Bacterial Genetics Flashcards
Describe the structure of the bacterial genome.
Genetics - The study of genomes and DNA/RNA, genome replication, gene expression,
genetic variation and distribution
All of the DNA in a bacterial cell in the bacterial genome.
It includes:
- chromosomes (single copy, circular, essential for life)
- mobile genetic elements (MGE) such as plasmids (autonomously replicating circular DNA) and prophages (viruses integrated into the chromosome)
EXAMPLE OF A BACTERIAL GENOME
e.g. MRSA strain 252 chromosome is 2.9 million bp, and carries integrated prophage, transposons, pathogenicity islands, antimicrobial resistance elements, etc.
Encodes ~ 2800 genes
MGEs
- Genetic material that can move around within a genome.
- Many MGEs encode virulence, antimicrobial resistance of host-significant genes. Acquisition of MGEs by horizontal transfer can lead to new bacterial variants with enhanced virulence, resistance or host range.
- MGEs are where most differences between different strains of genomes found
How can we utilise a bacterial genome?
Genome determines what the bacteria is capable of. Bacteria can only do what their genome allows them to do.
From a whole genome sequence, we can predict cell function. We identify patterns and homology to known genes and motifs. Bacteria are important causes of infection as well as key components of the microbiome. Bacteria are important industrial organisms. Bacteria have small, single-copy genomes that are relatively simple and easy
to study. Therefore, most of our fundamental understanding of genetics comes from
studying bacteria.
Putative Gene: It’s a segment of DNA whose protein, and its function, is not known, but based on its open reading frame, it is believed to be a gene (eg. toxins, virulence factors, metabolic pathways). We can Identify ‘missing’ genes or unexpected genes (eg. Campylobacter jejuni capsule). However, many putative ‘genes’ have no known or predicted function
Gene regions with predicted functional regions (eg. Transmembrane region, ATP binding region)
Start and stop regions for those genes
Regions where gene regulators and RNA polymerase bind
Integrated mobile genetic elements
Describe bacterial replication, and how it can lead to errors and evolution.
Replication of bacterial DNA is the first step in cell division. DNA polymerase enzymes catalyse the reaction to synthesise the new genome.
Sometimes, the DNA polymerase can make errors during replication, such as single nucleotide polymorphisms (SNPs). Some errors will be fixed, while other not. They can accumulate.
Some errors can be advantageous, detrimental or neutral to the bacterial cell in a particular habitat. This is how bacterial cells evolve, through survival of the fittest.
We can use whole genome sequencing to reveal the evolutionary history of a species and how it is spreading.
- Identify specific mutations for evolutionary success e.g HA-MRSA CC22 clone causes >75% of UK MRSA
- Geography/location by colour
- Estimate year of evolutionary change
HA-MRSA CC22
- originated in the UK Midlands
- Emergence correlates with pre-licencing trials of ciprofloxacin (a type of fluoroquinolone antibiotic) prior to 1987
- Subsequent spread throughout Europe, Australia, Singapore…
Describe the three ways in which MGEs are transferred across bacteria.
1) BACTERIAL TRANSFORMATION:
The donor cell has usually lysed and had its DNA chopped up. This DNA is released (with the desired gene) into the environment and taken up by the recipient cell. The recipient cell may incorporate that DNA into its DNA, exchanging its genes for the other bacterium’s genes.
2) BACTERIAL TRANSDUCTION:
Here, bacteriophages (viruses of bacteria) are induced, causing either infection of the bacteria by viruses or popping out the virus in the genome. This synthesises phage particles. They then package phage DNA into these particles and release them by lysis (usually by killing the cell) through the lytic pathway. The phage particles then inject their DNA/genes into the recipient cell.
LYTIC PATHWAY - When a virus starts to dictate what happens in the bacteria, so it makes lots of copies of itself
3) BACTERIAL CONJUGATION:
This is how big plasmids can move about. Plasmids encode for the conjugation ability. These plasmids encode genes needed to make a pore or pillus (a tube) that connects the two bacteria (direct contact). The plasmid then replicates on its own and tranfers across from the donor to recipient cell.
Describe plasmids and bacteriophages as MGEs.
PLASMIDS
Plasmids are a type of MGE. They are autonomously replicating circular DNA.
They are not essential for the host bacterium. Antimicrobial resistance genes in pathogenic bacteria are normally carried on plasmids. Have restriction sites where DNA can be added.
BACTERIOPHAGES
Bacteriophages are viruses of bacteria.
They can either lyse bacteria or their genome just sits in the bacterial chromosome (prophage). A prophage can encode important virulence genes.
PROPHAGES
e.g. Cholera toxin
Diphtheria toxin
Botulism toxin
Panton-Valentine leukocidin
What is generalised transduction?
When a temperate bacteriophage ‘accidentally’ packaged host bacterial DNA or plasmids into phage particles and delivers it to new bacteria.
Give two examples of bacterial immunity to protect themselves from foreign DNA (most importantly phage).
- Restriction Modification
Instead of protecting themselves from the phage particles, they protect themselves from the phage DNA. One mechanism that can be used is called Restriction-Modification (RM).
Restriction enzymes are made up of several units. In this example, the enzyme has a specificity subunit that binds to a specific palindromic sequence of DNA. The restriction subunit will restrict/cut through the DNA (both strands) at the specific site. There is a variant of the enzyme that doesn’t have the restriction subunit, only made up of the modification and specificity subunit. This variant binds to the same region of the DNA, and methylates the DNA sequence, which protects it from the restrictive variant of the enzyme - protection from self-digestion.
- 3 subunits in total
- CRISPR gene editing
A phage injects its dsDNA into the bacteria.
