MOLECULAR TAXONOMY..

Molecular Methods in Microbial Taxonomy : Molecular Methods in Microbial Taxonomy

What is Taxonomy? : What is Taxonomy? Taxonomy [Greek taxis, arrangement or order, and nomos, law, or nemein, to distribute or govern] is the Science of biological classification. Consists of three separate but interrelated parts: Classification Arrangement of organisms into groups or taxa (s., taxon) based on mutual similarity or evolutionary relatedness. Nomenclature Assign names to taxonomic groups in agreement with published rules. Identification The practical side of taxonomy, involves process of determining that a particular isolate belongs to a recognized taxon.

Why is Taxonomy Important : Why is Taxonomy Important Taxonomy is important for several reasons. To organize huge amounts of knowledge about organisms because all members of a particular group share many characteristics. It allows us to make predictions and frame hypotheses for further research based on knowledge of similar organisms. It places them in useful groups with precise names so that researchers can work with them and communicate efficiently. Accurate identification of microorganisms. Allows the construction of databases that can be used for a rapid identification of organisms in the routine lab Allows the validation and determination of the taxonomic resolution of new potential (identification) tools.

Microbial Classification Approaches : Microbial Classification Approaches Artificial approach Natural approach Phylogenetic approach Adansonian approach  Macromolecular approach

Slide 5 : Artificial Approach: Aristotle, during fourth century B.C. classified the living organisms into two kingdoms the Animals and the Plants.  Natural Approach: In the middle of 18th century Linnaeus (1758) devised a system, that was more useful. This system expressed the biological nature of the objects that it classified, There was scanty knowledge of microbes in this system also.

Slide 6 : Phylogenetic Approach : The post Darwinian biologists thought in terms of evolutionary affinities between organisms, In the 19th century the concept of a natural system accordingly changed if phylogenetic ones.  A German, Ernst H. Haeckel separated the unicellular organisms from plants and animals, placing them under a third kingdom, Protista. The fungi, protozoa, bacteria and algae were included in this kingdom.A modern system of this type is devised by R.H. Whittaker (1969). We have already referred to this five kingdom system of the living world. The microorganisms have been included in the kingdoms Monera, Protista and Fungi in this system. Carl Woese three Domain System also come under this Approach..

Adansonian Approach: Numerical computer or phenetic taxonomy) : Adansonian Approach: Numerical computer or phenetic taxonomy) This was first suggested by a French biologist, Michel Adanson, a contemporary of Linnaeus, in the 18th century. Further, concept was first developed by Robert R. Sokal  & Peter H. A. Sneath in 1963 and later elaborated by the same authors Here the taxonomic arrangement is based upon quantification of the similarities and differences among organisms.There are used as many characters as possible, each of which is of an equal weight.  It is assumed that if each phenotypic character is given equal weighting, it should express numerically the taxonomic distances between organisms, in terms of the number of characters they share, relative to the total number of characters examined.The results are fed into a computer so as to define the similarities and differences between microorganisms and thus to indicate possible natural groupings

Slide 8 : Characters Used in Classification: The following are the types of characters used in classification:1. Morphological characters - These concern cell shape and size, staining reactions. presence or absence of spores or reproductive forms, type of motility etc.2. Cultural Characters - These include the cultural requirements for multiplication (e.g. nutrients. oxygen, temperature, etc.) and the way growth occurs in liquid media, and particularly on solid media (e.g. colony form)3. Biochemical Characters - These include the more specific biochemical characteristics such as metabolic end-products and the presence or absence of a particular enzyme or pathway. 4.Serological Characters - These concern the nature of the surface antigens as revealed by suitable specific antibodies. 5. Molecular Characters -These include the sequences of bases in the DNA, GC ratios and nucleic acid  hybridisation.

