Understanding Microbial Communities Flashcards
Prokaryotes: the unknown majority
Cannot be cultured in the lab - growth rate, nutritional needs, communities needed
Limited morphological diversity unlike eukaryotes
Playermorphic (different structures under different conditions eg salt content, pH shifts etc)
How we define microbial community - poly microbial communities in biofilms
Need taxonomic identification of groups in mixtures/biofilms
FISH - separate and visualise
Coloured eg red vs green
What is where
Ratios
EPS
Characterisation of microorganisms
Classical - enrichment, pure culture, physiological characterisation
Problem
Culturing induced shifts
Fast growing organisms are favoured, nutrient load above natural level, inadequate culture conditions (gradient needed), Unknown growth factors
Cultivation as a prerequisite for characteristics
Sea water 0.001-01% cultivation effeciency
Freshwater lake 0.1-1%
Estuary 0.1-3%
Activated sludge 1-15%
Sediment 0.25%
Soil 0.3%
TIME CONSUMING AND DIFFICULT
Need for cultivation independent methods
Suitable marker genes
Ribosomal RNA, ATPase, elongation factor thermo unstable (molecular clock)
Functional genes
Dissimilatory sulphite reductase, adenosine 5-phosphosphate reductase, RuBisCo
But need signals to detect
Why ribosomal RNA
Present in every living organism
Same function - evolutionary conserved molecule, direct comparison of sequence, reconstruction of phylogeny
Large and small subunits - prokaryotes SSU 30s containing 16S rRNA, LSU 50s containing 5s and 23s rRNA
16s rRNA
First described for e.coli
Majority highly conserved
But areas of high variability so different levels of specificity
Can use for phylogenetic reconstruction
Universal phylogenetic tree derived from comparative 16s/18s rRNA sequencing
Bacteria
Archea (closer to eukaryotes)
Eukaryotes
Can get more and more detail from this
The rRNA approach
Sample
dNA extraction
PCR amplification
Cloning
Sequencing
Comparative sequence analysis
Phylogenetic affiliation
Huge data to use
Fluorescence in situ hybridisation (FISH)
Sequence database
Prove design
Prove testing
Insitu hybridisation
Detection
Sample
Full cycle approach
Cultivate independent
Phylogenetic diversity
Abundance if defined groups
Spatial, temporal variability
Why FISH
Only tool in microbio to determine true cell numbers
Target naturally amplified molecule rRNA within a the cell (100-1000 copies per cell)
Variable as well as conserved regions (broad/narrow specificity)
Flourescently labelled oligonucleotides (probes) (detection of stained whole single cells in natural context)
FISH using monolabelled oligonucleotides
Sample
Fixation
Fixed cells permeabilised
Hybridisation
Flourecently labelled oligonucleotides (probes)
Washing
Quantification
Flow cytometry or epiflourecence microscopy
rRNA databases
Prerequisite for probe design is a comprehensive database (all three domains and all types of RNA)
RDP- comprehensive database for bacterial rRNA
SILVA rRNA database project (aligned small subunit and large subunit sequence for all 3 domains)
rRNA databases issues
No comprehensive archeal sequemve database
No comprehensive 18s rRNA database
No 23s rRNA database
No 5s rRNA database
Tedious manual sequence retrieval from NCBI/EBI by blasts/acc number
ARB can only handle ~150000 sequences
Probe design criteria
Probe length 15-25 nucleotides (longer = more difficult to get into cell and ribosomal subunit)
Sequence
Dissociation temp (Td = 4N(G+C)+2N(A+T)
Number of diagnostic mismatches
Quality of mismatches
Position of mismatches
Neighbourhood bases
Intra molecular base pairing
Secondary structure of target
Method of hybridisation
Probe design
Genomic tree
Node points = differences in sequence
Make more specific
Position of mismatches
New probes need at least a single mismatch to all non target organisms
Important to keep mismatches ventral as mismatch at 3’ or 5’ end is only weakly destabilising
Quality of mismatches
Destabilising a-a, a-c, t-t, t-c, c-c
Slightly destabilising g-t, g-a, g-g
Aware
- GC stretches (impact efficiency)
- formation of hair pin structures
Optimisation the hybridisation conditions
Td = 4N(G+C) + 2N (A+T)
For dissociation temp
Formamide
First set experiments - tweaking salt, formamide, temp etc til targets labelled and non targets not
Competitor concept
Not as commonly used
2 species, very similar sequences so can’t distinguish between the 2
Two probes targeting other species, no label but block labelling of other probe
Stronger signal on original target species
Accessibility of sequence
Colour coded of 16s and 23s how accessible
Most variable regions most inaccessible
Species levels need to get to these regions
Solution:
Limitations
Permeabilitisayion of cell membrane
Low rRNA content affecting sensitivity
Problems accessing target site
Optimisation of hybridisation/washing conditions
Quantification
Species differentiation
Physiological information missing
Signal can be reduced by nucleotides acting as quenches
Unspecific binding of probes producing background noise
Helper probes
Can increase accessibility
Unlabelled oligonucleotides that bind adjacent or near target region
Need to be few nucleotides longer than probe to ensure tight binding beyond melting point of probe
Probe design difficult. Need equal broader specificity
Quantification
Flow cytometry for single cells in suspension
For complex samples/biofilms, rely on microscopy - numbers, biovolume, surface coverage
LNA-FISH
Locked nucleic acid = LNA
Inaccessible RNA
LNA nucleotides mixed with DNA or RNA residues
Hybridise according to Watson-crock base pairing tiles
Increase sensitivity and specificity
PNA-FISH
Pna = peptide nucleic acid
Synthetic DNA analogues, -ve charges sugar phosphate backbone replaced with non charged peptide backbone
Rapid and specific binding
Shorter probe lengths, easier accessibility to target sequence
Less need for stringent hybridisation conditions eg temp, ionic charges, enzymatic changes
CARD-FISH (catalysed reporter deposition)
Some environmental samples, single oligonucleotides with only one flouraphore did not provide strong signal
Increased using horseradish peroxidase labelled oligonucleotides
Staining results from secondary incubation with flourescently labelled tyrannise
Bound peroxidase molecules catalyse deposition of labelled tyrannise within cells targeted by HRP- probe
20x brighter signal
More stringent cell permeabilisation steps needed to enable larger enzyme labelled oligonucleotides to cross cell membrane- cell lysis and alter community
CLASI-FISH
CLASI = combinational labelling and spectral imagining
Uses combos of probes to label bacteria in mixed populations
Uses spectral imaging to visualise
Overcomes issue if reduced number of fluoraphores
Limitation- multiple hybridisation protocol, stringent conditions, increased permeabilisation
(AKA DOPE-FISH)
Computer tags and labelled/groups
How can you understand physiology and metabolic state?
Cellular activity often assigned to be linked to ribosome content
But cells can be highly active but have low ribosome content
Dead- remains stable for long time
MAR-FISH
Uptake radioactively labelled substrates into cells
Deleted by microautoradiography (MAR) with stimultaneous bacterial identification with FISH
decay from incorporated substrate can be visualised along with cells metabolising it
Developed further with Raman-FISH and NanoSIMS FISH = allow potential detection of metabolic activities in single cells eg degradation of contaminants
ISRT-FISH
In situ reverse transcription FISH
Specific mRNA are amplified by qPCR to cDNA
Targeted by fluorescent probes
Used to study metabolically active groups and control of gene expression in populations
Not really in biofilms
Examples of FISH applications
Food industry
Medical - rapid screening
Spatial and temporal understanding of poly microbial communities
