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Cavanaugh, Colleen

The environmental distribution and phylogeny of “Korarchaeota,” a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9°N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity.

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.

Recent evidence suggests that deep-sea vestimentiferan tube worms acquire their endosymbiotic bacteria from the environment each generation; thus, free-living symbionts should exist. Here, free-living tube worm symbiont phylotypes were detected in vent seawater and in biofilms at multiple deep-sea vent habitats by PCR amplification, DNA sequence analysis, and fluorescence in situ hybridization. These findings support environmental transmission as a means of symbiont acquisition for deep-sea tube worms.

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.

Chemoautotrophic symbioses, in which endosymbiotic bacteria are the major source of organic carbon for the host, are found in marine habitats where sulfide and oxygen coexist. The purpose of this study was to determine the influence of pH, alternate sulfur sources, and electron acceptors on carbon fixation and to investigate which form(s) of inorganic carbon is taken up and fixed by the gamma-proteobacterial endosymbionts of the protobranch bivalve Solemya velum. Symbiont-enriched suspensions were generated by homogenization of S. velum gills, followed by velocity centrifugation to pellet the symbiont cells. Carbon fixation was measured by incubating the cells with 14C-labeled dissolved inorganic carbon. When oxygen was present, both sulfide and thiosulfate stimulated carbon fixation; however, elevated levels of either sulfide (>0.5 mM) or oxygen (1 mM) were inhibitory. In the absence of oxygen, nitrate did not enhance carbon fixation rates when sulfide was present. Symbionts fixed carbon most rapidly between pH 7.5 and 8.5. Under optimal pH, sulfide, and oxygen conditions, symbiont carbon fixation rates correlated with the concentrations of extracellular CO2 and not with HCO3− concentrations. The half-saturation constant for carbon fixation with respect to extracellular dissolved CO2 was 28 ± 3 μM, and the average maximal velocity was 50.8 ± 7.1 μmol min−1 g of protein−1. The reliance of S. velum symbionts on extracellular CO2 is consistent with their intracellular lifestyle, since HCO3− utilization would require protein-mediated transport across the bacteriocyte membrane, perisymbiont vacuole membrane, and symbiont outer and inner membranes. The use of CO2 may be a general trait shared with many symbioses with an intracellular chemoautotrophic partner.

Deep-sea mussels of the subfamily Bathymodiolinae (Bivalvia, Pteriomorphia, Mytilidae) are dominant members of hydrothermal vent communities across the globe and have been found within every known hydrothermal vent biogeographic province. Thus, the apparent rarity of bathymodiolines at vents of the Juan de Fuca Ridge (JdF) is a notable exception. We examined mussels collected from the JdF over a span of 18 yr to clarify the classifications of mussel hosts and their symbiotic bacteria, and the relationships between sparsely distributed individuals. Several specimens, previously classified as Bathymodiolus and Adipicola, were reassigned as Adipicola sp. JdF based on new phylogenetic evidence and previous morphological studies. Adipicola sp. JdF are the most deeply branching members of Adipicola identified to date. Ultrastructural, molecular phylogenetic, and stable carbon isotope analyses indicated that Adipicola sp. JdF mussels harbor bacterial chemoautotrophic symbionts that appear to be extracellular and that are closely related to symbionts of other bathymodioline hosts. This study demonstrates that a sparsely distributed, yet cohesive, population of Adipicola has inhabited JdF vents for at least the past 18 yr. Moreover, the presence of extracellular symbionts in Adipicola sp. JdF at hydrothermal vents stands in contrast to the evolutionary patterns proposed for bathymodiolines in general. Adipicola sp. JdF represents an exception to the trend of bathymodiolines harboring extracellular symbionts living exclusively in shallow marine wood and bone habitats (<1000 m), indicating that bathymodiolines are more flexible with regard to habitat and symbiont characteristics than previously considered.