Serological Characters : Serological Characters Antigen Susceptibility: Cell wall (O), flagellar (H), and capsular (K) antigens are used to aid in classifying certain organisms at the species level, to serotype strains of medically important species ,to identify serotypes of public health importance. Serotyping is also sometimes used to distinguish strains of exceptional virulence such as,V. cholerae (O1 is the pandemic strain) and E. coli (enterotoxigenic, enteroinvasive, enterohemorrhagic, and enteropathogenic serotypes). Phage Susceptibility: Phage typing (determining the susceptibility pattern of an isolate to a set of specific bacteriophages) has been used primarily as an aid in epidemiologic surveillance of diseases caused by Staphylococcus aureus, mycobacteria, P.aeruginosa, V. cholerae, and S. typhi. Susceptibility to bacteriocins has also been used as an epidemiologic strain marker. In most cases recently, phage and bacteriocin typing have been supplanted by molecular methods.

Drawbacks for Phenotypic and Biochemical Based Microbial Taxonomy….. : Drawbacks for Phenotypic and Biochemical Based Microbial Taxonomy….. Traditional methods of bacterial identification(Phenotypic , Ecological, Biochemical) suffer from many drawbacks. Such as Biochemical Based: Can be used only for organisms that can be cultivated in vitro. Some strains exhibit unique biochemical characteristics that do not fit into patterns that have been used as a characteristic of any known genus and species. Highly related species cannot be phenotypically differentiated. Corresponding databases are often limited, hampering accurate identification.

Slide 11 : The same trait may evolve more than once. For example, bifidobacteria and lactobacilli both make lactic acid. Traits that interest humans are sometimes overemphasized. For instance, the ability of some organisms to cause botulism led to grouping unrelated clostridia under the species name Clostridium botulinum. Some organisms are “Metabolically inactive" (no one has yet been able to discover what they do) example, Many anaerobes fall into this category, thus making anaerobic taxonomy particularly difficult. They does not take into consideration the phylogenetic linkages among the bacteria, and it does not distinguish precisely enough between the closely related species The phenotypic, chemical, enzymatic and serological characteristics, which cover only 5-10% of the genome, the molecular genetic methods deal directly or indirectly with the genome polymorphism,also entire genome(DNA-Sequencing).

Slide 12 : Phenotype Based : Organisms are identified on the basis of phenotype, but, from the taxonomic standpoint, it is subject to error. Different enzymes (specified by different genes) may catalyze the same reaction. If a metabolic gene is functional, negative reactions can occur because of the inability of the substrate to enter the cell, because of a mutation in a regulatory gene, or by production of an inactive protein. There is not necessarily a one-to-one correlation between a reaction and the number of genes needed to carry out that reaction. For instance, six enzymatic steps may be involved in a given pathway. If an assay for the end product is performed, a positive reaction indicates the presence of all six enzymes, whereas a negative reaction can mean the absence or non function of one to six enzymes. Several other strain characteristics can affect phenotypic characterization; Growth rate, Incubation temperature, Salt requirement, and pH. Plasmids that carry metabolic genes can enable strains to carry out reactions atypical for strains of that species. The same set of “definitive” reactions cannot be used to classify all groups of organisms, and there is no standard number of specific reactions that allows identification of a species.

Reasons for Characterizing Species into Strains….. : Reasons for Characterizing Species into Strains….. There are a number of reasons why it may be necessary to characterize a microbial isolate beyond species level and determine its sub-species, strain, or even sub-strain. To relate individual cases to an outbreak of infectious disease. To study variations in the pathogenicity, virulence and antibiotic resistance of individual strains within a species. To trace the source of contaminants within a manufacturing process. To study the microbial ecology of complex communities, such as biofilms. To characterize microorganisms with important industrial applications.

Molecular Characters,viz.MOLECULAR TAXONOMY : Molecular Characters,viz.MOLECULAR TAXONOMY The advent of molecular biology in the 1980s contributed a set of powerful new tools that have helped microbiologists to detect the smallest variations within microbial species and even within individual strains. Because techniques that directly measure the degree of genetic similarity among organisms are not subject to above mentioned problems, molecular methods are now central for determining phylogeny and nomenclature. As Nucleotides are less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, one can research both DNA and RNA, and the main techniques that have been used in the systematics comprises of Restriction maps Construction and Restriction Analysis through RAPD, RFLP,AFLPs(AMPLIFIED FRAGMENT LENGTH POLYMORPHISMS). DNA-DNA hybridization DNA-RNA hybridization Sequencing of DNA

