Xiaozhen Mou

Bacterial carbon processing by generalist species in the coastal ocean

The assimilation and mineralization of dissolved organic carbon (DOC) by marine bacterioplankton is a major process in the ocean carbon cycle. However, little information exists on the specific metabolic functions of participating bacteria and on whether individual taxa specialize on particular components of the marine DOC pool. Here we use experimental metagenomics to show that coastal communities are populated by taxa capable of metabolizing a wide variety of organic carbon compounds. Genomic DNA captured from bacterial community subsets metabolizing a single model component of the DOC pool (either dimethylsulphoniopropionate or vanillate) showed substantial overlap in gene composition as well as a diversity of carbon-processing capabilities beyond the selected phenotypes. Our direct measure of niche breadth for bacterial functional assemblages indicates that, in accordance with ecological theory, heterogeneity in the composition and supply of organic carbon to coastal oceans may favour generalist bacteria. In the important interplay between microbial community structure and biogeochemical cycling, coastal heterotrophic communities may be controlled less by transient changes in the carbon reservoir that they process and more by factors such as trophic interactions and physical conditions.

Transporter genes expressed by coastal bacterioplankton in response to dissolved organic carbon

Coastal ocean bacterioplankton control the flow of dissolved organic carbon (DOC) from terrestrial and oceanic sources into the marine food web, and regulate the release of inorganic carbon to atmospheric and offshore reservoirs. While the fate of the chemically complex coastal DOC reservoir has long been recognized as a critical feature of the global carbon budget, it has been problematic to identify both the compounds that serve as major conduits for carbon flux and the roles of individual bacterioplankton taxa in mediating that flux. Here we analyse random libraries of expressed genes from a coastal bacterial community to identify sequences representing DOC-transporting proteins. Predicted substrates of expressed transporter genes indicated that carboxylic acids, compatible solutes, polyamines and lipids may be key components of the biologically labile DOC pool in coastal waters, in addition to canonical bacterial substrates such as amino acids, oligopeptides and carbohydrates. Half of the expressed DOC transporter sequences in this coastal ocean appeared to originate from just eight taxa: Roseobacter, SAR11, Flavobacteriales and five orders of γ-Proteobacteria. While all major taxa expressed transporter genes for some DOC components (e.g. amino acids), there were indications of specialization within the bacterioplankton community for others (e.g. carbohydrates, carboxylic acids and polyamines). Experimental manipulations of the natural DOC pool that increased the concentration of phytoplankton- or vascular plant-derived compounds invoked a readily measured response in bacterial transporter gene expression. This highly resolved view of the potential for carbon flux into heterotrophic bacterioplankton cells identifies possible bioreactive components of the coastal DOC pool and highlights differing ecological roles in carbon turnover for the resident bacterial taxa.

Metagenomic Identification of Bacterioplankton Taxa and Pathways Involved in Microcystin Degradation in Lake Erie

Cyanobacterial harmful blooms (CyanoHABs) that produce microcystins are appearing in an increasing number of freshwater ecosystems worldwide, damaging quality of water for use by human and aquatic life. Heterotrophic bacteria assemblages are thought to be important in transforming and detoxifying microcystins in natural environments. However, little is known about their taxonomic composition or pathways involved in the process. To address this knowledge gap, we compared the metagenomes of Lake Erie free-living bacterioplankton assemblages in laboratory microcosms amended with microcystins relative to unamended controls. A diverse array of bacterial phyla were responsive to elevated supply of microcystins, including Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria of the alpha, beta, gamma, delta and epsilon subdivisions and Verrucomicrobia. At more detailed taxonomic levels, Methylophilales (mainly in genus Methylotenera) and Burkholderiales (mainly in genera Bordetella, Burkholderia, Cupriavidus, Polaromonas, Ralstonia, Polynucleobacter and Variovorax) of Betaproteobacteria were suggested to be more important in microcystin degradation than Sphingomonadales of Alphaproteobacteria. The latter taxa were previously thought to be major microcystin degraders. Homologs to known microcystin-degrading genes (mlr) were not overrepresented in microcystin-amended metagenomes, indicating that Lake Erie bacterioplankton might employ alternative genes and/or pathways in microcystin degradation. Genes for xenobiotic metabolism were overrepresented in microcystin-amended microcosms, suggesting they are important in bacterial degradation of microcystin, a phenomenon that has been identified previously only in eukaryotic systems.

Flow-Cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-Independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations

ABSTRACT Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 μM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, α-Proteobacteria); Caulobacter (α-Proteobacteria); and Brachymonas and Xenophilus (β-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, α-Proteobacteria) and novel γ-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally.

