Lesson 4: Classification of Microorganisms February 3, 2015

Taxonomy Taxonomy—the science of classifying organisms (taxa—categories of organisms) Provides a reference for identifying organisms Carlos Linnaeus introduced a formal system of classification, dividing living organisms into two groups, Plantae and Animalia Used Latin names to provide a “common” language for all organisms

Three Domain Classification Study of ribosomes allowed scientists to divide cell types into three groups Remember ALL cells contain ribosomes rRNA (16S RNA) are different for each group Eukarya, Bacteria, and Archaea Bacteria and Archaea are very similar Bacteria contains peptidoglycan and Archaea do not Molecular Clock measures evolution in terms of mutations in the nucleotide sequences rRNA sequences mutate slowly (highly conserved) and provide the most confident results when looking at evolution

Clinical Application Several classes of antibiotics work by inhibiting protein synthesis The disruption of protein synthesis leads to the death of a cell Streptomycin and Gentamicin attaches to the 30S subunit and Erythromicin and Chloramphenicol attaches to the 50S subunit Antibiotics do not interfere with Eukaryotic protein synthesis

Mitochondrion degenerates Figure 10.1 The Three-Domain System. Eukarya Fungi Animals Origin of mitochondria Bacteria Origin of chloroplasts Amebae Mitochondria Slime molds Archaea Cyanobacteria Plants Ciliates Extreme halophiles Proteobacteria Green algae Chloroplasts Methanogens Dinoflagellates Hyperthermophiles Diatoms Gram-positive bacteria Euglenozoa Thermotoga Giardia Horizontal gene transfer occurred within the community of early cells. Mitochondrion degenerates Nucleoplasm grows larger Vertical Gene Transfer Lateral Gene Transfer

Table 10.1 Some Characteristics of Archaea, Bacteria, and Eukarya

Phylogenetics Each species retains some characteristics of its ancestor Grouping organisms according to common properties implies that a group of organisms evolved from a common ancestor Anatomy (Gram stain, Flagella, Shape) Metabolic Processes (Oxygen usage, Fermenter) rRNA

Scientific Nomenclature Common names Vary with languages and with geography Spanish Moss Tillandsia usneiodes Binomial nomenclature (genus + specific epithet) Used worldwide Genus capitalized and species lowercase Escherichia coli and Homo sapiens

Source of Specific Epithet Scientific Names Scientific Binomial Source of Genus Name Source of Specific Epithet Klebsiella pneumoniae Honors Edwin Klebs The disease Pfiesteria piscicida Honors Lois Pfiester Disease in fish Salmonella typhimurium Honors Daniel Salmon Stupor (typh-) in mice (muri-) Streptococcus pyogenes Chains of cells (strepto-) Forms pus (pyo-) Penicillium chrysogenum Tuftlike (penicill-) Produces a yellow (chryso-) pigment Trypanosoma cruzi Corkscrew-like (trypano = borer; soma = body) Honors Oswaldo Cruz

Taxonomic Hierarchy Domain Kingdom Phylum Class Order Family Genus Species

Figure 10.5 The taxonomic hierarchy. All organisms Eukarya Archaea Bacteria Domain Fungi None assigned for archaea None assigned for bacteria Kingdom Ascomycota Euryarcheota Proteobacteria Phylum Hemiascomycetes Methanococci Gammaproteobacteria Class Saccharomycetales Methanococcales Enterobacteriales Order Saccharomycetaceae Methanococcaceae Enterobacteriaceae Family Methanothermococcus Saccharomyces Escherichia Genus S. cerevisiae Species M. okinawensis E. coli Baker’s yeast Methanococcus E. coli

Critical Thinking When writing the genus and species for the first time in a paper the FULL genus must be used. Why? Escherichia coli and Entamoeba coli

Classification of Prokaryotes Prokaryotic species: a population of cells with similar characteristics Culture: grown in laboratory media Clone: population of cells derived from a single cell All cells in a clone SHOULD be identical but mutations may occur Strain: genetically different cells within a clone Usually denoted by numbers, letters, or names that follow the specific epithet E. coli O104:H4 (PATHOGENIC) VS. E. coli K12 (non-pathogenic)

