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90 Cards in this Set

  • Front
  • Back
Pathovar
(pv) pathogenicity with specific host
Biovar
(bv) biochemical or physiological properties
Serovar
(sv) antigenic components
Phagovar
(phv) susceptibility to different phages
Morphovar
(mv) special morphological features
Formal subspecies
Based on minor but consistent phenotypic or genetic differences between strains within a species
Treponema pallidum subspecies
pallidum (venereal syphilis)
endemicum (endemic nonvenereal syphilis)
pertenue (nonvenereal, non endemic - localized distribution)
Type strain
Refers to a strain that is selected as the reference strain for a species. It is usually the first strain described and isolated that represents the species. The type strain is very important and is maintained in a viable state in various culture collection centres,
Type species
Refers to a species selected as the reference species for the genus. It is usually the first species described and isolated that represents the genus. The type species is very important and maintained in a viable state in various culture collection centers.
Two types of classification systems
Phenetic classication and phyletic classification
Phenetic classification
Bacteria are placed into groups (taxa) based on shared (mutual) phenotypic and genotypic (phenetic) characters. However, most phenetic classification systems rely primarily on phenotypic rather than genotypic characterics. Also referred to as taxonomy, and an artificial system since grouping is based on arbitrary chosen criteria and does no indicate how different taxa may have evolved. Members of the same taxon may display similar characters, but it is not possible to determine how closely they are related to each other in terms or evolution (ie, whether all the organisms in the taxon evolved from a common ancestral organism)
Phyletic classification
(phylogeny, phylogenetics, natural classification) Bacteria are placed into groups based primarily on genetic similarity or genotype. Genotypic similarity indicates that the genome sequence is similar and that it is likely that the organisms are descended from a common ancestor. The phylogeny of bacteria is currently an area of extensive research (will be discussed later).
Ideal classification
A truly phenetic classifcation that shows the phylogeny of bacteria and phenotypic characteristics of the phyletic groups (polyphasic approach) - process is quite complicated since the phylogeny of bacteria is, in many cases, quite different from their taxonomy.
Bergey's classification
One of the oldest and most comprehensive classification of bacteria that has been used worldwide, based on phenotypic characteristics, newer editions incorporate genotypic characters. Latest complete edition - 1984-1989, 4 volumes (Bergey's Manual of Systematic Bacteriology).
Bergey's Manual of Determinative Bacteriology
Shorter, one-volume version of Bergey's. Follow same form of classification but gives more concise descriptions of organisms - genotypic characters also absent. The latter is used primarily for diagnostic work.
How are prokaryotes categorized in Bergey's
Based primarily on cell wall structure
Eubacteria with gram-negative cell walls
-outer membrane and thin peptidoglycan layer
-nutritionall diverse: chemo/photoautotrophs, chemo/photoheterotrophs
Eubacteria with gram-positive cell walls
-relatively thick, composed of peptidoglycan and teichoic acids
-mostly chemoautotrophs
Eubacteria lacking cell walls
(Mycoplasma, Mollicutes)
-phyletically related to gram-positives
Archaebacteria cell walls
-protein or polysaccharide cell walls, peptidoglycan absent
-distinctive glycerol phytanyl6 ether lipids in membranes - eukaryotes have glycerol fatty acyl ester lipids
-chemoheterotrophs and chemoautotrophs
What is Bergey's classifcation designed for? And how?
Primarily for use in identification of bacteria, i.e. diagnostic or determinative studies. How? It is well suited for this since the assignment of a few key characters allow one to determine the group to which the organism belongs (quick diagnostic and identification) - especially important in medical field.
Which Bergey groups appear to be monophyletic (likely common ancestral organism)?
Group 1 - Spirochetes
Group 5 - Facultative anaerobic gram-negative
Group 18 - Endospore forming gram-positive cocci and rods
Which Bergey groups appear to be polyphyletic (diverse phyletic origin)?
Group 4 - Gram-negative aerobic/microaerophilic rods and coci
Group 10 - Anoxygenic phototrophic bacteria
Summary of Bergey's System
-placement into groups dependent on a few phenotypic characteristics
-only a few characters define a taxon
-"groups" are not a formal taxon - no identification of relationship between different groups
-in some cases, normal hierarchical placement into families, general, and species
Disadvantage of Bergey's
There is no way of telling whether the organisms comprising a group are phyletically related. Since relatively few characters are assessed, it is also not possible to determine the degree of phenetic or phyletic relatedness among members of a taxon, i.e. how closely or distantly 2 organisms in the same group are related.
