Microbial Ecology / Microbial Communities Flashcards
Which of the following statements is TRUE?
a) Molecular methods show that less than 10% of bacteria can be cultured from any environmental sample
b) DAPI is a general stain for staining viruses in natrual samples. It stains RNA
c) The brown fluorescent protein (BFP) makes cells autofluoresce, allowing them to be tracked when introduced into the environment
d) Molecular methods show that less than 0.1% of bacteria can be cultured from any environmental sample
d) Molecular methods show that less than 0.1% of bacteria can be cultured from any environmental sample
Which of the following statements is TRUE?
a) Metagenomics measures the diversity of different molecules or compounds produced by bacteria in a microbial community
b) Metatransprotomics is used to analyse total protein synthesis by bacteria in a microbial community
c) Metatranscriptomics is used to analyse total DNA in a microbial community
d) Metatranscriptomics is used to analyse the diversity and abundance of different proteins in a microbial community
e) Metagenomics analyses DNA or genetic sequences in a microbial community
e) Metagenomics analyses DNA or genetic sequences in a microbial community
Which of the following statements is FALSE?
a) The human microbiome project reported that “the vaginal microbiome provides a new born baby with its first dose of microbes at birth”
b) The human microbiome project reported that “the diets of humans and in particular fat and fibre intake, will have no dramatic impacts on the makeup of our microbiome”
c) The human microbiome proect reported that “monitoring the gut microbiome can be used as an early detection of Crohn’s disease”
d) Microbiome studies have shown that a Mediterranean diet promotes gut bacteria that can be directly linked to ‘healthy ageing’ in older people”
e) A microbiome study on ATM keypads in NYC reported that “bacterial assemblages on ATM keypads are dominated by taxonomic groups known to be associated with human skin communities”
b) The human microbiome project reported that “the diets of humans and in particular fat and fibre intake, will have no dramatic impacts on the makeup of our microbiome”
When considering phylogenetic trees generated by 16sRNA gene sequences from bacteria; which of the following statements is FALSE?
a) Phylogenetic trees are a graphical depiction of the relationship around sequences of the organisms under study
b) The tips of brances on a phylogenetic tree represent species/strains that exist now from which sequence data was obtained
c) Nodes on a phylogenetic tree are points in evolution where the ancestor divereged into two new organisms, each of which then began to evolve to become more similar to each other again and this is reflected in a single shared pathway with many branches
d) The branch length in phylogenetic trees represents the number of changes that occurred along the branch
e) Ancestor relationships among the gene sequences are revealed by the branching order
c) Nodes on a phylogenetic tree are points in evolution where the ancestor divereged into two new organisms, each of which then began to evolve to become more similar to each other again and this is reflected in a single shared pathway with many branches
Which of the following statements is TRUE?
a) Viability PCR can differentiate between live and dead cells. It is based on Viability PCR dyes like PMAxx which are membrane-impermeable, which makes them live cell specific
b) The Green Fluorescent Protein (GFP) is naturally occurring and is derived from the 16s rRNA gene sequences from the jellyfish Aequorea victoria. It fluoresces GREEN when exposed to blue light
c) The three important steps in PCR reactions which occur in the sequence: Anneal, Denature and Elongation; result in millions of copies of a simple single fragment being generated for each primer pair that is used
d) Carl Woese proposed the use of PCR to target antimicrobial resistant genes from environmental samples
e) Phylogenetic analysis of microbial communities which uses DNA sequences, relies on PCR to get sufficient copies of a gene for reliable sequencing
c) The three important steps in PCR reactions which occur in the sequence: Anneal, Denature and Elongation; result in millions of copies of a simple single fragment being generated for each primer pair that is used
Which of the following statements is FALSE?
