Download Student Symposium, June 6th, 2008

2008 Center for Microbial Oceanography:
Research and Education (CMORE)
Summer Course “Microbial Oceanography: Genomes to Biomes”
Student Symposium, June 6th, 2008
University of Hawaii
Rebecca Case
Sophie Clayton
Alejandro Andrés Murillo Córdova
Li Deng
Caroline Fortunato
Melissa Laghdass
Christopher Reisch
Claudia Hernandez Rondón
Sandra Schöttner
Christine Shulse
Yuna Seo
Nellie Shaul
Danielle Morgan-Smith
Elena Stoica
Frank K. Thomson, III
Laure-Anne Ventouras
Harvard University
MIT/WHOI Joint Program
Pontificia Universidad Católica de Chile
The University of Bristol
University of Maryland
CNRS/UMR-France
University of Georgia
Estación de Fotobiología Playa Unión-Argentina
Max Planck Institute for Marine Microbiology
Scripps Institution of Oceanography
The University of Tokyo
Scripps Institution of Oceanography
Old Dominion University
University of Liverpool
Old Dominion University
Massachusetts Institute of Technology
REBECCA CASE
AFFILIATION: Harvard University
TITLE: Bacterial-algal interactions and the sulfur cycle
ABSTRACT:
Algae produce and store the major biogenic source of sulfur in the marine environment,
dimethylsulfoniopropionate (DMSP). DMSP is an osmolyte in algae and is made accessible for
microbial degradation in the ocean through lysis of algal cells. DMSP is degraded by bacteria
through the demethylation/demethiolation pathway or the cleavage pathway. The former
pathway degrades DMSP to sulfate, while the latter pathways product is dimethyl sulfide (DMS).
DMS is an important intermediate in the sulfur cycle and influences climate through the
formation of cloud condensation nuclei (CCN). Model calculations have shown a (theoretical)
negative feedback between DMS producing communities and global mean temperature,
suggesting these communities could play a role in climate.
SOPHIE CLAYTON
AFFILIATION: MIT/WHOI Joint Program
TITLE: Investigating controls on the seasonal cycle of /Synechococcus/ biomass
at MVCO using a simple box model
ABSTRACT:
It is generally thought that the interaction of physical and biological processes in the open ocean
results in a relatively constant background of small phytoplankton cells. This has not, however,
been fully investigated in coastal ecosystems. Recent observations from the
Martha's Vineyard Coastal Observatory (MVCO), made using an automated submersible flow
cytometer, have shown that this does not seem to be the case there (Sosik & Olson, 2006). Time
series of picoplankton biomass, comprised mainly of /Synechococcus/ and picoeukaryotes, from
2003 to the present, show that there is an increase in picoplankton biomass in the spring,
sustained throughout the summer, followed by a decrease in the autumn. Temperature has been
suggested to play a role in limiting growth during the winter months, but the combination of
physical and biological processes operating to regulate picoplankton biomass during the spring
and autumn transition periods is not well understood. The aim of this study, therefore, is to use a
simple box model which incorporates temperature-dependent growth, nutrient limitation and a
closure term representing the sum of all loss processes to Synechococcus (e.g. grazing, mortality,
advection,etc...) to investigate the controls on these seasonal variations at MVCO. The model is
optimized to find the parameters which best fit the observations, and the model-data misfit
assessed quantitatively. The structure of the model is modified, and the subsequent model-data
misfits compared, to test hypotheses about which environmental factors control the seasonal
cycle of /Synechococcus/ biomass.
ALEJANDRO ANDRÉS MURILLO CÓRDOVA
AFFILIATION: Pontificia Universidad Católica de Chile
TITLE: Cylindrospermopsis raciborskii has model organism for PSP toxins pathway synthesis
study
ABSTRACT:
In Chile, annually in summer an algal bloom (red tide) occur in the sea in austral regions and the
microorganism responsible is the dinoflagellate Alexandrium Catenella a eukaryotic microalga
that produce saxitoxin (STX) and their analogs, this neurotoxins act blocking the voltage gate
sodium channel and can be accumulated by bivalves, producing the “Paralytic Shellfish
Poisoning” (PSP) syndrome. In the case of fresh water sources the algal blooms with de
concomitant toxin contamination are produced by cyanobacteria. Cyanobacteria are
photosynthetic bacteria that are capable of produce a wide variety of injurious compounds toxics
or irritants. The species of cyanobacteria that are STX producers are Aphanizomenon flos-aquae,
Anabaena circinalis, Lyngbya wollei, Cylindrospermopsis raciborskii and Planktothrix agardhii.
