Grieg F. Steward

Dynamics of Bacterial Community Composition and Activity during a Mesocosm Diatom Bloom

ABSTRACT Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment (≈24 μg of chlorophylla liter−1). At this time bacterial abundance abruptly decreased from 2.8 × 106 to 0.75 × 106 ml−1, and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-μm size fraction towards the >1.0-μm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized α-Proteobacteria- andCytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, β-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.

Bacteria-organic matter coupling and its significance for oceanic carbon cycling

This paper synthesizes current ideas on the role of the microbial loop in carbon fluxes in the ocean and proposes some directions for future research. Organic matter flux into bacteria is highly variable, which can significantly influence the pathways of carbon flow in the ocean. A goal for future research is to elucidate the mechanistic bases of bacteria-organic matter coupling. This research should take into consideration the micrometer-scale distribution of bacteria and the composition, structure, and dynamics of the organic matter field in the bacteriums microhabitat. The ideas on the interactions of bacteria with the particulate organic phase need to be revised in view of recent findings of highly abundant, previously unknown particles ranging in size from nanometers to hundreds of micrometers. The “hot-spots” in the distribution of organic matter and remineralized nutrients can influence the rates as well as the direction of biogeochemical fluxes. Slow-to-degrade dissolved organic matter (DOM) may be produced because of loose bacteria-organic matter coupling resulting in DOM storage. Its use at a later time and place has profound implications for carbon fluxes and food web dynamics. A fundamental research need for the future is to understand the ecological interactions among the members of the microbial loop in an appropriate microhabitat context. While this goal was previously intractable, new molecular and optical techniques should make it possible to understand the biogeochemical activities of the microbial loop in terms of the ecology and evolution of pelagic microbial communities.

Glucose fluxes and concentrations of dissolved combined neutral sugars (polysaccharides) in the Ross Sea and Polar Front Zone, Antarctica

We hypothesized that dissolved carbohydrates would be large components of the labile dissolved organic carbon (DOC) pool and would support much bacterial growth in Antarctic waters, especially the Ross Sea, since previous work had observed extensive phytoplankton blooms with potentially high production rates of carbohydrates in Antarctic seas. These hypotheses were tested on cruises in the Ross Sea and Antarctic Polar Front Zone as part of the US JGOFS program. Concentrations and fluxes of free glucose (the only free sugar detected) were very low, but dissolved polysaccharides appeared to be important components of the DOC pool. Concentrations of dissolved combined neutral sugars increased >3-fold during the phytoplankton bloom in the Ross Sea and were a large fraction (ca. 50%) of the semi-labile fraction of DOC. The relatively high concentrations of dissolved combined neutral sugars, which are thought to be quite labile, appear to explain why DOC accumulated during the phytoplankton bloom was degraded so quickly once the bloom ended. Some of the polysaccharides appeared to be more refractory, however, since dissolved combined neutral sugars were observed in deep waters (>550 m) and in early spring (October) in the Ross Sea, apparently having survived degradation for >8 months. The molecular composition of these refractory polysaccharides differed from that of polysaccharides sampled during the phytoplankton bloom. Fluxes of DOC were low in the Ross Sea compared to standing stocks and fluxes of particulate material,

Bacterial community composition during two consecutive NE Monsoon periods in the Arabian Sea studied by denaturing gradient gel electrophoresis (DGGE) of rRNA genes

Abstract Horizontal and vertical variations in bacterial community composition were examined in samples collected during two Joint Global Ocean Flux Study (JGOFS) Arabian Sea cruises in 1995. The cruises, 11 months apart, took place during two consecutive NE Monsoon periods (January and December). Bacteria were harvested by filtration from samples collected in the mixed layer, mid-water, and deep sea at stations across the study area. Total bacterial community genomic DNA was analyzed by PCR amplification of 16S rRNA gene fragments, followed by denaturing gradient gel electrophoresis (DGGE). In total, 20 DGGE bands reflecting unique or varying phylotypes were excised, cloned and sequenced. Amplicons were dominated by bacterial groups commonly found in oceanic waters (e.g., the SAR11 cluster of α -Proteobacteria and cyanobacteria), but surprisingly none of the sequenced amplicons were related to γ -Proteobacteria or to members of the Cytophaga-Flavobacter-Bacteroides phylum. Amplicons related to magnetotactic bacteria were found for the first time in pelagic oceanic waters. The DGGE banding patterns revealed a dominance of ≈15 distinguishable amplicons in all samples. In the mixed layer the bacterial community was dominated by the same ≈15 phylotypes at all stations, but unique phylotypes were found with increasing depth. Except for cyanobacteria, comparison of the bacterial community composition in surface waters from January and December 1995 showed only minor differences, despite significant differences in environmental parameters. These data suggest a horizontal homogeneity and some degree of seasonal predictability of bacterial community composition in the Arabian Sea.

Development and Testing of a DNA Macroarray To Assess Nitrogenase (nifH) Gene Diversity

ABSTRACT A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml−1, a half-saturation of signal at 0.26 ng ml−1, and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.

Nitrogenase Gene Amplicons from Global Marine Surface Waters Are Dominated by Genes of Non-Cyanobacteria

Cyanobacteria are thought to be the main N2-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

Phylogenetic screening of ribosomal RNA gene-containing clones in Bacterial Artificial Chromosome (BAC) libraries from different depths in Monterey Bay.