a)The full length 16s rRNA gene consists of 9 variable regions and is approximately 1,500 base pairs long. Therefore primers are typically chosen which cover only a portion of the 16s rRNA gene
b) The Taq Polymerase that is used in PCR reactions comes from the thermophilic bacterium Thermus aquaticus
c) Immobilisation enzymes such as lactases, which converts lactose into glucose, can be used in the production of lactose free milk for lactose intolerant people
d) Saccharomyces cerevisiae is a bottom fermenting yeast which is typically used in lager fermentations. It tends to fall to the bottom of the fermenter during the alcoholic fermentation process
e) S. cerevisiae uses fermentation even in the presence of oxygen, when glucose concentrations are sufficiently high. This use of fermentation by the yeast in the presence of oxygen and at high glucose concentrations is referred to as the Crabtree Effect
d) Saccharomyces cerevisiae is a bottom fermenting yeast which is typically used in lager fermentations. It tends to fall to the bottom of the fermenter during the alcoholic fermentation process
What is Microbial Ecology?
How microbial populations assemble to form communities and how these communities interact with each other and their their environment
What are the major components of microbial ecology?
- Biodiversity
- Microbial activity
What are some Culture Independent methods of analysing microbial communities?
- General staining
- Fluorescent In Situ Hybridisation (FISH)
- PCR
- Microarrays & Microbial Diversity: Phylochips
- Environmental Genomics
What is DAPI?
A general fluorescent stain that binds stongly to adenine-thymine (AT) rich regions in DNA
Stains the bacteria with blue fluorescence
What colours does the LIVE/DEAD BacLight Bacterial Viability Kit for microscopy and quantitative assays dye cells?
RED - dead
GREEN - live
Where does the dye in Live Bacterial Gram Staining Kits bind to?
To N-acetylglucosamine in the peptidoglycan layer of gram +ve bacteria to give a red fluorescence
What colour or colours does the Live Bacterial Gram Staining Kit dye Gram +ve bacteria?
Fluorescent blue INTERIOR
&
Fluorescent red CELL SURFACE
What colour or colours does the Live Bacterial Gram Staining Kit dye Gram -ve bacteria?
Fluorescent blue only
Are Viability PCR dyes like PMAxx membrane-impermeable or membrane-permeable?
Membrane-impermeable
Makes them DEAD CELL specific
Where does the Viability PCR dye like PMAxx bind to in the cell?
Covalently bind to DNA
What does the modification of cells with PMAxx do?
Prevents amplification in subsequent PCR reactions
What FIVE steps are involved in the basic v-PCR workflow?
- Dye addition
- Incubation
- Light Exposure
- DNA Extraction
- Quantitive PCR (qPCR)
What features does the crystal jellyfish Aequorea victoria display in response to light?
BIOLUMINESCENT (glows in the dark) FLUORESCENT (glows in response to UV light)
What protein is a naturally occuring 27kDa protein derived from the jellyfish Aequorea victoria?
Green Fluorescent Protein (GFP)
Fluoresces GREEN when exposed to blue light
Why would GFP be cloned into a bacterial vector?
The green fluorescent protein makes cells autofluorescent and is a means of tracking cells introduced into the environment
What do PCR methods of microbial community analysis show?
Data for PCR based phylogenetic analyses
* allows phylotypes to be described
What are the THREE steps in PCR?
- Denaturation (94-98°C)
- Annealing (50-65°C)
- Elongation (72-78°C)
What is the target of PCR?
16S rRNA gene
What is Phylogenetics?
Analysis of molecular sequencing data to study evolutionary relationships among groups of organisms
What is Phylogeny?
The evolutionary history of a group of organisms
How is the path of microbial evolution determined?
Indirectly through phylogeny from nucleotide sequence data
What is Evolution?
A process of inherited nucleotide sequence change
Why are rRNA genes used in phylogenetic analysis?
- Universally distributed
- Functionally constant
- Sufficiently conserved
What did Carl Woese propose about rRNA?
The use of rRNA genes as targets for phylogenetic analyses
Which rRNA makes up Prokaryotes?
