Week 11 Flashcards
What are the two different routes for identifying important bacteria in a process?
Culture-dependent Work
Culture-independent Work
Both paths are important for modern microbiological work!
What is culture dependant work?
The isolation and study of microbes that you can grow in lab
This method is the oldest method and the one which has lead to the majority of historical microbiological findings
What is the simplest way for culture dependant work?
Using sterile technique plate sample onto rich medium
Isolation of bacteria from an environment on bacterial growth media (Rich Media)
Give diverse mix of microbes, not necessarily what you want
How can you modify culture dependant work?
Isolation of bacteria from an environment on selective growth media eg microbes we want use DMS as sole C source, use minimal media with DMS as sole C source
What can be an issue with carbon restrictive culture dependant work?
Even when microbes are isolated on agar plates with DMS as sole C source many will not catabolise it.
All plates are made up to contain agar which can be used as a C source
What is enrichment culturing?
Inoculating your environmental sample in liquid with DMS as sole C (no agar)
After a period of growth then use this to inoculate a fresh liquid media DMS as sole C
Repeat -this removes microbes unable to use the DMS since they will be outcompeted
Plate on DMS Plates
What is ithe outcome of enrichment culturing?
Decreased variability but high proportion of true DMS catabolisers
How do you know what you have got?
It is possible to identify the microbes through the use of diagnostic genes (highly conserved between organisms) eg 16S rRNA for prokaryotes
How do you identify what has been grown?
Extract gDNA from bacterial isolate
Amplify 16S or 18S rRNA gene using PCR
Sequence 16S rRNA gene & Bioinformatics (e.g. BLASTN)
What will culture-dependant work give you (through example of DMS)?
Model microbes that catabolise DMS (for C assimilation)- e.g. Methylophaga thioxydans DMS010
Study physiology, e.g. DMS010 uses DMS and MeSH
Study the genetics of DMS usage (e.g. genes involved, transcription/translation and conservation in other bugs)
Essential if you desire progressing to a biochemical understanding (extremely difficult to identify new genes by
Culture-Independent Work)
Must be mindful that is only one bug! A miniscule fraction of what is out there!
What are the advantags of culture dependent work?
Gives you model systems on which you can study the process (e.g. what environmental factors affect the process).
Can sequence the genome of the organism to indicate its genetic potential (helps to understand how it does work).
Can develop genetic systems on the microbes to further understand the process (mutate genes, study gene expression etc.).
Can cryopreserve the microbes for future use
What are the disadvantages of culture dependant work?
Less than 1% of bacteria are cultivable under lab conditions
Bacteria that grow in the lab may not be representative of the major players in the environment
Why can less than 1% of bacteria grow in lab conditions?
Media missing unknown essential components
Many microbes cannot grow axenically (on their own)
Many microbes grow in liquid but not on plates
Many microbes grow too slowly
Many microbes may require growth in host cells (pathogens/symbionts)
What is culture independant work?
The study of microbes in an environment/sample without their isolation on agar plates.
These methods are relatively newer and fully utilise high throughput sequencing and bioinformatics
How can you work out what microbes are in an environment?
Take sample of site of interest (e.g. 1-20 L seawater or ~ a g of sediment)
Isolate microbes (e.g. filter the 1-20 L seawater or spin down sediment)
Isolate metagenomic DNA from microbial community microbes (e.g. carry out a gDNA prep)
Carry out 16S or 18S rRNA PCR on metagenomic DNA (community DNA) and observe single DNA species on agarose gel
What needs to be done after isolating the 16S genes with agrose gel?
even though this looks like a single band it is not! All 16/18S genes are the same length. NEED TO IDENTIFY THEM
What are the two choices identifying the bacteria present in the gel?
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 {up to date}
What are the pros of denaturing gradient gel electrophoresis?
Allows direct visualisation of microbial diversity
Allows identification of major community members (low number)
Relatively cheap
Takes little time
What is needed to perform a Denaturing Gradient Gel Electrophoresis (DGGE)?
PCR requires a special 5’ primer with a GC clamp (stable and doesn’t denature)
What happens during Denaturing Gradient Gel Electrophoresis (DGGE)?
Seperation of DNA fragments of the same length but woth different base-pair sequences
Based on the decreased electrophoreitc mobility of a partially melted DNA molecule
Polyacrylamine gels containing a linearly increasing gradient of DNA denaturants
Gradient is usually formamide and urea in a polyacrylamide gel
What are the advantages of Denaturing Gradient Gel Electrophoresis (DGGE)?
Excise bands of interest and sequence
Gives you taxonomic info of bacteria abundant in sample
What taxonomic info can be gained from Denaturing Gradient Gel Electrophoresis (DGGE)?
Works best for major bands
Will give an idea of community´s complexity
Will demonstrate if an enrichment has occurred in enrichment studies
What are the limitations of Denaturing Gradient Gel Electrophoresis (DGGE)?
