Robert M. Morris

SAR11 clade dominates ocean surface bacterioplankton communities

The most abundant class of bacterial ribosomal RNA genes detected in seawater DNA by gene cloning belongs to SAR11—an α-proteobacterial clade. Other than indications of their prevalence in seawater, little is known about these organisms. Here we report quantitative measurements of the cellular abundance of the SAR11 clade in northwestern Sargasso Sea waters to 3,000 m and in Oregon coastal surface waters. On average, the SAR11 clade accounts for a third of the cells present in surface waters and nearly a fifth of the cells present in the mesopelagic zone. In some regions, members of the SAR11 clade represent as much as 50% of the total surface microbial community and 25% of the subeuphotic microbial community. By extrapolation, we estimate that globally there are 2.4 × 1028 SAR11 cells in the oceans, half of which are located in the euphotic zone. Although the biogeochemical role of the SAR11 clade remains uncertain, these data support the conclusion that this microbial group is among the most successful organisms on Earth.

Untangling Genomes from Metagenomes: Revealing an Uncultured Class of Marine Euryarchaeota

Mystery of an Unextreme Microbe Metagenomics has given us glimpses into the huge diversity of microorganisms that are the engines of Earths elemental cycling. These kinds of surveys can supply a good idea of the dominant organisms in ecosystems, but, because the majority of environmental microbes are difficult to culture and the most abundant organisms swamp metagenomes, it is difficult to discern the functional significance of other contributors. Iverson et al. (p. 587) sampled the Puget Sound and developed methods to reconstruct individual genomes from metagenomes, which allowed closure of a genome from the enigmatic marine group II Euryarchaeota. This Archaean is evidently motile, shows signs of extensive gene-swapping with bacteria, and offers some hints to the origin of proteorhodopsin—a molecule that some marine bacteria use to harvest energy from sunlight. Reconstruction of whole genomes from a complex microbial community has revealed an evolutionary surprise. Ecosystems are shaped by complex communities of mostly unculturable microbes. Metagenomes provide a fragmented view of such communities, but the ecosystem functions of major groups of organisms remain mysterious. To better characterize members of these communities, we developed methods to reconstruct genomes directly from mate-paired short-read metagenomes. We closed a genome representing the as-yet uncultured marine group II Euryarchaeota, assembled de novo from 1.7% of a metagenome sequenced from surface seawater. The genome describes a motile, photo-heterotrophic cell focused on degradation of protein and lipids and clarifies the origin of proteorhodopsin. It also demonstrates that high-coverage mate-paired sequence can overcome assembly difficulties caused by interstrain variation in complex microbial communities, enabling inference of ecosystem functions for uncultured members.

Seasonal dynamics of SAR11 populations in the euphotic and mesopelagic zones of the northwestern Sargasso Sea

Bacterioplankton belonging to the SAR11 clade of a-proteobacteria were counted by fluorescence in situ hybridization (FISH) over eight depths in the surface 300 m at the Bermuda Atlantic Time-series Study (BATS) site from 2003 to 2005. SAR11 are dominant heterotrophs in oligotrophic systems; thus, resolving their temporal dynamics can provide important insights to the cycling of organic and inorganic nutrients. This quantitative time-series data revealed distinct annual distribution patterns of SAR11 abundance in the euphotic (0–120) and upper mesopelagic (160–300 m) zones that were reproducibly correlated with seasonal mixing and stratification of the water column. Terminal restriction fragment length polymorphism (T-RFLP) data generated from a decade of samples collected at BATS were combined with the FISH data to model the annual dynamics of SAR11 subclade populations. 16S rRNA gene clone libraries were constructed to verify the correlation of the T-RFLP data with SAR11 clade structure. Clear vertical and temporal transitions were observed in the dominance of three SAR11 ecotypes. The mechanisms that lead to shifts between the different SAR11 populations are not well understood, but are probably a consequence of finely tuned physiological adaptations that partition the populations along physical and chemical gradients in the ecosystem. The correlation between evolutionary descent and temporal/spatial patterns we describe, confirmed that a minimum of three SAR11 ecotypes occupy the Sargasso Sea surface layer, and revealed new details of their population dynamics.

