Microbial Diversity 1-3 (Jason)

Question 1

What are the three domains of life and the taxonomic rank order

Answer 1

Domains:
Bacteria
Archaea
Eukarya

Taxonomic rank
Domain
Phylum
Class
Order
Family
Genus
Species

Question 2

What is a microbial species

Answer 2

• Defined as a group of microbes with many stable properties which are very different from other species.
• Largely based on genetic differences.
• Individuals of the same species are genetically very similar but not necessarily identical.
• The term strain refers to variants of the same species.

Question 3

Define the terms biovars, morphovars, serovars, and type strain

Answer 3

Biovars - strains that differ biochemically or physiologically.
Morphovars - strains that differ morphologically.
Serovars - strains that differ in antigenic properties.
Type strain - used as a reference point for the species. Often most fully characterised member of the species but not necessarily the most common.

Question 4

Describe phenetic classification of microbes and its problems

Answer 4

Morphological characteristics

• Shape and size are easy to measure.
• Structural features depend on the expression of many genes - usually genetically stable.
• Doesn’t usually vary greatly with environmental changes.
• Good indication of phylogenetic relatedness up to a point.

Physiological and metabolic

• Directly related to gene products (enzymes and transport proteins).
• Provide an indirect comparison of microbial genomes.
• Often groups microbes by function e.g. Lactobacillus spp. produce lactic acid.
• May group microbes by medically important data e.g. resistance or sensitivity to antibiotics, or by nutrient cycles they’re involved in.

Ecological characteristics
- Organised based on lifecycle patterns e.g. symbiotic relationships, ability to cause disease, and habitat preferences.

Problems

• Plasmids can move between cells and carry phenotypic traits with them.
• Antibacterials exploit the differences between the bacterial cell and the host’s eukaryotic cell however plasmids will often carry antibiotic resistance genes.
• To study phenotypes you need to grow the microbes however less than 1% of microbes in many environmental samples can be cultured.

Question 5

Describe genotypic classification of microbes and its problems

Answer 5

• Compares genetic similarity between organisms and provides an idea of relatedness at the DNA level.
• Allows classification of microbes that cannot be cultured.

Nucleic acid base comparison

• Examines the proportion of each nucleic acid base in the DNA.
• A-T pairs have two hydrogen bonds and G-C pairs have three hydrogen bonds. As a result it takes 50% more energy to break a G-C pair than an A-T pair.
• The proportion of G-C pairs in DNA can be revealed by measuring the energy needed to break all of the base pairs.
• Higher G-C content means a higher melting temperature.
• If G-C content differs by more than 10% it suggests the microbes are not closely related.
• However, G-C content tells you the amount of each base pairs but nothing about the actual DNA sequence.

Nucleic acid hybridisation

• Gives a direct measure of sequence similarity between genomes.
• The more similar the organisms are the more similar the DNA sequences will be.
• The DNA of one organism is heated to denature the double-stranded DNA and separate it into single strands. The resulting single strands are fixed onto a membrane.
• The DNA from a second organism is cut into short fragments and radioactively labelled.
• The cut DNA is denatured to produce single-stranded fragments which are added to the membrane containing the DNA of the first organism.
• The labelled DNA will hybridise to complementary sequences on the membrane. The extent of hybridisation can be measured by the quantity of label remaining on the membrane after the unhybridised DNA is removed by washing the membrane.

Problems

• To comprehensively compare many microbes you have to test hybridisation between each and every microbe.
• Tells you what proportion of DNA is similar but not what the similarities/ differences are.

Question 6

Describe phylogenetic classification and its problems

Answer 6

rDNA analysis

• The degree of similarity in rRNA sequences between two organisms that indicates evolutionary relatedness.
• The 16S rDNA genes in a sample are amplified using PCR. This makes many copies of the 16S rDNA gene but not other genes.
• PCR products are then sequenced and analysed by a computer.
• Sequences are aligned(to make sure you’re comparing the same part of each gene) and compared to identify differences and give a measure of evolutionary distance which is used to construct a phylogenetic tree and assess relatedness.
• Fast and straightforward to do as well as being useful for analysing microbes that cannot be cultured.
• Disadvantage: only works with pure cultures.

Denaturing gradient gel electrophoresis (DGGE)

• Due to its negatively charged backbone, DNA moves through a gel by electricity.
• The gel has a gradient of a denaturant which denatures the DNA at high enough concentrations.
• The denaturing concentration depends on the DNA sequence. Similar DNA sequences will move similar distances along the gel.

Fluorescent in-situ hybridisation (FISH)

• The sample of FISH-labelled microbes is separated out into tiny droplets, with one cell per drop.
• Each drop moves past a laser which measures the fluorescent label on the probe.
• The machine gives the droplet an electric charge to guide it into a specific vial.
• This allows you to count the number of each type of labelled microbe, measure how environmental changes affect the number of microbes at each taxonomic level, and separate out microbes of a particular taxonomic level for further study.