Hongmei Jing

Detection of Microcystin-Producing Cyanobacteria in Missisquoi Bay, Quebec, Canada, Using Quantitative PCR

ABSTRACT Toxic cyanobacterial blooms, as well as their increasing global occurrence, pose a serious threat to public health, domestic animals, and livestock. In Missisquoi Bay, Lake Champlain, public health advisories have been issued from 2001 to 2009, and local microcystin concentrations found in the lake water regularly exceeded the Canadian drinking water guideline of 1.5 μg liter−1. A quantitative PCR (Q-PCR) approach was developed for the detection of blooms formed by microcystin-producing cyanobacteria. Primers were designed for the β-ketoacyl synthase (mcyDKS) and the first dehydratase domain (mcyDDH) of the mcyD gene, involved in microcystin synthesis. The Q-PCR method was used to track the toxigenic cyanobacteria in Missisquoi Bay during the summers of 2006 and 2007. Two toxic bloom events were detected in 2006: more than 6.5 × 104 copies of the mcyDKS gene ml−1 were detected in August, and an average of 4.0 × 104 copies ml−1 were detected in September, when microcystin concentrations were more than 4 μg liter−1 and approximately 2 μg liter−1, respectively. Gene copy numbers and total microcystin concentrations (determined by enzyme-linked immunosorbent assay [ELISA]) were highly correlated in the littoral (r = 0.93, P < 0.001) and the pelagic station (r = 0.87, P < 0.001) in 2006. In contrast to the situation in 2006, a cyanobacterial bloom occurred only in late summer-early fall of 2007, reaching only 3 × 102mcyDKS copies ml−1, while the microcystin concentration was barely detectable. The Q-PCR method allowed the detection of microcystin-producing cyanobacteria when toxins and toxigenic cyanobacterial abundance were still below the limit of detection by high-pressure liquid chromatography (HPLC) and microscopy. Toxin gene copy numbers grew exponentially at a steady rate over a period of 7 weeks. Onshore winds selected for cells with a higher cell quota of microcystin. This technique could be an effective approach for the routine monitoring of the most at-risk water bodies.

Vertical profiles of bacteria in the tropical and subarctic oceans revealed by pyrosequencing.

Community composition of Bacteria in the surface and deep water layers were examined at three oceanic sites in the Pacific Ocean separated by great distance, i.e., the South China Sea (SCS) in the western tropical Pacific, the Costa Rica Dome (CRD) in the eastern tropical Pacific and the western subarctic North Pacific (SNP), using high throughput DNA pyrosequencing of the 16S rRNA gene. Bioinformatic analysis rendered a total of 143600 high quality sequences with an average 11967 sequences per sample and mean read length of 449 bp. Phylogenetic analysis showed that Proteobacteria dominated in all shallow and deep waters, with Alphaproteobacteria and Gammaproteobacteria the two most abundant components, and SAR11 the most abundant group at family level in all regions. Cyanobacteria occurred mainly in the surface euphotic layer, and the majority of them in the tropical waters belonged to the GpIIa family including Prochlorococcus and Synechococcus, whilst those associated with Cryptophytes and diatoms were common in the subarctic waters. In general, species richness (Chao1) and diversity (Shannon index H′) were higher for the bacterial communities in the intermediate water layers than for those in surface and deep waters. Both NMDS plot and UPGMA clustering demonstrated that bacterial community composition in the deep waters (500 m ∼2000 m) of the three oceanic regions shared a high similarity and were distinct from those in the upper waters (5 m ∼100 m). Our study indicates that bacterial community composition in the DOC-poor deep water in both tropical and subarctic regions were rather stable, contrasting to those in the surface water layers, which could be strongly affected by the fluctuations of environmental factors.

