Cell-specific beta-N-acetylglucosaminidase activity in cultures and field populations of eukaryotic marine phytoplankton.

It is widely appreciated that eukaryotic marine phytoplankton can hydrolyze a variety of compounds within the dissolved organic matter (DOM) pool in marine environments. Herein, cultures and field populations of marine phytoplankton were assayed for beta-N-acetylglucosaminidase activity, a terminal enzyme of chitin degradation. A traditional bulk assay, which can assess hydrolytic rate, but is not cell-specific, was complemented with a cell-specific assay that images the activity associated with single cells using an enzyme labeled fluorescence (ELF) substrate. beta-N-acetylglucosaminidase activity was widespread across various taxa of marine phytoplankton, and activity was observed both under controlled culture conditions and in field populations. The number of cells with enzyme activity varied with the nutritional physiology of the test species in three of the 17 cultures tested. In these three cases the number of cells with activity in the low nutrient medium was higher than in nutrient replete medium. Taken together, these data suggest that a broad group of marine phytoplankton may be a relevant part of chitin-like DOM degradation and should be incorporated into conceptual models of chitin cycling in marine systems.     

Dynamics of marine bacterial and phytoplankton populations using multiplex liquid bead array technology

Heterotrophic bacteria and phytoplankton dominate the biomass and play major roles in the biogeochemical cycles of the surface ocean. Here, we designed and tested a fast, high‐throughput and multiplexed hybridization‐based assay to detect populations of marine heterotrophic bacteria and phytoplankton based on their small subunit ribosomal DNA sequences. The assay is based on established liquid bead array technology, an approach that is gaining acceptance in biomedical research but remains underutilized in ecology. End‐labelled PCR products are hybridized to taxon‐specific oligonucleotide probes attached to fluorescently coded beads followed by flow cytometric detection. We used ribosomal DNA environmental clone libraries (a total of 450 clones) and cultured isolates to design and test 26 bacterial and 10 eukaryotic probes specific to various ribotypes and genera of heterotrophic bacteria and eukaryotic phytoplankton. Pure environmental clones or cultures were used as controls and demonstrated specificity of the probes to their target taxa. The quantitative nature of the assay was demonstrated by a significant relationship between the number of target molecules and fluorescence signal. Clone library sequencing and bead array fluorescence from the same sample provided consistent results. We then applied the assay to a 37‐day time series of coastal surface seawater samples from the Southern California Bight to examine the temporal dynamics of microbial communities on the scale of days to weeks. As expected, several bacterial phylotypes were positively correlated with total bacterial abundances and chlorophyll a concentrations, but others were negatively correlated. Bacterial taxa belonging to the same broad taxonomic groups did not necessarily correlate with one another, confirming recent results suggesting that inferring ecological role from broad taxonomic identity may not always be accurate.     

A computer study of the evolution of aerobic heterotrophic bacterial populations in sewage and river waters

The biodegradation of six surface active agents tested. The biochemical characteristics of the aerobic heterotrophic bacteria present in Montpellier waste waters and in Rhône water, before and after degradation, were studied by a standardized microbiological method. Four hundred strains isolated from these populations were compared to 29 reference strains by computer analysis. The reference strains were first grouped in classes (single linkage). Then the strains were compared first to these classes, and then to each reference strain. Whereas the bacterial populations were widely distributed before biodegradation, after degradation they were restricted to a few prevailing genera:Pseudomonas, Alcaligenes, andKlebsiella.     

Diel periodicity in dark uptake of ammonium by natural populations of marine phytoplankton

We discovered the phenomenon that dark uptake of ammonium increasesat night and maximizes around midnight. The maximum values atmidnight are 2 or 3 times higher than the values in the daytime.     

Geographical distribution and diversity of bacteria associated with natural populations of Drosophila melanogaster

Drosophila melanogaster is one of the most widely used model systems in biology. However, little is known about its associated bacterial community. As a first step towards understanding these communities, we compared bacterial 16S rRNA gene sequence libraries recovered from 11 natural populations of adult D. melanogaster. Bacteria from these sequence libraries were grouped into 74 distinct taxa, spanning the phyla Proteobacteria, Bacteroidetes, and Firmicutes, which were unevenly spread across host populations. Summed across populations, the distribution of abundance of genera was closely fit by a power law. We observed differences among host population locations both in bacterial community richness and in composition. Despite this significant spatial variation, no relationship was observed between species richness and a variety of abiotic factors, such as temperature and latitude. Overall, bacterial communities associated with adult D. melanogaster hosts are diverse and differ across host populations.     

