Microbial Genetics

• As we have looked at, information for all cellular function is from DNA, mRNA carries that info to ribosomes, rRNA codes for proteins constructed at ribosomes

• So – in an environmental/geological context, what is that info used for??

Genomic info Gives us:

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Changing the genetic code:• Mutation: Change in at least one nucleotide

– Point mutations: 1 nucleotide change– Framesift mutations: base-pair change

• Think on how sequence is ‘read’– ATTGGCCATAGG– ATTGGCCATAGG Codon grouping with a start and

stop coding– Frameshift changes that mess up codon groups, mess

up start/stop info – completely change the protein…– Result Silent (codons are degenerate – amino acids

coded by several possible sequences), Missence (amino acid substitution), Nonsense (shorter or longer protein)

Mutation agents:• ‘Natural’ error in replication• Chemical agent:

– acids – esp. nitrous acid by modifying structure of adenine– Base analog – organic base similar enough to substitute in– Mutagens – cause base-pair problems or disruption of

chain• Physical agent:

– UV light – disrupts base pairing – thymine dimerization (T-T bonds form)

– Radiation damage – high energy particles disrupt electronic structure and bonding

• Viruses and gene transfer – adding snippets of ‘foreign’ code

Genetic repair

• Organisms have the machinery to repair damaged DNA

• Some organisms have adapted to handle doing this often, as is the case for a particular bacteria that can survive in abnormally high radiation: Deinoccocus radiodurans (can survive 1.5 million rads – 3000x what would kill us)

Microbial Identification

• All organisms have unique genetic codes describing their composition and function

• The diffference between these codes is a measure of how similar these things are to each other

• IF all organisms evolved from a common ancestor, then all codes of current life came FROM that common ancestor

• – this idea is the study of phylogentics

• Tree of Like – Ernst Haekel, 1866

Tree of Life

http://www.sciencemag.org/cgi/content/full/311/5765/1283/FIG2

• Global phylogeny of fully sequenced organisms. The phylogenetic tree has its basis in a cleaned and concatenated alignment of 31 universal protein families and covers 191 species whose genomes have been fully sequenced (14). Green section, Archaea; red, Eukaryota; blue, Bacteria. Labels and color shadings indicate various frequently used subdivisions. The branch separating Eukaryota and Archaea from Bacteria in this unrooted tree has been shortened for display purposes.

Construction of phylogenies

• What piece of the genetic code is most appropriate for determining these relationships – what should that part of DNA be responsible for???– Homologous function (common to all organisms)– Larger is better (more info)– Rate of mutation accumulation relevant to evolution– Does it all make sense – i.e., when you look at the

phylogeny does it make sense? – function and evolution??

Comparative sequence analysis

• Looking to compare similarity (in base 4) of sequences1. ATTGGCCACG2. ATTCGCCTCG3. TGGCGCCTTT4. ATTGGGCACG

• Determine the ‘degree of similitude’ between these and represent that graphically

• Literally hundreds of ways to do this…

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What piece of DNA do we use

• 16S rRNA coding – info that is responsible for the 16S subunit in ribosomes– All bacteria and archea have 70S ribosomes

with a 16S subunit– All bacteria and archaea need this to make

proteins– Some parts accumulate mutation slow, other

parts fast (evolution resolved over long time but also with good resolution)

After original work of Carl Woese and colleagues

OK – How do we get genetic data?• DNA extraction• Since DNA is more stable, easier to extract and

preserve than is rRNA• Need to get the DNA ‘out’ of the organism

– Bead beating– Chemical– Thermal

• Purify this material through chemical and physical extraction/filtration

• A multitude of techniques exist to do this – often specific for the type of sample

• Why??

Now that you have the DNA…• For most techniques, even for the high cell

densities of biofilms or cultures, there is nowhere near enough material to actually analyze

• While the genetic code was known about for many years, it was a special discovery by a surfer-scientist, Kary Mullis, that kick-started modern molecular biology – PCR (for which he received the 1993 Nobel Prize in Chem)

• Based on the thermal tolerance of a DNA polymerase first isolated from the thermophile Thermus aquaticas (thus Taq polymerase)

• The first part of Mullis's idea was to use pairs of short, single-stranded DNA pieces as probes to bracket the exact DNA sequence in which you happened to be interested. You could then cleverly employ iterative biochemical processes to get that DNA sequence to 'reproduce the hell out of itself'. You could start with the tiniest samples of even impure DNA and wind up within hours with a test-tube full of whatever gene or DNA sequence you had targeted. 'I would be famous,' Mullis recalls thinking at the moment of discovery, 'I would get the Nobel Prize.'

• The discovery of PCR is here presented as a great American epiphany - its recipient struck by a flash of inspiration on the road, not to Damascus, but to Mendocino. Sometimes Mullis says the creative spirit came on an evening in April of 1983, sometimes in May. Anyway, the buckeyes were in bloom; Mullis's little silver Honda Civic was purring through the vineyards and redwoods of the Anderson Valley; and his mind wandered. Life is sweet, he thought: 'I am a big kid with a new car and a full tank of gas. I have shoes that fit. I have a woman sleeping next to me and an exciting problem, a big one.' At mile-marker 46.58 on Highway 128 - he had both the presence of mind and the sense of history to note the exact spot, if not the month - the epiphany arrives. 'Holy shit,' Mullis cries out, and his girlfriend almost, but not quite, wakes up. He pulls the Honda to the side of the road to write down his ideas and check his calculations. Within feverish minutes, the problem is solved, and Mullis is left with the mop-up operation of getting PCR actually to work. This takes almost two years, and the original report was famously rejected by both Nature and Science. Mullis was not fazed: '"Fuck them," I said.'