Characterization of the Proteomic Profiles of the Brown Tide Alga Aureoumbra lagunensis under Phosphate- and Nitrogen-Limiting Conditions and of Its Phosphate Limitation-Specific Protein with Alkaline Phosphatase Activity

ABSTRACT The persistent bloom of the brown tide alga Aureoumbra lagunensis has been reported in coastal embayments along southern Texas, but the molecular mechanisms that sustain such algal bloom are unknown. We compared the proteome and physiological parameters of A. lagunensis grown in phosphate (P)-depleted, P- and nitrogen (N)-depleted, and nutrient-replete cultures. For the proteomic analysis, samples from three conditions were subjected to two-dimensional electrophoresis and tandem mass spectrometry analysis. Because of the paucity of genomic resources in this species, a de novo cross-species protein search was used to identify the differentially expressed proteins, which revealed their involvement in several key biological processes, such as chlorophyll synthesis, antioxidative protection, and protein degradation, suggesting that A. lagunensis may adopt intracellular nutrient compensation, extracellular organic nutrient regeneration, and damage protection to thrive in P-depleted environments. A highly abundant P limitation-specific protein, tentatively identified as a putative alkaline phosphatase, was further characterized by enzyme activity assay on nondenaturing gel and confocal microscopy, which confirmed that this protein has alkaline phosphatase activity, is a cytoplasmic protein, and is closely associated with the cell membrane. The abundance, location, and functional expression of this alkaline phosphatase all indicate the importance of organic P utilization for A. lagunensis under P limitation and the possible role of this alkaline phosphatase in regenerating phosphate from extra- or intracellular organic phosphorus.

Co-occurrence of phycocyanin- and phycoerythrin-rich Synechococcus in subtropical estuarine and coastal waters of Hong Kong.

Phylogenetic diversity of Synechococcus with different pigmentation in subtropical estuarine and coastal waters of Hong Kong was revealed by the phylogeny of cpcBA and cpeBA operons encoding for phycocyanin (PC) and phycoerythrin (PE). Synechococcus containing only PC (PC-rich Synechococcus) dominated at the estuarine station in summer, whereas PE-rich marine Synechococcus containing both PC and PE (PE-rich Synechococcus) dominated in the coastal waters. Our PC sequences are closely related to freshwater strains but differed from Baltic Sea strains, implying that they were from river discharge. Among PE-rich Synechococcus, clones grouping with strains containing only phycoerythrobilin (PEB-only) were abundant in July, while clones grouping with strains possessing a low content of phycourobilin (PUB) in addition to PEB (low PUB/PEB) were more abundant in January at both stations. Clones of high PUB/PEB types were only presented at the coastal station, but were not detected at the estuarine station. The much higher diversity of both PC-rich and PE-rich Synechococcus, as compared with the Baltic Sea, and the occurrence of the high PUB/PEB strains indicate the high dynamic nature of this subtropical estuarine-coastal environment with strong mixing of water masses ranging from Pearl River plume to oceanic South China Sea water. Our results of phylogenetic study agreed well with flow cytometric counts, which revealed the coexistence of PC-rich and PE-rich Synechococcus in the subtropical coastal waters and the dominance of the former type in the estuarine waters during summer high freshwater discharge. These results indicate that picocyanobacteria, particularly PC-rich Synechococcus, which has long been overlooked, are an important part of the primary production, and they could play an important role in the microbial food web in estuarine ecosystems.

Diversity and Spatial Distribution of Hydrazine Oxidoreductase (hzo) Gene in the Oxygen Minimum Zone Off Costa Rica

Anaerobic ammonia oxidation (anammox) as an important nitrogen loss pathway has been reported in marine oxygen minimum zones (OMZs), but the community composition and spatial distribution of anammox bacteria in the eastern tropical North Pacific (ETNP) OMZ are poorly determined. In this study, anammox bacterial communities in the OMZ off Costa Rica (CRD-OMZ) were analyzed based on both hydrazine oxidoreductase (hzo) genes and their transcripts assigned to cluster 1 and 2. The anammox communities revealed by hzo genes and proteins in CRD-OMZ showed a low diversity. Gene quantification results showed that hzo gene abundances peaked in the upper OMZs, associated with the peaks of nitrite concentration. Nitrite and oxygen concentrations may therefore colimit the distribution of anammox bacteria in this area. Furthermore, transcriptional activity of anammox bacteria was confirmed by obtaining abundant hzo mRNA transcripts through qRT-PCR. A novel hzo cluster 2x clade was identified by the phylogenetic analysis and these novel sequences were abundant and widely distributed in this environment. Our study demonstrated that both cluster 1 and 2 anammox bacteria play an active role in the CRD-OMZ, and the cluster 1 abundance and transcriptional activity were higher than cluster 2 in both free-living and particle-attached fractions at both gene and transcriptional levels.