The pathway of synthesis of STX or genes related with the toxin remains unknown for all the
producer organisms, but we know that the precursors are the same for cyanobacteria and
dinoflagellate. Among the STX producers microorganisms the cyanobacteria are de best model
of study, especially de specie Cylindrospermopsis raciborskii, because it has strains that produce
toxin; STX and their analogs or cylindrospermopsin and strains non toxin producers and all the
strain are genetically homogeneous. Our objective is to characterize the toxin producer strains
genetically and functionally to identified de enzymatic machinery involved in STX production.
LI DENG
AFFILIATION: The University of Bristol
TITLE: The role of cyanophages in regulating bloom-forming freshwater cyanobacteria
ABSTRACT:
Cyanobacteria are important and diverse members of aquatic systems both in marine and
freshwater environments. Cyanobacterial viruses (cyanophages) in marine systems have been
shown to play roles in mortality of their hosts, as well as driving evolution of core metabolic
functions even photosynthesis. Thus I wondered what roles cyanophages might play in
freshwater systems where cyanobacterial genera were predominately filamentous and bloomforming, rather than unicellular non-bloom-forming cells. To this end, I used multiple genetic
loci (T4-phage portal protein, major capsid protein) to gain an overall understanding of the
phylogenetic diversity of these genes within natural phage communities in a couple of European
lakes. I next isolated 35 cyanophages that infect bloom-forming freshwater cyanobacteria
(Microcystis, Anabaena and Planktothrix) using viral-fraction waters from Lake Zurich,
Switzerland and lakes in the Cotswold Water Park, U.K. Morphological characterization
revealed a variety of morphotypes, including commonly observed tailed phage morphotypes
(e.g., podo-, myo-, siphoviruses), as well as the first filamentous cyanophage. Host range
characterization showed that some cyanophages were capable of infecting cyanobacterial hosts
across three genera. The ability to infect a wide range of host taxa which dominant water bodies
in variable depth and variable time extends the potential reproductive period for lytic
propagation of cyanophages, and also has implications for the transfer of genetic information
between phylogenetically disparate cyanobacterial lineages. A primary example of such viralmediated HGT might be the discovery of a phycocyanin gene previously thought to be
horizontally transferred in the host cells and was found in a cyanophage isolate A-CP1 by PCR
screen with substantive controls to minimize the probability of amplifying a host gene.
Phycocyanin is ubiquitous accessory photosynthesis gene thought to be important in host
metabolism during phage infection, but also previously hypothesized to have been horizontally
transferred is my phage a smoking gun for a mechanism?
CAROLINE FORTUNATO
AFFILIATION: University of Maryland
TITLE: Microbial community observation and prediction in the Columbia River, its estuary and
the adjacent coastal ocean
ABSTRACT:
Microbial communities are dynamic and vary over many environmental gradients. The goal of
this study is to determine the composition of the microbial communities of the Columbia River
system and how the different populations of this community are distributed from river to estuary
and across the coastal ocean. A 16S rRNA gene based microbial community record will be
created using the community fingerprinting technique, denaturing gradient gel electrophoresis
(DGGE). Once a map of microbial communities is established, shifts in the distribution of
individual microbial populations over space and time will then be connected with environmental
factors and linked to forecast models of coastal margin environmental conditions. Currently,
cruise data from August 2007 is being used to construct a system-wide map of microbial
population distributions, with future plans to examine seasonal and interannual changes in these
populations.
MELISSA LAGHDASS
AFFILIATION: CNRS/UMR France
TITLE: External sources of organic carbon in marine oligotrophic environment: degradation by
different functional and phylogenetic groups of the heterotrophic prokaryotes and impact on the
metabolic balance
ABSTRACT:
The metabolic balance (ration between respiration and production of the organic matter) depends
directly of the ecosystem functioning. Studies suggest that the respiration exceed the production
in the oligotrophic ocean causing a visible deficit of organic carbon. This deficit can be explain
either by a decoupling between production and respiration or by an external source of organic
matter that would provide the respiration. A temporal monitoring of this deficit is also essential
in order to understand the origin of this deficit. Besides, the heterotrophic prokaryotes contribute
to the essential of the respiration in the oligotrophic environment. Their metabolic activity plays
also a key role in the metabolic balance. The variability of the prokaryotic respiration depends
essentially of the bioavailability of organic matter and the nutrient concentration. Furthermore,
the nature of organic matter would play a role in the presence and activity of phylogenetic
groups. The identification of these groups is very important in order to understand their role in
the metabolic balance. In situ observations started in the NW Mediterranean Sea (MOLA
station) on February 2007. Samples are carrying out once a month to determine the metabolic
balance and identify the phylogenetic prokaryotes of heterotrophic prokaryotes. The metabolic
balance is evaluated by winckler method. Using molecular biology, oligonucleotidic probes
specific of bacterial groups or target probes will create. So, by combining microautoradiography
and in situ hybridization from created probes, activity of these groups could determine. An
experimental approach based on the chemostat culture is too envisaged to know the response of
different bacterial groups according to the sources of organic matter.
