Lecture 19: Microbiology - Who does what, where, and why? Flashcards
Give examples of why bacteria are important
1. Rhizobia form symbiosis with legumes
and fix atmospheric nitrogen to ammonia
2. Pathogens cause disease. e.g.,
salmonella
3. Phytoplankton drive marine carbon
fixation and are important in DMSP
synthesis
4. Actinomycetes e.g., Streptomyces, are
important producers of antibioticsHow do we know which microbes are important?
- Take the bacteria that convert 90% of
DMSP produced into DMS as an
example
a. Understand the basics - they eat DMS
and take a methyl group as a source of
carbon
What are the two routes for identifying bacteria (that are using DMS)?
- Culture-dependent work
2. Culture-independent work
What is culture-dependent work?
- “Isolation and study of microbes that you
can grow in a lab”
a. take sample (seawater) plate it out on
rich medium - yeast extract, provides
everything needed - Oldest method, has lead to many
microbiological findings - CON: Diverse mix of microbes, not
specific
How can you isolate the bacteria you want?
1. Use minimal media with DMS as sole
C source
2. Some organisms, called “agaroses” can
sink into the agar and release enzymes
to use agar as C source
How do you get around bacteria that use agarose as a carbon source?
- Enrichment Culturing
a. Inoculate bacteria to a liquid culture
where DMS is only source of carbon
b. move to new liquid DMS culture, keep
repeating
c. plate on DMS plates
How do you get around bacteria that use agarose as a carbon source?
- Enrichment Culturing
a. Inoculate bacteria to a liquid culture
where DMS is only source of carbon
b. move to new liquid DMS culture, keep
repeating
c. plate on DMS plates
How do you know what bacteria you have?
- Diagnostic genes (conserved between
organisms)
a. e.g., 16s RNA gene or 18s RNA gene
What does culture-dependent work give you?
- model organisms that catabolise DMS (e.g., Methylophaga thioxydans DMS010)
- Study physiology (DMS010 uses DMS and MeSH
- Study genetics of DMS usage (e.g., gene involved, conservation in other bacteira, transcription/translation)
- Essential if you desire progressing to biochemical understanding (Hard to do with culture-independent work)
- NEED TO BE CAREFUL IT IS ONLY ONE BACTERIA
What are the disadvantages of culture dependent work?
- Less than 1% of bacteria are cultivatable
under lab conditions. Why?
a. media missing unknown essential
components
b. microbes cannot grow axenically (on
their own)
c. microbes grow in liquid by not on
plates
d. Microbes gro too slowly
e. Microbes may require growth in host
cells (pathogens/symbionts) - Bacteria that grow in lab may not represent major players in the environment
What is culture-independent work?
- “Study of microbes in an environment/sample without their isolation on agar plates”
Relatively newer methods, fully utilising high throughput sequencing and bioinformatics
How does culture independent work?
1. Take sample of environment (ie. 1-20L of
seawater or a g of soil)
2. Isolate microbes (e.g., filter 1-20L sample
or spin down soil)
3. Isolate metagenomic DNA from
microbial community microbes (e.g.,
carry out a gDNA prep)
4. Carry out 16s or 18s rRNA PCR on
metagenomic DNA (community DNA)
and observe single DNA species on
agarose gelWhat do you do once you have your PCR product/metagenomic DNA from culture independent analysis?
- Denaturing Gradient Gel Electrophoresis (DGGE) - way of visualising distinct rRNA gene products (old fashioned)
- High throughput sequencing - amplicon sequencing of the PCR product to show community diversity
What is Denaturing Gradient Gel Electrophoresis (DGGE)?
- Allows direct visualisation of microbial
diversity - Allows identification of major community
members (low number) - Relatively cheap
- Takes little time
- Requires special 5’ primer with GC clamp
- Separation of DNA frags. of same length
with different BP sequences - Based on decreased electrophoretic
mobility of a partially melted DNA
molecule
What are the pros and cons of DGGE
+Works best for major bands
+Gives idea of community complexity
+ Demonstrate if an enrichment has
occurred in enrichment studies
-Extremely tricky for outputs
-If microbe is not abundant it may not
give you any info
What is “high throughput sequencing of rRNA gene PCR products”?
- Allow complete characterisation of all
components of a microbial community
depending on sequencing depth - E.g., can detect if a pathogen is in a
sample at very low abundance - Now quite cheap (£50) and v. little time
(month)