Comparative metaproteomics reveals ocean-scale shifts in microbial nutrient utilization and energy transduction

Bacteria and Archaea play critical roles in marine energy fluxes and nutrient cycles by incorporating and redistributing dissolved organic matter and inorganic nutrients in the oceans. How these microorganisms do this work at the level of the expressed protein is known only from a few studies of targeted lineages. We used comparative membrane metaproteomics to identify functional responses of communities to different nutrient concentrations on an oceanic scale. Comparative analyses of microbial membrane fractions revealed shifts in nutrient utilization and energy transduction along an environmental gradient in South Atlantic surface waters, from a low-nutrient gyre to a highly productive coastal upwelling region. The dominant membrane proteins identified (19%) were TonB-dependent transporters (TBDTs), which are known to utilize a proton motive force to transport nutrients across the outer membrane of Gram-negative bacteria. The ocean-wide importance of TonB-dependent nutrient acquisition in marine bacteria was unsuspected. Diverse light-harvesting rhodopsins were detected in membrane proteomes from every sample. Proteomic evidence of both TBDTs and rhodopsins in the same lineages suggest that phototrophic bacterioplankton have the potential to use energy from light to fuel transport activities. We also identified viral proteins in every sample and archaeal ammonia monooxygenase proteins in the upwelling region, suggesting that Archaea are important nitrifiers in nutrient-rich surface waters.

Comparative Proteomics of Dehalococcoides spp. Reveals Strain-Specific Peptides Associated with Activity

ABSTRACT Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.

Expression of Reductive Dehalogenase Genes in Dehalococcoides ethenogenes Strain 195 Growing on Tetrachloroethene, Trichloroethene, or 2,3-Dichlorophenol

ABSTRACT Reductive dehalogenase (RD) gene transcript levels in Dehalococcoides ethenogenes strain 195 were investigated using reverse transcriptase quantitative PCR during growth and reductive dechlorination of tetrachloroethene (PCE), trichloroethene (TCE), or 2,3-dichlorophenol (2,3-DCP). Cells grown with PCE or TCE had high transcript levels (greater than that for rpoB) for tceA, which encodes the TCE RD, pceA, which encodes the PCE RD, and DET0162, which contains a predicted stop codon and is considered nonfunctional. In cells grown with 2,3-DCP, tceA mRNA was less than 1% of that for rpoB, indicating that its transcription was regulated. pceA and DET0162 were the only RD genes with high transcript levels in cells grown with 2,3-DCP. Proteomic analysis of PCE-grown cells detected both PceA and TceA with high peptide coverage but not DET0162, and analysis of 2,3-DCP-grown cells detected PceA with high coverage but not TceA, DET0162, or any other potential RD. Cells grown with PCE or 2,3-DCP were tested for the ability to dechlorinate PCE, TCE, or 2,3-DCP with H2 as the electron donor. 2,3-DCP-grown cells were unable to dechlorinate TCE but dechlorinated PCE to TCE without a lag, and PCE-grown cells dechlorinated 2,3-DCP without a lag. These results show that 2,3-DCP-grown cells do not produce TceA and that DET0162 is transcribed but its translation product is not detectable in cells and are consistent with PceAs being bifunctional, also serving as the 2,3-DCP RD. Chlorophenols naturally occur in soils and are good candidates for the original substrates for PceA.

Prevalence of the Chloroflexi-Related SAR202 Bacterioplankton Cluster throughout the Mesopelagic Zone and Deep Ocean†

ABSTRACT Since their initial discovery in samples from the north Atlantic Ocean, 16S rRNA genes related to the environmental gene clone cluster known as SAR202 have been recovered from pelagic freshwater, marine sediment, soil, and deep subsurface terrestrial environments. Together, these clones form a major, monophyletic subgroup of the phylum Chloroflexi. While members of this diverse group are consistently identified in the marine environment, there are currently no cultured representatives, and very little is known about their distribution or abundance in the worlds oceans. In this study, published and newly identified SAR202-related 16S rRNA gene sequences were used to further resolve the phylogeny of this cluster and to design taxon-specific oligonucleotide probes for fluorescence in situ hybridization. Direct cell counts from the Bermuda Atlantic time series study site in the north Atlantic Ocean, the Hawaii ocean time series site in the central Pacific Ocean, and along the Newport hydroline in eastern Pacific coastal waters showed that SAR202 cluster cells were most abundant below the deep chlorophyll maximum and that they persisted to 3,600 m in the Atlantic Ocean and to 4,000 m in the Pacific Ocean, the deepest samples used in this study. On average, members of the SAR202 group accounted for 10.2% (±5.7%) of all DNA-containing bacterioplankton between 500 and 4,000 m.

