2007 ‫טקס חלוקת מלגות ע"ש אורן ברקו ז"ל‬
Environmental genomics of marine cyanobacteria
Sigrid Penno-Winters, PhD student at the Hebrew University at Jerusalem,
Interuniversity Institute for Marine Science Eilat
Supervisor Prof. Anton Post
Subject of research
The importance of marine picophytoplankton (< 2µm) as primary producers in vast areas of the world's
oceans has been recognized over the past decade. Two prokaryotic genera dominate the picophytoplankton
community in oligotrophic waters – the unicellular, non-nitrogen fixing cyanobacteria Synechococcus and
Prochlorococcus. They can contribute up to 80% of the total primary productivity in such waters. Although
the isolation of Synechococcus and Prochlorococcus in laboratory cultures has allowed the study of their
physiology and genetics, there is still little known about the genetic diversity of natural communities and
adaptation to diverse environmental factors. The main objective of my research is cultivation-independent
characterization of the diversity of Synechococcus and Prochlorococcus genotypes in the oligotrophic Gulf of
Eilat. Using an environmental genomic approach I investigate the genetic diversity of Synechococcus and
Prochlorococcus as well as of other members of the picoplankton community along nutrient gradients as
experienced along depth profiles and over the seasonal cycle. The hypothesis is that, as a result of these
gradients, phylogenetically closely related populations develop, which exhibit a genetic make-up suited to
address environmental conditions.
Methods
Water samples are taken at precise depth in different seasons at station A (29°28’N, 34°55’E) in the Gulf of
Eilat using a CTD-Rosette containing 12 liter Go-Flo bottles on board the IUI research vessel. Continuous
profiles of temperature, salinity, light and fluorescence are measured by the CTD, a LICOR photocell and a
fluorimeter. Furthermore, monthly profiles of Chl a, oxygen, pH and N and P nutrient concentrations are
measured by the IUI monitoring program and are available as ancillary data. The spatial and temporal
distribution of picophytoplankton communities is characterized by flow cytometry using a FACScan.
The water samples are prefiltered through a 6 µm mesh and than filtered onto a 0.45 µm filter which is than
used for genomic DNA extraction. For diversity studies of Synechococcus and Prochlorococcus genotypes
the N-regulatory gene ntcA is amplified by Polymerase Chain Reaction (PCR) using degenerated,
cyanobacterial specific primers. ntcA clone libraries are designed using standard cloning methods.
Synechococcus versus Prochlorococcus clones are selected either by colony PCR using specific primer or by
RFLP (restriction fragment length polymorphism). Positive clones are sequenced at the DNA analyses unit at
the Hebrew University of Jerusalem. Phylogenetic analyses of the environmental sequences along with
sequences retrieved from the gene database GenBank (NCBI) are done using PAUP software (Swofford,
2003). PCR based sequences of a single genes or gene fragment from natural samples provide limited
phylogenetic information. Therefore fosmid libraries are designed using the Copy Control Fosmid cloning kit
(Epicenter) allowing the study of environmental genome fragments of up to 40 kb. Randomly picked clones
are endsequenced in cooperation with the Marine Biology Laboratory at Woods Hole (USA) and the Genome
Center at the Goettingen University (Germany). Resulting sequences are submitted for BlastX analyzes using
GenBank (NCBI) in order to identify the organism present in the environmental sample .
Furthermore, the fosmid libraries are screened with specific probes for N-metabolic genes of Synechococcus
and Prochlorococcus using hybridization methods. Synechococcus and Prochlorococcus fosmid clones of
interest, identified by BlastX analysis or by hybridization are used for shotgun cloning in order to sequence
the entire fosmid insert. Therefore the purified fosmid DNA is physically sheared using a Hydroshear or
Nebulizer and subcloned into Topo-TA cloning vector (Invitrogen). Retrieved sequences are assembled using
Gap4 (R. Staden, 1995) and the entire sequence is compared to published Synechococcus and
Prochlorococcus genomes (GenBank, NCBI).
Results and significance of research
The analysis of the monthly nutrient concentrations and picophytoplankton cell counts from the IUI
monitoring program over the past years show the seasonal changes of mixed, nutrient enriched waters in the
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winter with eukaryotic algae abundance, the Synechococcus bloom at the onset of the stratification in spring
and the dominance of Prochlorococcus in oligotrophic waters in the summer.
