lecture 3 Flashcards
microbial systematics
define systematics
–> Study of the diversity of organisms and their relationships
it links phylogeny (evolutionary history) with taxonomy (classifying)
by characterising, names and placing organisms into groups based
on their natural relationships
what is bacterial taxonomy?
bacterial taxonomy focused on what the organisms did (phenotypes) by studying what compounds they grow on or what temperature they grow at. We focused on phenotypic comparisons because microbes carry very little characteristics and so it’s very difficult to determine one from the other.
why do we now use molecular techniques to drive taxonomies?
We now use molecular techniques to drive taxonomies and reflect the evolutionary relationship between organisms, because the molecules within a cell carry a signal of evolutionary history.
Systematic taxonomic ranks
Domain (bacteria, archaea, Phylus Class Order Family Genus Species Subspecies if relevant
why is there no unified species concept in bacteria?
Higher organisms species are separated by their ability to interbreed and produce viable
offspring
Problematic in microbiology due to:
• Asexual reproduction
• Lateral gene transfer
• Phenotypic and genotypic plasticity of microorganisms
current definition of bacterial species
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
define polyphasic bacterial taxonomy
In order to go through the process of naming a new organism, we have to work out what are the characteristics we want to use. So we use a polyphasic bacterial taxonomy which tells us what it can do, what its genes tell us about it and its history.
Phenotypic analysis:
- morphological (cell’s shape, what it looks like when/if it grows on a solid media)
- metabolic (products, eats, needs to grow)
- physiological and chemical characteristics (what temperature/solidity it grows at, if it needs light, some could have the same metabolic process but use different genes)
Genotypic analysis:
Comparing individual genes together or whole genomes (so we’re looking at those different genes that allow the different characteristics)
- so we can have different organisms that use the same substrate but use different pathway to degrade it and get the energy from it, this is the distinguishing factor between them.
Phylogenetic analysis:
Framework of evolutionary relationships based on the genotype and phenotype
problem: a single mutation can change apparent definition
of a species
(E. coli by definition produces the enzyme ß-galactosidase, would a single mutation of this ability mean it was no longer e coli?)
morphology of bacteria
• Gram stain
–> cell shape, size and arrangement (single, chains, etc.),
pleomorphism (existing in different shapes) formation of
cysts, spores (shape, size, etc.)
• Presence of flagella, capsules, staining reactions, etc
motility of bacteria
• Determination by phase-contrast microscopy
nutrition of bacteria
Testing for ability to grow on a range of compounds as sole
sources of carbon, energy and nitrogen
what is the staining procedure?
- spread culture in thin film over slide
- let it dry in air so that cells are desiccated on slide
- heat fix the cell on the slide by passing the slide through a flame, the stuck cells can now be stained
- flood slide with stain, leave it for 1 minute, rinse and dry it
gram staining procedure
- flood the smear with crystal violet (stain is toxic and stains every cell) all cells turn violet
- add iodine solution for 1-3 minutes to fix the crystal violet onto the cells
- if you add alcohol briefly, it will decolourise the G- cells while the G+ cells retain their purple colour
- counterstain with safranin for 1-2 min, the G- cells with go pink and the G+ cells will stay purple
(occasional faded out cells are either lysed or it’s a subcell that’s stuck on the side)
–> iodine make both purple, alcohol decolourises G- cells, safranin stains G- cells pink.
biochemical test to determine if they decompose simple carbohydrates
- (if a cell uses glucose and if it produces acid, so if it is anaerobic or aerobic) if it does produce acid, it will turn the pH indicator from red to yellow.
- This tells us that it produces acids from lipids, so it ferments the glucose and breaks it into 2 parts that will both be an acid (fermentation produces acids like acetate
- After the fermentation process, it could produce carbon dioxide as an end product of that degradation which tells us more about its metabolism
biochemical tests for enzymes that decompose large molecules
- if we have a plate with starch in it and grow an organism on it, and flood the plate with iodine, the iodine will stain any starch left in the media. A halo production of starch is a positive result of starch
- we can do the same with other substances that organisms degrade like DNA, protein, amylose, phospholipids, lipids (using DNase, protease, amylase, phospholipase, lipases)
- we see a halo because the cell is excreting the substance outside of its body in order to break it down and take it in (for example as short amino acids not big proteins) to utilise it inside the cell
cultural characteristics
colony based characters (colony shape, margin, elevation, surface appearance, opacity,
texture, pigmentation, odour and appearance of growth)
physiological properties (4)
- growth ph (acidophile or alkaliphile)
- barophile (requires high pressure like at the bottom of the ocean)
- halophile (requires very salty conditions)
- oxygen (aerobe, anaerobe, microaerophile)
different types of oxygen requiring microorganisms (5)
- obligate aerobe (o2 required)
- facultative anaerobe (better with o2, can also be anaerobic)
- olbigate anaerobe (doesn’t tolerate o2)
- aerotolerant anaerobe (can tolerate o2)
- microaerophilic (o2 needed but only low concentrations tolerated)
relationships with temperatures (4)
- psychrophile <15 (adapted by protein structure and membrane fluidity)
- mesophile 15-45
- thermophile >50
- hyperthermiphile >80
molecular analyses (FAME) - Fatty Acid Methyl Ester
no need to memorise
Determination of fatty acid profile of membrane lipids
- growth under ‘standard’ conditions, extraction of lipids,
chemically modify to methyl esters, analysis of FAME products
by gas chromatography
- differences in chain length
- presence of double bonds, rings, branched chains or hydroxy
groups
- compare chromatograms to database for best match
drawback of molecular analyses (FAME) - Fatty Acid Methyl Ester
(no need to memorise)
fatty acid profile depends on growth conditions (temp,
medium, growth phase) which need to be standardised.
not all strains can be cultivated under those conditions
genotyping analyses types (4)
- dna.dna hybridisation
- dna profiling
- multilocus sequence typing (MLST)
- gc base ratios
dna.dna hybridisation
no need to memorise
Genome wide comparison of sequence similarity.
Useful for discrimination of strains of a species
> 70% = same species;
25% = same genus;
<10% usually different genera
dna profiling
no need to memorise
ribotyping, rep-pcr, aflp…
Producing DNA fragment patterns for comparative analysis
Multilocus sequence
typing (MLST)
(no need to memorise)
Characterising strains within a species
Sequencing of several
‘housekeeping’ genes
assign different alleles to strains
procedure:
- new isolate or clinical sample
- dna isolation
- amplify 6/7 target genes by pcr
- sequencing
- determine alleles
- compare with other strains of the same species (linkage distance)
GC base ratios
no need to memorise
Percentage of GC bases in genome
Range 20-80%, Similar
organisms have ~same GC content, but very different organisms can also have close values
dna profiling - AFLP
no need to memorise
• 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
Deposition of type cultures
no need to memorise
Validity described species have to be deposited with at least two culture collections so that anyone else can get access to it and use it.
3 international culture collections: NCIMB, ATCC, DSMZ.