CHRISTOPHER REISCH
AFFILIATION: University of Georgia
TITLE: Purification and Characterization of DMSP Dependent Demethylase (DmdA) from
icMarine Bacteria
ABSTRACT:
The algal metabolite dimethylsulfoniopropionate (DMSP) is ubiquitous in marine environments,
ranging in concentration from 5-200 nM in surface waters. DMSP is the precursor of the volatile
compound dimethylsulfide, whh is the primary natural source of sulfur in the atmosphere.
Degradation of DMSP occurs through two competing pathways, yielding DMS in the cleavage
pathway or methanethiol through the demethylation pathway. Recently, the enzyme responsible
for the demethylation of DMSP, designated DmdA, was discovered in two marine isolates,
Silicibacter pomeroyi DSS-3 and Pelagibacter ubique HTCC1062. To further our understanding
of DMSP demethylation, recombinant DmdA from P. ubique was purified. The purified enzyme
exhibited strict substrate specificity for DMSP. The apparent Vmax of DmdA was 7.6µmols
min-1 mg-1 with an apparent Km for DMSP of 11.5 mM. In cell extracts of S. pomeroyi DSS-3
the apparent Km for DMSP was 10.2 mM, similar to that of purified recombinant DmdA. The
intracellular concentration of DMSP in chemostat-grown S. pomeroyi DSS-3 was found to range
from 66-93 mM, depending on growth conditions. These results suggest that cells actively
accumulate DMSP as an osmolyte. Moreover, initiation of DMSP demethylation by DmdA only
occurs at very high intracellular concentrations.
CLAUDIA HERNÁNDEZ RONDÓN
AFFILIATION: Estación de Fotobiología Playa Unión-Argentina
Title: Solar radiation effects on bacterioplankton of Patagonia.
ABSTRACT:
The decrease on stratospheric Ozone levels and the rise of incident solar radiation (mainly on
UVB range) affects aquatic systems. Even under normal levels, solar and UVB radiation
generates variety of responses (typically deleterious) on metabolic activity, physiology and
diversity of microorganisms. Therefore, the bacterioplankton response is related with light
penetration and with biogeochemical composition of each ecosystem. Chubut area (43° S,
Argentinean Patagonia) possesses strong winds that turn in low cloud cover and consequently
high levels of solar radiation along the year. A decade of punctual studies at this zone evidenced
the impact of solar radiation (PAR and UVR).At the Chubut River “mouth” there is high primary
and fishery production present. Photoinhibition and repair processes related to cell size were
reported mainly for phytoplankton. In the case of heterotrophic bacteria, there are not internal
protection mechanisms to deal with solar stress. I developed punctual experiments at the pier
with river and seawater (according to tides) along summer. Previous results showed mainly UVB
effects on bacterioplankton abundance, and growth. At the same time samples to check bacterial
diversity changes by solar stress are a work on progress.
SANDRA SCHÖTTNER
AFFILIATION: Max Plank Institute for Marine Microbiology
TITLE: Microbial habitats in cold-water coral reef ecosystems
ABSTRACT:
The potential of key ecosystems like cold-water coral reefs to foster enhanced species diversity
in the ocean is undisputed. However, little is known about the putative role that cold-water coral
reefs play as biodiversity hotspots for the “unseen majority” in the oceans: prokaryotic
communities. Our current efforts evolve around the central question, if cold-water corals act as
ecosystem engineers in their reef environment by shaping microbial diversity, activity and
functionality via habitat differentiation and release of organic matter. Pilot studies in 2006/2007
revealed that bacterial communities may associate very specifically with distinct microbial
habitats generated by the reef-building coral Lophelia pertusa, such as the coral skeleton surface,
mucus and tissue when compared to ambient sediments and seawater. In particular, coral mucus
was characterized by distinct and very diverse bacterial patterns, and has shown to act as possible
energy vector from corals to microbes in reef waters. Whether these trends have a general
validity for cold-water coral reef ecosystems is currently under investigation in more extensive
studies targeting the diversity, dynamics and potential function of prokaryotes along horizontal
and vertical gradients in different reef systems off Northern Norway and Chile.
