RE: [compost_tea] The soil metagenome and methods of discovery

From: John Dunbar <jdunbar_at_dunbarco.com>
Date: Wed, 18 Aug 2004 13:49:51 -0500

Interesting material on the unknowns of the soil food web.

Coincidently, I was reading one of Paul Stamets' book on fungi this morning.
He made the same point: many fungi are difficult to culture independent of
their natural environment because these fungi require the help of many other
organisms, and scientists have not identified all "the moving parts" so to
speak, nor have they identified any possible phases when each are required.
Some day that will change/improve as scientists map, identify, understand
the nature(s) of soil. This APS material, although way over my head, has a
lot of interesting comments.

Thanks Steve Diver for your always interesting postings.

John

-----Original Message-----
From: Steve Diver [mailto:steved_at_ncat.org]
Sent: Wednesday, August 18, 2004 12:44 PM
To: compost_tea_at_yahoogroups.com
Subject: [compost_tea] The soil metagenome and methods of discovery


After attending the American Phytopathological Society
meetings in Anaheim I've been looking for useful
information on molecular techniques.

A great number of papers and posters at APS examined
beneficial microbes that enhance soil health or provide
biocontrol, as well as plant pathogens that cause disese,
using molecular techniques and genomic sequencing.

rDNA sequencing
DNA extraction and PCR analysis
real-time PCR analysis
PCR (Polymerase Chain Reaction)
gel electrophoresis
gene expression
phylogenetic mapping

Here are two related papers with background information
of interest. If you click on the DOI it should take you to
the abstract, but you may need to go thru a university
library to obtain the full-text article.

Regards,
Steve Diver

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The soil metagenome – a rich resource for the discovery
of novel natural products

Rolf Daniel

Current Opinion in Biotechnology
Volume 15, Issue 3 , June 2004, Pages 199-204

http://dx.doi.org/doi:10.1016/j.copbio.2004.04.005

Excerpts:


Introduction

"Soil is a very complex habitat dominated by the soil solid phase.
In contrast to water systems, soil is relatively recalcitrant to
mixing, but soluble components of the solid soil matrix may
dissolve in soil water and reprecipitate at other sites. The
soil microorganisms are localized in close association with soil
particles, such as complexes of clay–organic matter [1.].
Microorganisms can be found as single cells or microcolonies,
which are often embedded in a polysaccharide matrix. Their
metabolism and interactions with other organisms and with soil
particles is dependent on the conditions at the microhabitat
level, which often differ between microhabitats even over very
small distances. The microhabitats for soil microorganisms include
micropores and the surfaces of soil aggregates of various
composition and sizes [2. and 3.]. Thus, soil can be regarded
as very heterogeneous with respect to conditions for microbial
growth and for the distribution of microorganisms and matrix
substances. This heterogeneity results in a wide variety of
microbial niches and a high diversity of soil microorganisms.
The microbial diversity in soils exceeds that of other environments
and is far greater than that of eukaryotic organisms: one gram
of soil can contain up to 10 billion microorganisms of possibly
thousands of different species [4.]. The genetic complexity of
microbial soil communities has been estimated by reassociation
of community DNA. Not taking into account the genomes of rare
and unrecovered microorganisms, such analyses have shown that
the soil community size is equivalent to 6000–10 000 Escherichia
coli genomes [5. and 6.]. In another study, the analysis of the
reassociation kinetics of the total bacterial DNA in a 30 g
soil sample revealed that it contained more than 500 000
species [7.]. These results illustrate that the entirety of
the microbial genomes found in soil samples, termed the soil
metagenome throughout this review, harbors more genetic
information than is contained in the approximately 5000 to
6000 individual prokaryotic microorganisms available in the
different culture collections. Thus, the genetic diversity
of the soil metagenome is a rich and widely unexplored resource
for new industrial enzymes and bioactive compounds.

In this review, the recent progress in recovering novel
biocatalysts and other biomolecules from the soil metagenome
is summarized. An overview of novel cultivation-dependent and
molecular cultivation-independent approaches to access and
exploit the diversity of soil microbial communities with respect
to natural product discovery is given."

