What is microbial diversity How do we measure it

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What is microbial diversity? How do we
measure it?

• Dr. William Stafford
• wstafford_at_uwc.ac.za

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• Text Books
• Atlas Bartha (1998) Microbial Ecology. UWC
  Library Cat No. 576.15 ATL Level 5
• Chapman, J.L. and Rees, M.J. (2002). Ecology.
  Principles and Applications. Cambridge University
  Press. UWC Cat No. 577 CHA.
• Hillis, Moritz Mable (1996) Molecular
  Systematics 2nd edition. Sinauer. Pages 169-176
  205-212 249-266 (321-381) 410-415. UWC Library
  Cat No 574.88028MOL Level 14
• Prescott, Harley Klein (2002) Microbiology 5th
  Edition. McGraw-Hill. Pages 505-506 610
  619-629 421-447 675-684. UWC Library Cat No
  579.PRE. Level 7
• Begon, Harper Townsend (1990) Ecology. UWC
  Library Cat No 574.5 BEG Level 5

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• Molecular Analysis of the Nitrate-Reducing
  Community from Unplanted and Maize-Planted Soils
  Laurent Philippot, Séverine Piutti, Fabrice
  Martin-Laurent, Stéphanie Hallet, and Jean Claude
  Germon Appl. Environ. Microbiol. 2002. 68
  6121-6128.
• Soil Microbial Community Structure across a
  Thermal Gradient following a Geothermal Heating
  Event Tracy B. Norris, Jon M. Wraith, Richard W.
  Castenholz, and Timothy R. McDermott Appl.
  Environ. Microbiol. 2002. 68 6300-6309.
• Coexistence of Multiple Proteobacterial
  Endosymbionts in the Gills of the Wood-Boring
  Bivalve Lyrodus pedicellatus (Bivalvia
  Teredinidae) Daniel L. Distel, David J. Beaudoin,
  and Wendy Morrill Appl. Environ. Microbiol. 2002.
  68 6292-6299.
• Molecular Analysis of Bacterial Community
  Structure and Diversity in Unimproved and
  Improved Upland Grass Pastures Allison E. McCaig,
  L. Anne Glover, and James I. Prosser Appl.
  Environ. Microbiol. 1999. 65 1721-1730.
• Bacterial rhodopsin Evidence for a new type of
  phototrophy in the sea Beja O, Aravind L, Koonin
  EV, Suzuki MT, Hadd A, Nguyen LP, Jovanovich SB,
  Gates CM, Feldman RA, Spudich JL, Spudich EN and
  DeLong EF. 2000. Science 2891902-1906.
• Impact of culture-independent studies on the
  emerging phylogenetic view of bacterial diversity
  Hugenholtz, P., et al. 1998 . J. Bacteriol.
  1804765-4774. http//www.mbio.ncsu.edu/JWB/MB409
  /lecture/lecture14/Hugenholtz.pdf

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• Morphological diversity
• Microbes are rods, cocci spirals, filaments,
  branched filaments, amorphous (irregular,
  star-shaped, stalked ), pleomorphic (different
  shapes under different conditions,). Most
  Bacteria Archaea are 1-5 microns in size, but
  they range from 0.1 to 660 um per cell
• Cells can be organized from simple pairs
  tetrads to filaments, sheets, rosettes, and true
  multicellular organisms. Many species form
  highly structured multi-species mats that
  resemble the tissues of animals and plants that
  carry out complex biochemical transformations
  eg.Biofilms

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• Structural diversity
• Most Bacteria have either a Gram-positive (single
  membrane, thick cell wall) or Gram-negative
  (double membrane, thin cell wall). There is a
  variety of cell wall types in the Archaea.
• There are a wide range of external structures
  flagella, pilli, holdfasts, stalks, buds,
  capsules, and sheaths, etc.
• There are also a wide variety of internal
  structure such as spores, daughter cells,
  thylakoids, mesosomes, nucleoid.

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• Metabolic diversity
• Chemoheterotrophs - both carbon energy are
  obtained from organic compounds.
• Chemoautotrophs - Cell carbon is obtained by
  fixing CO2. Energy is obtained from inorganic
  sources such as sulfur or nitrogen compounds,
  iron, hydrogen, etc.
• Photoheterotrophs - Cell carbon is obtained from
  organic compounds, but energy is taken from
  light..
• Photoautotrophs (photosynthetic) - Cell carbon is
  obtained by fixing CO2. Energy is from light.

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• Ecological diversity
• Marine or freshwater
• Temperature from -5C to 118C - Pyrodictium is
  grown in autoclaves!
• pH 0 to 11 - pH0 is 1M HCl!
• Symbiosis - inter and intra cellular with
  eukaryotes or other microbes, and complex
  communities such as microbial mats and biofilms
• A variety of environments from halophilic Archaea
  in microscopic brine pockets in subterranean salt
  domes to hydrogen utilizing Bacteria in deep
  groundwater

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• Behaviour diversity
• Motility taxis - microbes get to where they
  want to be via phototaxis, chemotaxis,
  magnetotaxis, etc. Many open-water organisms have
  gas vacuoles used to adjust their depth
• Various life developmental cycles - e.g.
  sporulation, swarmer phases, etc.
• Biochemical responses - microbes express the
  genes needed to compete for the resources that
  are available at that time.
• Communication between cells of the same
  different species - symbiosis, mat formation,
  quorum sensing, etc.