CHRISTINE SCHULSE
AFFILIATION: Scripps Institution of Oceanography
TITLE: Evolutionary origins and genetic regulation of omega-3 polyunsaturated fatty acid
synthase gene clusters in marine bacteria
ABSTRACT:
Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as EPA
(20:5) and DHA (22:6), is constrained to a narrow subset of marine γ-proteobacteria. The
availability of multiple genomes from both PUFA+ and closely related PUFA- strains allows for
a comparative investigation as to the evolutionary origins of PUFA synthase gene clusters (pfaAE). It is possible that the presence of the pfa gene cluster in different organisms that share the
same environment came about as a result of lateral gene transfer. Despite potential physiological
advantages conferred by PUFA production under low temperature or high pressure regimes
(membrane fluidity/phase dynamics), results to date suggest alternative selective advantages
possibly unrelated to thermal- or piezotolerance. Currently, little is known regarding PUFA
synthesis in these bacteria other than that it proceeds via a novel polyketide synthesis
mechanism. In order to explore the genetic regulation and coordinated production of PUFAs, a
recombinant EPA producing E. coli strain has been engineered and is being utilized to explore
the biochemical pathway of PUFA synthesis. Multifunctional domain profiling of pfa gene
products is revealing the biochemical structure and molecular machinery responsible for PUFA
production and comparisons amongst Pfa synthases reveal the basis for EPA versus DHA
discrimination. Further biochemical exploration of the different functional domains in the Pfa
proteins via domain swapping and combinatorial synthesis will provide a recipe of domains
necessary for PUFA synthesis. Ultimately, the collation of genomic, genetic, and biochemical
data will be used to further explore the ecological function of bacterial PUFA production in the
marine environment.
YUNA SEO
AFFILIATION: The University of Tokyo
TITLE: Particle Capture by Marine Bacteria
ABSTRACT:
In aquatic environments large numbers of submicron particles are generally present, and their
turnover and fate are of considerable ecological importance in geochemical cycles. Efficient
degradation of submicron particles can be attained if the particles are retained on the surfaces of
bacterial cells rather than remaining suspended in the aqueous phase. I hypothesized that some
bacterial groups possess particle-capture ability. To verify this hypothesis, I took two
approaches. First, paramagnetic particles were added to natural seawater samples to separate out
bacterial groups with PC capabilities. Method development was carried out in combination with
community structure analysis of the separated bacteria by denaturing gradient gel electrophoresis
and multidimensional scaling. Bacterial groups recovered by this new approach were different
from either the representative ones in the original seawater sample or large particle-associated
bacterial groups. Second, atomic force microscopy was utilized to directly observe the submicron
particles within natural bacterial populations. The numbers of cells retaining submicron particles
were high in the inner part of Tokyo Bay and at depths of 500 to 2000 m in the open ocean. I
suggest that there are bacterial groups in natural seawater that possess PC ability.
NELLIE SHAUL
AFFILIATION: Scripps Institution of Oceanography
TITLE: Investigating the biotic formation and degradation of methoxylated polybrominated
diphenyl ethers (MeO-PBDEs) in marine ecosystems.
ABSTRACT:
Many halogenated organic compounds were considered to be of anthropogenic origin, but there
is now evidence for their production in nature. For example, MeO-PBDEs have been shown to
accumulate in whale tissues, and radiocarbon evidence indicates a natural, ubiquitous, marine
origin. We are currently investigating answers to the following questions: What are the
ubiquitous biotic marine source(s)? Are the natural analogs accumulating in the marine
environment? Can bacteria in the marine environment degrade these natural MeO-PBDEs?
Investigation of these questions will occur in several phases: extraction and analysis of dissolved
and particulate organic matter to detect MeO-PBDEs; search GenBank environmental databases
for biosynthetic clusters similar to what has been found in sponge systems to identify pelagic
biotic sources; and incubate natural assemblages of bacteria with 13C-labeled MeO-PBDEs to
investigate incorporation and degradation. Examining sources and fates of halogenated aromatic
compounds in the environment remains as a vital component to our understanding of
contaminant biogeochemistry. If we are to understand and quantify the persistence and
transformation of industrial analogs, we must first characterize biotic production and distribution
of related compounds in environmental systems. This research would help to fulfill vital niches
in contaminant biogeochemistry, marine microbial ecology, and remediation.