Excerpts from ‘Dancing Naked in the Mind Field’ by Kary Mullis

PCR - Polymerase Chain Reaction• PCR is an in vitro technique for the amplification of a region of DNA

which lies between two regions of known sequence. • PCR amplification is achieved by using oligonucleotide primers.

– These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence.

• The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template. – This results in the synthesis of new DNA strands which are

complementary to the parent template strands. – These new strands have defined 5' ends (the 5' ends of the

oligonucleotide primers), whereas the 3' ends are potentially ambiguous in length.

• http://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

Primer selection• Primer is an oligonucleotide sequence – will target

a specific sequence of opposite base pairing (A-T, G-C only) of single-stranded nucleic acids

• For example, there is a– ¼ chance (4-1) of finding an A, G, C or T in any given DNA

sequence; there is a – 1/16 chance (4-2) of finding any dinucleotide sequence (eg.

AG); a – 1/256 chance of finding a given 4-base sequence.

• Thus, a sixteen base sequence will statistically be present only once in every 416 bases (=4 294 967 296, or 4 billion): this is about the size of the human or maize genome, and 1000x greater than the genome size of E. coli.

Primer Specificity

• Universal – amplifies ALL bacterial DNA for instance

• Group Specific – amplify all denitrifiers for instance

• Specific – amplify just a given sequence

Forward and reverse primers

• If you know the sequence targeted for amplification, you know the size which the primers should be anealing across

• If you don’t know the sequence… What do you get?

DNA Polymerase• DNA Polymerase is the enzyme responsible for

copying the sequence starting at the primer from the single DNA strand

• Commonly use Taq, an enzyme from the hyperthermophilic organisms Thermus aquaticus, isolated first at a thermal spring in Yellowstone National Park

• This enzyme is heat-tolerant useful both because it is thermally tolerant (survives the melting T of DNA denaturation) which also means the process is more specific, higher temps result in less mismatch – more specific replication

RFLP• Restriction Fragment Length Polymorphism

• Cutting a DNA sequence using restriction enzymes into pieces specific enzymes cut specific places

Starting DNA sequence:5’-TAATTTCCGTTAGTTCAAGCGTTAGGACC3’-ATTAAAGGCAATCAAGTTCGCAATAATGG

Enzyme X5’-TTC-3”-AAG-

Enzyme X5’-TTC-3”-AAG-

5’-TAATTT3’-ATTAAA

5’-CCGTTAGTT3’-GGCAATCAA

5’-CAAGCGTTAGGACC3’-GTTCGCAATAATGG

RFLP• DNA can be processed by RFLP either directly (if

you can get enough DNA from an environment) or from PCR product

• T-RFLP (terminal-RFLP) is in most respects identical except for a marker on the end of the enzyme

• Works as fingerprinting technique because different organisms with different DNA sequences will have different lengths of DNA between identical units targeted by the restriction enzymes– specificity can again be manipulated with PCR primers

Liu et al. (1997) Appl Environ Microbiol 63:4516-4522

Electrophoresis

• Fragmentation products of differing length are separated – often on an agarose gel bed by electrophoresis, or using a capilarry electrophoretic separation

DGGE• Denaturing gradient gel electrophoresis

– The hydrogen bonds formed between complimentary base pairs, GC rich regions ‘melt’ (melting=strand separation or denaturation) at higher temperatures than regions that are AT rich.

• When DNA separated by electrophoresis through a gradient of increasing chemical denaturant (usually formamide and urea), the mobility of the molecule is retarded at the concentration at which the DNA strands of low melt domain dissociate. – The branched structure of the single stranded moiety of the

molecule becomes entangled in the gel matrix and no further movement occurs.

– Complete strand separation is prevented by the presence of a high melting domain, which is usually artificially created at one end of the molecule by incorporation of a GC clamp. This is accomplished during PCR amplification using a PCR primer with a 5' tail consisting of a sequence of 40 GC.

Run DGGE animation here – from http://www.charite.de/bioinf/tgge/

RFLP vs. DGGEDGGE

• Advantages– Very sensitive to variations in

DNA sequence– Can excise and sequence

DNA in bands• Limitations

– Somewhat difficult– ”One band-one species” isn’t

always true– Cannot compare bands

between gels– Only works well with short

fragments (<500 bp), thus limiting phylogenetic characterization

RFLP• Advantages

– Relatively easy to do– Results can be banked for

future comparisons• Limitations

– Less sensitive phylogenetic resolution than sequencing

– Each fragment length can potentially represent a diversity of microorganisms

– Cannot directly sequence restriction fragments,making identification indirect

FISH• Fluorescent in-situ hybridization

– Design a probe consisting of an oligonucleotide sequence and a tag

– Degree of specificity is variable!– Hybridize that oligonucleotide sequence to the

rRNA of an organism – this is temperature and salt content sensitive

– Image using epiflourescence, laser excitation confocal microscopy

• Technique DIRECTLY images active organisms in a sample

16S gene

16S rRNA

CellCellmembranemembrane

DNA

16S gene**

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Fluorescent in situ hybridisation(FISH) using DNA probes

TAGCTGGCAGTAUCGACCGUCACGU

Fluorescein

AU

ProbeProbe(( 20 bases) 20 bases)

Fluorescent in site hybridization

10 µm

DAPI FER656

B Drift Slime Streamer

Oligunucleotide design

FISH variations

• FISH-CARD – instead of a fluorescent probe on oligo sequence, but another molecule that can then bond to many fluorescent probes – better signal-to-noise ratio

• FISH-RING – design of oligo sequence to specific genes – image all organisms with DSR gene or nifH for example

Clone Library

• http://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf

http://www.ifa.hawaii.edu/UHNAI/NAIweb/presentations/astrobiol6.pdf