How to proceed with identification
The characters of the unknown are compared with those that are classified, Unknown is identified if there is a positive correspondence with a classified organism.
Characteristics of a good identification system (3)
1. Few as possible tests - full characterization too time consuming
2. Test should be discriminatory - able to distinguish different organisms
3. Tests should be reliable - consistent results (reproducible)
Describe numerical taxonomy
An approach to cluster strains on the basis of large sets of unweighted phenetic data.
-quantifies relatedness between organisms
-introduced 200 years ago by Adanson for classifying plants and was adapted for bacteria by Sheath in 1957.
-requires many computations and only became feasible with the development of computers
How is phenetic relatedness between organisms quantified in numerical taxonomy?
In terms of % similarity (%S)
Outline of methods involved in numerical taxonomy
1. The strains (OTU - operational taxonomic units) to be classified are tested for a wide range of characters ( about 100-150, no less than 100). For each OTU, a character is scored as + or - (character state) and the results are summarized. The table is somewhate like a diagnostic table and compares the characters of all organisms in the test group.
2. Pair-wise comparison of OTU's - the results for each OTU is compared with that or every other OTU in a test group. The number of characters shared between each pair of OTU is assessed. Calculate a similarity coefficient (%similarity) that quantitates the degree of similarity between each pair of OTU).
3. The results are tabulated in a similarity matrix which shows the %S between each OTU pair. SJ and SSM values range from 0 to 1.0 (i.e. 0-100%).
4. The OTU pairs are ranked starting with those that show the highest %S value.
5. Single-linkage analysis: link different OTU pairs into successively larger groups, starting from highest to lowest %S.
6. Construct Phenograms (dendrograms) - this is the most common way of presenting results of ranking. The % S that links OTU's into a group is depicted in line graph.
What are phenons
OTU's in numerical taxonomy that show greater similarity and form clusters linked by a specified %S distinguished from other clusters. These are analogous to taxa. They may be identified according to %S that links the organism in a phenon, e.g. 90%S phenon
What is the consensus on the relationship between phenons and ranks in conventional taxonomy
There is no consensus, but some have suggested that an 85% similarity phenon may be analogous to the GENUS taxon
What do dendrograms show
The degree of relatedness between members in a phenon and between different phenons
What is a polythetic system (comment 1.)
Grouping of organisms depends upon the assessment of a large number of characters. The characters are unweighted, and are equally important for the purpose of grouping. Because a large number of characters are utilized, the groupings generated in this system are stable.
What is numerical taxonomy very good at (comment 2.)
Defining the strains that belong to a species and for differentiating between species and genera (i.e. lower ranking taxa)
What has numerical taxonomy been used to confirm? (comment 2.)
It has been used to confirm the relationship of many major bacteria and to determine the placement of newly discovered organisms into classification schemes.
How can numerical taxonomy show which characters are important for defining a group? (comment 3.)
Test OTUs for different characters and determine which characters are most prevalent (or are prevalent) in all OTUs. In 33 strains of Pseudomonas, 17 characters were present in 100% of OTUs, 8 characters in 90-99%, 5 characters in 80-89%
What kind of system is numerical taxonomy? (comment 4.)
A phenetic system. Although the organisms are grouped primarily be phenotypic characteristics, a wide range of phenotypic similarity indicates a similarity in genotype.
What is Mol %G+C (%GC)
Molar content of G and C residues given as a percentage of the total bases in DNA
What are the ranges of %GC in Plants and animals, Eukaryotes and Prokaryotes
Plants and animals = 35-50
Eukaryotes = 35-65
Prokaryotes = 23-78
Why is GC% useful?
Gives a rough indication of DNA sequence, organisms that are closely related through evolution will have similar chromosome sequence and consequently similar GC%. e.g. Strains belonging to a same species that have evolved from the same ancestral organism should have very similar GC%
When is GC% meaningful?
When used in conjunction with a phenetic description of a species.
Which type of classification species should have a very similar %GC?
