Microbial Ecology/Communities 2 Flashcards
(27 cards)
What is a phylogenetic tree
Graphic depiction of the relationship among sequences of the organisms under study.
Branches define the order of descent & the ancestry of the node.
Branch length represents the number of changes that occurred along the branch.
Process of generating the phylogenetic tree from 16s rRNA
- Isolate DNA
- Amplify 16s gene by PCR
- Run on agarose gel, check for correct size
- Sequence
- Align sequence & generate tree
What are the three domains of life
- Domain bacteria
- Domain archaea
- Domain eukarya
(1&2 are prokaryotes, 3 are eukaryotes)
What are archaea
Single celled microorganisms with structures similar to bacteria.
What kind of environments do archaea live in
Environments low in O2 - this makes them obligate anaerobes
Do archaea have a nucleus or other cell organelles
No nucleus or other organelles
What is the theory of endosymbiosis
It proposes that eukaryotic life evolved from archaea.
Organelles such as mitochondria & chloroplasts in eukaryotic cells evolved from bacteria that prokaryotic cells engulfed through endophagocytosis.
The cells & the bacteria inside evolved a symbiotic relationship (endosymbiotic relationship).
What are the 2 basic elements of fluorescent in situ hybridisation (FISH)
- A DNA probe
- A target sequence
What are indirect probes labelled with (FISH)
Indirect probes are labelled with modified nucleotides that contain a hapten
What are direct probes labelled with (FISH)
Direct probes are labelled with nucleotides that have been modified to contain a fluorophore
Steps in FISH (Fluorescent In Situ Hybridisation)
- DNA is labelled directly or indirectly before hybridisation
- Labelled DNA probe and the target DNA are denatured
- Combining the denatured probe and target allows annealing of the complementary DNA sequences
- If probe labelled indirectly - extra step for visualisation of non fluorescent hapten using enzymatic/immunological detection
What methods are used for single cell identification after the FISH method
- Epifluorescence microscopy
- Flow cytometry
Steps of FISH for bacterial pathogen identification in patients
- Collect infected tissue sample from patient
- Synthsise complementary oligonucleotide for pathogen with fluorescent tag chemically attached
- Treat tissue sample to make membranes of all cells permeable to the fluorescently tagged oligonucleotide
- Add fluorescently tagged oligonucleotide to patient sample
- Fluorescently tagged oligonucleotide will bind only to the pathogenic DNA
- Plate sample & observe under microscope. Pathogenic cells will fluoresce
What are genetic stains
Variety of fluorescent staining methods that employ the power of nucleic acid probes & thus are highly specific in their staining properties
2 examples of genetic stains
- phylogenetic staining
- fluorescent in situ hybridisation (FISH)
What can targeted gene sequencing be used for (3)
- To detect specific microbes in sample
- To assess potential metabolic activity in sample
- To use after RNA analysis allows real time assessment of metabolic activity of processes in the sample
What genes can be checked for in targeted PCR
Antibiotic resistance genes
What is the beta lactamase resistance gene
blaIMP
What are the tetracycline resistance genes
tetA, tetB, tetC, tetM, tetO & tetS
What are the vancomycin resistance genes
vanH, vanA, vanX, vanZ
What happens in metagenomics (environmental genomics)
- Characterises the entire genetic content of the environment
- Sequencing & analysis of microbial genomes
- Info allows community structure & function to be studied
Metagenomics
Analyses DNA/genetic sequences in a community - PCR or array analysis
Metatranscriptomics
Analyses sequence of total mRNA in a community - gene expression (PCR)
(transcription = DNA to mRNA)
Metaproteomics
Measures diversity of different proteins in a community - protein separation media & extract the proteins
