The Genome Revolution Flashcards
(84 cards)
1
Q
Is DNA a right-handed or left-handed double helix ?
A
Right
2
Q
What direction does DNA synthesis occur in ?
A
5’ to 3’
3
Q
What provides the energy for the creation of the phosphodiester bond ?
A
Energy released from cleavage of 2 phosphate (PO43-) groups from dNTP (deoxynucleotide triphosphate)
4
Q
Where does most of the protein binding occur ?
A
The major groove
5
Q
How many bases are in one helix turn ?
A
10
6
Q
How is supercoiling formed ?
A
topoisomerase, stabilised by proteins
7
Q
What is a bacterial genome ?
A
Genome: total catalogue of genetic material in a cell, made of DNA
- Chromosome: main genetic element, circular, supercoiled
- Plasmid: extra-chromosomal
8
Q
What things can genomes tell us ?
A
- Genome content (GC content)
- Genome architecture (position of ori, ter, rRNA
- Presence of lysogenic phages, plasmids, transposable elements, antibiotic resistance genes
- Horizontal gene transfer, genome formation (ancient and recent evolutionary changes)
- Specialisation (bacillus spp. can sporulate)
- Virulence factors (toxins, immune modulators, fimbriae (causes UTIs), adhesins) host-pathogen interactions
- Metabolic potential (ability to utilise specific substrates for growth)
- Environmental potential (ability to survive in extreme environments)
- Biotechnological potential (use in bioremediation, biofuel production)
9
Q
What does a high GC content indivate ?
A
o Both about the environment they live in and the percentage can indicate regions that were horizontally transferred
10
Q
Why do specific mutualists have a smaller genome ?
A
Specific mutualists have smaller genomes as they have lost genes that allow survival in other niches
11
Q
What is an example that backs up ‘Genomes indicate potential but not function’ ?
A
- Campylobacter jejuni (lacks RpoS (sigma factor required for stress-response)
- Persister cells (identical cells responding differently to stress)
12
Q
How are species diagnosed ?
A
- Diagnosed by biochemical tests, morphology, physiology, conserved gene sequences
13
Q
What is a specie ?
A
a monophyletic and genomically coherent cluster of individual organisms that show a high degree of overall similarity in many independent characteristics and is diagnosable by a discriminative phenotypic property
14
Q
What is the sequence-based definition of a specie ?
A
- 16S rRNA sequencing: similarity, phylogeny
- Average nucleotide identity > 95%
- DNA-DNA hybridization (DDH) >70%
15
Q
What are core, accessory and pan genes within the same specie ?
A
- Core: genes present in all individuals
- Accessory: “dispensable’ genes (strain-specific or in a subset of strains)
- Pan: core and accessory
16
Q
What is Maxam-gilbert sequencing ?
A
- Chemical methods
- Cleavage of specific bases breakages of DNA by piperidine
17
Q
What is sanger sequencing ?
A
- Use E. coli DNA polymerase I to synthesis DNA in vitro
- Synthesis stops by incorporation of dideoxygnucleotides (Hydrogen instead of a hydroxyl on the deoxyribose sugar)
- Separation of different sized fragments
- PCR with fluorescent, chain-determining ddNTPs, capillary gel electrophoresis, laser excitation and detection by sequencing machine
18
Q
What is ion torrent sequencing ?
A
- Ion semiconductor sequencing
- Release of a proton every time nucleotides are incorporated, change in pH and electrical signals
19
Q
What is nanopore sequencing ?
A
- Nucleotide-specific change in current every time a nucleotide passes through the pore
20
Q
What is Next gen sequencing ?
A
- Massively parallel high throughput genome sequencing fragment DNA into small pieces and sequence them all
- Multiple sequences can be sequenced at once
21
Q
What is bioinformatics ?
A
functional studies of genome assembly and annotation
22
Q
What are some sources of genetic diversity ?
A
- Spontaneous mutagenesis
- Errors introduced during DNA repair
- Homologous recombination (deletion/duplication/inversion)
- Transposable elements
- Exposure to mutagens (chemical agents), radiation
- Horizontal gene transfer (HGT)
23
Q
What is horizontal gene transfer ?
A
the movement of genetic material between organisms other than by the transmission of DNA from parent to offspring
24
Q
What are some reasons/evidence for HGT ?
A
- Genome diversification
- Evolution
- Adaptation
- Survivability
- Selection
25
What is a mobilome ?
a collection of mobile genetic elements (MGEs) within a genome
26
Examples of mobilome ?
- Lysongenic phages (virus that integrates its genetic material into the bacterial genome)
- Transposons
- Plasmids
27
What does mobilomes lead to ?
These lead to chromosomal rearrangements and chromosomal islands (horizontally acquired and integrated into the bacterial chromosome, they usually contain advantages)
- Extracellular DNA (eDNA)
28
What are the 4 mechanisms of HGT ?
vesiduction, conjugation, transformation, transduction
29
What does transformation mean during HGT ?
uptake of eDNA from the environment incorporated into chromosome by recombination genetic changes
o Donor recipient
o Recipient cells must be ‘competent’ i.e., have the ability to take up eDNA
30
What is the difference in transformation in gram positive and gram negative bacteria ?