Genospecies from phyletic classication. Taxospecies from phyletic classification are grouped primarily on the basis of phenotypic characteristics (showing high degree of phenotypic similarity) and do not necessarily share the same %GC.
What should be the %GC range for a species? For a genus?
5% or less range for strains of a same species (e.g. Pseudomonas is around 1% or less); and 10% or less for species of the same genus.
Why can't we use %GC alone to define a species
Similarity of GC% only corroborates with their phylectic association, and GC% doesn't give enough information on the sequence of DNA (i.e. same GC% does not mean that the sequences will be similar). Consider two sequence samples - AGTC and GTCA. They have the same GC% but are completely different sequences. Similarity %GC can only be used to support a grouping based on phenotypic characterizaiton.
Similarity in GC% does not necessarily translate in a similarity in sequence, but what does a difference in GC% value indicate? How has this been useful?
It indicates a difference in sequence. It has been useful for differentiation between phenotypically similar bacteria.
e.g.1 Cytophags and myxobacteria are both aerobic, gram-negatic, chemoheterotrophic rods that exhibit gliding motility but their GC composition clearly shows that they are phyletically distinct (33-34%GC for cytophags, 68-72%GC for myxobacteria)
e.g.2 Staphylococcus and micrococcus are both gram-positive cocci, but Staph. GC% = 30-40 and Micro. GC%=65-75
How can DNA be denature?
Alkaline (pH greater than 12) conditions or heat
How is the melting temperature of dsDNA (Tm is correlated to the GC content of DNA?
DNA with higher GC-content is more stable, since more H-bonds hold strands together (GC pairs held together by 3 H-bonds, while AT basepairs by 2 H-bonds)
Steps in determining GC%
1. Extracted dsDNA is heated and the change in A260nm is monitored - ssDNA absorbs more UV260nm than the same amount of dsDNA (effect of hyperchromicity). Tm is determined from the inflection point as A260nm increases during the transition from ds to ss form.
2. GC content is related to Tm by the empirical equation %GC=2.44(Tm-69.4), which was derived from determining Tm's of DNA whose %GC was found chemically.
What is DNA:DNA hybridization?
A measure of sequence similarity between DNA of different organisms - as similar sequences of DNA of different organisms will be able to form heterologous hybrids more easily than those of less similar sequence.
How do you obtain ssDNA?
Rapidly cool DNA that has been heated >Tm
How do you rehybridized ssDNA?
Purified DNA is heated to >Tm, and slowly cooled to 15-30°C below Tm, ssDNA re-associates to form dsDNA. Referred to as annealing or homologous hybridization.
What happens if heated ssDNA from 2 different organisms is mixed and allowed to cool slowly?
Homologous and heterologous hybrids may be formed. The extent of heterologous hybridization depends upon sequence similarity between the DNA of the two organisms.
What does a high level of hybridization indicates?
Similarity between the DNAs of two organisms and that the organisms are closely related genetically.
What is the procedure of determining DNA:DNA hybridization?
DNA is extracted from organisms, denatured and the ssDNA is immobilized on membrane filters. The filters are treated with protein to block any other DNA binding sites on filter (additional DNA can only bind by hybridization with immobilized DNA). Filters prepared this way can be stored indefinitely (freeze). One strain of the group is selected as the reference organism. ssDNA from the reference organism is radioactively labelled. The radioactive reference DNA is incubated with filters containing bound DNA at Tm-15-30°C to allow hybridization to take place (several hours to overnight). After washing, the radioactivity retained on filter is counted, directly related to hybridization (The amount of radioactivity retained in homologous hybridization = 100% hybridization). Less radioactivity is retained in heterologous hybridization because of differences in sequences of the bound and reference DNA. Heterologous hybridization is reported as a percentage of the homologous hybridization.
What is the amount of heterologous hybridization directly dependent upon?
It is directly dependent upon the sequence similarity of bound and reference DNA. In order for hybridization to even take place, there must be at least 70-80% sequence similarity. e.g. 0% hybridization indicates a similarity of 0-70% and it is only when there is at least 70-80% sequence similarity that any hybridization will occur.
What is a disadvantage of DNA:DNA hybridization?
Since there must be 70-80% overall sequence similarity for hybridization to occur, only DNA from closely related organisms will be able to hybridize.
How can the sensitivity of DNA:DNA hybridization be increased?
