Bacterial Genetics Flashcards
What is genetics?
The study of genomes and DNA/RNA, genome replication, gene expression, genetic variation & distribution
Describe the flow of genetic info when forming a protein
To form a protein, DNA transcribed into mRNA which is translated into a protein
How does replication error occur during evolution?
DNA polymerase can make errors
e,g, single nucleotide polymorphisms (SNPs)
What is a common plasmid transfer mechanism?
Bacterial Conjugation
Why is the study of bacterial genetics so significant?
- bacteria cause infection & are key components of
microbiome - important industrial organisms
- small single-copy genomes that are relatively simple &
easy to study
–> manipulating tools for genomes originate from
bacteria
What is the function of proteins?
Perform most cell functions including catalysis and synthesis of other structures therefore genome sequence controls all cell functions
How is bacterial transformation used in the lab?
Bacterial transformation is exploited in the lab to artificially move purified plasmids into bacteria
What is the significance of next generation sequencing?
e.g. Illumine benchtop machines
can sequence a bacterial isolate for £50 a day
- routine research laboratory
- introduction into microbiology diagnostic labs
Outline how plasmid transfer occurs
- Donor cells attaches to recipient cell via pillus which
draws cells together - Cells contact one another
- One of strand of plasmid DNA transfers to recipient
- The recipient synthesises a complementary strand to
become F+ cell
- the donor synthesises a complementary strand, restoring its complete plasmid
What are MGEs?
Mobile gene element that move around within genomes
How is the genome used in the lab?
New technologies leads to new understanding
whole genome sequencing + new computing power
Haemophillus influenza was the first bacterial genome sequenced
What is the significance of prophages?
The prophage can encode important virulence genes
e.g. cholera, diphtheria, botulism toxins & panton-
valentine leukocidin
What is CRISPR?
Clustered Regularly Interspaced Short Palindromic Repeats
- in 40% of bacteria
How does bacterial genome replication occur?
Replication is the first step in bacterial cell division
DNA polymerase catalyses the reaction to synthesise new genome
Requires primers and DNA template strand
How is genetic manipulation of eukaryotes carried out?
CRISPR is the most exciting new technology for eukaryotic cells gene editing and is a potential for treating genetic disorders e.g CF
What are bacteriophages?
MGE Viruses of bacteria
- can lyse & kill bacteria
- or genome sits in bacterial chromosomes (prophage)
What is the significance of bacterial gene regulation?
Bacteria are single celled organisms that are highly responsive to environmental triggers
e.g.
- nutrients, Oxygen, Iron
- Temperature, Bacterial pheromones, Mammalian cells &
hormones
Describe the bacterial genome
All DNA in a bacterial cell includes
- chromosomes - circular, single copy
- plasmids - autonomously replicating circular DNA
- prophage - viruses integrated into chromosomes
What is the consequence of genome replication errors?
Some errors can be fixed but errors can be accumulated
Errors can be advantageous, detrimental or neutral to the bacterial cell in a particular habitat
What is the significance of MGEs?
Horizontal transfer of MGEs
Many MGEs encode virulence, antimicrobial resistance or host specific genes.
Acquisition of MGEs leads to new bacterial variants with enhanced virulence or resistance in their host ranges
Describe the MRSA strain genome
2.9m base pairs in chromosomes
carries integrated prophage, transposons, pathogenicity islands and antimicrobial resistance elements
Encodes roughly 2800 genes
Outline how knockout is constructed
- Clone virulence gene into ‘suicide vector’ plasmid
- Clone antibiotic resistance marker into gene to disrupt
it - Transform it into bacterial cell
- Recombination via RecA (rare)
- Place onto agar with antibiotic and select for rare
isolate that has resistance marker
When are bacterial virulence factors expressed?
Many bacterial virulence factors are only expressed in vivo or in conditions mimicking in vivo
What does the genome determine?
Bacterial capabilities - can predict function by identifying patterns homology of known genes & motifs
What is the significance of studying bacterial gene functions?