Determination and quantification of alpha,beta,gamma,delta-unsaturated aldehydes as pentafluorobenzyl-oxime derivates in diatom cultures and natural phytoplankton populations: application in marine field studies

Reactive alpha,beta,gamma,delta-unsaturated aldehydes and oxo-acids produced by marine diatoms upon cell damage interfere negatively with the reproduction success of their grazers. A simple, sensitive and specific method based on gas-chromatography coupled to mass spectrometry (EI or CI/EC) was developed for the quantification of these deleterious substances in laboratory diatom cultures and in natural phytoplankton populations. For aldehyde quantification, diatom containing samples are damaged in the presence of O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA.HCl) which leads to an in situ derivatisation without inhibition of the biosynthesis of the aldehydes. The oxime derivates of oxo-acids were in addition reacted with N-tert-butyldimethylsilyl-N-methyl-trifluoracetamide (MTBSTFA).     

Variance within homogeneous phytoplankton populations, III: Analysis of natural populations

A theoretical framework for interpreting flow cytometric histograms from homogeneous phytoplankton populations was developed in part I of this series of articles and applied to chlorophyll fluorescence histograms from clonal cultures in part II. In this paper, we demonstrate the application of this framework to the analysis of cell volume distributions found in a natural assemblage of phytoplankton from the Gulf of California. Flow cytometric analyses of a surface water sample incubated for a period of 61 h revealed the sequential growth and decline of three distinct subpopulations. Cell volume distributions for each subpopulation measured at different times were analyzed, and the theoretical density function described in parts I and II was fitted to these distributions. The range of cell volumes within each subpopulation was similar to that predicted for asynchronous populations.     

Phased cell division in natural populations of marine dinoflagellates from shipboard cultures

In June 1975 samples were preserved every 2 h for 26 h from water collected from Santa Monica Bay, California and which was incubated on shipboard. The percentage of dividing dinoflagellate cells in the samples was determined by microscopic examination after applying a nuclear stain. The maximum proportion of dividing cells was at 03.30 h, 05.30 h, and 09.30 h for Ceratium dens Ostf. & Schmidt, C. furca (Ehrenb.) Claparède & Lachmann, and Dinophysis fortii Pavillard, respectively. Mean doubling times for these species, calculated from the percentage of dividing cells, were 3.3,4.6, and 1.4 days. Temporal stratification in the timing of cell division may reflect a selective process for minimizing interspecific competition.     

Distribution and diversity of natural product genes in marine and freshwater cyanobacterial cultures and genomes

Natural products are a functionally diverse class of biochemically synthesized compounds, which include antibiotics, toxins, and siderophores. In this paper, we describe both the detection of natural product activities and the sequence identification of gene fragments from two molecular systems that have previously been implicated in natural product production, i.e., nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), in diverse marine and freshwater cyanobacterial cultures. Using degenerate PCR and the sequencing of cloned products, we show that NRPSs and PKSs are common among the cyanobacteria tested. Our molecular data, when combined with genomic searches of finished and progressing cyanobacterial genomes, demonstrate that not all cyanobacteria contain NRPS and PKS genes and that the filamentous and heterocystous cyanobacteria are the richest sources of these genes and the most likely sources of novel natural products within the phylum. In addition to validating the use of degenerate primers for the identification of PKS and NRPS genes in cyanobacteria, this study also defines numerous gene fragments that will be useful as probes for future studies of the synthesis of natural products in cyanobacteria. Phylogenetic analyses of the cyanobacterial NRPS and PKS fragments sequenced in this study, as well as those from the cyanobacterial genome projects, demonstrate that there is remarkable diversity and likely novelty of these genes within the cyanobacteria. These results underscore the potential variety of novel products being produced by these ubiquitous organisms.     