Classification of Eukaryotes Eukaryotic species: a group of closely related organisms that breed among themselves Categorized based on unicellular (Protista and some Fungi) or multicellular (Animalia, Plantae, and some Fungi) Protists may be classified into clades which are genetically related groups (similar to strains of bacteria)

Classification of Eukaryotes Animalia: multi-cellular; no cell walls; chemoheterotrophic Plantae: multi-cellular; cellulose cell walls; usually photoautotrophic Fungi: chemoheterotrophic; unicellular or multicellular; cell walls of chitin; develop from spores or hyphal fragments Protista: a catchall kingdom for eukaryotic organisms that do not fit other kingdoms Grouped into clades based on rRNA

Classification of Viruses VIRUSES ARE NOT ALIVE! Are not comprised of cells Require a host to live and reproduce (obligate intracellular parasites) Viral species: population of viruses with similar characteristics that occupies a particular ecological niche

Classification and Identification Microorganisms Classification: placing organisms in groups of related species Lists of characteristics of known organisms Identification: matching characteristics of an “unknown” organism to lists of known organisms Clinical lab identification Microorganisms are identified for practical purposes such as determining treatment for infection

Clinical Identification Methods Morphological characteristics: useful for identifying eukaryotes but can be used for prokaryotes Shapes of bacterium; colony characteristics Differential staining: Simple staining, Gram staining, and acid-fast staining Based on cell membrane differences Biochemical tests: determines presence of bacterial enzymes Catalases, peroxidases, agglutination tests, fermentation tests, etc.

Figure 10.8 The use of metabolic characteristics to identify selected genera of enteric bacteria. Can they ferment lactose? No Yes Can they use citric acid as their sole carbon source? Can they use citric acid as their sole carbon source? No Yes No Yes Shigella: produces lysine decarboxylase Salmonella: generally produces H2S Can they ferment sucrose? Do they produce acetoin? No Yes No Yes Escherichia spp. E. coli O157 Citrobacter Enterobacter

Figure 10.9 One type of rapid identification method for bacteria: Enterotube II from Becton Dickinson. One tube containing media for 15 biochemical tests is inoculated with an unknown enteric bacterium. After incubation, the tube is observed for results. Phenylalanine Glucose Ornithine Adonitol Arabinose Lysine Lactose Sorbitol Indole Dulcitol Urease Citrate Gas H2S V–P The value for each positive test is circled, and the numbers from each group of tests are added to give the ID value. 2 + 1 4 + 2 + 1 4 + 2 + 1 4 + 2 + 1 4 + 2 + 1 2 1 7 Comparing the resultant ID value with a computerized listing shows that the organism in the tube is Proteus mirabilis. Atypical Test Results Confirmatory Test ID Value Organism 21006 Proteus mirabilis Ornithine– Sucrose 21007 Proteus mirabilis Ornithine– 21020 Salmonella choleraesuis Lysine–

Book References Bergey’s Manual of Determinative Bacteriology Provides identification schemes for identifying bacteria and archaea Morphology, differential staining, biochemical tests Bergey’s Manual of Systematic Bacteriology Provides phylogenetic information on bacteria and archaea Based on rRNA sequencing

Serology Serology is the science that studies serum and immune responses that are evident in serum (does not contain blood cell or clotting factors) Combine known anti-serum plus unknown bacterium Rabbit immune system injected with pathogen produces antibodies against that pathogen Strains of bacteria with different antigens are called serotypes, serovars, biovars Slide agglutination test

Figure 10.10 A slide agglutination test. Positive test Negative test

ELISA Enzyme-linked immunosorbent assay Direct or Indirect Direct ELISA looks for the presence of bacterium in the serum Indirect ELISA looks for the presence of antibodies in the serum Both use antibodies linked to enzyme Enzyme contains substrate that produces color

(a) A positive direct ELISA to detect antigens Figure 18.14.4 The ELISA method. 4 Enzyme's substrate ( ) is added, and reaction produces a product that causes a visible color change ( ). 4 Enzyme's substrate ( ) is added, and reaction produces a product that causes a visible color change ( ). (a) A positive direct ELISA to detect antigens (b) A positive indirect ELISA to detect antibodies