Temporal Expression of Respiratory Genes in an Enrichment Culture Containing Dehalococcoides ethenogenes

ABSTRACT Multiple reductive dehalogenase (RDase), hydrogenase (H2ase), and other respiration-associated (RA) oxidoreductase genes have been identified in cultured representatives of Dehalococcoides. Although their products are likely to play key roles in the environmentally important process of reductive dechlorination, very little information is available about their regulation and specific functions. Here we show increased expression and temporal variability in the expression of five RDase genes and in the expression of genes for a putative formate dehydrogenase (Fdh) and two H2ases, including a periplasmic [Ni/Fe] H2ase (Hup) and a cytoplasmic [Fe] H2ase (Vhu). mRNA transcripts extracted from tetrachloroethene-dechlorinating mixed cultures corresponding to Fdh, the H2ase Hup, and the RDase targets TceA and DET0162 were expressed most highly, with average levels 34 (± 7.5)-, 23 (± 6.7)-, 16 (± 3.3)-, and 13 (± 3.3)-fold higher, respectively, than that for RNA polymerase (RpoB). H2ase and RA transcripts reached their respective expression maxima within the first 2 h after feeding. RDase transcripts, however, were most highly expressed after 3 h and exhibited greater temporal variability than other transcripts. Comparison with D. ethenogenes strain 195 pure culture expression levels indicated that RDase DET1545 was more highly expressed in mixed cultures, where, on average, its transcript level was sixfold higher than that of RpoB. While the specific functions of several of these gene products remain elusive, the high expression levels and temporal variability reported here suggest that these groups of enzymes are metabolically important for the respiration of chlorinated ethenes in mixed cultures containing Dehalococcoides.

Isolation of an aerobic sulfur oxidizer from the SUP05/Arctic96BD-19 clade

Bacteria from the uncultured SUP05/Arctic96BD-19 clade of gamma proteobacterial sulfur oxidizers (GSOs) have the genetic potential to oxidize reduced sulfur and fix carbon in the tissues of clams and mussels, in oxygen minimum zones and throughout the deep ocean (>200 m). Here, we report isolation of the first cultured representative from this GSO clade. Closely related cultures were obtained from surface waters in Puget Sound and from the deep chlorophyll maximum in the North Pacific gyre. Pure cultures grow aerobically on natural seawater media, oxidize sulfur, and reach higher final cell densities when glucose and thiosulfate are added to the media. This suggests that aerobic sulfur oxidation enhances organic carbon utilization in the oceans. The first isolate from the SUP05/Arctic96BD-19 clade was given the provisional taxonomic assignment ‘Candidatus: Thioglobus singularis’, alluding to the clade’s known role in sulfur oxidation and the isolate’s planktonic lifestyle.

Characterization of the Community Structure of a Dechlorinating Mixed Culture and Comparisons of Gene Expression in Planktonic and Biofloc-Associated "Dehalococcoides" and Methanospirillum Species†

ABSTRACT This study sought to characterize bacterial and archaeal populations in a perchloroethene- and butyrate-fed enrichment culture containing hydrogen-consuming “Dehalococcoides ethenogenes” strain 195 and a Methanospirillum hungatei strain. Phylogenetic characterization of this microbial community was done via 16S rRNA gene clone library and gradient gel electrophoresis analyses. Fluorescence in situ hybridization was used to quantify populations of “Dehalococcoides” and Archaea and to examine the colocalization of these two groups within culture bioflocs. A technique for enrichment of planktonic and biofloc-associated biomass was developed and used to assess differences in population distribution and gene expression patterns following provision of substrate. On a per-milliliter-of-culture basis, most D. ethenogenes genes (the hydrogenase gene hupL; the highly expressed gene for an oxidoreductase of unknown function, fdhA; the RNA polymerase subunit gene rpoB; and the 16S rRNA gene) showed no statistical difference in expression between planktonic and biofloc enrichments at either time point studied (1 to 2 and 6 h postfeeding). Normalization of transcripts to ribosome (16S rRNA) levels supported that planktonic and biofloc-associated D. ethenogenes had similar gene expression profiles, with one notable exception; planktonic D. ethenogenes showed higher expression of tceA relative to biofloc-associated cells at 6 h postfeeding. These trends were compared to those for the hydrogen-consuming methanogen in the culture, M. hungatei. The vast majority of M. hungatei cells, ribosomes (16S rRNA), and transcripts of the hydrogenase gene mvrD and the housekeeping gene rpoE were observed in the biofloc enrichments. This suggests that, unlike the comparable activity of D. ethenogenes from both enrichments, planktonic M. hungatei is responsible for only a small fraction of the hydrogenotrophic methanogenesis in this culture.