Lecture 16 Molecular Analysis of Microbial Communities

1
Lecture 16Molecular Analysis of Microbial
Communities

• Review
• Microbial communities
• Questions
• Species question
• Analysis
• Applications
• Recombinant DNA technology
• Method
• Application

2
Review

• Any questions ?
• Any answers ?
• Maximum parsimony
• Bootstrapping
• Species identification
• 2 approaches
• Parasite identification
• 2 main aims
• Cryptic species
• Hybridization and introgression
• Significance
• Asymmetrical
• Speciation

3
Microbial communities

• The unsung heroes
• Extremely important
• Cycling of geochemical elements and nutrients
• Biotechnology
• Species rich
• Little known
• Only 1 culturable
• Main questions
• Who is who?
• Species concept
• Who is there?
• Species ID
• How many of them?
• Species diversity
• What are they doing?
• Ecological diversity
• Distribution

4
Who is who?Microbes and the Species Concept

• 22 species concepts
• Morphological (typological)
• Similar morphology
• Type specimens
• Biological
• Actually or potentially interbreeding
• Recognition
• Recognizing mates
• Ecological
• Similar niche
• Phylogenetic
• Monophyletic group

• Application to microbes
• Morphological
• Not much of a morphology
• Shape, size, flagellae
• Biological
• Mostly clonal, sex rare
• May occur between morphological species
• Different phyla!
• Ecological
• Functional physiology
• Proteins
• Fatty acid
• Phylogenetic
• Mainly used today
• 16S differentiation
• lt3 divergence

5
(No Transcript)
6
16S rRNA

• Small subunit of ribosome
• Single stranded
• Pairs up with itself
• Useful for phylogenetic reconstruction
• Conserved across all life
• Many copies in each cell
• Different levels of variability
• Primer design

7
16S rRNA variation

• Design primers in conserved regions
• Obtain sample
• Soil, water filtrate, etc
• Many different species
• PCR amplify variable regions
• Many different sequences
• All the same length
• All in the same PCR solution
• Questions
• How many species are there?
• Who is there?

8
Detecting sequence variation

• RFLP
• Several bands / species
• Number of bands number of species
• tRFLP
• Terminal RFLP
• One PCR primer labeled
• RFLP
• Only terminal fragment visible
• Detect variation in length
• One band per species

• Other techniques as well

9
tRFLPs of mixed community PCR products

• 4 marine sediment samples
• DNA extracted
• 16S rRNA
• tRFLP (3 enzymes)
• Allows characterization
• Diversity
• Number of bands
• Differentiation
• Proportion of different bands

10
Diversity of methanotrophs in rice fields

• Methane
• Major greenhouse gas
• Rice fields are big contributor
• Methanotropic bacteria
• Oxidize methane ? CO2
• Need oxygen

• Comparison between soil and roots
• T-RFLP pmo gene
• Distribution
• Oxygen supply

Horz et al, AEM 2001
Soil
Rice root associated
11
Alternative sequence detectionDGGE (denaturing
gradient gel electrophoresis)

• Denaturation of DNA depends on
• Temperature
• Chemical concentration
• Denaturants
• Sequence
• GC content
• Sequence
• GC clamp
• 20bp GC
• keeps DNA together
• 20 bp normal (specific) primer
• Electrophoresis
• Much slower when denatured
• Differences in denaturing concentration needed to
  cause separation of strand

12
DGGE in mixed communities

• Sulfate reducing bacteria in stratified Danish
  fjord
• No mixing
• Boundary _at_ 14m
• Difference in 16S rDNA
• Shows different communities above and below
  stratification

Teske et al, AEM 1996
13
Who is there?

• Now we know how many of them
• estimate
• But who exactly is there?
• How can we identify species?
• Need to separate sequences
• Cut band out of gel
• cloning
• Recombinant DNA technology
• Based on microbiological techniques
• Allow to grow a colony (lots of cells) from one
  cell
• Mainly E. coli
• Bacteria have plasmids

14
Bacterial plasmids

• Circular DNA molecule
• Self replicating
• May contain genes
• Used for gene transfer among bacteria
• Can insertsequences
• Can be extractedseparately
• Much smallerthanchromosome

15
How do we insert sequences into plasmids?

• Restriction enzymes
• Cut at different sites
• Palindromic
• Symmetrical
• Cut site may be asymmetrical
• Sticky ends
• May link to other DNA

16
Recombinant DNA
17
Cloning vectors

• Engineered bacterial plasmids containing
• Origin of replication
• Specific recognition sequence
• Antibiotic resistance gene
• Only bacteria with plasmid grow on medium with
  antibiotic
• B-galactosidase gene
• Turns colonies blue
• Cloning sites
• Restriction / ligation

18
No plasmid
No insert
Agar plate antibiotic
19
Back to microbial communities

• Collect sample
• Soil, compost, water
• Extract DNA
• PCR
• Gene of interest (16S rRNA)
• Clone
• Extract DNA from bacterial colonies
• Sequence

20
Same principle for any DNA

• Genomic libraries
• Cut whole genomic DNA
• Insert into plasmids
• Grow bacteria
• Library contains all DNA of the genome
• In theory
• How do we find the gene we are interested in?

21
Detection of specific sequences

• Hybridization with specific probe
• Identify colonies
• Find colonies on plate

22
Primer isolation(e.g. microsatellites)

• Extract DNA
• Restriction
• 300 800 bp
• Cloning
• Ligation
• Tranformation
• Hybridization
• Identify clones with microsatellites
• Sequence
• Design primers
• Optimize PCR

23
Key Concepts

• Microbial communities
• Importance
• Main questions
• Microbes and the species concept
• Phylogenetic species concept (PSC)
• 16S rRNA
• Amplify across variable region
• Conserved primer sites

• Detecting sequence variation
• tRFLP
• DGGE
• Examples
• Recombinant technology
• Plasmids and vectors
• How do they work
• Separation of individual sequences
• Genomic libraries
• Isolation of specific genes
• Primer isolation