1. The number of Guanine units approx equals the number of Cytosine units and the number of Adenine units approx equals the number of Thymine units G = C, A = T 2. The composition of DNA varies from one species to another

Lecture 4 Classification of Bacteria Flashcards by Kimberly Cassandra Bingbing

Practice and science of orderly classification of organisms into hierarchical units termed taxa

Taxonomy

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Three interrelated parts to taxonomy

Identification
Nomenclature
Classification

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Microscopic living organisms were formerly classified based on

Phenotypic expression

Morphology
Distinct attributes (morphology, metabolism, physio, cell chemistry (fatty acid composition), motility)

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Increasingly, methods of classification have come to rely on

Genotypic analysis

Led to reclassification/nomenclature

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Genetic information of bacteria is coded in

Deoxyribonucleic acid (DNA) base sequence

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Bacteria undergoes frequent variation by _, _, _, and _ in different environments leading often to relatively rapid evolution

Mutation, conjugation, transduction, and selection

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DNA structure

Polymers of nucleotides

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Nucleotides

Phosphate group
Deoxyribose sugar (“sugar phosphate backbone” of a DNA strand)
Nitrogenous base [Purines (A, G) and Pyrimidines (C, T)]

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A small segment of DNA is alternating molecules of _ and _ covalently bonded together to form one strand of the double-stranded DNA molecule

Deoxyribose and phosphate

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DNA Structure of Prokmkaryotes
A bacterial cell is composed of

2 complementary strands of DNA wound in a helical structure
Chargaff’s rule

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A DNA has

Hydrogen-bonded bases
Sugar phosphate backbone

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Chargaff’s rule

The number of Guanine units approx equals the number of Cytosine units and the number of Adenine units approx equals the number of Thymine units
G = C, A = T
The composition of DNA varies from one species to another

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DNA Base Composition

Guanine - Cytosine
Adenine - Thymine

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Base composition is expressed as the

Mole percentage of guanine-cytosine to the amount of DNA

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GC + AT = 100% of DNA

E.g. GC content is 40%, AT content is 60%

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All Enterobacteriaceae have GC percentages ranging from

50-54% (includes Escherichia coli and Salmonella)

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Genetic methods used to classify organisms

DNA profiling
DNA-DNA hybridization
Multilocus sequence typing (MLST)
Percentage of G + C in an organism’s DNA (GC ratio)

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Evolution of a genetically related group of organisms

Phylogeny

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Study of relationships between collection of things that are derived from a common ancestor
Means of inferring or estimating relationships between organisms

Phylogenetic analysis

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Evolutionary history inferred from phylogenetic analysis is usually depicted as

Branching, tree-like diagrams that represent an estimated pedigree of the inherited relationships among molecules (“gene trees”), organisms or both

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Phylogenetic analysis complements

Phenotypic and genotypic analysis, attempting to create a framework of evolutionary relationships

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Availability of sequencing data has permitted taxonomy to

Increasingly reflect phylogenetic relationships among microorganisms

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Importance of Taxonomy

Permits accurate identification of organisms
Provides precise names (efficient communication)
Groups similar organisms (allows predictions regarding members of the same group)

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Three primary lineages of evolution

Bacteria
Archaea
Eukarya

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Analysis of _ _ _ _ _ _ suggested that cellular life has evolved along 3 primary lineages

Small subunit ribosomal RNA gene sequences

The three domains are usually placed above the

Kingdom level

Two domains _ and _ are exclusively microbial and prokaryotic, and the third domain _ contains the eukaryotes

Bacteria and Archaea Eukarya

Levels of Classification

Domain Kingdom Phylum Class Order Family Genus Species Do Kings Play Chess on Fridays, Generally Speaking? DKPCOFGS

A group of similar individuals capable of interbreeding naturally and are reproductively isolated from other groups

Species

In microbiology, Bacteria and Archaea do not undergo

True reproduction

A collection of strains that share many similar properties but differ significantly from other strains

Bacterial species

A more precise definition based on genetic data: it is expected to share 70% or greater binding in standardized DNA-DNA hybridization studies or over 97% gene sequence identity for 16s ribosomal RNA (rRNA)

Bacterial species

Naming of microorganisms 18th century botanist Binomial system Latin or Latinized Greek derivations, printed in Italics

Carolus Linnaeus

Naming of microorganisms Two parts

Capitalized generic name Specific epithet E.g. Pseudomonas aeruginosa, Bacillus anthracis, Escherichia coli

Naming of microorganisms Organizations

The International Code of Nomenclature of Bacteria The International Journal of Systematic and Evolutionary Microbiology TICNB, TIJSEM

Numerical Taxonomy People

Peter H.A. Sneath (1957) and Robert R. Sokal (1963)

The grouping by numerical methods of taxonomic units into taxa on the basis of their characteristics Approach of systematics involving numerical evaluation of the similarities or affinitiesbetween taxonomic units, arrangement of units based on their affinity Requires the study of as many aspects of the biology of organisms (operational taxonomic units)

Numerical Taxonomy

Numerical Taxonomy Premise

All OTUs (operational taxonomic units) have equal importance; differential characteristics are used for identification

Stages in Numerical Taxonomy

1. Strain selection - pure cultures, inclusion of replicates, reference cultures 2. Test selection - variety of tests (min 50), use of rapid methods 3. Recording results - analysis of test error and rejection of poorly reproducible results 4. Data coding 5. Computer analyses - calculation of similarities, cluster analysis 6. Interpretation of results - definition of clusters, ID scheme, selection of representative strains for allied studies STRDCI

