Microbial Diversity and Assessment (II)

Guangyi Wang, Ph.D.POST103B

guangyi@hawaii.edu

Spring, 2007

http://www.soest.hawaii.edu/marinefungi/OCN403webpage.htm

• General introduction and overview– Taxonomy [Greek taxis, arrangement or order,

and nonos, law, or nemein, to distribute or govern]-the science of biological classification.

Three interrelated parts: classification, nomenclature, & identification.

classification - arrangement of organisms into groups or based on mutual similarity or evolutionary relatedness.

Nomenclature - the branch of taxonomy concerned with the assignment of names to taxonomic groups according to the published rules.

identification – the practical side of taxonomy, the process of determining that a particular isolate belongs to a recognized taxon.

– Why should we care about taxonomy?• Easy to organize huge amount of knowledge

about (micro)organisms.• To make predictions and fame hypotheses for

further research based on knowledge of similar organism (animal research and human health?)

• Places microorganism in meaningful, useful groups with precise names so that microbiologists can work with them and communicate efficiently.

• Essential for accurate identification of microorganisms (clinical microbiology?).

• It closely related to ecology, epidemiology and other scientific disciplines.

Two Different Possible Goals(1) Convenient ordering scheme. Use of a "key"

Phenetic system: groups organisms based on mutual similarity of phenotypic characteristics. May or may not correctly match evolutionary grouping.

Example: Group flagellated (motile) organisms in one group, non-motile organisms in another group.

(2) Scheme displaying evolutionary relationships.

Phylogenetic system: groups organisms based on shared evolutionary heritage.

Example: Mycoplasma (no wall) and Bacillus (walled Gram+ rods) are not obviously similar, would not be grouped together phenetically. But evolutionarily they are similar, more so than either to Gram- organisms

Strain:•descended from a single organism •different isolates may be same species but are different strains; often have slight differences

Terminology

Type strain:

•the first strain isolated or best characterized

•kept in collections; e.g., ATCC (American Type Culture Collection) maintains the following frozen or freeze-dried stocks: (number of species in parentheses)

Algae (120); Bacteria (14400); Fungi (20200); Yeasts (4300); Protozoa (1090); Cell lines: animal (2300); Cell lines: plant (25); Viruses: animal (1350); Viruses: plant (590); Viruses: bacteria (400)

many similar strains = species

strain, species, genus, family, order, class, division, kingdom

Example:

Genus: Escherichia

Species: coli

Family: Enterobacteriaceae

Class: Scotobacteria

Division: Gracilicutes

Kingdom: Procaryotae

Classical TaxonomyPhenetic systems (natural classification system): group organisms together based on the mutual similarity of the phenotypic characteristics.

Major characteristics used in classical taxonomy:Classical characteristics: morphological characteristics, physiological and metabolic characteristics, ecologic characteristics; genetic analysis.

Bacterial metabolism is exceptionally diverse. Many chemical substrates can serve as a source of energy for bacterial growth and production.

Chemosynthesis (by bacteria) is generally not as important as photosynthesis in producing organic matter, but is clearly important in understanding elemental cycling in the oceans.

Classification of bacterial based on metabolism

Molecular TaxonomyBasic assumptions

•Genes mutate randomly•Many mutations are "neutral" -- do not lead to any obvious disadvantage to the strain.•Once a mutation is established, all progeny of parent cell carry that particular mutation. For example, in figure below, if template "A" is erroneously replicated to a "C" in the opposite strand instead of T, then one generation later the error will be "locked into place", and all progeny with that DNA will be forever altered (unless a reversion mutation occurs at some later time).

•Two organisms that differ by only a few bases have diverged more recently in evolutionary time than organisms that differ by more bases.

•The following example shows how an evolutionary tree is constructed for four hypothetical organisms whose DNA sequence in one homologous region is known.

Organism A and B differ by one base substitution. C and D also differ by one base substitution. But A and C differ by three substitutions, and A and D by four. B and C differ by three substitutions, and B and D also by four. In terms of evolutionary history, A and B appear to be very similar,as do C and D. A-B and C-D are more distantly related.

Computers excel at taking such data and creating trees that accurately illustrate the divergence between different organisms, with linear distance being proportional to the number of accumulated errors. Here are a couple of ways a computer could represent the separation between these four organisms:

Phylogeny can answer questions such as:

Goals of molecular phylogeny

• How many genes are related to my favorite gene?• Was the extinct quagga more like a zebra or a horse?• Was Darwin correct that humans are closest

to chimps and gorillas?• How related are whales, dolphins & porpoises to cows?• Where and when did HIV originate?• What is the history of life on earth?