Slide 15 : Sequencing of sub-units 16S and 23S of rRNA(Sequencing of conserved genes because it is generally stable yet changes over long periods of time) DNA fingerprints (PFGE :Pulsed field gel electrophoresis ,RAPD, rep-PCR  ,protein patterns, MLEE analysis, plasmid patterns) DNA finger-printing with different probes or PCR primers Proteins Sequencing of conserved proteins Physical, kinetic, and regulatory properties Nucleic acids Base composition(G+C content)

Indirect analysis of the genome : Indirect analysis of the genome ELISA Electrophoretical mobility of total protein extract Ribosomal Proteins Pattern obtained by two-dimensional PAGE and HPLC There is a considerable variability in the ribosomal protein patterns of some species - S. venazuele, S. violaceus, S. parvulus, S. hygroscopicus, while the ribosomal protein patterns of S. lavendulae and S. avidinii are quite similar. This suggests that the two species are closely related . Multilocus Enzyme Electrophoresis is used widely in the investigations of genetic heterogeneity in bacterial populations and for clarifying the intraspecies diversity of the prokaryotes.

Direct analysis of the genome : Direct analysis of the genome G + C content determined by Formula: Estimated by determining the melting temperature of the DNA. Higher G + C gives a higher melting temperature. GC Ratios are usually of minimal value in the overall taxonomic characterization of an organism because 2 organisms can have identical GC ratios yet Unrelated both taxonomically & phylogenetically also, variety of base sequences are possible.

DNA FINGERPRINTINGRestriction Enzymes Assays : DNA FINGERPRINTINGRestriction Enzymes Assays REA is based on the ability of restriction enzymes to identify DNA sequences that are from 4 to 8 bases in length and to cleave the double helix at these sites. The number of fragments into which a given restriction enzyme cuts a bacterial genome depends on the frequency of restriction sites and ranges from less than 10 to several thousand. The length of a given fragment depends on the distance between restriction sites. A restriction enzyme digesting a given genome generates a reproducible pattern of bands; each band corresponds to a restriction fragment of a certain molecular weight. The pattern usually varies slightly between gels. Since every microbe will be giving a Different RFLP patterns, its Pattern of BANDS obtained corresponds to its DNA FINGERPRINT, and so is the Technique DNA FINGERPRINTING. Because standard agarose gel electrophoresis fails to efficiently resolve fragments that are >50,000 bases long, new methods have recently been developed that separate the very large fragments generated by enzymes that cut at rare sites. Pulsed field gel electrophoresis achieve superior separation of very large fragments .

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLP) : RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLP) RFLP fingerprinting technique is regarded as the most sensitive method for strain identification and several bacterial strains have been widely studied using this technique. RFLP is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of Restriction enzyme sites.

Methodology Involoved…. : Methodology Involoved…. The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction enzyme(EcoR1), which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a Restriction digest. The resulting DNA fragments are then separated by length through a process known as Agarose gel electrophoresis, and transferred to a membrane via the Southern blot procedure.  Hybridization of the membrane to a labelled DNA probe then determines the length of the fragments which are complementary  to the probe. An RFLP occurs when the length of a detected fragment varies between organisms. Each fragment length is considered an allele, and can be used in genetic analysis.

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RFLP continued… : RFLP continued… RFLP is still a technique used in marker assisted selection. Terminal restriction fragment length polymorphism (TRFLP or sometimes T-RFLP) is a molecular biology technique initially developed for characterizing bacterial communities in mixed-species samples. The technique has also been applied to other groups including soil fungi. TRFLP works by PCR amplification of DNA using primer pairs that have been labeled with fluorescent tags. The PCR products are then digested using RFLP enzymes and the resulting patterns visualized using a DNA sequencer. The results are analyzed either by simply counting and comparing bands or peaks in the TRFLP profile, or by matching bands from one or more TRFLP runs to a database of known species Means RFLP can work in Combination with both PCR and Electrophoresis related Techniques for Bacterial Identification.