The diversity of Synechococcus and Prochlorococcus populations in different seasons and depth with diverse
nitrogen sources and availabilities was studied using the ntcA gene. This gene, unique to cyanobacteria and
present as a single copy in the genomes plays a central role in nitrogen metabolism regulation in the cells. It
proved to be an excellent diversity marker giving a much higher resolution in phylogenetic analyses than
compared to standard diversity markers such as the 16S rRNA gene. An unexpected large diversity of distinct
Synechococcus types including 4 new genotypes, but a rather uniform Prochlorococcus population exhibiting
one genotype was found in the surface waters of the Gulf of Eilat. A multi-annual survey identified certain
Synechococcus genotypes as generalists being present year around. Other genotypes showed seasonal
alteration being more abundant and therefore better adapted to either the nutrient enriched environment in the
winter/spring or to the nutrient poor (oligotrophic) waters in the summer. Contrary, one Prochlororoccus
genotypes was identified throughout the year. Additional ntcA diversity studies in the Mediterranean Sea and
the Arabic Sea showed the dominance of a different Prochlororoccus genotype in surface waters. Concluding,
the existence and the seasonal or local alteration of different Synechococcus and Prochlorococcus genotypes
can be linked to the nutrient status of the environment they inhabit and therefore support the theory of
coexisting ecotypes well adapted to certain microhabitats. The results of these studies led to two publications
(Journal of Limnology and Oceanography and Journal of Environmental Microbiology) and a third
publication currently in preparation.
Environmental, high molecular weight genomic DNA from two different seasons and depths,
characterized by different nutrient and light availabilities were successfully cloned into fosmid vectors. Blast
search analysis of so far over thousand fosmid clone endsequences provide for the first time an overview of
organism within the size range of 0.45 to 6 µm present in the Gulf of Eilat. The majority of organisms belong
to the eubacteria phylum (54%) and much less to the phyla of eukaryotes (5%), archaea (4%) and viruses
(2%). 35% of the retrieved sequences could not be identified by the Blast search or are of unknown origin.
Most of the eubacteria belong to the order of proteobacteria (53%) with all families (α, ß, γ, δ, ε) present,
though to a different extent. Other orders of eubacteria were identified as planktomycetes (11%),
actinomycetes (5%), flavobacteria (5%), cyanobacteria (5%), chloroflexales (4%) and others. Formerly
described cyanobacteria species like Trichodesmium, Synechococcus and Prochlorococcus are present as well
as others like Nodularia, Lyngbya, Cyanothece and Nostoc which so far have been never described in the
Gulf .
Only recent studies showed the occurrence of archaea in open oceans which is confirmed in this study with
the majority belonging to the order of euryarcheota (88% of total archaea). Marine viruses are smaller than
0.2 µm and are known to be very abundant. They are underrepresented in our analysis due to the selected size
fraction. Nevertheless viruses were detected, mainly bacteriophages and cyanophage.
In the near future additional endsequences of two surface water fosmid libraries, one from a mixed and one
from a stratified water column will complete the database of present picoplankton groups in the Gulf of Eilat.
Through comparative analysis of fully sequenced genomes of Synechococcus and Prochlorococcus strains we
were able to identify gene clusters essential for the uptake of different nitrogen sources like ammonium, urea,
nitrite, nitrate and cyanate. The different strains showed great variation in presence or absence of these genes/
gene clusters. Synechococcus and Prochlorococcus environmental fosmid clones containing some of the
above mentioned genes/ gene clusters were identified and fully sequenced. The comparison of environmental
genome fragments to known genomes revealed the presence of specific genotypes with the genetic potential
to utilize certain N sources. Some fosmid clones which derived from a water sample of 140 m in a stratified
water column were identified as so called low light (LL) adapted Prochlorococcus with some genotypes been
described previously in the Gulf but others are identified for the first time. Within our ntcA clone libraries
from depth until 200 m LL Prochlorococcus were never detected. For more detailed information some of the
LL Prochlorococcus fosmid clones are currently fully sequenced .
Overall my research addresses the hypothesis that diversity in cyanobacterial genotypes (biodiversity) is
matched by plasticity in the gene pool that mediates environmental stress responses (niche adaptation). The
study will have an impact to better understand the importance, success and genetic potential of
picophytoplankton species in oligotrophic waters where they contribute significant to primary production. It
will provide better in sight in the environmental conditions that affect diversity, succession and productivity
of the marine phytoplankton.
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