Extremely tricky for the outputs
If your microbe is not abundant it may not give you any info
What is an overview of High throughput sequencing of rRNA gene PCR products?
Also called aplicon sequencing
Can allow the complete characterisation of all components of the microbial community depending on sequencing depth
Detect if a pathogen is in a sample at very low abundance
Now relatively cheap (£50 a sample) and takes little time (a month)
What is the method for High throughput sequencing of rRNA gene PCR products?
Take sample
Prep gDNA
Carry out PCR on gDNA using 16S or 18S primers and purify PCR product
Amplicon sequence the product:
Prepare a sequencing library (normally Illumina) –a process that adds short DNA adaptors enabling sequencing
Sequenced en masse (typically Illumina MiSeq)
Given all sequence reads ~30 million rRNA gene sequences
Bioinformatics analysis to classify different microbes in the community
What is the output for High throughput sequencing of rRNA gene PCR products?
Can say % of pretty much any bacterium present in a sample
Much more information than from DGGE
What is the function of enrichment cultures?
Gives a clue into which bugs are undergoing the process of interest eg catabolishing DMS
Same method as in previosu flashcard it promotes growth of the bacterial species that undergo a large amount of bacterial growth
What does the use of DGGE and amplicon sequencing with enrichment cultures?
When combined these techniques allow you to identify microorganisms important in your process of interest
These techniques are universal
What are metagenomics?
This is the process of applying high throughput sequencing on metagenomic DNA.
Will give taxonomic information on the community (based on all genes in data)
What are the overview of metagenomics?
Gives info on the metabolic potential of the community via indicating the abundance of diagnostic gene that drive processes, e.g. ddd genes for DMSP lysis
Potential to identify the key microbes driving your process, e.g. the most abundant microbe that has your process gene
What is relative abundance?
The percent composition of an organism of a particular kind relative to the total number of organisms in the area.
A relative abundance of 80% means that Piscirickettsiaceae are predicted to comprise 80% of the bacteria in the sample.
This can also be applied to genes, but in this case you gene of interest is normalised to genes that are known to be single copy in genomes, e.g. recA in bacteria
What are primary metabolites?
Part of the basic metabolic processes of the cell or its physical structure they are essential for survival
What are the general categories for primary metabolites?
Organic acids and sugars
Aminoacids
Nucleosides
Fatty acids and other lipids
Vitamins
Their intermediates in biosynthesis/degradation pathways
What are specific examples of primary metabolites?
Fermentation of alcohol
Shikimic acid pathway, present in all organisms except animals involved in the generation of precursors of aromatic amino acids, vitamin cofactors and other metabolites
Vitamin B12 or cobalamin, is biosynthesised by some bacteria and archaea. We are not able to make it and have to incorporated it through the diet
What are the aims for primary metabolites?
Primary metabolites are essential for the normal growth, development, and reproduction of the organism
They are also the building blocks for secondary metabolism and other non-essential processes
What are secondary metabolites?
Also called specialised metabolites or natural products
Small organic molecules not directly involved in the normal growth, development or reproduction of the organism
What are uses of secondary metabolites?
They mainly mediate environmental interactions both with other bacteria and fungi, as well as with higher organisms (chemical ecology)
Technically non-essential, but can confer selective advantages for the producer organism
What are examples of the way secondary metabolites can be used?
Competition
Communication
Toxicity/virulence
Nutrient scavenging
Protection against toxic chemicals/ UV
Where do secondary metabolites come from?
Natural products are derived from primary metabolites, but have their own specialised biosynthetic pathways
What is an overview of the use of secondary metabolites in gut bacteria?
1 trillion bacteria, or 95% of all the bacteria in our bodies
All use secondary metabolites for competition and cooperation between other bacteria living in the gut
What is an overview of the microbriome in the rhizosphere?
Differential distribution in the different areas of the soil and the plant, and also inside the insects, both as symbiotic and beneficial partners or as pathogens.
Plants produce nutrients in form of exhudates and other compounds that repel or attract specific microbes.
Produce different molecules, including plant hormones, stimulate immune system, volatile compounds
Microbes also communicate among themselves both in antagonistic (e.g. antibiotics) and synergistic ways (e.g. quorum sensing molecules)
What is an overview of biosynthetic production of secondary metabolites?
Bacterial chromosome the genes involved in biosynthesis or secondary metabolites are located contiguously forming
BIOSYNTHETIC GENE CLUSTERS (BGCs)
What are an overview of biosynthetic gene clusters?
Composed of one or several operons
Expression of the genes in the cluster is coordinated by specific regulation responding to environmental factors
Genes encode enzymes that create the backbone of the molecules and introduce tailoring modifications
BGCs can span from 5 kb bp to well over 100kb