16S
Which rRNA makes up Eukaryotes?
18S
Which part of the rRNA is free to mutate and evolve faster?
Loops
What is the 16S rRNA gene sequencing process?
- Bacterial colonies
- DNA extraction
- PCR
- PCR product cleanup
- Cycle sequencing
- Sequencing product cleanup
- Sequence detection
- Data assembly & analysis
What do the TIPS of the branch on a Phylogenetic Tree represent?
Species / Strains that exist now
What do the NODES on a Phylogenetic Tree represent?
Points in evolution where the ancestor diverged into two new organisms
What do the BRANCHES define in Phylogenetic Trees?
The order of descent and the ancestory of the node
What does the BRANCH LENGTH represent on a Phylogenetic Tree?
The number of changes that occurred along the branch
What are universal phylogenetic trees based on?
rRNA gene sequence analysis
How are Archaea described?
Obligate anaerobes living in environments low in oxygen
What are the structures called that are fossils of Archaean cells?
Stromatolites
3.5 billion years ago
What are the structures called that are fossils of Archaean cells?
Stromatolites
3.5 billion years ago
What is the Theory of Endosymbiosis?
- Proposes that Eukaryotic life evolved from Archaea
- Organelles such as mitochondria and chloroplasts evolved from certain types of bacteria engulfed through endophagacytosis
How are INDIRECT probes labelled in Fluorescent In Situ Hybridisation (FISH)?
Labelled with modified nucleotides that contain a hapten
How are DIRECT probes labelled in Fluorescent In Situ Hybridisation (FISH)?
Labelled with nucleotides that have been modified to contain a fluorophore
What are TWO genetic staining methods?
- Phylogenetic Staining
- Fluoroescent In Situ Hybridisation (FISH)
What is Targeted Gene Sequencing used for?
- Can be used to detect specific microbes in a sample
- Allows assessment of the potential metabolic activity in any sample
- Utilised following RNA analysis allows real-time assessment of the metabolic activity of one or a series of processes in a sample
What are the steps in the Environmental Genomics Approach to Metagenomics?
- Microbial community
- Restriction digest total DNA
- Sequence
- Assembly & Annotation
What are the steps in the Community Sampling Approach to Metagenomics?
- Extract total community DNA
- Amplify single gene
- Sequence & generate tree
What information does the Environmental Genomics Approach to Metagenomics provide?
Total gene pool of the community:
1. ID of all gene categories
2. Discovery of new genes
3. Linking of genes to phylotypes
What information does the Community Sampling Approach to Metagenomics provide?
Single-Gene phylogenetic tree:
1. Phylogenetic snapshot of most members of the community
2. ID of novel phylotypes
What is Metagenomics?
Analyses DNA or genetic sequences in a community - representation (PCR or array analysis)
What is Metatranscriptomics?
Analyses sequence of total mRNA in a community - gene expression (PCR)
What is Metaproteomics?
Measures diversity and abundance of different proteins in the community
What is Metabolomics?
Measure the diversity of molecules or compounds that are produced
What is a Phylochip?
Microarray that focuses on phylogenetic members of microbial community
What colour does a phylochip microarray light up if there are sulfur reducing bacteria species present in the community?
Red
What is the largest group of microbes in the gut?
Firmicutes
What is Xenobiotic?
Any synthetic chemical not normally naturally produced by an organism
Which compounds are typically the pesticides most resistant to degradation?
Highly chlorinated
Which enzyme is involved in the cleavage of cathecol to cis, cis-muconate by an intradiol ring-cleavage?
Dioxygenase
How much O2 is required in soil air in the Bioventing process?
Above 2%
What are Polybrominated Diphenyl Ethers (PBDEs) used in?
Flame retardant materials
What TWO bacterial strains are able to degrade PBDEs under aerobic conditions?
- Sphingomonas strain S33
- Pseudomonas capacia