Lowering the temperature of hybridization, so that poorly formed hybrids with many mismatches would be counted.
How do you compare the thermostability of heterologous hybrids?
Heterologous hyrbdrids will be less stable and will dissociate to ssDNA and eluted from the membrane at lower temperatures than homologous duplexes. This temperature is referred to as Tm(e).
What is Tm(e)?
Directly related to the amount of mismatches and correlates with the degree of hybridization, i.e. the higher the Tm(e), the greater is the hybridization and sequence similarity.
What are the % hybridization suggestions for genetic species and genus?
>70% = genetic species
40-60% = genetic genus
*genetic or genomic
What happens when the rules for genetic species and genus according to DNA:DNA hybridization are applied to current nomenclature?
Destabilizing effect - Some common bacterial pathogens would need name changing and this would cause much confusion in identification and treatment of infection provoked by these bacteria.
What does the fact that the amount of hybridization decreases with more distantly-related species (even if they belong to the same genus) indicate?
During the course of evolution, the sequences in overall DNA change dramatically and that DNA:DNA hybridization can evaluate close, but not distant, relationships.
What is rRNA:DNA hybridization?
Measure of sequence similarity between the rDNA (ribosomal genes) of different organisms.
What are ribosomal genes?
Conserved genes that usually make up less than 1% of the total genome. They encode 16S rRNA (1650 bases), 5S rRNA (120 bases) and 23S rRNA (3300 bases). Relatedness of different organisms is determined by comparing their rRNA gene sequences by rRNA:DNA hybridization.
What is the procedure of rRNA:DNA hybridization?
1. Radioactive rRNA is prepared from the reference organism.
2. Radioactive rRNA is incubated with filter immobilized ssDNA from the reference (homologous) organism and test (heterologous) organisms at Tm-15% to allow hybridization (must wash really well after). The rRNA acts as a probe and specifically selects and hybridizes with rRNA genes in DNA. The counts retained when rRNA hybridizes with homologous DNA represent 100% hybridization.
3. In heterologous hybridization, the amount of hybridization is given as a perccentage of the homologous rRNA:DNA hybridization.

*Just like DNA:DNA hybridization, there must be 70-80% similarity between rRNA and DNA in order for hybridization to occur.
What does rRNA:DNA hybridization make possible?
It makes it possible to quantify the phyletic relatedness between more distantly related organisms, i.e. organisms that fail to show positive DNA:DNA hybridization.
Who is Palleroni and what did he do?
Palleroni studied the relationship among around 40 Pseudomonas species using DNA:DNA and rRNA:DNA hybridization in the 1970's. Determined 5 groups using rRNA:DNA info:
1. aeruginosa group
2. pseudomallei group
3. acidovorans group
4. diminuta group
5. maltophila group
See notes and handouts for more information
Who is Woese and what did he do?
Woese (Univ. of Illinois) devised a method that more directly assessed sequences of similarity of rRNA of different organisms. His work is summarized in "Bacterial evolution" (Microbial.Rev. 1987, v.51, p.221-271). From prokaryotic ssu (30S ribosomal subunit), he isolated 16S (1500 bases) and from the Eukaryotic ssu (40S ribosomal subunit) - the 18S rRNA (2300 bases)
Steps in constructing an oligonucleotide catalogue
At the time it was possible to sequence small oligonucleotides, but not the complete molecule.

1. Isolate ssu-RNA from ribosome and digest with RNAase T1. This enzyme cuts the 3' side of all guanine residues to generate oligonucleotides of differen sizes. The fragments are labelled with 32P and separated by electrophoresis and purified. The sequence of each oligonucleotide with >5 bases is determined. A list of these sequences in called the oligonucleotide catalogue. For 16S rRNA, the sequences of about 100 olignucleotides constitutes a catalogue.

2. Oligonucleotide catalogues of 16S rRNA's of different organisms are compared (e.g. Bacillus). Some oligonucleotides present in 1 species, orthers are common to 2 or more. This is expected if the species evolved from a common ancestral organism. They would share common sequences and also show distinct sequences that have been accumulated during evolution. The greater the number of shared sequences, the greater sequence similarity and the more closely related are the two organisms.

3. Calculation of similarities: in a test group, the oligonucleotide catalogue of an organism is compared with that of every other organism in the group in a pair-manner. A binary association coefficient (Sab) is calculated for each pair (Sab=2Nab(Na+Nb)).