DNA/DNA reassociation/Hybridization…. : DNA/DNA reassociation/Hybridization…. In this example, which is a control experiment (the radiolabeled sample is reannealed with unlabeled DNA from the same strain), the degree of reassociation is highest and treated as 100%. If a different strain is reannealed with the radiolabeled DNA, it will show a lower degree of reannealing (compared with the 100% attributed to the control), indicative of the similarity between the two strains being tested. Strains with reannealing values of 70% or greater are considered to be the same species.

Slide 24 : DNA-RNA HYBRIDIZATION

Slide 25 : In 1961, McCarthy and Bolton presented a means of comparing genetic material through DNA-DNA hybridization a method for bacterial systematics. DNA-DNA hybridization or DNA-DNA reassociation technique is based on a comparison between whole genome of two bacterial species. Used in bacterial classifification for delineation of species . Each DNA molecule is made of two strands of nucleotides. If the strands are heated, they will separate—and as they cool, the attraction of the nucleotides will make them bond back together again. To compare different species, scientists cut the DNA of the species into small segments, separate the strands, and mix the DNA together. DNA from two organisms is sheared into small segments, and a small amount of radiolabeled DNA from one organism is added to a large amount of unlabeled DNA from the other organism(TEST ORGANISM). Since the concentration of labelled DNA is low, complementary strands of labelled DNA do not "find each other"; however, they do "find" complementary segments of unlabeled DNA. The amount of radioactivity incorporated into double strands is compared with that incorporated when the organism's own unlabeled DNA is used in excess.

Slide 26 : A very useful convention has been that an incorporation ratio of >70% under standard conditions indicates that both organisms belong to the same species. An incorporation ratio of 70% does not mean that the organisms share an overall sequence similarity of 70%; the actual similarity is much higher. However, it must be noted that this technique gives the relative % of similarity but not the actual sequence identity. The technique is based on the fact that at high temperatures, DNA can be denatured, but the molecule can be brought back to its native state by lowering down the temperature (reassociation). It is based on three parameters i) G + C mol % , ii) the ionic strength of the solution iii) the melting temperature of DNA hybrid (Tm). Tm is the only variable parameter out of three (as ionic strength can be kept constant). Therefore, more the similarity between the heteroduplex molecule, more temperature will be required to separate it (high Tm value). Interpretation Of Result is Done on Basis of Various Temperature BELOW Tm: About 250 C below Tm:- Shows RELATEDNESS At 300 – 500 C below Tm- Show DIVERSITY in Species. At Incubation 100 -- 150 C – Shows Close Relation between Organisms. Include the strain with 70% or greater DNA-DNA hybridization values with 5°C less ΔTm values and both the values must be considered.

DNA melting curve : DNA melting curve

Tm and DNA base composition : Tm and DNA base composition

RNA Sequencing.. : RNA Sequencing.. 16srRNA SEQUENCING What are the characteristics of 16S rRNA that make it useful as an analyte for bacterial Identification or also Good Evolutionary Chronometer? Ubiquitous.(found in all cells) Highly Conserved Molecule Contains variable and hyper-variable regions of sequence Extensively studied and represented in databases . Easily Isolated and present in Thousands of Copies Can be analyzed easily to determine Exact nucleotide bases in its make up. Functionally Similar between organisms rRNA`s participate in Protein Synthesis. Sequence changes slowly good for looking across long periods of time.

16S RNA : 16S RNA Secondary structure of the 16S rRNA molecule from the small ribosomal subunit of the bacterium Escherichia coli. The bases are numbered from 1 at the 5' end to 1,542 at the 3' end. Every tenth nucleotide is marked with a tick mark, and every fiftieth nucleotide is numbered. Tertiary interactions with strong comparative data are connected by solid lines.

Conservation and variation in small subunit rRNA : Conservation and variation in small subunit rRNA This diagram shows conserved and variable regions of the small subunit rRNA (16S in prokaryotes or 18S in eukaryotes). Each dot and triangle represents a position that holds a nucleotide in 95% of all organisms sequenced, though the actual nucleotide present (A, U, C, or G) varies among species.

