of microorganisms constitutes one of the three domains (major branches)
of life. Discovered by Carl Woese in the late 1970's, archaea were
originally thought to be those organisms that lived only in extreme
environments. These organisms share many common biological features
that set them aside from the rest of the bacteria that microbiologists
had studied for years. For instance, their outer membranes are made
of ether-linked rather than ester-linked fatty acids like the rest
of biological life, and the way that they process their DNA and RNA
in the cell is much more like a eukaryote. By comparing gene features
that are common to all forms of life, in this case the ribosomal RNA,
Dr. Woese found that the extreme environment loving microorganisms
appeared to share a common evolutionarily origin - and that they were
sufficiently different from the bacteria and eukaryotes that he proposed
a third form (Domain) of life, the Archaebacteria. Since then the
name has been shortened to Archaea, and these organisms once thought
to only inhabit extreme environments are found in nearly every environment
that we look! They are the dominant life form in many extreme environments,
but the group is much more diverse that originally recognized, such
that relatives of the hyperthermophiles (organisms living at temperatures
> 80) have been found in seawater, lakes, and soils throughout the
- Organisms living in aquatic
habitats are classified by size (megaplankton, mesoplankton, microplankton,
nanoplankton, picoplankton) and biological group (zooplankton, phytoplankton,
bacterioplankton). The bacterioplankton encompass both bacteria and
archaea that are free-living in aquatic habitats.
or chemicals that microbes produce during their daily life functions.
These can include proteins/enzymes, ribonucleic acids, fatty acids,
antibiotics, gases, minerals, and other chemicals. Detection of these
metabolites and chemicals can provide clues as to which processes
the organisms are involved with, which chemical transformations they
are performing, and how they respond to changes in their environment.
Gradient Gel Electrophoresis - a molecular biology tool used to separate
DNA fragments by their sequence specific melting (denaturation) point.
DGGE has increasingly been used to visualize the diversity of ribosomal
RNA genes present in natural microbial samples from all sorts of habitats.
After a sample is run through the gel, individual sequences, representing
different species show up as distinct bands - therefore providing
a fingerprint of the diversity in a sample. You can run 15-20 samples
on a gel at a time, and compare the fingerprints within and between
that thrive in niche environments where most organisms are not adapted
for survival. Such environments include but are not limited to sulfur
springs (lacking oxygen), thermal vents (heat extremes), brine channels
(extremes in salt), and polar environments (cold extremes).
cosmid, which is like a plasmid, but it capable of containing much
larger pieces of DNA, up to 50 kb compared to about 10 kb in a plasmid.
Like plasmids, fosmids are circular. However, unlike plasmids, E.Coli
can only ever consume (and therefore replicate) one fosmid, which
yields a much lower copy number when cloning. We can get larger pieces
of DNA, but less of them, with fosmids vs. plasmids.
have many genes, but they are not always "turned on," or expressed.
Changes in an organism's environment can affect which genes are turned
"on" or "off." For microorganisms the expression of a specific gene
may occur within minutes or even seconds following exposure to a given
environmental change. For example, a large group of proteins, known
as heat shock proteins (HSPs), were found to be over-expressed following
exposure of E. coli cells to a temperature increase of only
5 degrees C. The HSP mRNA is expressed, which then codes for the proteins
that help the cell deal with the heat exposure (HSPs degrade proteins
that are denatured, and protect ones that are needed).
of the entire DNA sequence (all of the genes present) in an organism
or group of organisms.
are long pieces of DNA (20,000-150,000 bases long - contig originates
from contiguous - meaning one piece), while libraries are a form of
archiving the large pieces of DNA. When a library is constructed,
individual contigs are cloned into individual Escherichia coli (E.
coli) cells, which grow up into colonies on agar plates when plated.
Each colony contains many cells, though all those cells are "clones,"
since they are the result of multiple cellular divisions of the same
cell. We select these clones (manually, or these days with robots),
and place them into multi-well plates that can archive 96-384 different
clones at a time.
an incredibly abundant group of microorganisms inhabiting the worlds'
ocean - not often thought of as an extreme environment. They are most
commonly found in deep sea waters from ~150m (450 ft), all the way
to the sea-floor (1000-4000 m), where they can make up from 10-40%
of the cells found (Karner, M.B., E.F. DeLong, and D.M. Karl 2001.
Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature
409:507-510). This distribution makes it somewhat difficult to study
them. Further complicating their study is the fact that we haven't
figured out how to culture them yet.
developed in the early 1990's which adapts previous DNA hybridization
approaches to a microscale format in which the DNA for thousands of
genes can be spotted on a substrate (a microscope slide, silica chip
or other surface) in a grid (array) in which the address of each gene
is known. Nucleic acids (mRNA converted to cDNA, or DNA itself) from
samples of interest are then labeled with fluorescent dyes and allowed
to find perfect matches with genes on the array, the process of hybridization.
Microarrays are most commonly used to study gene expression - to visually
detect which genes in an organism or group of organisms are "turned
on" (expressed) or "turned off" (latent).
reading frame (ORF)
- Regions in prokaryotic genomes
that code for proteins are called open reading frames, or ORFs. An ORF
is a DNA fragment whose 5' end starts at a start codon, has a series
of consecutive triplets (codons) and ends at a stop codon. All protein-coding
genes are contained in ORFs, but not all recognized ORFs contain proteins.
organisms that are uniquely adapted to cold water, sediments, soils,
snow, and ice, and grow optimally at temperatures less than 10°C.
of the shotgun arrays is to identify functional genes of interest
directly from gene expression/microarray hybridization experiments
(those that have "interesting" differential expression signals,
are highly expressed etc.), and thereby circumvent the sequencing
step, which is time consuming and costly. The shotgun
libraries are prepared, then the inserts from the random libraries
are amplified by PCR, purified, and spotted directly on the microarrays.
These are called shotgun arrays, since though we know which subclone
the spots come from, we do not know anything other than the phylogenetic
identity of the genome from which the subclones were generated.
DNA is sheared
randomly by partial digestion with restriction endonucleases, mechanical
shearing by forcing through a small bore needle, or as in our case
a process called nebulization. The ends of the randomly sheared DNA
are treated to make them "clean" or blunt ended - then these fragments
are ligated into plasmids which are transformed into E. coli,
then picked into their own 96-well plates and processed to sequence
the DNA. In our application we have prepared shotgun libraries of
already cloned large-contig DNA (from the APEL), since it's very difficult
to sequence directly off of the fosmid DNA as a template (it's too
large, and difficult to get in high enough concentrations). The plasmids
are in high copy number in each E. coli cell, making it easy
to get high yields of plasmid DNA from each clone.
of salts present in ocean water results in a depression of the freezing
point. That is, the salts present interrupt the formation of the ice
crystal structure of water, and lower (depress) the temperature at
which water freezes.