Genes are necessary for phenotype and pathogenesis especially for complex virulence pathways involving more than one gene
Why are bacterial genomes manipulated?
To make tools for industrial production of proteins
To make tools for studying bacteria / gene function
Can all DNA be transferred into bacteria?
No not all DNA can be transferred into bacteria as Bacterial immunity protects itself from foreign DNA
Give examples of bacterial virulence factors being selectively expressed in vivo
Vibrio cholera expresses cholera toxin and pilin necessary for colonisation only in human intestinal tract
Corynebacterium diphtheriae only produces diphtheriae toxin in low iron conditions e.g. in vivo
Describe how gene expression is regulated via the Lac Operon
- Absence of lactose - lac repressor binds to operator,
blocking transcription - Presence of lactose - lactose binds to lac repressor,
releasing it from operator, allowing RNA Pol. to
proceed with transcription at a slow rate
- cAMP and CAP complex increases RNA pol. activity
- but have no effect in lactose absence
What is general transduction?
Temperate bacteriophages ‘accidentally’ packages host bacterial DNA or plasmids into phage particles and delivers it to new bacteria
What is the significance of RNA sequencing & transcriptional circuits?
Only genes important for survival & virulence are expressed
Gene regulation pathways that respond to in vivo signals are targets for therapeutics
How does restriction modification occur?
SRM molecule binds to DNA
R (restriction) digests
M (modification) methylates and protects DNA
What is the consequence of the bacteriophage mechanism?
- Genes encoded on phage begin forming proteins
- Proteins formed assemble structures into phage
particles - Phage particles formed suck up phage genome copies
into heads - Bacteria killed and phage is released
- Phage particles find a new host’s outside to latch onto
& infect DNA into new bacterial cell
That DNA can now circuarise and integrate into chromosomes
When is isogenic mutant pairs / knockouts used?
Used to study bacterial / gene function
Give examples of homologous regions in bacterial genomes
Putative genes
- encode proteins with predicted functions
- toxins, virulence factors, metabolic pathways
Gene regions with predicted functional regions
- transmembrane regions, ATP binding region
- start & stop codons
- RNA pol gene regulatory binding site
- Integrated mobile genetic elements
What are Plasmids?
Type of MGE that replicate autonomously and have circular DNA
Aren’t essential for host Bacteria
How do MGEs move between bacteria?
Bacterial Transformation
- release of DNA and antibiotic resistant gene into
recipient cell
Bacterial Transduction
- Release of phage from a phage-infected donor cell into
recipient cell
Bacterial conjugation
- Transposin plasmid from donor cell into recipient cell
What are the different sites present on plasmids?
- Origin of replication
- Antibiotic resistant gene
- selectable marker
- promoter
- 5’ primer site
- restriction sites
- inserted gene
- 3’ primer site
Outline how RNA sequencing is done in a lab
- Grow bacteria in conditions of interest
- Extract bacteria (extract bacterial mRNA & convert to
DNA) - Sequence DNA and quantitate numbers of each genes
transcript
What is a gene?
Hereditary protein / DNA template / base sequence with potential to be translated into a specific protein
Outline the bacteriophage mechanism
Induced by stress (UV light infection)
1. Phage genome pops out of chromosome & circularises
2. Circular phage pops back in chromosome
3. Replicates & makes many more copies
=> lysogenic phase
Which bacteria are plasmids used as cloning vectors for?
E. coli ampR
E. coli ori
LacZ
Outline the key features of DNA
- double helix of polynucleotide
- DNA sequence composed of 4 nucleotide bases A,T,G,C
- form complementary base pairs via hydrogen bonds AT
and GC
Give an example of how genomes are manipulated
Cloning genes
- transfer of insulin genes via recombinant plasmid
- Digest plasmid with restriction enzyme
- Use same restriction enzyme to cut DNA strands
==> forms sticky ends - Seal gap with ligase enzyme
Which region on the plasmid is highly targeted by multiple restriction enzymes?
The cloning region