Characteristics used in numerical taxonomy of bacteria Category of test - Test

Colonial morphology - colony size, shape; fluorescent pigments Micromorphology - Gram stain, structural features Growth characteristics - growth in broth, aerobe, anaerobe

New approaches to bacterial taxonomy

Molecular subtyping (for definitive ID of bacteria)

Permits identification of bacteria below species level Refined ID based on DNA fingerprint E.g. 600 E. coli O157:H7 subtypes, more than1800 Salmonella Typhimurium subtypes

Molecular subtyping

Provides means of tracking an organism Describes its molecular epidemiology Defines its transmission routes

Molecular subtyping

Importance of molecular subtyping

Supports quality control in food manufacture (probiotics) Applied in tracking source of bacterial contamination in food manufacturing Food borne cases of illness and outbreaks of infectious disease can be tracked using DNA fingerprinting to ID etiological agent - links vetmed and public health IDs transmission routes (MRSA between humans and animals)

Three general approaches to molecular subtyping

Restriction Fragment Length Polymorphism (RFLP) analysis PCR based amplification (of conserved repetitive sequences in bacterial genomes) DNA sequencing - no single subtyping method is routinely used

Bacterial subtyping methods Sequential development of analytical methods - Molecular basis of subtyping methods

1st generation methods - Plasmid DNA profiling 2nd generation methods - Restriction endonuclease digestion of total DNA 3rd generation methods - Pulsed field gel electrophoresis (PFGE); PCR based amplification 4th generation methods - Multilocus variable number tandem repeat analysis (MLVA); Multilocus sequence typing (MLST); DNA sequencing

Purification of all plasmids from a bacterium Separation on agarose gel Comparison to a collection of bacterial isolates Used successfully to identify serovars of Salmonella May be limited (some bacteria may not contain plasmids) Important when characterizing genetic markers associated with antibiotic resistance

Plasmid profiling

Enzymes which scan along a length of DNA looking for particular sequence of bases that they recognize (4-6 base pairs) Enzyme attaches to DNA and cuts each strand of the double helix (molecular scissors)

Restriction enzymes

Total genomic DNA (chromosome and plasmid) purification Subjected to enzymatic digestion with a restriction endonuclease Enzyme cleaves at specific recognition sites Produces multi band pattern or restriction fragment length polymorphic pattern Detected after electrophoresis on agarose gel RFLP patterm can be complex, limits this subtyping method Plasmids can be lost in a strain

Restriction endonuclease analysis (REA)

Single stranded DNA or RNA anneal (attach) to complementady DNA or RNA PCR and DNA sequencing rely on this phenomenon - primers are short DNA fragments that anneal to a specific location on a genome

DNA hybridization

Similar or identical nuclear sequence

Homology

Can be applied to any genome Examples: - Random amplification of polymorphic DNA (RAPD) - PCR-RFLP analysis

PCR-based subtyping

PCR-based subtyping example Does not require prior knowledge of organism's DNA sequence Uses a random primer in PCR to generate DNA fingerprint Rapid protocol but lacks reproducibility

Random amplification of polymorphic DNA (RAPD)

PCR-based subtyping example Applied to target gene with high degree of polymorphism Amplified PCR subjected to digestion using RE

PCR-RFLP analysis

First bacterial genome to have its DNA sequence

Haemophilus influenzae

Needs construction of an extensive library of randomly generated DNA fragments Short fragements are cloned into a suitable vector (circular DNA) DNA sequence is then determined using Sanger DNA sequencing (1st gen sequencing method) Bioinformatics computing tools are used to search the DNA sequences from the library More recent advances in sequencing technologies and computational analysis have led to development of faster analytical approaches - Next generation sequencing (NGS) takes days to perform

DNA sequencing

A massively parallel sequencing technology that offers ultra high throughput, scalability, and speed (multiple strands of DNA can be sequenced at the same time) Used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA

Next Generation Sequencing (NGS)

Whenever a new base is added to the replicating DNA strand, a corresponding fluorescent signal is emitted which is detected in real time

Next Generation Sequencing (NGS) Principle

Allows real time sequencing of long DNA strands A long strand of DNA is fed through the nanopore As the DNA passes through the nanopore, it disrupts an electric current flowing across the membrane. Different bases (A, T, C, G) in the DNA affect the current in unique ways; current is read by the machine and reveals the sequence of the DNA.

Nanopore sequencing

Small membrane pores that are about the size of a single DNA molecule

Nanopores

The study of genetic material from environmental samples like soil, water, feces, animal gut contents, without needing to culture the organisms in a lab Applications: - Microbiomes - Disease Detection

Metagenomics

Metagenomics Applications To understand the diverse community of microbes living in animals' guts. This can give insights into animal health, digestion, and nutrition

Microbiomes

Metagenomics Applications To identify pathogens (disease-causing organisms) in animals quickly and accurately. Helps in diagnosing infections or outbreaks Antimicrobial resistance, environmental monitoring

Disease Detection

Steps in capillary DNA sequencing

1. DNA extraction from bacterial cells 2. Cloning of short DNA fragments into a vector 3. SS-DNA + DNA polymerase, primers, deoxynucleotides, fluorescently labelled chain terminators 4. Primer anneals to ssDNA, strand elongation occurs as complementary dNTPs are incorporated, elongation stops 6. Capillary gel electrophoresis 7. Chain termination fluoresce - read by laser scanning, data integrated to chromatograph

Pulsed field Gel Electrophoresis (PFGE) Multiple Locus Variable-number tandem repeat Analysis (MLVA)

PFGE - DNA can be seen under UV light, digital cam takes photo of gel and stores pic in computer MLVA - data output is an electropherogram, shows DNA standards of known size in red, PCR products in blue green black

Lecture 4 Classification of Bacteria Flashcards

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