What are Sab analogous to?
Similarity coefficient. In this case, each specific ologinucleotide is analogous to a character in numerical taxonomy except these are genotypic, not phenotypic characters.
What is the Sab directly related to?
It is directly related to the similarity in the sequences of ssu-RNA's of organisms a and b and therefore the sequence similarity of the genes encoding the ssu-RNA. If a and b are the same organism, Sab = 1.0. The closer the value is to 1, the more similar are the sequences of the ssu RNA gene.
How many organisms have had oligonucleotide catalogues prepared for?
>400 organisms, and Sab values determined though pair-wise comparisons of catalogues.
What was the conclusion of Woese's study? (11 organisms)
Using Sab comparison of organisms, Woese postulated the existence of a third kingdom of organisms, the archaebacteria, which according to the data were different from the eubacteria as eubacteria are different from the eukaryotes.
What is common between organisms belonging to a domain?
They are more similar to each other than they are to sequences of organisms belonging to other domains. One can therefore conclude that the organisms in a domain are phyletically closer related to each other than to the organisms in other domains, i.e. they arose from a common ancestral organism.
How many organisms did Woese place into 1 of 3 domains?
>400, and when more organisms were classified it was possible to place them into smaller groups within each domains.
What is the ultimate goal of microbial systematics?
Relate microbial taxonomy to evolution, thus placing microbiology on a level footing with botany and zoology.
What is the problem with taxonomic units of prokaryotes?
They are arbitrarily defined by humans to facilitate communication, but they do not always correlate with the units evolved in nature.
The construction of a hierarchical system is necessarily a compromise that takes into account of what?
-inability to understand fully the mode and tempo at which invidual bacterial cells evolved
-the restriction of basing taxonomic unit on a minority of existing organisms, hence on a small fraction of biodiversity only
-the subjectiveness which taxonomist consider characters to be "important" or less significant
What is the problem of defining a taxa at all levels exclusively on the basis of genomic information (presence of a taxon specific sequence/nucleotides?)
As more and more organisms are sequence, the abundance of information might begin to blur the sequence signatures of genomic taxa that could be defined today.
What is the problem with the lowest rank in hierarchical structure, the species?
Bacterial species is not a coherent collection of identical strains, but is composed of different clones that differ slightly from each other in genotype and phenotype (spontaneous mutation).
What is the error rate of cellular DNA replication and what does it cause?
The error rate of the cellular DNA replication (approx. 10^-8 per gene per generation) and it generates within a SINGLE COLONY mutants that differ (ina few nucleotides) from their ancestral cell. A few generations later, individual clones may have evolved to constitute the beginning of an evolutionary line within the species.
What is a fundamental concept of taxonomy?
The system that should be chosen should be PRACTICAL and SERVES THE USER.
What do difference in the rate of evolution within individual lines make it impossible to do?
To define a single cut off point for a taxon at any level, especially because differences at the genomic level do not need to be accompanied by difference at the genotypic (? - phenotypic) level.
Even if the genetic level provides us with the possibility of creating a classification system based exclusively on sequences, what should be taken under consideration?
The guidelines and standards of such classification system should not, for practical reasons, be placed so high that they are outside the reach of the majority of taxonomists.
What is the major emphasis of the polyphasic system?
Establish a framework of phylogenetic groupings and to delineate subsequently these groupings based on suitable phenotypic characteristics.

From a taxonomic point of view, the restriction of the wealth of information contained in and expressed by the genome to the mere analysis of the primary structure of macromolecules or a single property of unknown significance would not take advantage of the evolutionary process at all levels of information.
What needs to take place to allow reliable and manageable identification and classification?
Decisions about the rank of an organism in a hierarchical system is made on the basis of contributions of genotypic and phenotypic properties that distinguish it from neighboring groups.
What is an important pre-requisite of a hierarchical system?
That it remains flexible and open to change in reflecting the course of evolution of properties as accurately as possible and also to facilitate classification in accordance to the natural relationship of organisms.
How can a meaningful polyphasic approach be obtain?
It should provide a high degree of STABILITY without abolishing flexibility.
What is the basis for a polyphasic system?
Well-founded polygenetic branching pattern deribved from hybridization and sequence analysis.