Colleen M. Cavanaugh

Evidence for a core gut microbiota in the zebrafish.

Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status.

Characterization of an Autotrophic Sulfide-Oxidizing Marine Arcobacter sp. That Produces Filamentous Sulfur

ABSTRACT A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4′,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is “Candidatus Arcobacter sulfidicus.” For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO2 fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO2 fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by “Candidatus Arcobacter sulfidicus” or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.

Phylogenetic and metabolic diversity of bacteria associated with cystic fibrosis

In patients afflicted with cystic fibrosis (CF), morbidity and mortality are primarily associated with the adverse consequences of chronic microbial bronchial infections, which are thought to be caused by a few opportunistic pathogens. However, recent evidence suggests the presence of other microorganisms, which may significantly affect the course and outcome of the infection. Using a combination of 16S rRNA gene clone libraries, bacterial culturing and pyrosequencing of barcoded 16S rRNA amplicons, the microbial communities present in CF patient sputum samples were examined. In addition to previously recognized CF pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus, >60 phylogenetically diverse bacterial genera that are not typically associated with CF pathogenesis were also detected. A surprisingly large number of fermenting facultative and obligate anaerobes from multiple bacterial phyla was present in each sample. Many of the bacteria and sequences found were normal residents of the oropharyngeal microflora and with many containing opportunistic pathogens. Our data suggest that these undersampled organisms within the CF lung are part of a much more complex microbial ecosystem than is normally presumed. Characterization of these communities is the first step in elucidating potential roles of diverse bacteria in disease progression and to ultimately facilitate advances in CF therapy.

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses.

Optimal methods for estimating kinetic isotope effects from different forms of the Rayleigh distillation equation

Abstract The interpretation of stable isotope values hinges on precise, accurate estimates of kinetic isotope effects (α), which are equal to 1k/2k, where 1k and 2k are the reaction rate constants for the two isotopes. Kinetic isotope effects are commonly determined by monitoring the reactant concentration (C) and isotope ratio (R) relative to their initial values (C1 and R1, respectively). Values of α are estimated from the C and R values by using the Rayleigh distillation equation (RDE). (A) (R/R 1 )=(C/C 1 ) 1/α−1 We conducted simulation experiments to evaluate the precision of the many different published approaches of estimating α from linearized versions of the RDE and reached the following conclusions: (a) kinetic isotope effects estimated from the slope of the line ln(R) vs. ln(C) were accurate and precise; (b) regressing ln(Ri/Rj) on ln(Ci/Cj), where all i datapoints were compared to all preceding datapoints j resulted in inflated 95% confidence intervals; (c) forcing the regression of ln(R/R1) on ln(C/C1) through the origin resulted in 95% confidence intervals for α that covered the true value less than 90% of the time; and (d) regression methods that compensate for errors in both x and y values need to be used with caution. When combining multiple datasets, values of α were sensitive to the form of the equation and the level of error. If all datasets had the same level of error, the optimal estimate of α was achieved by a linear regression with dummy variables. However, when the three datasets had different levels of error, the optimal estimate of α and much narrower 95% confidence intervals were obtained by using the Pitman estimator. Our study demonstrates that some of the other methods jeopardize the accuracy and precision of empirically determined kinetic isotope effects, thus confounding the interpretation of stable isotope values in the environment.

Quantifying Nonspecific TEM β-Lactamase (blaTEM) Genes in a Wastewater Stream

ABSTRACT To control the antibiotic resistance epidemic, it is necessary to understand the distribution of genetic material encoding antibiotic resistance in the environment and how anthropogenic inputs, such as wastewater, affect this distribution. Approximately two-thirds of antibiotics administered to humans are β-lactams, for which the predominant bacterial resistance mechanism is hydrolysis by β-lactamases. Of the β-lactamases, the TEM family is of overriding significance with regard to diversity, prevalence, and distribution. This paper describes the design of DNA probes universal for all known TEM β-lactamase genes and the application of a quantitative PCR assay (also known as Taqman) to quantify these genes in environmental samples. The primer set was used to study whether sewage, both treated and untreated, contributes to the spread of these genes in receiving waters. It was found that while modern sewage treatment technologies reduce the concentrations of these antibiotic resistance genes, the ratio of blaTEM genes to 16S rRNA genes increases with treatment, suggesting that bacteria harboring blaTEM are more likely to survive the treatment process. Thus, β-lactamase genes are being introduced into the environment in significantly higher concentrations than occur naturally, creating reservoirs of increased resistance potential.