+ binding of DNA, uptake of single stranded DNA, homologous recombination
- binding, uptake, translocation
31
What is conjugation in terms of HGT ?
: Transfer of DNA from one cell to another mediated by conjugative plasmid in donor
o Donor recipient
o Requires cell-to-cell contact (mating)
o Recipient can receive conjugative plasmid, other plasmids, chromosomal genes
32
How is conjugation mediated in E. coli ?
With the F (fertility) plasmid
33
What is the tra genes, insertion sequences (IS) and Tn1000, oriV, oriT in E. coli ?
tra genes: F pilus assembly and F plasmid transfer
Insertion sequences (IS) and Tn1000: intergration with chromosome
oriV: origin of replication
oriT: origin of transfer
34
What is hfr strains in E. coli ?
high frequency recombination strain
35
How is the conjugative plasmid integrated into the chromosome ?
Homologous recombination
36
What are Integrative conjugative elements (ICEs)
- Integrate into chromosome
- Encode conjugation machinery, commonly type IV secretion system
- Transferred like conjugative plasmids
- Can carry AMR, virulence, metabolic genes than enhance survival
37
What is SaPI ?
Staphylococcus aureus pathogenicity islands
- Similar to ICE
- Have integration and excision genes (like ICEs)
- Do not have machinery for transfer; need helper prophage for transfer
- Can carry AMR, virulence, metabolic genes that enhance survival
38
What is transduction in terms of HGT ?
Transfer of DNA by a bacterial virus (bacteriophage)
39
What are the 2 types of transduction ?
o Generalised transduction (by lytic/virulent phages)
o Specialised transduction (by lysogenic/temperate phages)
40
What is Generalised transduction?
Any gene from donor chromosome transductant by lytic/virulent
41
What is Specialised transduction ?
Selected region of donor chromosome transductant by lysogenic/temperate phage
Phage is integrated at specific site in chromosome
Phage replication is controlled by bacterial chromosome
42
What is vesiduction ?
vesicle driven HGT
43
What is vesiculation and vesiductant ?
Vesiculation: releasing of a vesicle into the environment
vesiductant: the recipient of the vesicle
44
What is a biofilm ?
- Multicellular communities with members belonging to same/different groups adhered to a biotic/abiotic surface
- Composed of extracellular polymeric substances – eDNA, proteins, polysaccharide, membrane vesicles
45
What makes Biofilms a hotspot for HGT ?
Biofilms are a hotspot for HGT, due to eDNA being released
- Actively secreted or released from eukaryotic and prokaryotic cells (autolysis (self-destruction by enzymes) or fratricide (killing of genetically similar neighbour cells)
- Role in pathogenicity, fitness, evolution
- Nutrient source
- DNA recombination and repair
- Role in structural integrity of biofilms
- Resistance to host antimicrobial peptides
- Accompanied by secreted nuclease
46
What is a bacteriophage ?
Bacteriophage: Virus that infects and replicates within bacteria
- Derived from bacteria and the Greek phagein meaning to devour
- Archaea are also infected by viruses; these are sometimes are referred to as phages
47
How are bacteriophages classified ?
morphology, nucleic acid type
48
What are the types of bacteriophages ?
Head and Tail viruses, Icosahedral (head), filamentous
49
What is the size of bacteriophages?
are smaller than a few viruses and most bacteria
50
What is a lytic phage ?
infection from a bacteriophage
51
What is a lysogenic phage
replicate inside a bacteria
52
What are some characteristics of bacteriophages ?
- They are parasites requiring host machinery and metabolites/energy for replication
- Route for genetic exchange: HGT via transduction
- Can alter the properties/phenotype of infected cells when not in lytic phase
- Most abundant organisms in the biosphere: soil, water, us
- There can be more than one phage in a cell
- Lytic phages are virulent
- Most phages are temperate: capable of both lytic life cycle and lysogenic life cycle
53
What are differences between lytic and lysogenic bacteriophages ?
o Lytic (infects, replicates and bursts), no intergration into host DNA
o Lysogenic (intergration of DNA and replicates it without killing it immediately, a prohpgae
54
What is an episome ?
can replicate independently and also in association with chromosome if integrated, transferred through conjugation
55
What is a prophage ?
- Prophage: integrated into chromosome, dormant
- Can be separated from bacteria using a 0.45 micro filter
56
How do phages infect ?
specific receptors and affects host range
57
What does 'Genomes of phages have little sequence redundancy' mean?
- Very little repetition or unnecessary duplication of genetic material
58
What may be in the genomes of phages ?
both strands may have coding sequences, ‘Essential’ and ‘non-essential’ genes
59
What does gene expression in phages are orderly mean ?
- Gene expression is ordered and sequential to aid the lysogenic/lytic phase
60
What is the transcriptional read-through in phages ?
