1. Chapter 10 Lecture Outline
See separate PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes. 1

2. A Glimpse of History
1870s: Bacteria classified by shape (Ferdinand Cohn)
1908: Physiology rather than morphology (Sigurd Orla-Jensen)
1930s: Classification based on evolutionary relationships (Albert Kluyver, C. B. van Niel)
1970: Relationships determined by comparing physical traits, nucleotide sequences (Roger Stanier)
Late 1970s: Prokaryotes divided into two major groups based upon ribosomal RNA sequences (Carl Woese)
Led to current three domain system: Bacteria, Archaea, Eukarya

3. 10.1. Principles of Taxonomy
Taxonomy is the science that studies organisms to arrange them into groups, or taxa
Three separate but interrelated areas:
Identification
Process of characterizing in order to group
Classification
Arranging organisms into similar or related groups
Nomenclature
System of assigning names

4. 10.1. Principles of Taxonomy
Taxonomic Hierarchies
Species is basic unit: group of morphologically similar organisms capable of producing fertile offspring
Definition problematic for prokaryotes
Species is group of closely related isolates or strains
Informal groupings also used
May be genetically unrelated
Lactic acid bacteria
Anoxygenic phototrophs
Endospore-formers
Sulfate reducers
Kingdoms for prokaryotes
still in state of flux

5. 10.1. Principles of Taxonomy
Phylogeny is evolutionary relatedness
Yields three-domain system based on Carl Woese et. al.
Replaces R. H. Whittaker’s five-kingdom system (1969)

6. 10.1. Principles of Taxonomy
Three-domain system based on evolutionary relatedness
Replaces five-kingdom system
Plantae, Animalia, Fungi, Protista, Prokaryotae
Based on obvious morphological differences
Does not reflect recent genetic insights of ribosomal RNA data indicating plants and animals more closely related than Archaea to Bacteria

7. 10.1. Principles of Taxonomy
Bergey’s Manual of Systematic Bacteriology
Describes all known
species
Newest edition in five
volumes
Classifies according
to genetic relatedness
Previous edition
grouped according to
phenotype, so some
major differences
Names given according to
International Code of
Nomenclature of Bacteria

8. 10.2. Using Phenotypic Characteristics to Identify Prokaryotes
Microscopic morphology
Culture characteristics
Metabolic capabilities
Serology
Fatty acid analysis

9. 10.2. Using Phenotypic Characteristics to Identify Prokaryotes
Microscopic morphology is important initial step
Quickly determines size, shape, staining characteristics
Sometimes enough to diagnose eukaryotic infections
Gram stain distinguishes between Gram-positive and Gram-negative bacteria
May suggest sufficiently to start appropriate therapy
Special stains (e.g., acid-fast, endospore) useful

10. 10.2. Using Phenotypic Characteristics to Identify Prokaryotes
Culture characteristics can give clues
Streptococci colonies generally fairly small
Serratia marcescens colonies often red at 22°C
Pseudomonas aeruginosa often produces green pigment
Cultures also have distinct fruity odor
Differential media aids in identification
Streptococcus pyogenes (strep throat) yields β-hemolytic colonies on blood agar
E. coli (urinary tract infection) ferments lactose, forms pink colonies on MacConkey agar

11. 10.2. Using Phenotypic Characteristics to Identify Prokaryotes
Metabolic capabilities
Biochemical tests provide more certainty of identification
Catalase test
Many rely on pH indicators
Sugar fermentation
Urease production

12. 10.2. Using Phenotypic Characteristics to Identify Prokaryotes

13. 10.2. Using Phenotypic Characteristics to Identify Prokaryotes
Metabolic capabilities (continued…)
Basic strategy relies on dichotomous key
Flowchart of tests with positive or negative result
Simultaneous inoculating speeds process
Some tests accomplished without culturing (e.g., breath test to assay urease and identify Helicobacter pylori)

14. 10.2. Using Phenotypic Characteristics to Identify Prokaryotes
Metabolic capabilities (continued…)
Commercial kits available allow rapid identification via biochemical tests

15. 10.2. Using Phenotypic Characteristics to Identify Prokaryotes
Serology
Proteins, polysaccharides of prokaryotic cells can serve as identifying markers
Most useful include surface structures of cell wall, capsule, flagella, pili
Some Streptococcus species contain unique carbohydrate in cell wall
Serological tests use antibodies for detection (Chapter 18)

16. 10.2. Using Phenotypic Characteristics to Identify Prokaryotes
MALDI-TOF ( matrix- assisted laser desorption ionization time of flight mass spectrometry)
Measure the masses of various components using mass spectrophotometer
Sample spotted on sample plate with matrix
Laser beam vaporizes and ionizes sample
Time of flight: small ions travel faster than larger ones in tube
Mass spectrum a “fingerprint” or profile of the proteins and other macromolecules in the cell

17. 10.2. Using Phenotypic Characteristics to Identify Prokaryotes
MALDI-TOF provides rapid under 15 min. identification

18. 10.3. Using Genotypic Characteristics to Identify Prokaryotes
Detecting Specific Nucleotide Sequences
Tests can identify sequences unique to species or group
Nucleic acid probes
Nucleic acid amplification tests (NAATs)
Limitation is each detects only single possibility
Need to run multiple probes if organism being tested could be one of multiple different species or related groups

