1. Diversity of Soil Microbes

2. 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

3. 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

4. Approaches for Assessing Diversity
Microbial community
Culture
Nucleic acid extraction
Organism isolation
Molecular characterization
Phenotype

5. Nucleic acids as biomarkers

6. 16S rRNA structure

7. 16S rRNA Sequence Conservation Level nearly universal intermediate
hypervariable

8. Phylogenetic categories of organisms
Comparative analysis of 16S ribosomal RNA genes
Ancient Taxa:
The Kingdoms

9. Remotely Related Taxa: The Classes and Divisions (Phyla)
Not shown are candidate divisions, organisms detected by PCR-16S analysis,
but no currently isolated and cultured representative

10. Moderately Related Bacterial Taxa:
The Major Intradivisional
Groupings
(Orders and Families)

11. 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.

12. Approaches for Assessing Diversity
Microbial community
Culture
Nucleic acid extraction
Organism isolation
Molecular characterization
Phenotype
PCR

13. Molecular Characterization
DNA extract
PCR
PCR products
Separate molecules (cloning)
ATGCCG….
Determine Nucleotide Sequence
Comparison to data base for identification

14. 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.)

15. Relative abundance of bacteria in soil
Patterns: High = Proteobacteria, Acidobacteria, Verruomicrobia
Medium = Cytophagales, Actinobacteria, Firmicutes, Planctomycetes
Low = Nitrospira

16. Bacterial phyla abundance in arid soils

17. Bacterial phyla abundance in forest soils
FEMS Microbiol. Ecology (2002) 42:

18. 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

19. 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
Since all sphingomonas isolates were putatively identified as the same species we applied a second genetic approach call rep-PCR fingerprinting.
16s rRNA looks at changes in one gene
Where as rep-PCR looks at changes in the content of chromosomal DNA
And rep-PCR may provide insight into functional differences at the strain level.
Bacteria contain elements called repetitive sequences. rep-PCR takes advantage of these repetitive sequences by denaturing genomic DNA to expose the repetitive sequences. Primers bind the repetitive sequences.
DNA sequences between repetitive sequences are amplified.
After many cycles of this, DNA fragments of various lengths are generated and then resolved by gel electrophoreses yielding complex fragment patterns.
Similar bacteria strains show similar patterns.
Fragments are resolved by
gel electrophoresis yielding
complex fragment patterns
Similar strains show
similar patterns

20. Colored dots indicate different subspecies (strains)
REP-PCR Gel
Colored dots indicate different subspecies (strains)

21. REP-PCR Analysis of fluorescent Pseudomonas isolates from different sites and continents
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.

22. REP-PCR Analysis of fluorescent Pseudomonas isolates from different continents
Genotypes of isolates from different locations form separate clusters
Indicates genotypes were endemic

23. Summary: Geographic distribution
Fluorescent pseudomonads and others are globally- distributed
Development of endemic species occurs at finer scales
Spatial (geographic) isolation affects bacterial diversification

24. 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?

25. 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

26. 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

27. Growth and enrichment in
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

28. 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

29. Molecular Characterization
DNA extract
PCR
PCR products
Separate molecules by denaturing gradient gel electrophoresis (DGGE)

30. DGGE Analysis
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)

31. Rhizosphere variability over time
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:

32. 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

33. Functions possesed by the indicated group
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?

34. 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

35. Dilution experiment results: Biomass and diversity
No significant difference in biomass, but
Decreasing diversity as function of dilution
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

36. Dilution experiment results: Molecular analysis of bacterial diversity
Decreasing number of bands, decreasing diversity
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

37. Dilution experiment results: Activity as a function of diversity
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.

38. 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.