Regulation of bacterial metabolic activity by dissolved organic carbon and viruses

The regulation of bacterial metabolic activity by viruses and dissolved organic carbon (DOC) was examined using natural microbial communities in three treatments (active viruses, inactive viruses, and virus free) at two contrasting coastal sites (pristine vs. eutrophic) with substantial differences in environmental conditions during the wet and dry seasons. Our results showed that net growth rates and production of bacterioplankton were reduced primarily by viruses via repressing metabolically active bacteria with high nucleic acid (HNA) content which had a high capacity for incorporating carbon, while bacterial respiration was primarily regulated by DOC lability. The quality of organic matter played a more important role in regulating bacterial growth efficiency (BGE) than the supply of organic matter in eutrophic coastal waters. The lack of HMW-DOC and high carbon demand in the virus-free treatment resulted in a significant increase in cell-specific bacterial respiration, which was responsible for the lowest bacterial growth efficiency among the three treatments. The presence of viruses did not necessarily lower bacterial growth efficiency since virus-induced mortality alleviated bacterial carbon demand and enhanced carbon cycling. Virus-induced mortality was greater in relatively pristine waters than eutrophic waters, likely since the high supply of substrates alleviated the pressure of viral infection, through extracellular proteases produced by bacteria, which might result in the hydrolytic destruction or modification of viral capsids. An important implication of our results was that the input of riverine DOC and nutrients improved bacterial metabolic activity by alleviating virus-induced mortality of bacteria in estuarine and coastal waters.

Effect of Seawater–Sewage Cross-Transplants on Bacterial Metabolism and Diversity

Bioassays experiments were conducted to determine the metabolic and community composition response of bacteria to transplants between relatively pristine coastal seawater and sewage-impacted seawater. There were four treatments: (1) pristine seawater bacteriau2009+u2009pristine seawater (Pbu2009+u2009Pw), (2) sewage-impacted bacteriau2009+u2009sewage-impacted water (Sbu2009+u2009Sw), (3) pristine seawater bacteriau2009+u2009sewage-impacted water (Pbu2009+u2009Sw), and (4) sewage-impacted bacteriau2009+u2009pristine seawater (Sbu2009+u2009Pw). Sewage-derived DOC was more labile and readily utilized by bacteria, which favored the growth of high nucleic acid (HNA) bacteria, resulting in high bacterial production (BP, 113u2009±u20094.92 to 130u2009±u200915.8xa0μg C l−1u2009day−1) and low respiration rate (BR, <67u2009±u200911.3xa0μg C l−1u2009day−1), as well as high bacterial growth efficiency (BGE, 0.68u2009±u20090.09 to 0.71u2009±u20090.05). In contrast, at the relatively pristine site, bacteria utilized natural marine-derived dissolved organic matter (DOM) at the expense of lowering their growth efficiency (BGE, <0.32u2009±u20090.02) with low BP (<62u2009±u20096.3xa0μg C l−1u2009day−1) and high BR 133u2009±u200914.2xa0μg C l−1u2009day−1). Sewage DOM input appeared to alter the partitioning of carbon between respiration and production of bacteria, resulting in a shift toward higher BGE, which would not enhance oxygen consumption. Taxonomic classification based on 454 pyrosequencing reads of the 16S rRNA gene amplicons revealed that changes in bacterial community structure occurred when seawater bacteria were transferred to the eutrophic sewage-impacted water. Sewage DOM fueled the growth of Gammma-proteobacteria and Epsilson-proteobacteria and reduced the bacterial richness, but the changes in the community were not apparent when sewage-impacted bacteria were transferred to pristine seawater.

