3 - Microbial Systematics

Question 1

What is systematics?

Answer 1

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

Question 2

Bacterial taxonomy naming rules

Answer 2

• Genus/species names all written in italics
• Name reflects something about the organism

Question 3

Examples of bacterial taxonomy

Answer 3

Staphylococcus epidermidis
“bunch of grapes” clusters
coccus-shaped bacterium
isolated from skin

Bacillus thermophilus
rod-shaped bacterium
grows at high temperature

Question 4

Species def for microorganisms - lower organisms

Answer 4

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

Question 5

problems with no higher def of species in microbiology

Answer 5

• asexual reproduction (no breeding necessary)
• lateral gene transfer
• phenotypic and genotypic plasticity of microorganisms

Question 6

What is the modern method to classification of microorganisms

Answer 6

Polyphasic approach

Question 7

Different classes and examples of extremophiles, or physiological properties of microorganisms

Answer 7

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

Question 8

Polyphasic approach to classification

Answer 8

• Phenotypic analysis - morphological, metabolic, physiological and chemical characteristics
• Genotypic analysis - comparative at gene and genome level
• Phylogenetic analysis - framework of evolutionary relationships

Question 9

Phenotypic analyses info and potential problems

Answer 9

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?

Question 10

what is TYPE culture

Answer 10

the strain of which the description of a species is based

Question 11

Morphological analyses info (physical characteristics) for classification

Answer 11

• 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

Question 12

Motility analyses for classification

Answer 12

Determination by phase contrast micoscopy

Question 13

Nutritional analyses for classification

Answer 13

testing ability of microorganism to grow on a rfange of different compounds as carbon or energy sources, sources of nitrogen, etc.

Question 14

Differential staining used for differentiating between bacteria
- what does it do

Answer 14

Gram Stain
- shows different strcutures of cell walls in different bacteria
- produce Gram-positive or Gram-negative results
- help visualise bacterial/archael cells

Question 15

General Staining method

Answer 15

• 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

Question 16

Function of differential staining

Answer 16

render different kind sof cells or organelles different colours
- improve contrast between cell organelles and their background

Question 17

Gram staining procedure
- final result?

Answer 17

• 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

Question 18

Phenotype analysis - biochemical tests: Decomposition of simple carbohydrates

Answer 18

• 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

Question 19

Phenotype analysis - biochemical tests: test for specific enzymes

Answer 19

• 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)

Question 20

Analysis of different bacteria based on cultural characteristics (behaviour in culture/agar plate)

Answer 20

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

Question 21

toxic forms of O2, and enzymes used to break them dows

Answer 21

catalase - hydrogen peroxide - H2O2
Superoxide dismutase - breaks down superoxide (O2- anion) into molecular oxygen (O2) and H2O2 by reduction with Hydrogen.

Question 22

Molecular analyses method: FAME - Fatty Acid Methyl Ester analysis (Fatty Acid Profiling)
- info and drawbacks

Answer 22

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

Question 23

genotyping analysis: DNA profiling - AFLP - info

Answer 23

• 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

Question 24

genotyping analysis: multilocus sequence typing - info

Answer 24

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

Question 25

genotyping analysis: GC base ratios (guanine-cytosine base ratios)

Answer 25

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

Question 26

Obligate aerobe info

Answer 26

• O2 required
• contain enzymes to deal with toxic forms of oxygen

Question 27

Facultative anaerobe info

Answer 27

• Usually better growth with O2
• can also grow anaerobically
• enzyme to deal with toxic forms of oxygen

Question 28

Obligate anaerobe info

Answer 28

Does not tolerate O2
- no enzymes needed to deal with toxic O2 forms

Question 29

Aerotolerant anaerobe

Answer 29

• Can tolerate O2, not needed for life though
• Has superoxide dismutase to deal with toxic O2 forms
• e.g. lactobacillus

Question 30

Microaerophilic organism info

Answer 30

O2 required, but only low concentration tolerated
- enzymes for toxic O2 forms usually not present or required

Question 31

Why DNA sequencing is best