59 research outputs found
The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing.
Microbial ecology is plagued by problems
of an abstract nature. Cell sizes are so
small and population sizes so large that
both are virtually incomprehensible. Niches
are so far from our everyday experience
as to make their very definition elusive.
Organisms that may be abundant and
critical to our survival are little understood,
seldom described and/or cultured,
and sometimes yet to be even seen. One
way to confront these problems is to use
data of an even more abstract nature:
molecular sequence data. Massive environmental
nucleic acid sequencing, such
as metagenomics or metatranscriptomics,
promises functional analysis of microbial
communities as a whole, without prior
knowledge of which organisms are in the
environment or exactly how they are
interacting. But sequence-based ecological
studies nearly always use a comparative
approach, and that requires relevant
reference sequences, which are an extremely
limited resource when it comes to
microbial eukaryotes.
In practice, this means sequence databases
need to be populated with enormous
quantities of data for which we have
some certainties about the source. Most
important is the taxonomic identity of
the organism from which a sequence is
derived and as much functional identification
of the encoded proteins as possible. In
an ideal world, such information would be
available as a large set of complete, well curated,
and annotated genomes for all the
major organisms from the environment
in question. Reality substantially diverges
from this ideal, but at least for bacterial
molecular ecology, there is a database
consisting of thousands of complete genomes
from a wide range of taxa,
supplemented by a phylogeny-driven approach
to diversifying genomics [2]. For
eukaryotes, the number of available genomes
is far, far fewer, and we have relied
much more heavily on random growth of
sequence databases, raising the
question as to whether this is fit for
purpose
The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): Illuminating the Functional Diversity of Eukaryotic Life in the Oceans through Transcriptome Sequencing
Microbial ecology is plagued by problems of an abstract nature. Cell sizes are so small and population sizes so large that both are virtually incomprehensible. Niches are so far from our everyday experience as to make their very definition elusive. Organisms that may be abundant and critical to our survival are little understood, seldom described and/or cultured, and sometimes yet to be even seen. One way to confront these problems is to use data of an even more abstract nature: molecular sequence data. Massive environmental nucleic acid sequencing, such as metagenomics or metatranscriptomics, promises functional analysis of microbial communities as a whole, without prior knowledge of which organisms are in the environment or exactly how they are interacting. But sequence-based ecological studies nearly always use a comparative approach, and that requires relevant reference sequences, which are an extremely limited resource when it comes to microbial eukaryotes
Real-Time PCR Quantification of rbcL (Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase) mRNA in Diatoms and Pelagophytes
Transcriptional activity is often used as a surrogate for gene expression in environmental microbial communities. We developed a real-time PCR assay in which the ABI-Prism (PE Applied Biosystems) detection system is used for quantification of large-subunit ribulose-1,5-bisphosphate caboxylase/oxygenase (rbcL) mRNA in diatoms and pelagophytes both in cultures and from natural phytoplankton communities. Plasmid DNA containing rbcL inserts, as well as in vitro transcribed mRNA of the plasmids, was used to generate standard curves with a dynamic range of more than 6 orders of magnitude with high accuracy and precision (R(2) = 0.998). Expression levels in a cultured diatom (Phaeodactylum tricornutum) were quantified through one light-dark cycle by using traditional (35)S-labeled oligonucleotide hybridization and real-time PCR. The mRNA levels detected by the two techniques were similar and correlated well (R(2) = 0.95; slope = 1.2). The quantities obtained by hybridization were slightly, yet significantly, larger (t = 5.29; P = 0.0011) than the quantities obtained by real-time PCR. This was most likely because partially degraded transcripts were not detected by real-time PCR. rbcL mRNA detection by real-time PCR was 3 orders of magnitude more sensitive than rbcL mRNA detection by hybridization. Diatom and pelagophyte rbcL mRNAs were also quantified in a profile from an oligotrophic site in the Gulf of Mexico. We detected the smallest amount of diatom rbcL expression in the surface water and maximum expression at a depth that coincided with the depth of the subsurface chlorophyll maximum. These results indicate that real-time PCR may be utilized for quantification of microbial gene expression in the environment
Gene Diversity and Organization in \u3cem\u3erbcL\u3c/em\u3e-Containing Genome Fragments from Uncultivated \u3cem\u3eSynechococcus\u3c/em\u3e in the Gulf of Mexico
Factors controlling phytoplankton distribution and activity in the marine environment are often poorly understood. Knowledge of genomic content and organization may aid in understanding these processes. To this end, bulk environmental DNA was extracted from Gulf of Mexico seawater and cloned into a bacterial artificial chromosome (BAC) vector to generate a library of 3000 large-insert clones. This library was probed for various types of RubisCO gene (rbcL); 8 rbcL-containing clones (20 to 73 kb in size) were detected and sequenced. rbcL sequences indicated that they originated from marine α-Synechococcus spp. Genomic organization was highly similar to that of the sequenced genome of Synechococcus sp. strain WH 8102 in the region surrounding rbcL. Several differences from known α-Synechococcus were also observed, including putative monoamine oxidase and amino acid transport genes, and electron transport, photosynthesis and hypothetical protein genes similar to those of Prochlorococcus spp. (i.e. flavodoxin, thioredoxin reductase and iron-stress-induced chlorophyll-binding proteins). These findings indicate that the order and composition of the carbon fixation operon (cbbX to ccmK) is highly conserved in α-Synechococcus, while adjacent genomic composition indicates expanded metabolic capabilities obtained from gene-transfer events with Prochlorococcus spp. and other cyanobacteria
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