3 - Microbial Systematics Flashcards
What is systematics?
Study of the diversity of organisms and their relationships
- links phylogeny (evolutionary history) whith taxonomy
- bacterial taxonomy traditionally focused on phenotypic comparisons
- Recently, molecular analyses allow taxonomy to reflect evolutionary relationship between organisms
Bacterial taxonomy naming rules
- Genus/species names all written in italics
- Name reflects something about the organism
Examples of bacterial taxonomy
Staphylococcus epidermidis
“bunch of grapes” clusters
coccus-shaped bacterium
isolated from skin
Bacillus thermophilus
rod-shaped bacterium
grows at high temperature
Species def for microorganisms - lower organisms
a group of strains that show a high degree of overall similarity and differ considerably from related strain groups with respect to many independent characteristics
problems with no higher def of species in microbiology
- asexual reproduction (no breeding necessary)
- lateral gene transfer
- phenotypic and genotypic plasticity of microorganisms
What is the modern method to classification of microorganisms
Polyphasic approach
Different classes and examples of extremophiles, or physiological properties of microorganisms
very high temp (80>) - hyperthermophile
high temp (50>) - thermophile
low temp (15<) - psychrophile
normal temps (15-45) - mesophile
low pH - acidophile (pH <6)
high pH - alkaliphile (pH >8)
Pressure - barophile (live at bottom of ocean)
Salt toleration - halophile
Aerobe - O2 required (final e- acceptor)
Anaerobe - does not require O2
Microaerophile - low O2 conc. needed
Polyphasic approach to classification
- Phenotypic analysis - morphological, metabolic, physiological and chemical characteristics
- Genotypic analysis - comparative at gene and genome level
- Phylogenetic analysis - framework of evolutionary relationships
Phenotypic analyses info and potential problems
Anlyses observable traits of an organism
- results compared with standard or TYPE cultures
Problem: a single mutation can change apparent definition of the species
- e.g. E. coli by definition produces the enzyme ß-galactosidase and produces indole from tryptophan at 44°C
Would single mutations knocking out these abilities mean it was no longer E. coli?
what is TYPE culture
the strain of which the description of a species is based
Morphological analyses info (physical characteristics) for classification
- Gram staining used as method to determine species via cell wall structure
- cell shape, size and arrangement, pleomorphism, formation of cysts, spores etc. define
- presence of flagella is an indicator of some bacteria
Motility analyses for classification
Determination by phase contrast micoscopy
Nutritional analyses for classification
testing ability of microorganism to grow on a rfange of different compounds as carbon or energy sources, sources of nitrogen, etc.
Differential staining used for differentiating between bacteria
- what does it do
Gram Stain
- shows different strcutures of cell walls in different bacteria
- produce Gram-positive or Gram-negative results
- help visualise bacterial/archael cells
General Staining method
- spread culture in thin film over slide
- dry in air
- pass slide through flame to heat fix onto slide
- flood slide with stain
- rinse and dry
Microscopy - place drop of oil on slide - examine with 100X objective lens
Function of differential staining
render different kind sof cells or organelles different colours
- improve contrast between cell organelles and their background
Gram staining procedure
- final result?
- spread culture in thin film over slide
- dry in air
- pass life through Bunsen flame to fix onto slide
- flood heat-fixed smear with crystal violet for 1 min
- Add iodine solution for 1 min
- Decolourise with alcohol
- Counterstain with safranin for 1-2 mins
- Gram + cells - purple
- Gram - cells - pink to red colour
Phenotype analysis - biochemical tests: Decomposition of simple carbohydrates
- acid from glucose in anaerobic or aerobic conditions
- fermentation produces acids (lactate, acetate in respiration)
- changes pH, indicator can show colour change, shows change occurs
- CO2 collects in Durham tube - waste product of Carbon compounds in respiration
Phenotype analysis - biochemical tests: test for specific enzymes
- Enzymes that decompose large molecules are tested in agar plates
- halo production indicating positive result
- e.g. DNase, protease, amylase, phospholipase, lipases, etc.
(Figure 1 shows effects of amylase on starch in agar plate, E. Coli doesn’t utilise glucose)
Analysis of different bacteria based on cultural characteristics (behaviour in culture/agar plate)
Colony based charcteristics may include:
- Colony shape, margin, elevation, surface appearance, opacity, texture, pigmentation, odour and appearance of growth
- certain substances in culture may inhibit growth of some microorganisms
- e.g. selective media, sensitivity to antibiotics, dyes, toxins, etc. in culture may have effect in local bacteria populations
toxic forms of O2, and enzymes used to break them dows
catalase - hydrogen peroxide - H2O2
Superoxide dismutase - breaks down superoxide (O2- anion) into molecular oxygen (O2) and H2O2 by reduction with Hydrogen.
Molecular analyses method: FAME - Fatty Acid Methyl Ester analysis (Fatty Acid Profiling)
- info and drawbacks
Determination of fatty acid lipid membranes:
- analysis via gas chromatography
- differences in chain length
- presence of double bonds, ring, branched chains or hydroxy groups
- Compare chromatograms to database for best matching bacteria or organism
Drawbacks:
- fatty acid profile depends on growth conditions (temp, medium, growth phase) which need to be standardised
genotyping analysis: DNA profiling - AFLP - info
- AFLP = Amplified fragment length polymorphism
- PCR targeting repetitive elements in bacterial genome
- Used to distinguish closely related strains
Analyses of results is comparison of electrophoretic patterns
genotyping analysis: multilocus sequence typing - info
Procedure:
- new isolated or clinical sample
- DNA isolation
- amplify 6-7 target genes by PCR
- DNA sequencing
- determine alleles
- compare with other strands of the same species
- looks at characteristic strains within a species to compare relationships
- Sequencing of several ‘housekeeping’ genes assign different alleles to strains
- can compare same genes between different organisms to analyse similarities and relationship
genotyping analysis: GC base ratios (guanine-cytosine base ratios)
compare percentage of GC bases in the genome of the species
Range 20-80%, Similar organisms have ~same GC content, but very different organisms can also have close values
Obligate aerobe info
- O2 required
- contain enzymes to deal with toxic forms of oxygen
Facultative anaerobe info
- Usually better growth with O2
- can also grow anaerobically
- enzyme to deal with toxic forms of oxygen
Obligate anaerobe info
Does not tolerate O2
- no enzymes needed to deal with toxic O2 forms
Aerotolerant anaerobe
- Can tolerate O2, not needed for life though
- Has superoxide dismutase to deal with toxic O2 forms
- e.g. lactobacillus
Microaerophilic organism info
O2 required, but only low concentration tolerated
- enzymes for toxic O2 forms usually not present or required
Why DNA sequencing is best
