloned from the gills of a recently described deep-sea mussel species demonstrate that this symbiont represents a new taxon which is closely related to free-living, cultivable Type I methanotrophic bacteria. This symbiont is distinct from known chemoautotrophic symbionts. Thus, despite compelling similarities between the symbioses, chemoautotrophic and methanotrophic symbionts of marine bivalves have independent phylogenetic origins.

A simple method for quantification of uncultured microorganisms in the environment based on in vitro transcription of 16S rRNA.

A simple method for the quantification of uncultured microorganisms in the environment was developed. In vitro-transcribed 16S rRNA is used as a template for midpoint dissociation temperature (Td) determinations of specific oligonucleotide probes and as a standard in quantitative probing. It replaces the need for total nucleic acids extracted from pure cultures of the organisms to be quantified. A sense RNA of a size almost identical to that of native 16S rRNA can be transcribed from ribosomal DNA clones recovered in studies of the phylogenetic diversity of microbial communities. This in vitro-transcribed rRNA yields dissociation curves typical of oligonucleotides. They parallel curves determined with total nucleic acids but yield slightly higher Td values. Neither unspecific sticking of the probe nor probe washing off the DNA template at low temperatures fully accounted for the discrepancy in probe release from the two templates. This suggests that the native rRNA itself has melting characteristics different from those of its in vitro-transcribed counterpart. However, this difference does not affect the performance of in vitro-transcribed rRNA compared with total nucleic acids as a standard in quantitative hybridizations. No difference was found between the estimates of the relative quantity of a single bacterial species in a mixed community obtained with the two standards, regardless of whether DNA was removed from the samples. This protocol will allow the large-scale quantification of the ecological importance of uncultured microorganisms in natural environments for the first time.

Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of Solemya velum (Bivalvia: Mollusca).

The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP(2)Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of Solemya velum, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large subunit of RbuP(2)Case from tobacco (Nicotiana tabacum) cross-reacted on immunoblots with a protein of similar molecular mass occurring in extracts of the symbiont-containing gill tissue of S. velum. No cross-reactivity was detected in symbiont-free tissue extracts. The antiserum also cross-reacted in immunoblots with proteins of Thiobacillus neapolitanus, a free-living sulfuroxidizing chemoautotroph whose RbuP(2)Case has been well characterized. In protein A-gold immunoelectron microscopy studies, this antiserum consistently labeled the symbionts but not surrounding host gill tissue, indicating that the symbionts are responsible for the RbuP(2)Case activity.

Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis.

The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and between individual S. velum organisms suggested that one species of symbiont is dominant within and specific for this host species. Phylogenetic analysis of the 16S sequences of the symbionts indicates that they lie within the chemoautotrophic cluster of the gamma subdivision of the eubacterial group Proteobacteria.

Intracellular coexistence of methano- and thioautotrophic bacteria in a hydrothermal vent mussel.

The coexistence of two phylogenetically distinct symbiont species within a single cell, a condition not previously known in any metazoan, is demonstrated in the gills of a Mid-Atlantic Ridge hydrothermal vent mussel (family Mytilidae). Large and small symbiont morphotypes within the gill bacteriocytes are shown to be separate bacterial species by molecular phylogenetic analysis and fluorescent in situ hybridization. The two symbiont species are affiliated with thioautotrophic and methanotrophic symbionts previously found in monospecific associations with closely related mytilids from deep-sea hydrothermal vents and hydrocarbon seeps.

Evidence for methylotrophic symbionts in a hydrothermal vent mussel (bivalvia: mytilidae) from the mid-atlantic ridge.

Symbioses between chemolithoautotrophic bacteria and the major macrofaunal species found at hydrothermal vents have been reported for numerous sites in the Pacific Ocean. We present microscopical and enzymatic evidence that methylotrophic bacteria occur as intracellular symbionts in a new species of mytilid mussel discovered at the Mid-Atlantic Ridge hydrothermal vents. Two distinct ultrastructural types of gram-negative procaryotic symbionts were observed within gill epithelial cells by transmission electron microscopy: small coccoid or rod-shaped cells and larger coccoid cells with stacked intracytoplasmic membranes typical of methane-utilizing bacteria. Methanol dehydrogenase, an enzyme diagnostic of methylotrophs, was detected in the mytilid gills, while tests for ribulose-1,5-bisphosphate carboxylase, the enzyme diagnostic of autotrophy via the Calvin cycle, were negative. Stable carbon isotope values (deltaC) of mytilid tissue (-32.7 and -32.5% for gill and foot tissues, respectively) fall within the range of values reported for Pacific vent symbioses but do not preclude the use of vent-derived methane reported to be isotopically heavy relative to biogenically produced methane.

Cloning and sequencing of a form II ribulose-1,5-biphosphate carboxylase/oxygenase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila.

The bacterial symbiont of the hydrothermal vent tubeworm fixes carbon via the Calvin-Benson cycle and has been shown previously to express a form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene cbbM, which encodes this enzyme, has been cloned and sequenced. The gene has the highest identity with the cbbM gene from Rhodospirillum rubrum, and analysis of the inferred amino acid sequence reveals that all active-site residues are conserved. This is the first form II RubisCO cloned and sequenced from a chemoautotrophic symbiont and from a deep-sea organism.

Diversity and heterogeneity of epibiotic bacterial communities on the marine nematode Eubostrichus dianae.

The diversity of a microbial community covering the surface of a marine nematode was analyzed by performing a 16S ribosomal DNA (rDNA) restriction cutting and sequencing analysis. In two clone libraries constructed by using individual nematodes, 54 and 85 restriction patterns were identified, and only 13 of these patterns were common to both libraries. Sequence analysis indicated that the common patterns belonged to four groups related to sequences of cytophagas, sulfate-reducing bacteria, members of the gamma subclass of the class Proteobacteria, and caulobacters. At least two groups appeared to be permanent members of the community as they were also detected in a 16S rDNA library constructed 3 years previously by using 100 pooled nematode specimens. A surprising outcome was that very dominant filamentous bacteria were apparently not represented in the clone libraries, as quantitative probing showed that none of the common operational taxonomic unit groups displayed the expected overwhelming dominance. Nevertheless, our analysis revealed both an unexpectedly high level of bacterial diversity and heterogeneity in samples representing presumably very similar microenvironments.