Phylogenetic composition and distribution of picoeukaryotes in the hypoxic northwestern coast of the Gulf of Mexico

Coastal marine hypoxic, or low‐oxygen, episodes are an increasing worldwide phenomenon, but its effect on the microbial community is virtually unknown by far. In this study, the community structure and phylogeny of picoeukaryotes in the Gulf of Mexico, which are exposed to severe hypoxia in these areas was explored through a clone library approach. Both oxic surface waters and suboxic bottom waters were collected in August 2010 from three representative stations on the inner Louisiana shelf near the Atchafalaya and Mississippi River plumes. The bottom waters of the two more western stations were much more hypoxic in comparison to those of the station closest to the Mississippi River plume, which were only moderately hypoxic. A phylogenetic analysis of a total 175 sequences, generated from six 18S rDNA clone libraries, demonstrated a clear dominance of parasitic dinoflagellates from Marine alveolate clades I and II in all hypoxic waters as well as in the surface layer at the more western station closest to the Atchafalaya River plume. Species diversity was significantly higher at the most hypoxic sites, and many novel species were present among the dinoflagellate and stramenopile clades. We concluded that hypoxia in the Gulf of Mexico causes a significant shift in picoeukaryote communities, and that hypoxia may cause a shift in microbial food webs from grazing to parasitism.

Contrasting Bacterial Dynamics in Subtropical Estuarine and Coastal Waters

Seasonal succession and composition of both attached and free-living bacterial communities were studied in subtropical estuarine and coastal waters with contrasting hydrographic conditions. A higher abundance of attached bacteria was recovered in the estuarine waters containing high concentrations of dissolved organic carbon (DOC) resulting from the freshwater discharge in the adjacent Pearl River, and Proteobacteria, including α-, β-, and γ-groups, predominated the attached community at both stations. Free-living bacterial communities at both stations showed higher diversity and lower seasonality than their attached counterparts, and α-Proteobacteria accounted for the highest proportion at both stations. Redundancy analysis (RDA) demonstrated that, in addition to the obvious temperature effects, DOC and microphytoplankton (>20xa0μm Chl a) drive the temporal variation of attached bacteria at the estuarine and coastal stations, respectively. On the other hand, picophytoplankton (<2xa0μm Chl a) and dissolved oxygen concentration explained most of the free-living bacterial community succession at the estuarine station, while those at the coastal station were associated with micro- and picoplankton (Chl a fractions of <2 and >20xa0μm). These findings suggest that temperature and bottom–up effects play a more important role for the spatial–temporal variations of both attached and free-living bacterial communities in the subtropical estuarine and coastal waters.

Anthropogenic impact on diazotrophic diversity in the mangrove rhizosphere revealed by nifH pyrosequencing

Diazotrophs in the mangrove rhizosphere play a major role in providing new nitrogen to the mangrove ecosystem and their composition and activity are strongly influenced by anthropogenic activity and ecological conditions. In this study, the diversity of the diazotroph communities in the rhizosphere sediment of five tropical mangrove sites with different levels of pollution along the north and south coastline of Singapore were studied by pyrosequencing of the nifH gene. Bioinformatics analysis revealed that in all the studied locations, the diazotroph communities comprised mainly of members of the diazotrophic cluster I and cluster III. The detected cluster III diazotrophs, which were composed entirely of sulfate-reducing bacteria, were more abundant in the less polluted locations. The metabolic capacities of these diazotrophs indicate the potential for bioremediation and resiliency of the ecosystem to anthropogenic impact. In heavily polluted locations, the diazotrophic community structures were markedly different and the diversity of species was significantly reduced when compared with those in a pristine location. This, together with the increased abundance of Marinobacterium, which is a bioindicator of pollution, suggests that anthropogenic activity has a negative impact on the genetic diversity of diazotrophs in the mangrove rhizosphere.