The document discusses the classification of microorganisms. It covers terminology used in taxonomy, provides a brief history of taxonomy, and describes various methods used for microbial classification including morphological, biochemical, genetic, and numerical methods. The intuitive method relies on expert opinion while genetic methods include analyzing the G+C content of DNA and performing nucleic acid hybridization and sequencing to determine genetic relatedness.

1. Classification of Microorganisms Prepared by Rajesh M.Patel Assistant Professor, Department of Pharmaceutical Biotechnology, Ganpat University. Email:rajmit_120@yahoomail.com

2. Contents of the chapter Terminology Taxonmomy: Introduction and History Classification Nomenclature Taxonmic methods for classification of microorganisms Intuitive method Genetic methods % G+C content Nucleic acid hybridization DNA chip technique Numerical classification methodss

3. Terminology Taxonomy Classification of living organisms into groups Phylogenetic Classification System: Groups reflect genetic similarity and evolutionary relatedness Phenetic Classification System: Groups do not necessarily reflect genetic similarity or evolutionary relatedness. Instead, groups are based on convenient, observable characteristics.

4. Terminology Species: A collection of microbial strains that share many properties and differ significantly from other groups of strains Strain: a population of organisms descended from a single organism or pure culture isolate. OR a culture derived from a single parent that differs in structure or metabolism from other cultures of that species

5. Terminology Type strain: One of the first strain among the species, studied and fully characterised than other strains. Biovars procaryotic variant strains characterized by biochemical or physiological differences Morphovars procaryotic variant strains characterized by morpholigical characteristics Serovars procaryotic variant strains characterized by distinctive antigenic properties

6. Taxonomy Organizing, classifying and naming living things Formal system originated by Carl von Linn é (1701-1778) Identifying and classifying organisms according to specific criteria Each organism placed into a classification system

7. Taxonomy Taxa: order or arrangement N omos: law Nemein: to distribute or govern The branch of biology dealing with the classification of life.

8. Taxonomy / Systematics can be understand by general examples Nomenclature Providing a formal name Genus & species Ford Crown Victoria Chevy Impala Toyota Camry Honda Civic Classification Organization into groups Car Truck SUV Van Identification Distinguishing features Engine size Mileagae Number of passengers Type of transmission

9. Taxonomy: History 1700s 2 kingdoms: plant and animal 1800s 3 kingdoms: plant, animal, and protista 1950-1990s 5 kingdoms: plant, animal, protista, fungi, monera Present: 6 kingdoms: eubacteria, archaebacteria, protista, animal, plant, fungi Taxonomy consists of 3 parts Classification Identification Nomenclature

10. Classification Arrangement into groups based on mutual similarity or evolutionary relatedness Ordering of organisms into groups.

11. Classification Natural classification: arranges organisms into groups whose members share many characteristics and reflects as much as possible the biological nature of organisms. Linnaeus developed the first natural classification, based largely on anatomical characteristics. E.g. classification of humans as mammals denotes that they have hair, self-regulating body temperature,and milk-producing mammary glands in the female. Polyphasic classification

12. Taxonomy Domain K ingdom P hylum C lass O rder F amily G enus s pecies

13. 3 Domains Eubacteria true bacteria, peptidoglycan Archaea odd bacteria that live in extreme environments, high salt, heat, etc. (usually called extremophiles) Eukarya have a nucleus & organelles (humans, animals, plants)

14. 3 Domains system

16. Five-Kingdom System of Biological Classification Proposed in 1969 by Robert Whitaker : 1. Kingdom Procaryotae (Monera): Oldest known cells. Lived over 3.5 billion years ago. Lack a nucleus and membrane bound organelles. The other four kingdoms are eucaryotes . Have a true nucleus and membrane bound organelles. 2. Kingdom Protista: Mostly unicellular, lack tissue organization. Most have flagella during life. 3. Kingdom Fungi : May be unicellular (yeasts) or multicellular (molds). Many are saprotrophs. 4. Kingdom Plantae: Multicellular, photosynthetic. 5. Kingdom Animalia: Multicellular, heterotrophs that ingest food through a mouth or oral cavity.