Figure 10.12 The Western blot. If Lyme disease is suspected in a patient: Electrophoresis is used to separate Borrelia burgdorferi proteins in the serum. Proteins move at different rates based on their charge and size when the gel is exposed to an electric current. Lysed bacteria Polyacrylamide gel Proteins Larger Paper towels Smaller The bands are transferred to a nitrocellulose filter by blotting. Each band consists of many molecules of a particular protein (antigen). The bands are not visible at this point. Sponge Salt solution Gel Nitrocellulose filter The proteins (antigens) are positioned on the filter exactly as they were on the gel. The filter is then washed with patient’s serum followed by anti-human antibodies tagged with an enzyme. The patient antibodies that combine with their specific antigen are visible (shown here in red) when the enzyme’s substrate is added. The test is read. If the tagged antibodies stick to the filter, evidence of the presence of the microorganism in question—in this case, B. burgdorferi—has been found in the patient’s serum.

Bacteriophages are viruses that infect bacteria Figure 10.13 Phage typing of a strain of Salmonella enterica. Bacteriophages are viruses that infect bacteria

Flow Cytometry Uses differences in electrical conductivity between species Fluorescence of some species Cells selectively stained with antibody plus fluorescent dye

Figure 18.12 The fluorescence-activated cell sorter (FACS). A mixture of cells is treated to label cells that have certain antigens with fluorescent-antibody markers. 2 Cell mixture leaves nozzle in droplets. Fluorescently labeled cells 3 Laser beam strikes each droplet. Laser beam Detector of scattered light Laser 4 Fluorescence detector identifies fluorescent cells by fluorescent light emitted by cell. Electrode Fluorescence detector 5 Electrode gives positive charge to identified cells. Electrically charged metal plates 6 As cells drop between electrically charged plates, the cells with a positive charge move closer to the negative plate. 7 The separated cells fall into different collection tubes. Collection tubes 6

Genetic Identification DNA fingerprinting Electrophoresis of restriction enzyme digests rRNA sequencing Polymerase chain reaction (PCR)

Figure 10.14 DNA fingerprints. 2 3 4 5 6 7

Heat to separate strands. Figure 10.15 DNA-DNA hybridization. Organism A DNA Organism B DNA 1 Heat to separate strands. 2 Combine single strands of DNA. 3 Cool to allow renaturation of double-stranded DNA. 4 Determine degree of hybridization. Complete hybridization: organisms identical Partial hybridization: organisms related No hybridization: organisms unrelated

Figure 10.16 A DNA probe used to identify bacteria. Plasmid Salmonella DNA fragment 3 Unknown bacteria are collected on a filter. 1 A Salmonella DNA fragment is cloned in E. coli. 4 The cells are lysed, and the DNA is released. 2 Cloned DNA fragments are marked with fluorescent dye and separated into single strands, forming DNA probes. 5 The DNA is separated into single strands. 6 DNA probes are added to the DNA from the unknown bacteria. Fluorescent probe Salmonella DNA 7 DNA probes hybridize with Salmonella DNA from sample. Then excess probe is washed off. Fluorescence indicates presence of Salmonella. DNA from other bacteria

Figure 10.17ab DNA chip. (a) A DNA chip can be manufactured to contain hundreds of thousands of synthetic single-stranded DNA sequences. Assume that each DNA sequence was unique to a different gene. (b) Unknown DNA from a sample is separated into single strands, enzymatically cut, and labeled with a fluorescent dye.

Figure 10.17cd DNA chip. (c) The unknown DNA is inserted into the chip and allowed to hybridize with the DNA on the chip. (d) The tagged DNA will bind only to the complementary DNA on the chip. The bound DNA will be detected by its fluorescent dye and analyzed by a computer. In this Salmonella antimicrobial resistance gene microarray, S. typhimurium-specific antibiotic resistance gene probes are green, S. typhi-specific resistance gene probes are red, and antibiotic-resistance genes found in both serovars appear yellow/orange.

FISH Fluorescent in situ hybridization Used to identify specific sequences in DNA/Chromosomes Add DNA probe for S. aureus

Figure 10.18 FISH, or fluorescent in situ hybridization.