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 Nucleic acid extraction Organism isolation Molecular characterization Phenotype

Nucleic acids as biomarkers

16S rRNA structure

16S rRNA Sequence Conservation Level 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) 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)

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.

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

Molecular Characterization DNA extract PCR PCR products Separate molecules (cloning) ATGCCG…. Determine Nucleotide Sequence 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

Bacterial phyla abundance in arid soils

Bacterial phyla abundance in forest soils 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 www.bacbarcodes.com 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

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

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.

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

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

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

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

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 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 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

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?

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 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 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 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.