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Chapter9 Microbial taxonomy Three separate but interrelated parts: 1. Classification: the arrangement of organisms into groups or taxa based on mutual similarity or evolutionary relatedness. 2. Nomenclature: the branch of taxonomy concerned with the assignment of names to taxonomic groups in agreement with published rules. 3. Identification: the practical side of taxonomy, the process of determining that a particular isolate belongs to a recognized taxon. Taxonomy is important for several reasons 1. 2. 3. 4. It allows us to organize huge amounts of knowledge about organisms Allows us to make predictions and frame hypotheses for further research based on knowledge of similar organisms. It places microorganisms in meaningful, useful groups with precise names so that microbiologists can work with them and communicate efficiently. Identification of microorganisms accurately Taxomomic ranks Strain: one single isolate or line Type: sub-set of species Species: related strains Genus: related species Family: related genera Order; class; phylum; domain Related concepts A species: a collection of strains that have a similar G+C composotion and 70% or greater similarity as judged by DNA hybridization experiments. A biovars: variant procaryotic strains characterized by biochemical or physiological differences. Morphovars: differ morphologically Serovars: have distinctive antigenic properties Type strain: it is usually one of the first strains studied and often is more fully characterized than other strains Classification systems Phenetic classification: one that groups organisms together based on the mutual similarity of their phenotypic characteristics. Comparing as many attributes as possible. Numerical taxonomy: computers may be used to analyze data for the production of phenetic classification. Information about the properties of organisms is converted into a form suitable for numerical analysis and then compared by means of a computer. Phylogenetic classification: based on evolutionary relationships rather than general resemblance. defficult because of the lack of a good fossil record. Comparision of genetic material and gene products Major characteristics used in taxonomy Morphological characteristics Physiological and metabolic characteristics Ecological characteristics Genetic analysis Molecular characteristics Morphological characteristics Cell shape Cell size Cilia and flagella Cellular inclusions Color Mechanism of motility Endospore shape and location Spore morphology and location Colonial morphology Ultrastructural characteristics Staining behavior Physiological, metabolic and Ecological characteristics Carbon and nitrogen sources Cell wall constituents Energy sources Fermentation products Luminescence Motility Osmotic tolerance Storage inclusions General nutritional type Growth temperature optimum and range Mechanisms of energy conversion pH optimum and growth range Photosynthetic pigments Salt requirements and tolerance Secondary metabolites formed Sensitivity to metabolic inhibitors and antibiotics Genetic analysis The study of transformation and conjugation in bacteria is sometimes taxonomically useful. Plasmid-borne traits can cause errors in bacterial taxonomy if care is not taken. Transformation can occur between different procaryotic species but only rarely between genera. E.coli can undergo conjugation with the genera Salmonella and Shigella but not with Proteus and Enterobacter Molecular analysis Historical • guanine (G)+ cytosine (C) (% GC) Now • Hybridization • Gene characterization – sequencing – other DNA-DNA hybridization Strain 1 Heat + 0% Homology Strain 2 100% Homology DNA-DNA hybridization Groups bacterial strains into species Below species level • little or no relatedness 16S rRNA Sequencing similarity above species level allows relatedness comparisons of all bacteria closely related bacterial species may be identical development of clinical tests based on sequence Ribosomal RNAs as Evolutionary Chronometers Reasons: • • • • Ancient molecules Functionally constant Universally distributed Moderately well conserved in sequence across broad phylogenetic distances The 16S rRNA or 18S rRNA Technique The 16S rRNA or 18S rRNA Technique In Prokaryotes: • 5S rRNA is too small, contains limited info • 23S rRNA is too large, too difficult to manage • 16S rRNA has the right size for studies In Eukaryotes: • 18S rRNA is used for phylogenetic measurements The 16S rRNA or 18S rRNA Technique The 16S rRNA or 18S rRNA Technique Ribosomal Database Project (RDP): • http://www.cme.msu.edu/RDP Compare your sequences with the database to find out the organisms you identify Phylogenetic Trees from DNA Sequences Distance-Matrix Method for generating the trees Evolutionary Distance (ED) Computer compare the sequence differences and build the phylogenetic tree based on corrected ED Phylogenetic Trees from DNA Sequences Signature Sequences: unique to certain group of organisms Applications: Phylogenetic Probes “official” taxonomy and nomenclature Taxonomy: Bergey’s manual of systematic bacteriology or Bergey’s manual of determinative bacteriology in some sence official Nomenclature: all new names are validly published to gain standing in the nomenclature, either by being published in papers in the international Journal of Systematic Bacteriology or, if published elsewhere, by being announced in the Validation Lists Bergey’s mamual of systematic bacteriology The first edition: phenetic Procaryotic groups are divided into four volumes: (1)gram-negative bacteria of general, medical, or industrial importance. (2)gram-positive bacteria other than actinomycetes (3)gram-negative bacteria with distinctive properties, cyanobacteria, and archaea (4)actinomycetes Bergey’s mamual of systematic bacteriology The second edition: phylogenetic Five volumes: Volume 1: the archaea, and the deeply branching and phototrophic bacteria Volume 2: the Proteobacteria Volume 3: the low G+C gram-positive bacteria Volume 4: the high G+C gram-positive bacteria Volume 5: the Planctomycetes, Spirochaetes, Fibrobacteres, Bacteroidetes, and Fusobacteria Table19.9 p441 Table19.8 p436 Identification in the diagnostic laboratory • • • • Aids treatment Susceptibility – antibiotic selection Based on taxonomy Simple, low cost, rapid Steps in isolation and identification • Step 1. Streaking culture plates – colonies on incubation (e.g 24 hr) – size, texture, color, hemolysis – oxygen requirement Blood Agar Plate Isolation and identification Step 2. Colonies Gram stained • cells observed microscopically Gram negative Gram positive Heat/Dry Crystal violet stain Iodine Fix Alcohol de-stain Safranin stain Step 3. Isolated bacteria are speciated Generally using physiological tests Typical Culture Laboratory Bench Step 4. Antibiotic susceptibility testing Susceptible Not susceptible Bacterial lawn No growth Growth Antibiotic disk Rapid diagnosis without culture • WHEN AND WHY? • grow poorly – Isolation slow may not be clinically useful • can not be cultured – isolation impossible Rapid “Strep” Test Streptococcal antigenic extract Antibody Latex beads Bacterial DNA sequences amplified directly from human body fluids • Polymerase chain reaction (PCR) • Great success in rapid diagnosis of tuberculosis. Microscopy • spinal fluids (meningitis脑膜炎) • sputum (tuberculosis) • sensitivity poor Serologic identification • antibody response to the infecting agent • several weeks after an infection has occurred Questions 1. 2. 3. 4. 5. 6. 7. Why is Ribosomal RNAs used as Evolutionary Chronometers Why is 16S rRNA employed to study phylogenetics rather than the smaller 5S rRNR and the large 23S rRNA in Prokaryotics? How to sequence 16S rRNA from inside the cells? How to identify a prokaryotic or eukaryotic organism based on 16S or 18S rRNA? How to build Phylogenetic Trees from DNA Sequences? Why it is said the archaea closer to eukarya than bacteria is? What is signature sequence and how can it be used?