Lecture 4 Classification of Bacteria Flashcards
Practice and science of orderly classification of organisms into hierarchical units termed taxa
Taxonomy
Three interrelated parts to taxonomy
Identification
Nomenclature
Classification
Microscopic living organisms were formerly classified based on
Phenotypic expression
Morphology
Distinct attributes (morphology, metabolism, physio, cell chemistry (fatty acid composition), motility)
Increasingly, methods of classification have come to rely on
Genotypic analysis
Led to reclassification/nomenclature
Genetic information of bacteria is coded in
Deoxyribonucleic acid (DNA) base sequence
Bacteria undergoes frequent variation by _, _, _, and _ in different environments leading often to relatively rapid evolution
Mutation, conjugation, transduction, and selection
DNA structure
Polymers of nucleotides
Nucleotides
Phosphate group
Deoxyribose sugar (“sugar phosphate backbone” of a DNA strand)
Nitrogenous base [Purines (A, G) and Pyrimidines (C, T)]
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
DNA Structure of Prokmkaryotes
A bacterial cell is composed of
2 complementary strands of DNA wound in a helical structure
Chargaff’s rule
A DNA has
Hydrogen-bonded bases
Sugar phosphate backbone
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
DNA Base Composition
Guanine - Cytosine
Adenine - Thymine
Base composition is expressed as the
Mole percentage of guanine-cytosine to the amount of DNA
GC + AT = 100% of DNA
E.g. GC content is 40%, AT content is 60%
All Enterobacteriaceae have GC percentages ranging from
50-54% (includes Escherichia coli and Salmonella)
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)
Evolution of a genetically related group of organisms
Phylogeny
Study of relationships between collection of things that are derived from a common ancestor
Means of inferring or estimating relationships between organisms
Phylogenetic analysis
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
Phylogenetic analysis complements
Phenotypic and genotypic analysis, attempting to create a framework of evolutionary relationships
Availability of sequencing data has permitted taxonomy to
Increasingly reflect phylogenetic relationships among microorganisms
Importance of Taxonomy
Permits accurate identification of organisms
Provides precise names (efficient communication)
Groups similar organisms (allows predictions regarding members of the same group)
Three primary lineages of evolution
Bacteria
Archaea
Eukarya
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
- Strain selection
- pure cultures, inclusion of replicates, reference cultures - Test selection
- variety of tests (min 50), use of rapid methods - Recording results
- analysis of test error and rejection of poorly reproducible results - Data coding
- Computer analyses
- calculation of similarities, cluster analysis - 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
- DNA extraction from bacterial cells
- Cloning of short DNA fragments into a vector
- SS-DNA + DNA polymerase, primers, deoxynucleotides, fluorescently labelled chain terminators
- Primer anneals to ssDNA, strand elongation occurs as complementary dNTPs are incorporated, elongation stops
- Capillary gel electrophoresis
- 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