Metatranscriptomic signature of exogenous polyamine utilization by coastal bacterioplankton

The polyamines putrescine (PUT) and spermidine (SPD) are ubiquitous in seawater, but mechanisms that drive the degradation of these important nitrogen sources by marine bacteria remain unclear. We employed a comparative metatranscriptomics approach to compare gene transcription patterns between coastal bacterioplankton communities with and without amendments of PUT or SPD, in an effort to understand how bacterial communities and their genes shape polyamine biogeochemistry in the ocean. Statistically different transcript categories in the PUT (25 COG groups) and SPD (23 COG groups) samples, relative to controls that received no amendment (CTRL), indicated that genes encoding the cellular translation machinery and the metabolism of organic nitrogen and carbon became enriched in the community transcriptome when polyamine availability increased. Of the three known pathways for bacterial polyamine degradation, only genes in the transamination pathway were enriched in the PUT and SPD libraries, suggesting that this route dominated polyamine degradation. Taxonomic affiliation of significantly enriched diagnostic genes in the PUT and SPD libraries pointed to roseobacter- and SAR11-affiliated bacteria as the predominant taxa driving transformation in this coastal ocean, although other diverse marine bacterioplankton groups (Gammaproteobacteria, Betaproteobacteria, Actinobacteria and Bacteroidetes) also contributed to polyamine-related gene transcription.

Ammonia-oxidizing Archaea in Kamchatka Hot Springs

Mounting evidence suggests that ammonia-oxidizing archaea (AOA) may play important roles in nitrogen cycling in geothermal environments. In this study, the diversity, distribution and ecological significance of AOA in terrestrial hot springs in Kamchatka (Far East Russia) were explored using amoA genes complemented by analysis of glycerol dialkyl glycerol tetraethers (GDGTs) of archaea. PCR amplification of functional genes (amoA) from AOA and ammonia-oxidizing bacteria (AOB) was performed on microbial mats/streamers and sediments collected from three hot springs (42°C to 87°C and pH 5.5-7.0). No amoA genes of AOB were detected. The amoA genes of AOA formed three distinct phylogenetic clusters with Cluster 3 representing the majority (∼59%) of OTUs. Some of the sequences from Cluster 3 were closely related to those from acidic soil environments, which is consistent with the predominance of low pH (<7.0) in these hot springs. Species richness (estimated by Chao1) was more frequently higher at temperatures below 75°C than above it, indicating that AOA may be favored in the moderately high temperature environments. Quantitative PCR of 16S rRNA genes showed that crenarchaeota counted for up to 80% of total archaea. S-LIBSHUFF separated all samples into two phylogenetic groups. The profiles of GDGTs were well separated among the studied springs, suggesting a spatial patterning of archaeal lipid biomarkers. However, this patterning did not correlate significantly with variation in archaeal amoA, suggesting that AOA are not the predominant archaeal group in these springs producing the observed GDGTs.

Algicidal Activity of Streptomyces eurocidicus JXJ-0089 Metabolites and Their Effects on Microcystis Physiology

ABSTRACT Copper sulfate (CuSO4) has been widely used as an algicide to control harmful cyanobacterial blooms (CyanoHABs) in freshwater lakes. However, there are increasing concerns about this application, due mainly to the general toxicity of CuSO4 to other aquatic species and its long-term persistence in the environment. This study reported the isolation and characterization of two natural algicidal compounds, i.e., tryptamine and tryptoline, from Streptomyces eurocidicus JXJ-0089. At a concentration of 5 μg/ml, both compounds showed higher algicidal efficiencies than CuSO4 on Microcystis sp. FACHB-905 and some other harmful cyanobacterial strains. Tryptamine and tryptoline treatments induced a degradation of chlorophyll and cell walls of cyanobacteria. These two compounds also significantly increased the intracellular oxidant content, i.e., superoxide anion radical (O2 −) and malondialdehyde (MDA), but reduced the activity of intracellular reductants, i.e., superoxide dismutase (SOD), of cyanobacteria. Moreover, tryptamine and tryptoline treatments significantly altered the internal and external contents of microcystin-LR (MC-LR), a common cyanotoxin. Like CuSO4, tryptamine and tryptoline led to releases of intracellular MC-LR from Microcystis, but with lower rates than CuSO4. Tryptamine and tryptoline (5 μg/ml) in cyanobacterial cultures were completely degraded within 8 days, while CuSO4 persisted for months. Overall, our results suggest that tryptamine and tryptoline could potentially serve as more efficient and environmentally friendly alternative algicides than CuSO4 in controlling harmful cyanobacterial blooms. IMPORTANCE Cyanobacterial harmful algal blooms (CyanoHABs) in aquatic environments have become a worldwide problem. Numerous efforts have been made to seek means to prevent, control, and mitigate CyanoHABs. Copper sulfate (CuSO4), was once a common algicide to treat and control CyanoHABs. However, its application has become limited due to concerns about its general toxicity to other aquatic species and its long-term persistence in the environment. There is a great need for algicides with higher specificity and low environmental impacts. This study reports the isolation and characterization of two natural algicidal compounds from a streptomycete strain, Streptomyces eurocidicus JXJ-0089. Our results suggest that the identified algicides could potentially serve as more efficient and environmentally friendly alternative algicides than CuSO4 in controlling harmful cyanobacterial blooms.

