Diversity of Soil Microbes
Questions
What types of microbes comprise the microbial
community?
What are their actitvities?
What aspects of the environment control their
activities?
How is the composition of a community related to a
given soil and activity within the soil?
How does overall composition change?
How does representation of a certain group change?
How do perterbations of the soil impact the structure
of the community?
How do changes in composition affect activity
Microbial Classification
Genotype
Phylogenetic: analysis of genes encoding ribosomal RNA
Genomic: analysis of multiple genes or DNA sequences
in the genome
Phenotype
Cell structure: Gram stain, flagella, etc.
Metabolic traits: Enzyme activity
Approaches for Assessing Diversity
Microbial
community
Culture
Organism
isolation
Phenotype
Nucleic acid extraction
Molecular
characterization
Nucleic acids as biomarkers
16S rRNA structure
16S rRNA
Sequence Conservation Leve
nearly universal
intermediate
hypervariable
Phylogenetic categories of organisms
Comparative analysis of 16S
ribosomal RNA genes
Ancient Taxa:
The Kingdoms
Remotely Related Taxa: The Classes and Divisions (Phyla)
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Not shown are candidate divisions, organisms detected by PCR-16S analysis,
but no currently isolated and cultured representative
Moderately Related Bacterial Taxa:
The Major Intradivisional
Groupings
(Orders and Families)
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Highly Related Taxa: The Genera and Species
Example: Phylogeny of
Ammonia-oxidizing bacteria
Organisms of the same species
are >97% identical in 16S rRNA
gene.
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Approaches for Assessing Diversity
Microbial
community
Culture
Organism
isolation
Phenotype
Nucleic acid extraction
Molecular
characterization
PCR
Molecular
Characterization
DNA extract
PCR
PCR products
Separate molecules (cloning)
Determine Nucleotide Sequence
ATGCCG….
Comparison to data base for identification
Overview of the
Polymerase
chain reaction
(PCR)
For phylogenetic
analysis,
primers (P1, P2)
target regions of
16S rDNA that
are conserved
within a given
group of
organisms
(Bacteria,
Proteobacteria,
Pseudomonas,
etc.)
Relative abundance of bacteria in soil
Patterns: High = Proteobacteria, Acidobacteria, Verruomicrobia
Medium = Cytophagales, Actinobacteria, Firmicutes,
Planctomycetes
Low = Nitrospira
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Bacterial phyla abundance in arid soils
Bacterial phyla abundance in forest soils
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FEMS Microbiol. Ecology (2002) 42:347-357
Are some types of bacteria unique (endemic) to
a given location or soil?
Phyla and species are globally distributed (not endemic)
Are some strains (subspecies) endemic?
Requires analysis that affords a greater level of
resolution than 16S
--> Genotype analysis
Reptitive element (REP)-PCR Genomic fingerprinting
16S rRNA analysis =
changes in one gene
(DNA sequence) at one
site in the genome
REP = short sequences
that are occur in multiple
locations throughout the
bacterial genome
REP-PCR
assays
variation in sequence at
multiple sites throughout
the genome
Patterns
differentiate
bacteria at subspecies
level
Genomic DNA
denatured to expose
repetitive sequences
Primers bind the
repetitive sequences
DNA sequences
between repetitive
sequences are
amplified
DNA fragments of
various lengths are
generated
www.bacbarcodes.com
Fragments are resolved by
gel electrophoresis yielding
complex fragment patterns
Similar strains show
similar patterns
REP-PCR
Gel
Colored dots
indicate different
subspecies
(strains)
Species 1
Species 2
REP-PCR Analysis of fluorescent
Pseudomonas isolates from
different sites and continents
Fluorescent Pseudomonas:
Produce bright pigments in culture
Commoly isolated from soils and plant
rhizospheres
Metabolically diverse
Are strains globally mixed or endemic?
Examine REP-PCR patterns of bacteria isolated from
different continents, and different sites within
continents.
REP-PCR Analysis of
fluorescent Pseudomonas
isolates from different
continents
Genotypes of isolates from
different locations form
separate clusters
Indicates genotypes were
endemic
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Summary: Geographic distribution
•Fluorescent pseudomonads and others are globallydistributed
•Development of endemic species occurs at finer scales
•Spatial
(geographic)
diversification
isolation
affects
bacterial
Effects of plant growth on microbial community
composition
How do plants alter the composition of soil microbial
communities?