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• Evolutionary diversity (genetic diversity)
• Evolutionary diversity is usually expressed in
  terms of trees - branched graphs that trace the
  geneologies of organisms. When these trees are
  based on genetic diversity, they can be
  quantitative and objective.
• There are several Methods to determine genetic
  diversity

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Genome DNADNA hybridization

• Extent that the genomic DNAs of 2 species will
  hybridize is a general measure of how much
  sequence similarity there is between the genomes,
  and therefore how closely related they are.
• Two organisms are considered to be the same
  species if the DNADNA hybridization is 70 or
  greater, or different species of the same genus
  if they have measurable hybridization less than
  70.
• Cot1/2 is used to express the diversity of the
  population and the similarity between genomes

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DNA Molecular markers

• Some sequences are better than others - the most
  important factors to consider are
• Clock-like behavior, i.e. sequence divergence in
  the gene between two organisms should be
  proportional to how long ago they diverged.
  Clock-like behaviour depends mostly on functional
  constancy of the sequence - function change leads
  to large, directed sequence change.
• Phylogenetic range. The sequence must be present
  in all of the organisms to be analysed The gene
  must have enough variation in sequence to
  evaluate statistically but must be similar enough
  to that homologous residues can be identified.
• No horizontal transfer. This means that the gene
  must be acquired only by inheritance, not by
  horizontal transfer from another organism. An
  example of frequently horizontally- transferred
  genes are those encoding antibiotic resistance.
• Must have a large existing data set to compare
  your sequences with!

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• In most cases, the gene encoding the RNA in the
  small subunit of the ribosome (ssu rRNA) is the
  best choice because
• It is present in all cells with the same function
• It is conserved enough in sequence structure of
  be readily accurately aligned.
• It contains both rapidly slowly evolving
  regions - the fast regions are useful for
  determining closely-related species, whereas the
  slow regions are useful for determining distant
  relationships
• Horizontal transfer of rRNA genes apparently does
  not occur (also true for other genes in the
  central information processing pathways of the
  cell).
• There is a large database (about 10,000) of
  aligned sequences available

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DNA molecular markersProbe Hybridization

• Probe Hybridization using a specific labelled
  oligonucleotide to detect a known sequence. Used
  to detect the presence of certain taxa in cell
  communities
• (i) microscopically (FISH)
• (ii) environmental samples (Southern and Northern
  blots).
• (iii) Microarrays. Flourescently label cDNA
  (amplified from microbe(s) or environmental
  sample) are hybridized to an array of
  oiligonucleotides. These oligos have signature
  sequences to certain taxa.

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• DNA molecular markers PCR gene targeting
• PCR amplifies genes logarithmically-a single
  gene, is specifically amplified to a million
  molecules in 30 cycles! (denaturation, primer
  annealing, and DNA polymerization)
• PCR can be used as a
• Stand-alone diagnostic tool OR
• Preliminary step to amplify DNA for ARDRA, DGGE
  or cloning and DNA sequencing

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Design of Primers for gene targeting and
amplification

• Typically oligonucleotide 15-30mer
• Specific ONLY to one organism or group of
  organisms-Identifying the presence of known
  organisms in the environment.
• Universal or specific to a certain group of
  organisms -Identifying novel taxa and community
  analysis

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The sequence variation of the PCR product can be
investigated by

• ARDRA (amplified ribosomal DNA restriction
  analysis).
• PCR amplify ribosomal RNA gene and cut with
  restriction enzyme. Sequence variation will
  result in different size fragments (separated on
  agarose gel). Require two or more tetrameric
  restriction enzymes to detect gt97 sequence
  variation of 16S rRNA gene.
• For community analysis (samples of mixed species
  the PCR product must first be cloned so that
  individual DNA genospecies can be analysed.

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ARDRA (details)

• PCR amplify DNA molecular marker eg. (rRNA gene)
  from DNA obtained from different organisms
• Cut PCR products with restriction endonuclease-
  identify genotypes (OTU)

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SSCP and DGGE

• SSCP and DGGE are polyacrylamide gel
  electrophoresis methods that can detect down to
  1bp sequence difference. These methods are based
  on the differential melting temperature of DNA
  molecules with different sequence

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DGGE
DGGE (details)
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DNA sequencing

• DNA sequencing - Sequencing involves denaturing
  the DNA, annealing an oligonucleotide primer, and
  extending from this primer with DNA polymerase in
  the presence of dNTPs and small amounts of 'chain
  terminator' dideoxynucleotides (analogs of dNTPs
  that DNA polymerase cannot continue extending
  from)
• Each reaction gives 300-700 bases of sequence.