19. 10.3. Using Genotypic Characteristics to Identify Prokaryotes
Nucleic acid probes locate nucleotide sequence characteristic of species or group
Most methods first increase DNA in sample
E.g., inoculation on agar or in vitro DNA amplification
Fluorescence in situ hybridization (FISH) probes for 16S rRNA (Chapter 9)

20. 10.3. Using Genotypic Characteristics to Identify Prokaryotes
Nucleic acid amplification tests (NAATs) used to increase number of copies of specific DNA sequences
Allows detection of small numbers of organisms
Often from body fluids, soil, food, water
Detection of organisms that cannot be cultured
Polymerase chain reaction (PCR) common technique (Chapter 9)

21. 10.3. Using Genotypic Characteristics to Identify Prokaryotes
Sequencing Ribosomal RNA Genes
Ribosomal RNA (rRNAs) or encoding DNA (rDNAs)
Sequences relatively stable
Ribosome would not function with too many mutations
16S rRNA most useful because of moderate size
~1,500 nucleotides
16S (18S in eukaryotes)
RNAs are small subunit
(SS, or SSU) rRNAs
Sequence compared with
extensive databases
Can identify organisms that
cannot be grown in culture

22. 10.4. Characterizing Strain Differences
Characterizing strains important
Foodborne illnesses
Diagnosing certain diseases
Forensic investigations of bioterrorism, biocrimes

23. 10.4. Characterizing Strain Differences
Biochemical Typing
Group with characteristic pattern: biovar, or biotype
Serological Typing
E. coli distinguished by antigenic type of flagella, capsules, lipopolysaccharide molecules
E. coli O157:H7 (O antigen is lipopolysaccharide; K antigen is flagella)
Group with characteristic
antigens: serovar, or serotype

24. 10.4. Characterizing Strain Differences
Molecular Typing
Cut DNA samples with same restriction enzyme
Separate via gel electrophoresis
Patterns called restriction fragment length polymorphisms (RFLPs)
Different RFLPs indicate different strains
PulseNet is CDC database that tracks foodborne pathogens
Multilocus sequence typing (MLST) is newer method

25. 10.4. Characterizing Strain Differences
Phage Typing
Relies on differences in susceptibility to bacteriophages
Susceptibility pattern can be determined with bacteria and different bacteriophage suspensions
Largely replaced by molecular methods
Still useful in labs lacking equipment for genomic testing

26. 10.4. Characterizing Strain Differences
Antibiograms
Antibiotic susceptibility patterns
Clearing zones around antibiotic discs
Largely replaced by molecular techniques

27. 10.5. Classifying Prokaryotes
Classification historically based on phenotypic traits
Size, shape, staining, metabolic capabilities
But phenotypically similar organisms may be only distantly related; conversely, closely related organisms may appear dissimilar
New molecular techniques more accurate
Provide greater insights into evolutionary relatedness
DNA sequences viewed as evolutionary chronometers
Provide relative measure of time elapsed since divergence from common ancestor
Mutations accumulate over time
DNA sequencing allows construction of phylogenetic tree

28. 10.5. Classifying Prokaryotes

29. 10.5. Classifying Prokaryotes
Phylogenetic tree shows evolutionary relatedness
But DNA sequencing also highlights obstacle
Horizontal gene transfer complicates DNA comparisons
For example, bacterium Thermotoga maritima appears to have acquired ~25% of genes from archaeal species
Some scientists have
proposed a shrub with
interwoven branches

30. 10.5. Classifying Prokaryotes
16S rDNA Sequence Analysis
DNA hybridization
DNA Base Ratio (G + C Content)
Phenotypic Methods

31. 10.5. Classifying Prokaryotes
16S rDNA Sequence Analysis
Comparisons revolutionized classification
Sequences highly conserved since function critical
Lack of mutations allows identification of distant relatedness
Certain regions relatively variable, can determine recent divergence
Horizontal gene transfer appears rare
Culturing not necessary
May not resolve at species level since closely related prokaryotes can have identical 16S rDNA sequences
DNA hybridization a better tool in these cases

32. 10.5. Classifying Prokaryotes
DNA Hybridization
Relatedness of organisms can be determined by similarity of nucleotide sequences
Sequence homology measured by DNA hybridization
Extent of hybridization reflects degree of similarity
Complementary base pairing of single strands
If high percentage, considered related
70% similarity often considered same species
But Shigella and Escherichia should be grouped in same species based on DNA hybridization

33. 10.5. Classifying Prokaryotes
DNA Base Ratio (G + C Content)
Ratio of bases in DNA (A:T and G:C)
Base ratio expressed as G + C content, or GC content
If ratio deviates by more than a few percent, organisms not related
Similarity of GC content does not mean relatedness

34. 10.5. Classifying Prokaryotes
Phenotypic Methods
Have been largely replaced by 16S ribosomal nucleic acid sequence methods
Some taxonomists believe classification should be based on more than just genotypic traits
Phenotypic methods still important since provide foundation for prokaryotic identification