DANIELLE MORGAN-SMITH
AFFILIATION: Old Dominion University
TITLE: Quantification and community composition of deep-sea flagellates based on nuclear
morphology and fluorescence in situ hybridization
ABSTRACT:
The pelagic deep ocean is the largest habitat on earth, yet we know very little about the
distribution and activity of protists in this environment. Flagellates, for instance, which are
responsible for controlling bacterial abundance in the surface ocean, and which are major players
in the material and energy transfer of pelagic food webs, have not been studied to any significant
extent in the deep sea. For this project, water samples were taken in the eastern subtropical North
Atlantic at depths ranging from 100m to 5000m and were filtered onto membranes which
underwent tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) using
oligonucleotide probes of eukaryote-specific sequences. These filters were then scanned for
positive TSA-FISH and DAPI signals using an epifluorescence microscope with a robotic stage.
We compared filters across depth and geographic area in terms of abundance of eukaryotes and
by community composition based on DAPI nuclear morphology. In comparison to prokaryotes,
numbers of eukaryotes drop off disproportionately with depth.
ELENA STOICA
AFFILIATION: University of Liverpool
TITLE: In situ identification of planktonic ammonia-oxidizing bacteria (AOB) in the NW
Black Sea coast
ABSTRACT:
Ammonia oxidation, the first step in nitrification, is a key process in the marine N cycle that
results in the formation of nitrate trough activity of microbial (bacterial and archaeal) ammoniaoxidizers. Both aerobic and anaerobic ammonia oxidation have been studied in the central part of
the Black Sea, the largest marine anoxic basin in the world. However, no data on taxonomic
diversity and ecological roles of ammonia-oxidizers community are available for the NW Black
Sea, a dynamic region largely influenced by Danube River and anthropogenic factors. We
investigated the presence and relative abundance of the main prokaryotic taxa for Bacteria and
ammonia-oxidizing bacteria (AOB) by a combination of whole-cell fluorescence in situ
hybridization with rRNA-targeted oligonucleotide probes and catalyzed reporter deposition
(CARD-FISH). With horseradish peroxidase-labeled oligonucleotide probes we specifically
detected Bacteria, beta- and Gamma-Proteobacteria and the major groups of ammonia-oxidizing
bacteria (Beta-AOB and Gamma-AOB) in the Danube-Black Sea mixing zone during 2007.
CARD-FISH analysis indicated that planktonic AOB might constitute a significant fraction of
the bacterioplankton in the NW Black Sea coastal system, and show distinct spatial abundance
patterns in relation to the prevailing ecological conditions.
FRANK K. THOMSON, III
AFFILIATION: Old Dominion University
TITLE: Ships Ballast as a Potential Vector for the Transfer of Antibiotic Resistance Genes
Among Coastal Environments
ABSTRACT:
Ships ballast has been implicated as a vector in the dispersal of invasive species into new
environments. Numerous cases have been documented for the introduction of macroorganisms
via ballasting operations, but what is less known is the role of ballast water and residuals in the
spread of potentially harmful microbes, especially with reference to novel genotypes. Of
particular interest are the genes encoding for various forms of antibiotic resistance, many of
which are carried on mobile genetic elements. A three-year sampling effort has yielded over 300
putative isolates of Vibrio cholerae from ships' ballast tanks and various environmental sources,
of which 208 have been profiled for antibiotic susceptibility using twelve diverse antibiotics. The
results demonstrate widespread resistance to b-lactams (74.6%), with isolated instances of
resistance to other antibiotics. Plasmid extractions and restriction enzyme analyses have shown
the presence of an ostensible plasmid of approximately 38 kbp in many of these isolates.
Additional work is being done using restriction fragment length polymorphisms and PCR for
specific antibiotic resistance genes in an attempt to characterize these plasmids. Knowledge from
this study and future microcosm experiments will help us to ascertain the potential for horizontal
gene transfer in a ballast tank setting.
LAURE-ANNE VENTOURAS
AFFILIATION: MIT
ABSTRACT:
Despite extensive efforts to understand and predict the effects that recent anthropogenic activity
will have on the physico-chemical properties of the ocean, much is still unknown as to how the
marine biosphere will be impacted. To understand how these unnatural perturbations specifically
affect microbial communities, we will use a metagenomics approach to analyze the results of
mesocosm perturbation experiments. Changes at the gene expression level will be monitored
using 454-sequencing technology, while changes at the community structure level will be
monitored using a genome-proxy microarray technology that was developed in-house. Currently
I am working on applying the genome-proxy microarray to study the changes in microbial
community structure over time and depths at the ALOHA-HOT station. The genome-proxy
microarray allows sampling of both the phylogenetic and functional breadth of a community by
using probes that target the gen ome sections from a variety of uncultivated microbes. After
gaining good understanding of the background structure of the microbial assemblages of the
North Pacific Tropical Gyre, I will carry out the mesocosm perturbation experiments that will
either focus on changing temperature, pH, heavy metal or specific pollutant concentrations.