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Denature and anneal primerEnlongation and
termination
DNA sequencing (details)
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DNA sequencing (details contd.)

• Run sample on a high-resolution gel.
• A fluorometer at the bottom of the gel detects
  the termination dyes as they run past in each
  lane of the gel. The connected computer collects
  this data and 'reads' the sequence from the
  pattern of peaks.

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Phylogenetic trees

• The number of nucleic acid or amino acid
  differences between two organisms is proportional
  to the time since they diverged from a common
  ancestor.

1 2 3
1 AAGGCTA 2 AAGGGTA 3 AAGGATG Example
Rate of Evolution 1bp per 100 years
100years
200 years
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Treeing and evaluation methods.

• Counting differences between two sequences
  underestimates the number of changes that occured
  between them, because more than one evolutionary
  change at a single position (e.g. A -gt G -gt U)
  counts as only one difference between two
  sequences, and in the case of reversion counts as
  no change at all (e.g. A -gt G -gt A).
• In the Jukes Cantor method, any change is
  scored equivalently. A commonly-used alternative
  is the Kimura 2-parameter model, in which
  transversions and transitions are scored
  differently since Transitions are 2-20 times more
  common than transversions.
• Transition. Change of a pyrimidine nucleotide
  into to another pyrimidine or change of a purine
  nucleotide into an another purine nucleotide.
  Transversion. Change of a pyrimidine nucleotide
  into a purine nucleotide or vice versa.
  Transversions are 2-20 rarer than transitions.

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Treeing algorithms

• Neighbor-joining
• This method is a least-squares distance-matrix.
• A B C D E
• A - - - - -
• B 0.10 - - - -
• C 0.19 0.21 - - -
• D 0.25 0.25 0.25 - -
• E 0.24 0.26 0.25 0.05 -
• The closest neighbors in the distance matrix are
  D and E (0.05), so these branches are joined
• The distances from all other sequences to D and E
  are then averaged to reduce the distance matrix
• Now the closest neighbors are A and B, so join
  them
• That's it! If there were more sequences, you'd
  re-reduce the matrix as before, repeat the
  process over-and over until all of the nodes were
  resolved.

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• Parsimony
• The tree that requires the smallest number of
  sequence changes is the most likely tree. No
  distance matrix is calculated, instead trees are
  searched and each ancestral sequence calculated,
  then the number of "mutations" required are added
  up. Testing every possible tree is not usually
  possible, so a variety of search algorithms are
  used to examine only the most likely trees.
  Likewise, there are a variety of ways of counting
  (scoring) sequence changes.
• Maximum-likelihood
• This method starts with a cluster analysis to
  generate a "starting" tree, from which the
  substitution model is derived. It then goes back
  and calculates the tree that has the maximum
  likelihood of resulting in the observed sequences
  based on the model parameters.
• Sound complicated? It is, and ML tree
  construction is by far the most computationally
  intensive of the methods described. But it's
  generally also the best, in the sense that the
  trees are more consistent and robust

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Bootstrapping

• Bootstrapping is a method to evaluate the
  reliability of a tree.
• In a bootstrap analysis, trees are generated from
  random sampling (comparing randomly selected
  positions in the alignment 100 or 1000 times)
• The reliability of a particular branching
  arrangement in a tree is judged by the frequency
  that the branch appears in all of the resulting
  trees.

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Interpreting phylogenetic trees

• Scale.- Time or evolutionay divergence.
• Terminal nodes.-The ends of the branches of the
  evolutionary tree - typically the modern
  organisms.
• Internal nodes. -These represent the last common
  ancestors of all of the organisms bound by this
  node.
• Root.- This is the 'base' of the tree - the last
  common ancestor of all of the organisms. To do
  this you need an outgroup (organism falls outside
  of the group defined by the organisms of
  interest).
• Branches. The connections between nodes in the
  tree. These represent to evolutionary pathway
  between common ancestors (internal nodes) and
  modern organisms (terminal nodes).

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Some features of these molecular phylogenetic
trees

• There are 3 evolutionary groups Bacteria, Archaea
  and Eukarya
• All previous trees were subjective qualitative.
  This tree is quantitative and objective, based on
  statistical analysis of gene sequences, in this
  case small subunit ribosomal RNA.
• The tips of all the branches are modern
  organisms. Each node within the tree represents a
  common ancestor.
• There is no ranking of above (superior) or below
  (inferior) in the tree. Evolutionary distance
  (divergence) is measured along the lengths of the
  branches connecting species.
• Multicellular eukaryotes are a very small portion
  of evolutionary diversity - just the tip of one
  branch of the eukaryotes
• Prokaryotes fill 2/3rds of the tree (bacteria and
  archaea).
• The tree also offers final proof of the
  endosymbiont theory for the origin of
  mitochondria and chloroplasts since these
  organelles have their own DNA genes. The
  mitochondria are related to the purple Bacteria,
  and the chloroplasts are related to
  cyanoabacteria.