- Transcriptional read-through: RNA polymerase transcribes beyond termination in order to reach late gene regions
61
What is differential gene expression in phages ?
- Differential gene expression is the temporal regulation through transcription factors and promoter recognition
62
Phages include inteins, what are they ?
self-splicing protein segments
63
What is an example of phages in disease ?
- Bacteriphage T12 in streptococcus pyogenes (speA gene erythrogenic toxin A scarlet fever)
64
How are phages being used in medicine ?
Phage therapy (use viruses to kill bacterial infections), phage typing (bacterial strain identification), biocleansing (industrial piping in food manufacturing, nuclear power plants), food pathogen reduction (campylobacter jejuni), diagnostics, surface display of peptides/antibodies
65
What is bacteriophage T7 ?
a phage that infects E .coli, linear dsDNA, lytic phage, genes are ordered, uses its own RNA polymerase
66
What is the order of genes in bacteriophage T7?
early genes switch on protein synthesis machinery, anti-restriction enzymes, protein kinase, RNA polymerase
67
What are GP4 proteins ?
protein that stabilizes the genome of the phage, unwinds the DNA molecule for replication, makes primers (primase activity)
68
What are concatemers ?
short cuts of genome that joins together during infection
69
what is a pac site ?
joining site of concatemers, recognised by class III genes (last genes expressed), that chop up the concatemers then get arranged into the head of the newly formed bacteriophage heads, one genome per head (morphogenesis)
70
What is bacteriophage M13
Infects E. coli with an F pilus, single stranded DNA, circular, lysogenic
71
How does M13 infect ?
inserts entire genome (infecting strand)o Positive DNA molecule so it cant be a template for photosynthesis
o Needs converting to dsDNA
o Replicating form (RF) of DNA formed using host DNA pol I
- Use host RNA pol to make its proteins
72
What is Bacteriophage lamda ?
- Receptor is the lamdaB of E. coli
- DNA and head and tail
- Linear DNA with has cos ends – so can circularise in order for protection
- Can be lytic and lysogenic
- Lytic – circular DNA
- Lysogenic – linear DNA, lamda DNA is inserted into the host genome
73
How does a lytic phage work ?
- Adsorption
- Entry of linear DNA
- Pairing of cos elements nicked circle
- Ligated circle DNA by ligase
- Supercoiling by topoisomerase
- Replication
- Replication
- Rolling circle replication concatamers
- Cleavage of concatamers at cos sites: packaging into head
- Addition of tails
74
How does a lysogenic phage work ?
- Integration due to the POP sites (on the phage genome) and BOB on the host site
- The O region is identical, homologous recombination can happen at this site
- intergrase is needed
- after recombination a BOP and POB site is made at either ends of the host genome
75
What proteins regulate whether a virus causes lysogeny or lysis ?
CI vs Cro,
C1 protein turns of transcription of PL and PR, turns off production of Cro lysogeny
- Cro turns off transcription of PRM, turns off production of CI lysis
- They repress each other’s transcription classical bistable genetic switch
76
What does N protein do ?
N protein is made first. Binds to certain sites on the genome where transcription is stopped, allowing for transcription to continue past termination sites, so a long RNA forms
Promoter PL early left operon
Promoter PR early right operon
- N – antiterminator, allows long transcripts of PL and PR, Cro inhibits this
77
What does intergrase do ?
if made = lysogeny
78
What enforces lysogeny ?
So intergrase is made – this allows for lysogeny, anti-cro, C1 – represses lysis, anti-Q removes Q (no transcription of late genes)
- These all enforce lysogeny
79
What is PRM ?
promoter of maintenance of lysogeny
- C111 is made which stabilizes the binding at the promoters to ensure lysogeny proteins continue to be made.
- Host proteases can destroy the C11/C111 complex so lytic is allowed, this leads to SOS response so the genome will be excised
o If destroyed the PL and PR can be used seen as there is no C1 repressor so Cro is in excess so lysis is occurred, C11 is made but not enough for lysogeny.
o Cro binds to PRM C1 can’t be made, DNA replication continues from O and P, bacteria becomes a phage factory
80
How are phages controlled ?
- Extracellular modulators
o Nucleases
o Proteases
o Ion chelating molecules (citrate, EDTA)
o Receptor availability
- Intracellular methods
o Restriction enzymes
o Immunity systems (CRISPR/Cas)
o Super-infection immunity
81
What are Bdellovibrio bacteriovorus and BALOs?
- Gram negative
- Prey on gram-negative bacteria
- Attach, penetrate, grows inside it, degrades the interior and matures
82
What are vampirococcus ?
- Gram negative
- Prey: chromatium spp. (freshwater purple sulfur bacteria)
- Attach, inges (injects enzymes into the host), sucks nutrients out and release
83
What is daptobacter ?
- Gram-negative
- Prey: chromatium spp. (freshwater purple sulfur bacteria)
84
Why are bacterial predators hard to study ?
Hard to culture
