Microbial Community Analysis With thanks to: Boris Wawrik, Ph.D. Jerome Kukor, Ph.D. Lee Kerkhof, Ph.D.

Microbial ecology - Long term goals: Questions we ask: To gain a better understanding of the ecology of important microorganisms in environmental samples Questions we ask: Which bacteria are present in a sample? How many different types? Which bacteria are active and growing? What’s the ecology of microorganisms in the context of their environment ? How can we apply this knowledge (e.g. bioremediation, fermentation)?

Traditional Approach Culture Organisms Isolate Pure Cultures Study Metabolism of Cultures

Direct plating of seawater Electron microscopy Electron microscopy suggested much higher bacterial abundance in the marine environment (1920s) One ml of seawater typically yields 102-103 colonies This is a low number => bacteria were not considered important in the marine environment

Here are some reasons why Most cells are dead. 2) Many bacteria can not grow on the media. 3) In principle the media components are fine, the concentration is off. 4) The cells grow too slowly for you to assay. 5) Many cells become inactivated by fast growing colonies producing inhibitors. 6) Changes in conditions inhibit growth (e.g. temperature, pressure, placement on agar) 7) Cells clump. 8) Cells stick to pipettes, dilution tubes, sampling gear. 16

The Revolution : Polymerase Chain Reaction (PCR)

What can molecules tell us ? The Central Dogma of Molecular Biology DNA Who is there ? Who is not there ? What functional genes are there ? BUT can not tell you who is active RNA Who is active ? Who is expressing genes? Protein Enzyme activity Rate measurement (e.g. primary production by 14C carbon fixation)

Traditional vs. culture independent methods known novel Microbial Life, BOX 17.5 Dunbar et al., AEM No. 4, Vol 65, 1999, pp. 1662-69

Why use Ribosomal RNA Genes? 1. Everybody’s got ‘em 2. All perform the same function--protein synthesis 3. High homology--good for probing or PCR. 4. Good for telling us big picture lineages. 5. Many new rapid molecular biological methods to detect (Pauling and Zuckerkandl-1965; Woese, 1987)

Why is the small subunit rRNA gene so useful ? Conserved in parts – highly variable in other parts. Thus it a very good phylogenetic marker VERY large database of sequences Cell have many ribosomes which can be targeted with probes (e.g. FISH, &TRFLP) for community analysis 16S rRNA gene sequencing is now the gold standard for community analysis

Primer design: degenerate PCR Conserved sequence shared by all species * * * * * * Ambiguities in the sequence 5’-TWCGTSGARCTGCACGGVACCGGYAC-3’ IUPAC degeneracies: W = A or T S = G or C R = A or G V = C or G or A Y = C or T 2*2*2*3*2 = 48 different primers sequences

Some caveats: Not all methods yield the same results Different samples require different extraction methods It is best to try several methods and determine effort, yield, and purity Most people nowadays opt for extractions kits, because they are simple, rapid, reproducible and reasonable cheap Biggest problems: PCR inhibitors that co-extract Low DNA yields (e.g. clay)

Who are all these uncultivated bacteria ? There are regions that are highly similar among all bacteria These regions can be used to design universal 16S PCR primers Using these primers we can amplify the 16S sequences from a natural population This mixture of PCR products can be cloned and the inserts from individual colonies sequenced Microbial Life-’02 Perry et al. (Woese, Giovannoni, Ward, Stahl, Pace and others – late 1980s and early 1990s)

How do we estimate bacterial community composition ? Phylogenetic analysis DNA extraction Sequencing Library screening Primer design and PCR Comparison to other samples – hypothesis testing TA cloning

Examples of what you can do with 16S PCR technology DGGE TRFLP FISH SIP

DGGE (Denaturing Gradient Gel Electrophoresis) simple complex PCR products of mixed communities are loaded on a gel with a gradient of denaturant Typically 20-80% formamide double stranded DNA will run down the gel until it melts Melting determined by sequence and GC content Different sequences migrate different distances You obtain a ‘barcode’ of the community 20% 80%

DGGE (cont.) Advantages Disadvantages Can cut individual bands and clone or sequence them Can detect very small differences in DNA sequences Disadvantages High complexity samples give smears Requires specialized gel rig Acryl-amide is highly toxic

TRFLP (Terminal Restriction Fragment Length Polymorphism) Mixed population is amplified using a 16S primer with a fluorescent tag PCR product is cut with a 4bp cutting restriction endonuclease Different sequences will give different length fragments Sample is injected into a capillary sequencer to sort fragments by size cut with 4bp RE FU fragment size

TRFLP (cont.) Advantages Disadvantages Very sensitive Fast, easy and cheap Disadvantages Can NOT cut bands to get sequence data Requires capillary sequencer Hard to distinguish noise from little peaks sometimes

PCR is inherently NOT quantitative Amplification of some sequences maybe be sub-optimal Primer binding Secondary structure of template Reaction kinetics Amplification tends to lead to a 1:1 product ratio regardless of the starting DNA ratios Amplification of low abundance templates in a mixed template experiment will often be suppressed PCR can produce erroneous sequences Mis-incorporation of nucleotides by TAQ polymerase Formation of chimeric sequences LIBRARY CONTENT CANOT BE USED TO CALCULTE DIVERSITY INDICES

Many questions in ecology involve determining the active portion of a community Many species may be present but only a few might be active If you are looking for a functional gene, only some of the bacteria that contain this gene may be involved in actual substrate transformation Among the active ones, who is most dominant/active? Which bacteria are stimulated by a treatment (treatments may not kill other bacteria and 16S can detect them, although they are no longer active)?

Stable isotope probing Bacterial population 13C apple pie A population is grown on a substrate that contains 13C carbon Cells that eat the 13C labeled substrate will incorporate it into their DNA. Dormant cells will not DNA extracted and heavy (13C containing) DNA is separated from light (only 12C containing) DNA by CsCl density gradient centrifugation The heavy band is isolated and the community analyzed by PCR – TA cloning approach + grow on labeled substrate 12C DNA CsCl gradient extract DNA/RNA 13C DNA centrifugation

SIP (cont.) Who is there ? Who is eating apple pie ?

FISH (Fluorescent In Situ Hybridization) A cell population is fixed with formaldehyde The cell membranes are permeablized DNA or RNA probe is hybridized to cells In-Situ i.e. while the cells are still mostly intact The oligonucleotide contains a fluorescent label, which can be visualized by epifluorescence microscopy

FISH (cont.) Advantages Disadvantages Allows visualization of a particular population of cells (e.g. a species of interest) Gives quantitative information about a microbial population Can probe for DNA, mRNA and ribosomal RNA Disadvantages Cross-hybridization Different groups often do not add up to 100% of the population Relatively expensive and time consuming (bacterial population) (chromosome mapping)

Microautoradiography of labeled substrate and fluorescent in situ hybridization F F Allows for co-localization of radiolabel and phylogenetic probe 16

DAPI and Flo-probed cells exposed to 3H amino acids Cottrell and Kirchman 2000, AEM 66: 1692–1697

Take home messages: Molecular methods Most people prefer to work with DNA, because it is easiest and there are now many standard methods, reagents, and kits PCR based techniques have important limitations/biases DNA based methods can not determine who is active Phylogenetic analysis can not be used to calculate diversity indices (like the Shannon index) Molecular methods should be put into context of the biology and ecology of a system THE BETTER OUR METHODS THE MORE WE LEARN