Was the quagga (now extinct) more like a zebra or a horse?

Woese PNAS

There are two main kinds of information inherentto any tree: topology and branch lengths.

We will now describe the parts of a tree.

Molecular phylogeny: nomenclature of trees

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Molecular phylogeny uses trees to depict evolutionaryrelationships among organisms. These trees are basedupon DNA and protein sequence data.

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Tree nomenclature

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taxon

operational taxonomic unit (OTU)such as a protein sequence

OUT-One of the organisms being compared in a phylogenetic analysis.

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Tree nomenclature

branch(edge)

Node (intersection or terminating pointof two or more branches)

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Tree nomenclature

Branches are unscaled... Branches are scaled...

…branch lengths areproportional to number ofamino acid changes

…OTUs are neatly aligned,and nodes reflect time

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Tree nomenclature

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multifurcatinginternalnode

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Tree nomenclature: clades

Clade ABF (monophyletic group)

1) Monophyletic group is applied to a group of organisms that includes an ancestral species and all of its descendants; e.g. Aves, Mammalia. This group is a complete branch of the tree of life, the phylogeny of life. Such a branch is called a clade.

2) The Polyphyletic taxon is a group composed of a number of organisms which might bear some similarities, but does not include the most recent common ancestor of all the member organisms (usually because that ancestor lacks some or all of the characteristics of the group). The taxon shares derived characters which originated several times by convergence.

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Clade CDH

Fig. 11.4Page 366

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Clade ABF/CDH/G

Fig. 11.4Page 366

Examples of clades

Lindblad-Toh et al., Nature438: 803, 8 Dec. 2005, fig. 10

The root of a phylogenetic tree represents thecommon ancestor of the sequences. Some treesare unrooted, and thus do not specify the commonancestor.

A tree can be rooted using an outgroup (that is, ataxon known to be distantly related from all otherOTUs).

Tree roots

Tree nomenclature: roots

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Rooted tree(specifies evolutionarypath)

Unrooted tree

Fig. 11.6Page 368

Tree nomenclature: outgroup rooting

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(used to place the root)

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Fig. 11.6Page 368

Requirements for Molecular “clock”

•Universally distributed across the group chosen for study

•Functionally homologous in each organism or with identical function

•Properly align the two molecules in order to identify regions of sequence homology and sequence variance.

•Change at a rate commensurate with the evolutionary distance measure.

•e.g. cytochromes, iron-sulfur proteins (ferredoxins), ATPase, RecA, & Ribosome RNA.

•Attempt to use basic assumptions to establish history of evolutionary lineages

•Over long periods of time, assume mutations occur with roughly predictable frequency

•16S RNA is found in small ribosomal subunit (30S) of procaryotic ribosomes.

Use of 16S RNA sequence homology

•Since mitochondria and chloroplasts have these ribosomes also, it is found in all 3 kingdoms. •Most ribosomal RNA mutations are deleterious. Very few mutations are neutral. •Therefore evolution of 16S RNA is very slow. It is a very good molecule to use to compare organisms that may have diverged as far back as 3 or 4 billion years ago. •Visit the Ribosomal Database Project on the Web for access to data and software tools. This site lets users upload sequence information, generates alignments with other ribosomal genes, and returns aligned sequences with best matches, as well as generating phylogenetic trees.

CLASSIFICATION - 3 Domains (Woese, 1978): CurrentEubacteria - true bacteriaArchaebacteria - ancient bacteriaEukaryotes - protists, fungi, plants, animals

Three Domains of Life

ProKaryotes divided into two distinct groups very early on. The archaea and bacteria first diverged, the eukaryotes developed.

Bergey’s Manual of Systematic Bacteriology - 2nd edemphasis on 16S rRNA sequencephylogenetic classification

Vol. 1 Archae & Deeply Branching & PhototrophicBacteria

Vol. 2 ProteobacteriaVol. 3 The Low G+C Gram-positive BacteriaVol. 4 The High G+C Gram-positive BacteriaVol. 5 The Planctomycetes, Spirochaetes,

Fibrobacteria, Bacteroidetes & Fusobacteria

Phylogenetic Classifications

Bergey’s Manual of Systematic Bacteriology - 2nd ed

Phylogenetic Classifications

Detailed phylogenetic tree of the major lineages (18 phyla) of Bacteria based on 16S ribosomal RNA sequence comparisons

Phylogentic Overview of Bacteria

Archaea consist of 3 distinct groups

Summary

• Importance of taxonomy• Type of taxonomy• Understand phylogenetic tree• Main groups of bacteria