Background: Symbioses between chemoautotrophic bacteria and marine invertebrates are rare examples of living systems that are virtually independent of photosynthetic primary production. These associations have evolved multiple times in marine habitats, such as deep-sea hydrothermal vents and reducing sediments, characterized by steep gradients of oxygen and reduced chemicals. Due to difficulties associated with maintaining these symbioses in the laboratory and culturing the symbiotic bacteria, studies of chemosynthetic symbioses rely heavily on culture independent methods. The symbiosis between the coastal bivalve, Solemya velum, and its intracellular symbiont is a model for chemosynthetic symbioses given its accessibility in intertidal environments and the ability to maintain it under laboratory conditions. To better understand this symbiosis, the genome of the S. velum endosymbiont was sequenced. Results: Relative to the genomes of obligate symbiotic bacteria, which commonly undergo erosion and reduction, the S. velum symbiont genome was large (2.7 Mb), GC-rich (51%), and contained a large number (78) of mobile genetic elements. Comparative genomics identified sets of genes specific to the chemosynthetic lifestyle and necessary to sustain the symbiosis. In addition, a number of inferred metabolic pathways and cellular processes, including heterotrophy, branched electron transport, and motility, suggested that besides the ability to function as an endosymbiont, the bacterium may have the capacity to live outside the host. Conclusions: The physiological dexterity indicated by the genome substantially improves our understanding of the genetic and metabolic capabilities of the S. velum symbiont and the breadth of niches the partners may inhabit during their lifecycle. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-924) contains supplementary material, which is available to authorized users.

The mxaF gene, coding for the large ((\alpha)) subunit of methanol dehydrogenase, is highly conserved among distantly related methylotrophic species in the Alpha-, Beta- and Gammaproteobacteria. It is ubiquitous in methanotrophs, in contrast to other methanotroph-specific genes such as the pmoA and mmoX genes, which are absent in some methanotrophic proteobacterial genera. This study examined the potential for using the mxaF gene as a functional and phylogenetic marker for methanotrophs. mxaF and 16S rRNA gene phylogenies were constructed based on over 100 database sequences of known proteobacterial methanotrophs and other methylotrophs to assess their evolutionary histories. Topology tests revealed that mxaF and 16S rDNA genes of methanotrophs do not show congruent evolutionary histories, with incongruencies in methanotrophic taxa in the Methylococcaceae, Methylocystaceae, and Beijerinckiacea. However, known methanotrophs generally formed coherent clades based on mxaF gene sequences, allowing for phylogenetic discrimination of major taxa. This feature highlights the mxaF gene’s usefulness as a biomarker in studying the molecular diversity of proteobacterial methanotrophs in nature. To verify this, PCR-directed assays targeting this gene were used to detect novel methanotrophs from diverse environments including soil, peatland, hydrothermal vent mussel tissues, and methanotroph isolates. The placement of the majority of environmental mxaF gene sequences in distinct methanotroph-specific clades (Methylocystaceae and Methylococcaceae) detected in this study supports the use of mxaF as a biomarker for methanotrophic proteobacteria.

Longitudinal studies of the microbiota are important for discovering changes in microbial communities that affect the host. The complexity of these ecosystems requires rigorous integrated experimental and computational methods to identify temporal signatures that promote physiologic or pathophysiologic responses in vivo. Employing a murine model of infectious colitis with the pathogen Citrobacter rodentium, we generated a 2-month time-series of 16S rDNA gene profiles, and quantitatively cultured commensals, from multiple intestinal sites in infected and uninfected mice. We developed a computational framework to discover time-varying signatures for individual taxa, and to automatically group signatures to identify microbial sub-communities within the larger gut ecosystem that demonstrate common behaviors. Application of this model to the 16S rDNA dataset revealed dynamic alterations in the microbiota at multiple levels of resolution, from effects on systems-level metrics to changes across anatomic sites for individual taxa and species. These analyses revealed unique, time-dependent microbial signatures associated with host responses at different stages of colitis. Signatures included a Mucispirillum OTU associated with early disruption of the colonic surface mucus layer, prior to the onset of symptomatic colitis, and members of the Clostridiales and Lactobacillales that increased with successful resolution of inflammation, after clearance of the pathogen. Quantitative culture data validated findings for predominant species, further refining and strengthening model predictions. These findings provide new insights into the complex behaviors found within host ecosystems, and define several time-dependent microbial signatures that may be leveraged in studies of other infectious or inflammatory conditions.

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae(type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family,Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the familyMethylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of theMethylococcaceae, includingMethylobacter, Methylomonas,Methylomicrobium, Methylococcus, andMethylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed generaMethylocella and “Methylothermus,” which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57°C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.