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

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

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

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

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)

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

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

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

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

10.2. 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

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

10.2. Using Phenotypic Characteristics to Identify Prokaryotes

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)

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

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)

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

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

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

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)

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)

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

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

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

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

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

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

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

10.5. Classifying Prokaryotes

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

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

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

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

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

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