Cultivation-dependent approaches

"The diversity of soil microorganisms has been exploited for
many years based on the cultivation and isolation of microbial
species. Most natural products of economic value such as
antibiotics or other pharmaceuticals are derived from cultured
soil microorganisms, but the rate of discovery of novel natural
products derived from isolated microorganisms has significantly
decreased during the past couple of years [8.]. This is mainly
due to a high rediscovery frequency, as the same organisms are
recovered repeatedly by using standard cultivation techniques.
It has been estimated that 0.1–1% of the total soil population
can be cultured by applying these techniques. In a study by
Kellenberger [9.], approximately 107 cells were counted in
1 g of soil, but only 0.1% of the cells were culturable. Thus,
99.9% of the genetic diversity present in this population was
lost owing to difficulties in enriching and isolating
microorganisms. These unrecovered microorganisms represent
an unexplored reservoir of novel strains, which may produce
novel natural products."

Cultivation-independent molecular approaches

"To expand the basis for natural product discovery from soil
and other habitats and to circumvent some of the problems
associated with culturing microorganisms cultivation-independent
molecular approaches have been developed (Figure 1). These
alternative ways of accessing the soil metagenome include the
extraction of nucleic acids, PCR amplification, and the direct
cloning of soil DNA [17., 18., 19., 20., 21., 22. and 23.]."

etc...

++++++++++++++++++++++++++++++++++++++++++++++++++++++++

Biotechnological prospects from metagenomics

Patrick D Schloss and Jo Handelsman

Current Opinion in Biotechnology
Volume 14, Issue 3, June 2003, Pages 245-355

http://dx.doi.org/doi:10.1016/S0958-1669(03)00067-3

Excerpts:


Introduction

"Metagenomics is the culture-independent genomic analysis of
microbial communities. The term is derived from the statistical
concept of meta-analysis (the process of statistically combining
separate analyses) and genomics (the comprehensive analysis of
an organism's genetic material) [1]. Metagenomics can be used to
address the challenge of studying prokaryotes in the environment
that are, as yet, unculturable and which represent more than 99%
of the organisms in some environments [2]. This approach builds
on recent advances in microbial genomics and in the polymerase
chain reaction (PCR) amplification and cloning of genes that
share sequence similarity (e.g. 16S rRNA, nif, recA) directly
from environmental samples [3]. Whereas PCR amplification
requires prior knowledge of the sequence of the gene to design
primers for amplification, direct extraction and cloning of DNA
for genomics can theoretically access genes of any sequence or
function. Direct genomic cloning offers the opportunity to
capture operons or genes encoding pathways that may direct
the synthesis of complex molecules, such as antibiotics.
Sequence information about the genes flanking a gene of
particular interest can also be obtained, potentially providing
insight into the genomic environment of the gene or the
phylogenetic affiliation of the organism from which it was
derived. Moreover, a long-term goal of metagenomic analysis
is to reconstruct the genomes of uncultured organisms by
identifying verlapping fragments in metagenomic libraries
and `walking', clone to clone, to assemble each chromosome.

In this review, we discuss the two approaches to metagenomic
analysis, designated the `function-driven' and
`sequence-driven'
analyses and review key discoveries derived from metagenomics.
We close with an exploration of future technical and strategic
mprovements that will enhance the discovery rate and focus on
types of genes or organisms of particular interest for
biotechnological advances."


Enriching for genomes from metabolically active cells

"The symbionts of aphids and the marine sponge represent the
simplest type of community, containing a single species with
some genetic variation in the population. At the other extreme
of complexity is the soil, containing an estimated 109 prokaryotes
and more than 2000 genome types per gram of soil, with an average
genome type representing less than 0.05% of the soil community
[40]. With today's technology it would be difficult to obtain
complete coverage of all of the genomes in a soil community.
Therefore, carving out slices of the community, selected for
a common feature, reduces the complexity of the task and brings
into sight the possibility of complete coverage of a subset of
the soil community."

++++++++++++++++++++++++++++++++++++++++++++++++++++++++
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Received on Wed Aug 18 2004 - 15:05:33 EDT

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