17. Five-Kingdom Classification System ( Whittaker)

19. Scientific Nomenclature Developed in the eighteenth century. Bionomial nomenclature: Genus and specific epithet(species) Eg. Escherichia coli, Salmonella typhi, Bacillus subtilis Rules for naming are set by international committee’s International Code of Zoological Momenclature International Code of Botanical Nomenclature Bacteriological Code and Bergey’s Manual

20. 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

21. Bergey’s Manual of Systematic Bacteriology First edition published in 1923

22. Techniques For Determining Microbial Taxonomy The Intuitive method Genetic Homology DNA/RNA sequencing DNA chip technology Numerical Taxonomy

23. Techniques For Determining Microbial Taxonomy And Phylogeny

24. Morphological features are important . Morphology is easy to study and analyze, particularly in eukaryotic microorganisms and the more complex prokaryotes. Structural features depend on the expression of many genes, are genetically stable, and do not vary greatly with environmental changes. Many different morphological features are employed in the classification and identification of microorganisms . Light microscope : resolution limit of about 0.2 m reduces its usefulness in viewing smaller microorganisms and structures. The transmission and scanning electron microscopes, with their greater resolution, have immensely aided the study of all microbial groups Morphological characteristics

25. Morphological characteristics

26. Morphological characteristics Differential staining: Gram staining, acid-fast staining Biochemical tests: Determines presence of bacterial enzymes

27. Morphological characteristics Biochemical tests used to identify selected species of human pathogens isolated from marine mammals. Assume you isolated a gm-ve rod that produces gas from glucose, is urease negative and indole positive. What is the bacterium?

28. Morphological characteristics

29. Physiological and metabolic characteristics are very useful because they are directly related to the nature and activity of microbial enzymes and transport proteins. Since proteins are gene products, analysis of these characteristics provides an indirect comparison of microbial genomes. Physiological and Metabolic Characteristics

30. Physiological and Metabolic Characteristics

31. Ecological properties: affect the relation of microorganisms to their environment. Taxonomically valuable because even very closely related microorganisms can differ considerably with respect to ecological characteristics. Microorganisms living in various parts of the human body markedly differ from one another and from those growing in freshwater and marine environments. Some examples of taxonomically important ecological properties are life cycle patterns; the nature of symbiotic relationships; the ability to cause disease in a particular host; and habitat preferences such as requirements for temperature, pH, oxygen, and osmotic concentration. Ecological Characteristics

32. The Intuitive method It is rely on the study of the properties of the organisms by taxonomists for several years which decides to represent one or more species or genera. Demerit: The characteristics of an organism that is important to one person may not be so important to another. Different taxonomists arrive at very different groupings.

33. Nucleic acid base composition DNA contains four purine and pyrimidine bases: A, G, C, T. (G+C)/(A+T) ratio or G+C content, % of G+C in DNA, reflects the base. Mol % (G+C)= [(G+C)/(G+C+A+T)]*100 Estimated by determining the melting temperature of the DNA Higher G + C gives a higher melting temperature Methods to determine base composition 1.HPLC: Hydrolyse the DNA & Analyse with HPLC 2.Melting temperature (T m ): double stranded DNA, A=T, G = C, High G +C = high melting point.

34. Nucleic acid base composition Absorption of DNA:at  max 260 nm. Separation of strand Abs. When a DNA sample is slowly heated: absorbance as hydrogen bonds are broken and reaches a plateau when all the DNA has become single stranded The midpoint : Tm, a direct measure of the G +C content. Density of DNA also increases linearly with G+C content. %G +C: by centrifuging DNA in a CsCl density gradient Figure: A DNA Melting Curve. The Tm is indicated

35. Nucleic Acid Base Composition The G+C content of DNA from animals and higher plants : average 40%, ranges between 30 to 50%. Prokaryotic G+C content : 25 to 80%. If two organisms differ in their G + C content by >10%, : different base sequences. The G + C content of strains within a particular species is constant. Very different base sequences can be constructed from the same proportions of AT and GC base pairs. .

36. Nucleic Acid Base Composition Only if two microorganisms also are alike phenotypically does their similar G + C content suggest close relatedness.

