1. Mic 101: L8 SST The Bacteria: Systematic Classification

2. The History of Bacterial Classification (Tortora) The scientific study of the classification of living organisms is called taxonomy. The first differentiation was with Gram reaction With advancement of microscope and metabolic studies, bacteria were classified according to their morphology and metabolic patterns Clinicians classify bacteria according to the pathogenic ability, symptoms of the disease and ease of treating the infection. In 1969, Robert Whittaker proposed a classification of all living organisms depending on morphology, called the five kingdom classification. With advancement of molecular biology, scientists were more concerned with the evolutionary history of organisms. Carl Woese proposed a classification system with all organisms divided into 3 domains depending on their macromolecular and genetic composition as preserved by evolution. This is the latest three domain classification.

3. The Gram Reaction In 1884, Danish bacteriologist Hans Christian Gram developed a method of differentiating bacteria according to their cell wall composition. Gram collected samples from different sources and heat-fixed them on the glass slide He applied Crystal violet dye to the smear for one minute and washed with water. The he applied Iodine for 1 minute to the slide and then washed it with water. He put ethanol to the slide for 15-20 seconds and washed it with water. Finally he added Safranin to the slide for 1 minute, washed with water and dried in air. The bacteria with thick peptidoglycan layer in their cell wall appear purple because they retain the Crystal violet dye. These are called Gram positive. The bacteria with thin peptidoglycan layer and higher amount of LPS/LP in their cell wall loose the color of Crystal violet because the ethanol treatment dissolves the LPS/LP and washes crystal violet. So finally these bacteria take the color of safranin and look orange. These are called Gram negative.

4. Demonstration of Gram Reaction

5. Usefulness Of Gram Reaction The Gram positive and Gram negative bacteria differ in pathogenicity and sensitivity to antibiotic. So the Gram reaction is an easy way to know how the disease-causing bacteria can be controlled. Eg. Gram positive bacteria can be controlled with penicillin and cephalosporin more easily than Gram negative ones. The Gram reaction is fast (5-6 minutes), easy, inexpensive and needs only slides, stains, ethanol, water and microscope. So it can be applied almost anywhere.

6. Positive Bacillus, Clostridium, Staphylococcus, Streptococcus, Lactobacillus, Listeria Negative E. coli, Salmonella, Shigella, Klebsiella, Vibrio, Pseudomonas, Neisseria, Haemophilus Some common Gram positive and negative bacteria

7. Five Kingdom Classification: The Logic Whittakar proposed that prokaryotes, unicellular eukaryotes, multi-cellular eukaryotes, plants and animals are morphologically different and should belong to five different kingdoms in the living world. He assumed the prokaryotes are the earliest living forms (3.4 billion years ago) and eukaryotes have evolved from prokaryotes (1.4 billion years ago) from a complex process of evolution and endosymbiosis.

8. Five Kingdom Classification: The Description Kingdom Monera : Unicellular Prokaryote The beginning of life on earth Major members: Bacteria, Archaea, Cyanobacteria, Mycoplasma Kingdom Protista : Unicellular eukaryote Evolved nucleus Major members: Algae, Amoeba, Euglenoid, Flagellate, ciliate, slime mold Kingdom Fungi Mostly Multi-cellular eukaryote, get nutrition through absorption Major member: yeast, mold, fungi Kingdom Plantae Produce glucose by photosynthesis Major members: green algae and plants Kingdom Animalia Ingestive nutrition; consume pre-formed food Major members: helminths, nematodes, worms, arthropods, mollusks, echinoderms, vertebrates

9. Endosymbiosis Symbiosis means the interaction of two organisms living in mutual benefit in a way that their separate existence is difficult to distinguish. Endosymbiosis means one organism living inside the cell of another organism. Eg. An uncharacterized bacteria is found inside the protist Cyanophora paradoxa, but their separate identity can only be assayed through genome sequencing.

10. 3 Domain Concept Molecular biological techniques aiming the 16s RNA and organization of DNA show existence of 3 types of organisms among all living things: Domain Eubacteria are prokaryotes with looped rRNA and peptidoglycan in the cell wall. All known bacteria, Streptomyces, Actinomyces, Cyanobacteria are in this domain. Domain Archaea are prokaryotes with non-looped rRNA and are characterized by extreme tolerance of methane, salt, temperature and acidity. Methanogens, halophiles, picrophiles and pyrophiles are in this domain. Domain Eukarya are eukaryotes with non-looped rRNA, 80s ribosomes and membraneous organelles. All fungi, protozoa, algae, plants and animals are in this domain. The 3 Domain concept also accepts endosymbiotic hypothesis.

11. Three Domain Concept

12. Comparison between 3 domains

13. Explanation of the existence of Chloroplast and Mitochondria in Eukaryotes Some scientists think the organelles of eukaryotic cells evolved from endosymbiotic prokaryotic cells. During the 1.4 billions years of endosymbiosis, they lost their individuality and adapted to exist as organelles only. Chloroplast and mitochondria have 70s ribosome, circular DNA and are sensitive to chloramphenicol; these are features of prokaryotic cells. In addition, chloroplast and mitochondria divide independently of the eukaryotic genomic DNA. These evidence points to their evolution from pre-existing prokaryotes.

14. Significance of smallness The very small size of microbes has an impact on fitness as it affects metabolism, generation time, colonization ability and growth rate. Small size → relative to cell volume more surface is exposed to the environment. Nutrients and waste-products rapidly pass into and out of a cell as transport distances of a metabolite within an organism and between the organism and the surroundings are short.

15. Significance of Smallness Usually higher cellular metabolic rates and growth rates and larger populations (high numbers of rapidly metabolising cells) allow more efficient energy management relative to other larger cells and multi-cellular organisms. If living conditions are favourable → may cause major physiochemical changes in an ecosystem over a relatively short period of time.

16. Questions 1. Is a viral infection a type of endosymbiosis? Why do we still use Gram reaction? In which domain will you place an organism which is a prokaryote without peptidoglycan in cell wall? Which molecule is the basis of the 3 domain classification? What things are common between a mitochondria and bacteria?

17. Questions Why do Whittaker and Woese classified Fungi differently in their classification system? What do Archaea and bacteria have in common? What do Archaea and Eukarya have in common?