Detection, diversity and expression of aerobic bacterial arsenite oxidase genes

The arsenic (As) drinking water crisis in south and south-east Asia has stimulated intense study of the microbial processes controlling the redox cycling of As in soil-water systems. Microbial oxidation of arsenite is a critical link in the global As cycle, and phylogenetically diverse arsenite-oxidizing microorganisms have been isolated from various aquatic and soil environments. However, despite progress characterizing the metabolism of As in various pure cultures, no functional gene approaches have been developed to determine the importance and distribution of arsenite-oxidizing genes in soil-water-sediment systems. Here we report for the first time the successful amplification of arsenite oxidase-like genes (aroA/asoA/aoxB) from a variety of soil-sediment and geothermal environments where arsenite is known to be oxidized. Prior to the current work, only 16 aroA/asoA/aoxB-like gene sequences were available in GenBank, most of these being putative assignments from homology searches of whole genomes. Although aroA/asoA/aoxB gene sequences are not highly conserved across disparate phyla, degenerate primers were used successfully to characterize over 160 diverse aroA-like sequences from 10 geographically isolated, arsenic-contaminated sites and from 13 arsenite-oxidizing organisms. The primer sets were also useful for confirming the expression of aroA-like genes in an arsenite-oxidizing organism and in geothermal environments where arsenite is oxidized to arsenate. The phylogenetic and ecological diversity of aroA-like sequences obtained from this study suggests that genes for aerobic arsenite oxidation are widely distributed in the bacterial domain, are widespread in soil-water systems containing As, and play a critical role in the biogeochemical cycling of As.     

Effects of PCB on interspecific competition in natural and gnotobiotic phytoplankton communities in continuous and batch cultures

The toxicity of polychlorinated biphenyls (PCB) to the diatomThalassiosira pseudonana (formerlyCyclotella nana), grown in pure and mixed cultures, was greatest when in competition with other species. Continuous cultures were superior to batch cultures for studying competitive interactions, and PCB caused greater alteration of species composition in continuous cultures than it did in batch cultures. Natural phytoplankton communities from Vineyard Sound, maintained in continuous culture, responded to PCB stress the same as did gnotobiotic communities, withT. pseudonana showing similar responses in both communities. A PCB concentration of 0.1 μg/liter (0.1 part per billion), a level not uncommon in natural waters, did not affect algal growth in pure cultures but caused substantial disruption of continuous culture communities. The possible impact of PCB pollution on natural phytoplankton communities is discussed. Contribution No. 3181 from the Woods Hole Oceanographic Institution.     

Carbohydrate dynamics and growth rate of natural phytoplankton populations

Diurnal changes in carbohydrate content of natural phytoplankton populations differed from those found for cyanobacteria grown in continuous cultures. The carbohydrate accumulation rate was not constant during the light period. Also in contrast to results obtained using continuous cultures the photosynthetic characteristics changed during the light period. A close correlation was observed between changes in carbohydrate accumulation rate and the efficiency of photosynthesis over 24 hours. Seasonal changes in carbohydrate consumption rate over the dark period were proportional to changes in growth rate.     

Detection of the microbial activity of aerobic heterotrophic, anoxic heterotrophic and aerobic autotrophic activated sludge organisms with an electrochemical sensor

The microbial activity of aerobic heterotrophic, anoxic heterotrophic and aerobic autotrophic microorganisms in biological wastewater treatment was determined by means of an electrochemical bioactivity sensor. The development of the sensor resulted in a system which can determine the microbial activities that are relevant for effective wastewater treatment. The signals of the sensor system are proportional to the substrate degradation and it can show inhibiting effects on the biomass. The most important advantages of the system are: it is independent of O₂ consumption, the three most important types of metabolic activities in wastewater technology can be measured with one sensor, furthermore the measurement is suitable for automation and it is on-line. The result is a potential for the optimization of processes based on microbial activity.     

Variance within homogeneous phytoplankton populations, II: Analysis of clonal cultures

In part I of this series of articles, a framework was presented for interpreting histograms of volume or fluorescence as measured by a flow cytometer on homogeneous phytoplankton populations. In this paper, the analytical framework is applied to flow cytometric histograms from laboratory experiments involving clonal phytoplankton cultures. The density function derived in part I was modified to include a third parameter representing a linear shift of the origin. This modified density function was fitted to chlorophyll fluorescence histograms for populations believed to be asynchronous (grown in continuous light) and also to histograms from populations grown on a 14:10 (h:h) light/dark cycle. Near-synchronous subpopulations sorted from an asynchronous population were also analyzed. In populations in which underlying assumptions (asynchronous divisions, constant growth) are valid, curve fits provide estimates of the inherent variability among cells at age 0. The implication of fitting the density function to populations in which these assumptions are not valid is discussed.     

