Michael Wyman

Spatial Distribution and Transcriptional Activity of an Uncultured Clade of Planktonic Diazotrophic γ-Proteobacteria in the Arabian Sea

ABSTRACT The spatial distribution of an uncultured clade of marine diazotrophic γ-proteobacteria in the Arabian Sea was investigated by the development of a specific primer pair to amplify an internal fragment of nifH by PCR. These organisms were most readily detected in highly oligotrophic surface waters but could also be found in deeper waters below the nutricline. nifH transcripts originating from this clade were detected in oligotrophic surface waters and, in addition, in the deeper and the more productive near-coastal waters. The nifH sequences most closely related to the unidentified marine bacterial group are from environmental clones amplified from the Atlantic and Pacific Oceans. These findings suggest that these γ-proteobacteria are widespread and likely to be an important component of the heterotrophic diazotrophic microbial community of the tropical and subtropical oceans.

Nitrate/Nitrite Assimilation System of the Marine Picoplanktonic Cyanobacterium Synechococcus sp. Strain WH 8103: Effect of Nitrogen Source and Availability on Gene Expression

ABSTRACT The genes encoding the structural components of the nitrate/nitrite assimilation system of the oceanic cyanobacterium Synechococcus sp. strain WH 8103 were cloned and characterized. The genes encoding nitrate reductase (narB) and nitrite reductase (nirA) are clustered on the chromosome but are organized in separate transcriptional units. Upstream of narB is a homologue of nrtP that encodes a nitrate/nitrite-bispecific permease rather than the components of an ABC-type nitrate transporter found in freshwater cyanobacteria. Unusually, neither nirA nor ntcA (encoding a positive transcription factor of genes subject to nitrogen control) were found to be tightly regulated by ammonium. Furthermore, transcription of glnA (encoding glutamine synthetase) is up-regulated in ammonium-grown cells, highlighting significant differences in nitrogen control in this cyanobacterium. Nitrogen depletion led to the transient up-regulation of ntcA, nirA, nrtP, narB, and glnA in what appears to be an NtcA-dependent manner. The NtcA-like promoters found upstream of nirA, nrtP, and narB all differ in sequence from the canonical NtcA promoter established for other cyanobacteria, and in the case of nirA, the NtcA-like promoter was functional only in cells deprived of combined nitrogen. The ecological implications of these findings are discussed in the context of the oligotrophic nature of oceanic surface waters in which Synechococcus spp. thrive.

Rapid shifts in picoeukaryote community structure in response to ocean acidification

Rapid shifts in picoeukaryote community structure were observed during a CO2 perturbation experiment in which we followed the development of phytoplankton blooms in nutrient-amended mesocosms under the present day or predicted future atmospheric pCO2 (750 μatm, seawater pH 7.8). Analysis of rbcL clone libraries (encoding the large subunit of RubisCO) and specific quantitative PCR assays showed that two prasinophytes closely related to Micromonas pusilla and Bathycoccus prasinos were present, but responded very differently to high CO2/acidification. We found that the abundance of Micromonas-like phylotypes was significantly higher (>20-fold) under elevated CO2/low pH, whereas the Bathycoccus-like phylotypes were more evenly distributed between treatments and dominated the prasinophyte community under ambient conditions.

Denitrifying alphaproteobacteria from the Arabian Sea that express nosZ, the gene encoding nitrous oxide reductase, in oxic and suboxic waters

ABSTRACT Marine ecosystems are significant sources of the powerful greenhouse gas nitrous oxide (N2O). A by-product of nitrification and an intermediate in the denitrification pathway, N2O is formed primarily in oxygen-deficient waters and sediments. We describe the isolation of a group of alphaproteobacteria from the suboxic waters of the Arabian Sea that are phylogenetically affiliated with Labrenzia spp. and other denitrifiers. Quantitative PCR assays revealed that these organisms were very broadly distributed in this semienclosed ocean basin. Their biogeographical range extended from the productive, upwelling region off the Omani shelf to the clear, oligotrophic waters that are found much further south and also included the mesotrophic waters overlying the oxygen minimum zone (OMZ) in the northeastern sector of the Arabian Sea. These organisms actively expressed NosZ (N2O reductase, the terminal step in the denitrification pathway) within the OMZ, an established region of pelagic denitrification. They were found in greatest numbers outside the OMZ, however, and nosZ mRNAs were also readily detected near the base of the upper mixed layer in nutrient-poor, oxic regions. Our findings provide firm molecular evidence of a potential sink for N2O within well-ventilated, oceanic surface waters in this biogeochemically important region. We show that the Labrenzia-like denitrifiers and their close relatives are habitual colonizers of the pseudobenthic environment provided by Trichodesmium spp. We develop the conjecture that the O2-depleted microzones that occur within the colonies of these filamentous, diazotrophic cyanobacteria might provide unexpected niches for the reduction of nitrogen oxides in tropical and subtropical surface waters.

A putative link between phagocytosis-induced apoptosis and hemocyanin-derived phenoloxidase activation

Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.