37. G+C contents of microorganisms Organism % G + C Actinomyces 59–73 Bacillus 32–62 Clostridium 21–54 Escherichia 48–52 Micrococcus 64–75 Neisseria 47–54 Proteus 38-41 Pseudomonas 58-70 Salmonella 50-53 Staphylococcus 30-38 Aspergillus niger 52

38. Nucleic Acid Hybridization By mixing ssDNA from two different species and determining the percentage of the DNA that can form dsDNA hybrids The greater the percent hybridization, the closer the species

39. Nucleic Acid Hybridization Heat mixture of dsDNA. cool and hold at a temperature about 25°C. The base sequences will reassociate to form stable dsDNA. Whereas noncomplementary strands will remain single . I ncubation of the mixture at 30 to 50°C will allow hybrids of more diverse ssDNAs to form, at 10 to 15°C permits hybrid formation only with almost identical strands.

40. Nucleic Acid Hybridization Nitrocellulose filters with bound nonradioactive DNA strands are incubated with single-stranded DNA fragments made radioactive with 32P, 3H, or 14C. Radioactive hybridize with the membrane-bound ss-DNA. Wash to remove any nonhybridized ssDNA Measure the radioactivity. The quantity of radioactivity bound to the filter reflects the amount of hybridization and thus the similarity of the DNA sequences.

41. Nucleic Acid Hybridization The degree of similarity is expressed as the % DNA radioactivity retained on the filter compared with the % of homologous DNA radioactivity bound under the same conditions Two strains whose DNAs show 70% relatedness and less than a 5% difference in Tm are considered members of the same species. Very different DNA molecules will not form a stable, detectable hybrid. More distantly related organisms are compared by carrying out DNA-RNA hybridization.

42. DNA Chip technology Quick method to detect a pathogenic microorganisms in a host by identifying a gene that is unique to that pathogen. Protocol: DNA chip is made up of DNA Probes. A DNA sample should be collected from unknown organism is labelled with a fluorescent dye and added to the chip. Hybbridization between probe DNA and sample DNA is detected by fluorescence.

43. DNA chip Technology

44. Nucleic Acid Sequencing Genome structures directly compared only by sequencing DNA and RNA. The 5S and 16S rRNAs isolated from the 50S and 30S subunits, respectively. Purified, radioactive 16S rRNA is treated with the enzyme T1 ribonuclease. The fragments are separated and sequenced. 16S rRNA fragments from different procaryotes are aligned and compared using a computer, and association coefficients ( Sab values) are calculated.

45. Nucleic Acid Sequencing Complete rRNAs sequencing First, RNA is isolated and purified. Then, revers transcriptase is used to make cDNA. Next, the PCR amplifies the cDNA. Finally, the cDNA is sequenced and the rRNA sequence deduced from the results.

46. Numerical Taxonomy Peter H. A. Sneath and Robert Sokal have defined Numerical taxonomy: “the grouping by numerical methods on the basis of their character states.” Information about organisms is converted into numerical form and compared by means of a computer. Each characteristics given equal weight. At least 50 and preferably several hundred characteristics compared. Determine the presence or absence of selected characters in the group of organisms. Calculate simple matching coefficient ( SSM), Jaccard coefficient.

47. Numerical Taxonomy

48. Numerical Taxonomy The simple matching coefficients, or other association coefficients are then arranged to form a similarity matrix. The results of numerical taxonomic analysis are often summarized with a treelike diagram called a dendrogram. Organisms with great similarity are grouped together and separated from dissimilar organisms such groups of organisms are called phenons. Phenons formed at about 80% similarity often are equivalent to species.

49. Numerical Taxonomy Clustering and Dendrograms in Numerical Taxonomy. (a) A small similarity matrix that compares six strains of bacteria. The degree of similarity ranges from none (0.0) to complete similarity (1.0). ( b) The bacteria have been rearranged and joined to form clusters of similar strains. For example, strains 1 and 2 are the most similar. The cluster of 1 plus 2 is fairly similar to strain 3, but not at all to strain 4. ( c) A dendrogram showing the results of the analysis in part b. Strains 1 and 2 are members of a 90-phenon, and strains 1–3 form an 80-phenon. While strains 1–3 may be members of a single species, it is quite unlikely that strains 4–6 belong to the same species as 1–3.

50. THANK YOU