Marine picoplankton are central mediators of many oceanic biogeochemical processes, but much of their biology and ecology remains ill defined. One approach to better defining these environmentally significant microbes involves the acquisition of genomic data that can provide information about genome content, metabolic capabilities, and population variability in picoplankton assemblages. Previously, we constructed and phylogenetically screened a Bacterial Artificial Chromosome (BAC) library from surface water picoplankton of Monterey Bay. To further describe niche partitioning, metabolic variability, and population structure in coastal picoplankton populations, we constructed and compared several picoplankton BAC libraries recovered from different depths in Monterey Bay. To facilitate library screening, a rapid technique was developed (ITS-LH-PCR) to identify and quantify ribosomal RNA (rRNA) gene-containing BAC clones in BAC libraries. The approach exploited natural length variations in the internal transcribed spacer (ITS) located between SSU and LSU rRNA genes, as well as the presence and location of tRNA-alanine coding genes within the ITS. The correspondence between ITS-LH-PCR fragment sizes and 16S rRNA gene phylogenies facilitated rapid identification of rRNA genes in BAC clones without requiring direct DNA sequencing. Using this approach, 35 phylogenetic groups (previously identified by cultivation or PCR-based rRNA gene surveys) were detected and quantified among the BAC clones. Since the probability of recovering chimeric rRNA gene sequences in large insert BAC clones was low, we used these sequences to identify potentially chimeric sequences from previous PCR amplified clones deposited in public databases. Full-length SSU rRNA gene sequences from picoplankton BAC libraries, cultivated bacterioplankton, and nonchimeric RNA genes were then used to refine phylogenetic analyses of planktonic marine gamma Proteobacteria, Roseobacter, and Rhodospirillales species.

The seasonal development of the bacterioplankton bloom in the Ross Sea, Antarctica, 1994-1997

We report on investigations of bacterioplankton growth dynamics and carbon utilization in the full water column of the Ross Sea, Antarctica carried out on six cruises in 1994–1997, using epifluorescence microscopy, thymidine and leucine incorporation to estimate bacterial abundance and production, respectively. The Ross Sea experienced a bacterial bloom with an amplitude equaling similar blooms observed in the North Atlantic and North Pacific, reaching 3 � 10 9 cells l � 1 or 35 mmol C m � 2 in late January. Increases in bacterial biomass were driven both by increases in abundance and in cell volume. Cell volumes ranged from 0.03mm 3 cell � 1 in early spring to over 0.15mm 3 cell � 1 in midsummer. Larger cells were associated with faster division rates. Bacterial growth rates ranged 0.02–0.3 divisions d � 1 , equal to rates at lower latitudes. Bacterial biomass accumulated steadily in the upper water column at a net rate of 0.03 d � 1 . While there is clear evidence of a bacterial bloom in the Ross Sea, equal to bacterioplankton blooms observed in other oceanic systems, the magnitude of bacterial response relative to the phytoplankton bloom was modest. For example, euphotic zone bacterial production (BP) rates were equivalent to 1–10% of particulate primary production (PP) except in April 1997 when PP was very low and BP : PP was sometimes >1. BP integrated over the upper 300 m was a more substantial fraction of the overlying PP than BP in the euphotic zone alone, with bacterial carbon demand in the upper 300 m about 30% of the seasonal PP. There was significant seasonal variation of bacterial biomass below the euphotic zone, indicating dynamic bacterial growth in the lower layer, and a supply of labile organic matter for bacteria. Bacterial metabolism is apparently limited by DOC flux in the upper layer. There is little evidence of temperature limitation, independent of substrate concentration. The relatively small diagenesis of phytoplankton biomass in the

Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray

ABSTRACT Investigations of the distribution and diversity of nitrogen-fixing microorganisms in natural environments have often relied on PCR amplification and sequence analysis of a portion of one of the key enzymes in nitrogen fixation, dinitrogenase reductase, encoded by nifH. Recent work has suggested that DNA macroarrays provide semiquantitative fingerprints of diversity within mixtures of nifH amplicons (G. F. Steward, B. D. Jenkins, B. B. Ward, and J. P. Zehr, Appl. Environ. Microbiol. 70:1455-1465, 2004). Here we report the application of macroarrays for a study in the Chesapeake Bay. Samples from different locations in the bay yielded distinct fingerprints. Analysis of replicates and samples from different locations by cluster analysis showed that replicates clustered together, whereas different samples formed distinct clusters. There was a correspondence between the hybridization pattern observed and that predicted from the distribution of sequence types in a corresponding clone library. Some discrepancies between the methods were observed which are likely a result of the high nifH sequence diversity in the Chesapeake Bay and the limited number of sequences represented on this version of the array. Analyses of sequences in the clone library indicate that the Chesapeake Bay harbors unique, phylogenetically diverse diazotrophs. The macroarray hybridization patterns suggest that there are spatially variable communities of diazotrophs, which have been confirmed by quantitative PCR methods (S. M. Short, B. D. Jenkins, and J. P. Zehr, Appl. Environ. Microbiol., in press). The results show that DNA macroarrays have great potential for mapping the spatial and temporal variability of functional gene diversity in the environment.

Constraining bacterial production, conversion efficiency and respiration in the Ross Sea, Antarctica, January–February, 1997

Bacteria consume dissolved organic carbon at rates averaging about 50% of primary production across a wide spectrum of marine ecosystems. However, total utilization rates are poorly constrained due to a lack of data on conversion e