Molecular and physiological responses of two classes of marine chromophytic phytoplankton (diatoms and prymnesiophytes) during the development of nutrient-stimulated blooms

ABSTRACT Generic taxon-specific DNA probes that target an internal region of the gene (rbcL) encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) were developed for two groups of marine phytoplankton (diatoms and prymnesiophytes). The specificity and utility of the probes were evaluated in the laboratory and also during a 1-month mesocosm experiment in which we investigated the temporal variability in RubisCO gene expression and primary production in response to inorganic nutrient enrichment. We found that the onset of successive bloom events dominated by each of the two classes of chromophyte algae was associated with marked taxon-specific increases in rbcLtranscription rates. These observations suggest that measurements of RubisCO gene expression can provide an early indicator of the development of phytoplankton blooms and may also be useful in predicting which taxa are likely to dominate a bloom.

Transcriptionally active heterotrophic diazotrophs are widespread in the upper water column of the Arabian Sea

Pelagic nitrogen fixation makes an important contribution to the fixed nitrogen budget of the worlds oceans. Filamentous and unicellular cyanobacteria are significant players in this process but less is known of the potential activity of heterotrophic diazotrophs, although they are present and can be quite numerous in the nitrogen-deplete surface waters of the tropical and sub-tropical oceans. In this study we focused on the potential activity of several clades of heterotrophic nitrogen-fixers identified by phylogenetic analysis of 44 non-Trichodesmium-related, nifH (encoding the Fe-subunit of nitrogenase) clones from the Arabian Sea. Specific Northern slot blot protocols were developed to quantify nifH mRNAs from each clade and showed that two groups of Gammaproteobacteria, including the previously characterized UMB clade, and a third, novel phylotype affiliated with cluster III anaerobes, were actively expressing nitrogenase in the equatorial waters of this region. Transcripts (nifH mRNAs) from the latter clade were particularly abundant and were also detected in the suboxic waters of the oxygen minimum zone further north. Like the gammaproteobacterial groups, nifH expression by these organisms appeared to be insensitive to combined nitrogen concentrations and was readily detected in the nutrient-replete waters below the upper mixed layer as well as at shallower depths.

Lack of Control of Nitrite Assimilation by Ammonium in an Oceanic Picocyanobacterium, Synechococcus sp. Strain WH 8103

ABSTRACT In cyanobacteria, the transcriptional activator NtcA is involved in global nitrogen control and, in the absence of ammonium, regulates the expression of genes involved in the assimilation of alternative nitrogen sources. The oceanic picocyanobacterium Synechococcus sp. strain WH 8103 harbors a copy of ntcA, but in the present study, we show that unlike other marine cyanobacteria that have been investigated, this strain is capable of coassimilating nitrite when grown in the presence of ammonium. Transcript levels for the genes encoding the nitrate/nitrite-bispecific permease NrtP and nitrate reductase (NarB) were substantially down-regulated by ammonium, whereas the abundances of nitrite reductase (NirA) transcripts were similar in nitrite- and ammonium-grown cells. The growth of Synechococcus sp. strain WH 8103 in medium containing both ammonium and nitrite resulted in only minor changes in the expression profile in comparison to that of nitrite-grown cells with the exception that the gene encoding the high-affinity ammonium transporter Amt1 was down-regulated to the levels seen in ammonium-grown cells. Whereas the expression of nrtP, narB, and amt1 appears to be NtcA dependent in this marine cyanobacterium, the transcription and expression of nirA appear not to be. The ability to coassimilate nitrite and reduced-nitrogen sources like ammonium may be an adaptive trait that enables oceanic strains like Synechococcus sp. strain WH 8103 to exploit the low nitrite concentrations found in oceanic surface waters that are not available to their principal and more numerous competitor, Prochlorococcus.

Dynamics of ribulose 1,5-bisphosphate carboxylase/oxygenase gene expression in the coccolithophorid Coccolithus pelagicus during a tracer release experiment in the Northeast Atlantic

ABSTRACT We report a pronounced diel rhythm in ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene expression in a natural population of the coccolithophorid Coccolithus pelagicus sampled during a Lagrangian experiment in the Northeast Atlantic. Our observations show that there is greater heterogeneity in the temporal regulation of RubisCO expression among planktonic chromophytes than has been reported hitherto.

Quantitative PCR Profiling of Escherichia coli in Livestock Feces Reveals Increased Population Resilience Relative to Culturable Counts under Temperature Extremes.

The relationship between culturable counts (CFU) and quantitative PCR (qPCR) cell equivalent counts of Escherichia coli in dairy feces exposed to different environmental conditions and temperature extremes was investigated. Fecal samples were collected in summer and winter from dairy cowpats held under two treatments: field-exposed versus polytunnel-protected. A significant correlation in quantified E. coli was recorded between the qPCR and culture-based methods (r = 0.82). Evaluation of the persistence profiles of E. coli over time revealed no significant difference in the E. coli numbers determined as either CFU or gene copies during the summer for the field-exposed cowpats, whereas significantly higher counts were observed by qPCR for the polytunnel-protected cowpats, which were exposed to higher ambient temperatures. In winter, the qPCR returned significantly higher counts of E. coli for the field-exposed cowpats, thus representing a reversal of the findings from the summer sampling campaign. Results from this study suggest that with increasing time post-defecation and with the onset of challenging environmental conditions, such as extremes in temperature, culture-based counts begin to underestimate the true resilience of viable E. coli populations in livestock feces. This is important not only in the long term as the Earth changes in response to climate-change drivers but also in the short term during spells of extremely cold or hot weather.