Lecture 2 Flashcards
What is a phylogenetic tree?
Graphic depiction of the relationship among sequences of organisms under study
-consists of nodes and branches- like fam tree
-tips of the branch represent species/strains that exist now from which sequence data was obtained
-nodes are points in evolution where the ancestor diverged into 2 new organisms, each of which began to evolve alone a separate pathway.
What is ED?
Evolutionary Distance
What are the 3 domains of life?
Domain bacteria
Domain Archaea
Domain Eukarya
Why is virus not included in the domains of life?
Lack of rRNA
What is Archaea?
-Group of single-celled mo that like bacteria are prokaryotes that have no cell nucleus or any other ogranelles within their cells
-Have independent evolutionary history and numerous diff in their biochemistry compared to others so have own domain
-Obligate anaerobes - dont grow in presence of oxygen
What does the theory of endosymbiosis propose?
Proposes that Eukaryotic life evolved from Archaea
Proposes that organelles such as mitochondria and chloroplasts in euk cells evolved from certain types of bacteria that prokaryotic cells engulfed through endophagocytosis
These cells and bacteria trapped inside subsequently evolved a symbiotic relationship- in this endosymbiotic relationship, bacteria lived within the other pro cells
What is FISH and what are the basic elements used?
Fluorescent In Situ Hybridization
-basic elements are DNA probe and a target sequence.
What happens before hybridization in FISH?
-The DNA is labelled - indirect labelling and direct labelling
-INDIRECT probes are labelled with modified nucleotides that contain a hapten
-DIRECT labelling uses nucleotides that have been modified to contain a fluorophore
What happens in FISH?
-Before hybridisation, the DNA is labelled (direct or indirect)
-The labelled probe and target DNA are denatured
-Combining the denatured probe and target allows the annealing of the complementary DNA sequences
-If the probe has been labelled indirectly, an extra step is needed for visualisation of the nonfluorescent hapten that uses an enzymatic or immunological detection system
Pros of indirect and direct labelling?
FISH is faster with directly labelled probes
Indirectly labelled probes have the advantage of using several layers of antibodies to produce a brighter signal
Why is the sample fixed first in FISH?
To stabilize the cells and permeabilize the cell membrane
How is single-cell identification and quantification carried out on FISH sample?
By epifluoresence microscopy or flow cytometry
Process of FISH for bacterial pathogen identification?
1) Collect infected tissue sample from patient
2) Synthesize complementary oligonucleotide for suspected pathogen with fluorescent tag chemically attached.
3) Chemically treat tissue sample to make the membranes of all cells permeable to the fluorescently tagged oligonucleotide.
4) Add fluorescently tagged complementary oligonucleotide to sample
5) The fluorescently tagged oligonucleotide will bind ONLY to the pathogenic DNA
6) Plate sample and observe under micrscope. The pathogenic cells will fluoresce
What do fluorescent-staining methods use and what are examples of these stains?
-Employ the power of nucleic acid probes and thus are highly specific in their staining properties
-E.g phylogenetic staining and fluorescent in situ hybridization - ammonia oxidising bacteria (red) nitrite oxidising bacteria (green)
What is targeted gene sequencing used for?
-Can be used to detect specific microbes in a sample/environment
-Allows assessment of the potential metabolic activity in any sample
-Utilized following RNA analysis allows real-time assessment of the metabolic activity of one or a series of processes in a sample
Antibiotic resistance genes - what is involved in enzymatic drug modification?
Beta-lactamase gene, blalMP
Antibiotic resistence - what is involved in efflux of the drug, ribosomal protection or enzymatic drug modification?
Tetracycline resistence genes tetA, tetB, tetC, tetM, tetO, tetS
Antibiotic resistance - What is involved in target modification?
Vancomycin resistance genes vanH, vanA, vanX, vanZ
Annealing temps of TetA, tetB, tetC?
tetA - 50 bp:210
tetB - 50 bp: 659
tetC - 49 bp: 418
What antibiotic resistant gene does ampicillin have?
ß-lactamase gene
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 (Protein separation media and extract the proteins)
What is Metabolomics?
Measures the diversity of molecules or compounds that are produced (different extractions in solution followed by lazor detection of the analytes)
Bacterial assemblages known to be associated with human skin communities?
Actinobacteria, Bacteroides, Firmicutes, Proteobacteria
What was the most represented bacterial species on the ISS?
Staphylococcus species
What is purpose of bacterial biofilms?
-Protected against environmental stress due to their extrcellular matrix which could contribute ti persistent infections after treatment with antibiotics
-Capable of strongly attaching to surfaces, where their metabolic by-products could lead to surface material degradation
What can microgravity affect?
Microgravity can alter biofilm behaviour in unexpected ways, making the presence of biofilms in space a risk for both astronauts and spacefligjt hardware