An enzyme, CAS, will bind to the dsDNA and turn it into little bits of DNA integrated into the chromosome. These genes get integrated downstream of genes known as CRISPR.
The integrated genes can be transcribed into mRNA, and those mRNA fragments then get modified by other proteins into processed cRNAs.
These cRNAs then combine with a third protein to form the CAS cRNA complex.
This complex can now bind to the dsDNA because of the specificity of the sequence. The third enzyme in the complex digests the DNA, deactivating it.
CRISPR Background
- CRISPR is the most exciting new technology for gene editing of eurkaryotic cells, with potential for treating genetic disorders such as cystic fibrosis
- clustered regularly interspaced short palindromic repeats
- Found in 40% of bacteria
- Adaptive immunity or protection from foreign DNA or MGE previously encountered
- CRISPR technology has now been harnessed to genetically modify eukaryotic DNA at
specific target sequences
How is bacterial gene expression regulated?
Not all genes are expressed all of the time.
Also, we have regulators that can interfere with how RNA polymerase binds.
A classic example of gene regulation by a repressor protein is the lac operon.
WHEN LACTOSE PRESENT
- Repressor protein converted to inactive form
- Does not bind to operator region
- RNA Polymerase can then move along and past the operator and transcribe and translate the lacZ, lacY and lacA genes
WHEN LACTOSE ABSENT
- Repressor remains bound to operator region - operator becomes repressed
- Prevents moving of RNA Polymerase along the operon
- Prevents transcription and translation of genes
SIGNIFICANCE OF GENE EXPRESSION REGULATION
Bacteria are single-celled organisms that are highly responsive to environmental triggers.
Triggers include :
nutrients
oxygen
iron
temperature
bacterial pheromones - the way in which bacteria communicate with one another
mammalian cells, hormones
Etc.
EXAMPLES
Vibrio cholera expresses cholera toxin and pilin necessary for colonisation
- only in the human intestinal tract.
Corynebacterium diphtheria only produces diphtheria toxin in low iron conditions such as those found in vivo.
Why do we manipulate genomes?
- to make tools for the industrial production of proteins
- to make tools for studying bacteria or gene function
example - cloning genes by artificial ligation of DNA
Describe how we can clone genes using bacteria.
Take a plasmid and cut it at a specific sequence using a restriction enzyme. We then cut a piece of a human gene with the same restriction enzyme. This leaves bits of overhanging, linker DNA (sticky ends), which is used to bind the human DNA into a recombinant plasmid (via ligase).
The recombinant plasmid is then moved into a bacterium (most commonly used, E.Coli), which will then multiply and make lots of the protein encoded in the human gene.
Give a real-life scenario where technological advancements have led to greater understanding of the bacterial genome.
BACTERIAL GENOME SEQUENCING
The first bacterial genome sequence
- Haemophilus influenza
Next generation sequencing technology
- (eg. Illumina benchtop machines)
- can sequence a bacterial isolate for approx. £50 in one day. Routine research laboratory method (almost)
- introduction into microbiology diagnostic labs is just beginning
- Allows variation between bacteria to be viewed e.g comparison of of the first 5 S. aureus genome sequences
Can all DNA transfer into any bacteria?
NO.
Bacterial immunity to protect itself from foreign DNA, e.g. phage
Outline the nature of in vivo gene expression
Many bacterial virulence factors are only expressed in vivo, or in conditions mimicking those found in vivo. In vivo conditions are found in the host i.e the body.
Presumably, only genes important for survival and virulence are expressed in vivo, so it is a marker of their importance. Gene regulation pathways that respond to in vivo signals are targets for therapeutics.
In vivo transcriptional genomics
- Used to change the conditions in bacteria depending on what you’re interested in
- Grow and culture bacteria in conditions of interest
- Extract bacteria
- Extract bacterial mRNA
- Convert mRNA to DNA
- Sequence the DNA and quantitate the numbers of transcripts of each gene
Outline the process of genetic cloning
Plasmids as cloning vectors
- lacZ for selection of plasmids with insert
- Cloning region = target sites for multiple restriction enzymes
- Replication in E. coli ori
- Selection for plasmid presence in E. coli ampR
DNA Cloning - Any DNA can be cloned
- Isolate plasmid (vector) DNA and human DNA (containing gene of interest)
- Insert human DNA into plasmids
- Cut both DNAs w/same restriction enzyme
- Mix the DNAs; they join by base pairing (some plasmids join w/the gene of interest) - sticky ends
- Add DNA ligase to bond covalently
X-gal is a sugar that is added to the agar and turns the colonies blue when the bacteria carrying the lacZ gene product can break it down
PROCESS OF transforming the cloned gene on a vector into bacteria
Only those bacteria carrying a plasmid will grow on ampicillin
- Put plasmids into lacZ^- bacteria by transformation after PROCESS OF DNA CLONING
- Clone cells
- Plate cells onto medium w/ampicillin + X-gal
- Identify clones of cells containing recombinant plasmids by their ability to grow in presence of ampicillin and their white color
- Identify clone carrying gone of interest
CHECK which clones work correctly
- Culture the cells in conditions where only the gene you’ve taken up could grow (add the antibiotic if you’ve added an antibiotic resistant gene)
How are knockouts constructed?
KNOCKOUTS - When one of the organism’s genes are made inoperative (knocked out)
Clone virulence gene into “suicide vector” plasmid
Clone antibiotic resistance marker into the gene to disrupt it
Transform into bacterial cell
Recombination (via RecA protein, rare)
Plate onto agar with antibiotic and select for the rare isolate that has the resistance marker