STRUCTURE of 16srRNA:- : STRUCTURE of 16srRNA:- Primary Structure : RNA sequence Secondary Structure: Formation of double stranded regions (helices) Tertiary Structure: Folding of Primary and Secondary structures Quaternary Structures Interactions with other molecules (e.g. RNAs, proteins) For Sequencing Conservation Map are Analysed and primers are Designed to bind to conserved region on Sequencing. Sequence data is generated through hypervariable regions.

Method Outline : Method Outline Isolate bacterial DNA • • Amplify 16S rRNA gene • • rRNA gene (Region 1) Sequence a portion of 16S rRNA gene • • Compare sequence obtained with GenBank to find “Match ” Gen Bank is a freely available web-based database of 16S rDNA sequences Contains bacteria, fungi and other microrganisms valuable Data, Further BLAST “match” was done,for Species Characterization and Identification.

Ribotyping…. : Ribotyping…. Technique for Bacterial Identification exploits RNA based phylogenetic Characterizations. Method is so specific that it has been given Nick-Name ``MOLECULAR FINGERPRINTING`` because Restriction Patterns of a Particular Bacterial Species is ``UNIQUE`` It is both Rapid and Specific Technique i.e. why have many applications in clinical diagnostics and for microbial analyses of food, water and beverages. Principle involves the fingerprinting of genomic DNA restriction fragments that contain all or part of the genes coding for the 16S and 23S rRNA. Conceptually, ribotyping is similar to probing restriction fragments of chromosomal DNA with  cloned probes (randomly cloned probes or probes derived from a specific coding sequence such as that of a virulence factor)

Protocol for Ribotyping…. : Protocol for Ribotyping…. Bulk Dna/Dna Encoding 16srRNA Taken and PCR amplified Treated with Restriction Enzymes Separated by Electrophoresis Probed using Ribosomal RNA Probe Pattern generated from Fragments of DNA on the gel is Digitized Computer used for Comparisons of this pattern from reference organism Database.

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Recent Trends and Methods in Molecular Taxonomy… : Recent Trends and Methods in Molecular Taxonomy… PCR Based Molecular Methods…. Multilocus sequence typing (MLST) is a technique used for typing of multiple loci. The procedure characterizes isolates of bacterial species using the DNA sequences of internal fragments of multiple (usually six) housekeeping gene. Approximately 450-500 bp internal fragments of each gene are used, as these can be accurately sequenced on both strands using an automated DNA sequencer. For each housekeeping gene, the different sequences present within a bacterial species are assigned as distinct alleles and, for each isolate, the alleles at each of the loci define the allelic profile or sequence type (ST).

Methodology and MLST Advantage : Methodology and MLST Advantage MLST directly measures the DNA sequence variations in a set of housekeeping genes and characterizes strains by their unique allelic profiles. The principle of MLST is simple: the technique involves PCR amplification followed by DNA sequencing. Nucleotide differences between strains can be checked at a variable number of genes (generally seven) depending on the degree of discrimination desired. Primer sequences and protocols can be accessed electronically. It is reproducible and scalable. MLST is automated, and its data can be used to investigate evolutionary relationships among bacteria. MLST provides good discriminatory power to differentiate isolate The first MLST scheme to be developed was for Neisseria meningitidis , also Camplyobacter,S.aureus,S.pyogenes, Characterized.C. albicans isolates obtained from diverse ecological niches including human and animal hosts.

Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) : Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) Multiple-locus variable number tandem repeat analysis (MLVA) is a sub typing method that utilizes short, repetitive regions of DNA called variable-number tandem repeats (VNTR) which may be present in different numbers of repeats between strains. These variable number tandem-repeat (VNTR) regions have been identified in many species of bacteria. The first step involves amplifying the VNTR using PCR,It is used to amplify the conserved DNA outside the VNTR in addition to the variable VNTR region. The fragment size is dependant on the number of repeats for a sample. The size of the resulting fragment is determined by either horizontal or capillary electrophoresis .