Are certain bacteria preferentially selected for
colonization of the rhizosphere?
Do certain plants select for certain bacteria?
What has a greater impact on the composition of plant
rhizospheres? The plant type or the soil?
Rhizosphere: General effects
Density (abundance) of bacteria increases in rhizosphere
relative to bulk soil
Bacterial diversity in rhizosphere decreases relative to soil.
Increase in abundance of Proteobacteria relative
to other phyla
Comparison of rhizosphere vs. soil effects
Different populations of pseudomonads colonize the rhizosphere of
the same plants grown in different soils
Unique to Soil 1
Unique to Soil 2
Rhizosphere
Effects
Growth and enrichment in
plant rhizosphere
Sub-populations selected
for growth from each soil
Selection imposed by
rhizosphere
varies with plant, and plants
(root) age
Soil 1 Population
Soil 2 Population
Rhizosphere Effects
Plant selects for organisms from a pool that has
developed and established in the soil.
Different parts of the pool may be selected by different
plants
Since plant changes with time, selection also varies with
time for a given plant
Molecular
Characterization
DNA extract
PCR
PCR products
Separate molecules by denaturing
gradient gel electrophoresis (DGGE)
DGGE Analysis
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Basis for DGGE separation: DNA molecules, which differ
by only one nucleotide within a low melting domain will
have different melting points (temperature or chemical)
Rhizosphere variability over time
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DGGE profiles of Pseudomonas rhizosphere communities at the early (A) and late (B)
flowering stage and at the senescent growth stage (C). Different lanes (1–3) represent
rhizosphere sample from different pots. GM, transgenic plants without herbicide; G,
transgenic plants with Basta application; MM, wild-type plant without herbicide application;
B, wild-type plants with Butisan S application. Bands 2a, 2b, 2c and 2d had the same
mobilities as bands 1a (3a), 3b, 1f and 1d.
FEMS Microbiol. Ecol. 41:181-190
Perterbations: General effects on
soil microbial communities
Perterbations: Typically decrease diversity, select for a
proliferation of subpopulations
Examples:
Plants- rhizosphere
Animals - Earthworm casts
Geological - volcanic eruption
Climatic - Fire
Anthropogenic - Pollution, agricultural practices
Do bacterial community structure changes (decreased
diversity) induced by perturbations affect function?
Transformations
A
B
C
D
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Functions possesed by the indicated group
Stability in transformation of A -->D is supported by diversity
in organisms and establishment of redundancy in the
activities they possess.
How is decreased diversity (less redundacy) reflected in
community activity?
Community Dilution Experiment
Hypothesis: Reduced diversity, reduces redundancy,
and reduces the capability of soils to respond to stress
Approach:
Sterile soil inoculated with serial dilution of soil
suspension
Incubated 9 months
Measured biomass, assessed community diversity,
and activities
Dilution experiment results: Biomass and diversity
No significant difference in biomass, but
Decreasing diversity as function of dilution
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The overall biodiversity in sterile soil inoculated with dilutions of a
soil suspension (A, 10 0; B, 10 2; C, 10 4; D, 10 6). The number of
species within the individual populations measured (i.e. soil
bacterial DNA bands, cultivable bacterial morphotypes, cultivable
fungal morphotypes and protozoan species) was normalized
relative to the maximum number observed, summed and divided
by four (i.e. the number of taxonomic groups) to give the
biodiversity index. The bar represents ± one standard error, n=3
Dilution experiment results: Molecular analysis of
bacterial diversity
Decreasing number of bands, decreasing diversity
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The DNA banding pattern of soil bacteria obtained by DGGE analysis of
eubacterial-primer based amplicons from sterile soil inoculated with dilutions
of a soil suspension (A, 100; B, 102; C, 104; D, 106). The three lanes of each
treatment represent individual replicates, n=3
Dilution experiment results: Activity as a function of
diversity
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Effect of stresses, in the
form of Cu addition or heat
treatment, on the ability of
sterile soil inoculated with
dilutions of a soil suspension
(A, 10 0; B, 10 2; C, 10 4; D,
10 6) to decompose grass
residues at increasing time
intervals following the
application of the stress.
Bars represent one standard
error, n=3.
Community Dilution Experiment-Conclusion
Diversity could be experimentally manipulated
No detectable change in activity with the level of diversity
reduction achieved.
Minimum level of diversity (functional redundancy) to
support process stability in soil is unknown.