Temporal and Vertical Distributions of Bacterioplankton at the Gray's Reef National Marine Sanctuary

ABSTRACT Large spatial scales and long-term shifts of bacterial community composition (BCC) in the open ocean can often be reliably predicted based on the dynamics of physical-chemical variables. The power of abiotic factors in shaping BCC on shorter time scales in shallow estuarine mixing zones is less clear. We examined the diurnal variation in BCC at different water depths in the spring and fall of 2011 at a station in the Grays Reef National Marine Sanctuary (GRNMS). This site is located in the transition zone between the estuarine plume and continental shelf waters of the South Atlantic Bight. A total of 234,516 pyrotag sequences of bacterial 16S rRNA genes were recovered; they were taxonomically affiliated with >200 families of 23 bacterial phyla. Nonmetric multidimensional scaling analysis revealed significant differences in BCC between spring and fall samples, likely due to seasonality in the concentrations of dissolved organic carbon and nitrate plus nitrite. Within each diurnal sampling, BCC differed significantly by depth only in the spring and differed significantly between day and night only in the fall. The former variation largely tracked changes in light availability, while the latter was most correlated with concentrations of polyamines and chlorophyll a. Our results suggest that at the GRNMS, a coastal mixing zone, diurnal variation in BCC is attributable to the mixing of local and imported bacterioplankton rather than to bacterial growth in response to environmental changes. Our results also indicate that, like members of the Roseobacter clade, SAR11 bacteria may play an important role in processing dissolved organic material in coastal oceans.

T-RFPred: a nucleotide sequence size prediction tool for microbial community description based on terminal-restriction fragment length polymorphism chromatograms

BackgroundTerminal-Restriction Fragment Length Polymorphism (T-RFLP) is a technique used to analyze complex microbial communities. It allows for the quantification of unique or numerically dominant phylotypes in amplicon pools and it has been used primarily for comparisons between different communities. T-RFPred, Terminal-Restriction Fragment Prediction, was developed to identify and assign taxonomic information to chromatogram peaks of a T-RFLP fingerprint for a more comprehensive description of microbial communities. The program estimates the expected fragment size of representative 16S rRNA gene sequences (either from a complementary clone library or from public databases) for a given primer and restriction enzyme(s) and provides candidate taxonomic assignments.ResultsTo show the accuracy of the program, T-RFLP profiles of a marine bacterial community were described using artificial bacterioplankton clone libraries of sequences obtained from public databases. For all valid chromatogram peaks, a phylogenetic group could be assigned.ConclusionsT-RFPred offers enhanced functionality of T-RFLP profile analysis over current available programs. In particular, it circumvents the need for full-length 16S rRNA gene sequences during taxonomic assignments of T-RF peaks. Thus, large 16S rRNA gene datasets from environmental studies, including metagenomes, or public databases can be used as the reference set. Furthermore, T-RFPred is useful in experimental design for the selection of primers as well as the type and number of restriction enzymes that will yield informative chromatograms from natural microbial communities.

Identification of polyamine‐responsive bacterioplankton taxa in South Atlantic Bight

Putrescine and spermidine are short-chained aliphatic polyamines (PAs) that are ubiquitously distributed in seawater. These compounds may be important sources of dissolved organic carbon and nitrogen for marine bacterioplankton. Here, we used pyrotag sequencing to quantify the response of bacterioplankton to putrescine and spermidine amendments in microcosms established using surface waters collected at various stations in the South Atlantic Bight in October 2011. Our analysis showed that PA-responsive bacterioplankton consisted of bacterial taxa that are typically dominant in marine systems. Rhodobacteraceae (Alphaproteobacteria) was the taxon most responsive to PA additions at the nearshore site. Gammaproteobacteria of the families Piscirickettsiaceae; Vibrionaceae; and Vibrionaceae and Pseudoalteromonadaceae, were the dominant PA-responsive taxa in samples from the river-influenced coastal station, offshore station and open ocean station, respectively. The spatial variability of PA-responsive taxa may be attributed to differences in composition of the initial bacterial community and variations of in situ physiochemical conditions among sites. Our results also provided the first empirical evidence that Gammaproteobacteria might play an important role in PA transformation in marine systems.