Comparative study of the aerobic, heterotrophic bacterial flora of Chesapeake Bay and Tokyo Bay.

A comparative study of the bacterial flora of the water of Chesapeake Bay and Tokyo Bay was undertaken to assess similarities and differences between the autochthonous flora of the two geographical sites and to test the hypothesis that, given similarities in environmental parameters, similar bacterial populations will be found, despite extreme geographic distance between locations. A total of 195 aerobic, heterotrophic bacterial strains isolated from Chesapeake Bay and Tokyo Bay water were examined for 115 biochemical, cultural, morphological, nutritional, and physiological characters. The data were analyzed by the methods of numerical taxonomy. From sorted similarity matrices, 77% of the isolates could be grouped into 30 phena and presumptively identified as Acinetobacter-Moraxella, Caulobacter, coryneforms, Pseudomonas, and Vibrio spp. Vibrio and Acinetobacter species were found to be common in the estuarine waters of Chesapeake Bay, whereas Acinetobacter-Moraxella and Caulobacter predominated in Tokyo Bay waters, at the sites sampled in the study.     

Comparative study of the aerobic, heterotrophic bacterial flora of Chesapeake Bay and Tokyo Bay.

A comparative study of the bacterial flora of the water of Chesapeake Bay and Tokyo Bay was undertaken to assess similarities and differences between the autochthonous flora of the two geographical sites and to test the hypothesis that, given similarities in environmental parameters, similar bacterial populations will be found, despite extreme geographic distance between locations. A total of 195 aerobic, heterotrophic bacterial strains isolated from Chesapeake Bay and Tokyo Bay water were examined for 115 biochemical, cultural, morphological, nutritional, and physiological characters. The data were analyzed by the methods of numerical taxonomy. From sorted similarity matrices, 77% of the isolates could be grouped into 30 phena and presumptively identified as Acinetobacter-Moraxella, Caulobacter, coryneforms, Pseudomonas, and Vibrio spp. Vibrio and Acinetobacter species were found to be common in the estuarine waters of Chesapeake Bay, whereas Acinetobacter-Moraxella and Caulobacter predominated in Tokyo Bay waters, at the sites sampled in the study.     

Radiolaria associated with large diversity of marine alveolates

We have isolated cells of unculturable radiolarians from marine coastal waters. Individual cells were subjected to single cell whole genome amplification (SCWGA) and gene-targeted PCR. Using this approach we recover a surprisingly large diversity of sequences related to the enigmatic marine alveolate groups 1 and 2 (MALV I and MALV II) that most likely represent intracellular symbionts or parasites of the radiolarian cells. 18S rDNA phylogeny of the MALV sequences reveals 4 distinct clades of radiolarian associates here named Radiolarian Associated Sequences (RAS) 1-4. One clade of both phaeodarian and radiolarian associates and one clade of only phaeodarian associates are also identified. The MALV sequences cluster according to host type, i.e. sequences from associates identified in radiolarians, fish, copepods, ciliates or dinoflagellates are not intermixed but separated into distinct clades. This implies several independent colonizations of host lineages and links a large diversity of MALV to radiolarian-associated species. This demonstrates that radiolarians may be an important reservoir for MALV, making them a key group for understanding the impact of intracellular symbionts on the marine ecosystem. This study shows that applying SCWGA on unculturable cells is a promising approach to study the vast diversity and interactions of intracellular eukaryote organisms.     

Phenotypic and genotypic adaptation of aerobic heterotrophic sediment bacterial communities to mercury stress

The effects of mercury contamination of lake sediments on the phenotypic and genotypic mercury resistance of the indigenous heterotrophic aerobic bacterial communities were investigated. Strong positive correlations between mercury sediment concentration and the frequency of the gene coding for mercury volatilization (mer) (r = 0.96) or the phenotypic mercury resistance (r = 0.86) of the studied communities suggested that the inheritance via selection or genetic exchange of the mer gene had promoted bacterial adaptation to mercury. Failure to detect the mer gene in one mercury-contaminated sediment where phenotypic expression was low suggested that other mechanisms of resistance may partially determine the presence of mercury-resistant organisms in mercury-contaminated sediment or that the mercury in this particular sediment was very chemically limited in its availability to the microorganisms.