Slide 40 : MLVA TECHNIQUE

Repetitive sequence-based PCR (rep-PCR) : Repetitive sequence-based PCR (rep-PCR) Bacterial and fungal genomes contain numerous non-coding, repetitive DNA sequences separating longer, single copy, sequences and their arrangement varies between strains. The rep-PCR technique relies on amplifying these repetitive sequences to produce amplicons of varying length that can be separated by electrophoresis giving a fingerprint comprised of bands that fluoresce at different intensities after binding with an intercalating dye. Also developed into a commercial typing system giving rapid results and using dedicated software to aid typing. The system is widely used for typing human pathogens.

Multilocus Enzyme Electrophoresis(MLEE) : Multilocus Enzyme Electrophoresis(MLEE) Multilocus Enzyme Electrophoresis is a method of Characterizing micro-organisms by the relative Mobilities under Electrophoresis of a large number of Intracellular Enzymes. These differences in mobility are directly related to mutations at the gene locus that causes amino acid substitutions in the enzyme coded by the gene. Differences in the electrostatic charge between the substituted and original amino acid will affect the net charge of the enzyme and hence its Electrophoretic Mobility. Thus it is possible to relate mobility differences to different alleles at the gene locus for the enzyme in question. These Mobility Variants are called Electromorphs.The unique profile of Electromorphs produced for each strain of Organism is called an ELECTROMORPH TYPE(ET). MLEE is more Recently used in Epidemiological Typing Of Bacteria and other Micro-organisms.

Pulse Field Gel Electrophoresis…. : Pulse Field Gel Electrophoresis…. Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid (DNA) molecules by applying an electric field that periodically changes direction to a gel matrix. PFGE was developed in 1984 and has since become the gold standard for bacterial sub typing.  The procedure for this technique is relatively similar to performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 120 degrees either side. This procedure takes longer than normal gel electrophoresis due to the size of the fragments being resolved and the fact that the DNA does not move in a straight line through the gel.

Slide 44 : Uses: For determining strain genetic similarity in many bacteria. Subtyping has made it easier to discriminate among strains of Listeria monocytogenes and thus to link environmental or food isolates with clinical infections. Neisseria meningitidis Serogroup C isolates. PFGE to analyze a number of Serratia marcescens isolates that caused an outbreak in the intensive care unit of a Malaysian hospital. (S. marcescens is a common soil bacterium that was once thought to be innocuous but is now known to be capable of causing infections in immune compromised patients.) PFGE Advantages -Easily applied to different species -Patterns consistent within and between laboratories (strict adherence to standard conditions is necessary)-PFGE generally yields a high amount of pattern diversity    PFGE Drawbacks-Labour intensive-Relatively slow (approximately 24 hours completion)-Complex patterns challenging for inter-laboratory pattern comparisons-One mutation can yield differences in several fragments (PFGE cannot determine phylogenic relationships) -Dependant on isolated bacteria

Slide 45 : Electric current 18-20 hours buffer 14 C electrodes

How does PFGE differ from PCR identification of bacterial strains? : How does PFGE differ from PCR identification of bacterial strains? In using PCR to identify a bacterial strain, a single segment of DNA is amplified, typically an rRNA gene or a portion of it. The amplified fragment is then sequenced and the sequence compared to that of sequences in the databases. Since rRNA gene sequences are so conserved, this method does not have the resolution to distinguish individual strains of the same species, although it can be used for species identification. In PFGE, there is no amplification. Instead, restriction enzymes are used to digest chromosomal DNA to generate a characteristic pattern. Also, the detection method is staining of the DNA, not DNA sequencing.

Slide 47 : Taxonomic Information LEVEL 1: DNA, rRNA sequencing, hybridisations, genotyping, %G+C, ... LEVEL 2: proteins sequencing, SDS-PAGE, serology, zymograms, ... LEVEL 3: chemotaxonomy FAME, PyMS, polyamines, polar lipids, ... LEVEL 4: phenotyping BIOLOG™, API, antibiograms, salt tolerance, ...

So Do you Think Molecular Techniques are Best for Microbial Taxonomy???? : So Do you Think Molecular Techniques are Best for Microbial Taxonomy????