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.

Lateral Symbiont Acquisition in a Maternally Transmitted Chemosynthetic Clam Endosymbiosis

Deep-sea clams of the family Vesicomyidae live in symbiosis with intracellular chemosynthetic bacteria. These symbionts are transmitted maternally (vertically) between host generations and should therefore show a pattern of genetic variation paralleling that of the cotransmitted host mitochondrion. However, instances of lateral (nonvertical) symbiont acquisition could still occur, thereby decoupling symbiont and mitochondrial phylogenies. Here, we provide the first evidence against strict maternal cotransmission of symbiont and mitochondrial genomes in vesicomyids. Analysis of Vesicomya sp. mt-II clams from hydrothermal vents on the Juan de Fuca Ridge (northeastern Pacific) revealed a symbiont phylotype (designated symB(VII)) highly divergent from previously described symbionts of the same host lineage. SymB(VII)-hosting clams occurred at low frequency (0.02) relative to individuals hosting the dominant symbiont phylotype. Phylogenetic analysis of 16S rRNA genes from a wide range of symbionts and free-living bacteria clustered symB(VII) within the monophyletic clade of vesicomyid symbionts. Further analysis of 3 symbiont loci (23S, dnaK, and soxA) across 11 vesicomyid taxa unambiguously placed symB(VII) as sister to the symbiont of a distantly related host lineage, Vesicomya sp. from the Mid-Atlantic Ridge (98.9% median nucleotide identity across protein-coding loci). Using likelihood and Bayesian model discrimination methods, we rejected the strict maternal cotransmission hypothesis by showing a significant decoupling of symbiont and host mitochondrial (COI and mt16S genes) phylogenies. Indeed, decoupling occurred even when symB(VII) was excluded from phylogenetic reconstructions, suggesting a history of host switching in this group. Together, the data indicate a history of lateral symbiont transfer in vesicomyids, with symB(VII) being the most conspicuous example. Interpreted alongside previous studies of the vesicomyid symbiosis, these results suggest a mixed mode of symbiont transmission characterized by predominantly vertical transmission punctuated with instances of lateral symbiont acquisition. Lateral acquisition may facilitate the exchange of genetic material (recombination) among divergent symbiont lineages, rendering the evolutionary history of vesicomyid symbiont genomes much more complex than previously thought.

Intragenomic Variation and Evolution of the Internal Transcribed Spacer of the rRNA Operon in Bacteria

Variation in the internal transcribed spacer (ITS) of the rRNA (rrn) operon is increasingly used to infer population-level diversity in bacterial communities. However, intragenomic ITS variation may skew diversity estimates that do not correct for multiple rrn operons within a genome. This study characterizes variation in ITS length, tRNA composition, and intragenomic nucleotide divergence across 155 Bacteria genomes. On average, these genomes encode 4.8 rrn operons (range: 2–15) and contain 2.4 unique ITS length variants (range: 1–12) and 2.8 unique sequence variants (range: 1–12). ITS variation stems primarily from differences in tRNA gene composition, with ITS regions containing tRNA-Ala + tRNA-Ile (48% of sequences), tRNA-Ala or tRNA-Ile (10%), tRNA-Glu (11%), other tRNAs (3%), or no tRNA genes (27%). Intragenomic divergence among paralogous ITS sequences grouped by tRNA composition ranges from 0% to 12.11% (mean: 0.94%). Low divergence values indicate extensive homogenization among ITS copies. In 78% of alignments, divergence is <1%, with 54% showing zero variation and 81% containing at least two identical sequences. ITS homogenization occurs over relatively long sequence tracts, frequently spanning the entire ITS, and is largely independent of the distance (basepairs) between operons. This study underscores the potential contribution of interoperon ITS variation to bacterial microdiversity studies, as well as unequivocally demonstrates the pervasiveness of concerted evolution in the rrn gene family.

Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria

ABSTRACT 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.