W12 Bacterial genetics Flashcards
Genetics
The study of genomes and DNA/RNA, genome replication, gene expression,
genetic variation and distribution
. Bacterial Genome :
All of the DNA in a bacterial cell.
Includes
- Chromosome (single copy, circular, essential for life)
- Mobile genetic elements (MGE), such as
plasmids (autonomously replicating circular DNA)
Prophage (viruses integrated into the chromosome)
Bacterial Genome example
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
The genome determines
what the bacteria is capable of.
Bacteria can only do what their genome allows them to do
Predicting cell function examples
For example -
Putative genes encoding proteins with predicted functions (eg. toxins, virulence factors, metabolic pathways)
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
Predicting cell function - theory
From a whole genome sequence, we can predict cell function. We identify patterns and homology to known genes and motifs
We can Identify ‘missing’ genes or unexpected genes (eg. Campylobacter jejuni capsule)
However, many putative ‘genes’ have no known or predicted function
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
How do bacteria vary?
Comparison of the first 5 S. aureus whole genome sequences
chromosomes are represented linearly in grey, and homology between genomes is red
DNA polymerase can make errors.
e.g. single nucleotide polymorphisms (SNPs).
Some errors will be fixed. Errors can accumulate.
Errors can be advantageous, detrimental or neutral to the bacterial cell in a particular
habitat
→ Evolution and survival of the fittest.
The tree is based on
The tree is based on all of the SNP differences in each sequenced isolate (1000s in total)
Colours are geography of isolates
WGS to identify
identify specific mutations for evolutionary success
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…
Mobile Genetic Elements (MGE)
Horizontal transfer of MGE
Many MGEs encode virulence, antimicrobial resistance or host-specific genes
Acquisition of MGEs can lead to new bacterial variants with enhanced virulence or resistance or host range
How MGEs move between bacteria
Bac transformation:
Donor cell releases DNA (w/antibiotic-resistance gene) into recipient cell
Bac transduction:
Phage-infected donor cell releases phage to recipient cell
Bac conjugation:
Pilus joined onto recipient cell by DNA P
F plasmid transfers DNA w/relaxasomeTransferasome
So now both have F plasmid + pilus
Transposon in donor cell to recipient cell
Plasmids are a type of MGE
Autonomously replicating, circular DNA.
2 kb to >100 kb.
Not essential for the host bacteria
Antimicrobial resistance genes in pathogenic bacteria are often carried on plasmids.
Easy to manipulate in the laboratory - plasmid map
MGEs: Bacteriophage – viruses of bacteria
Can lyse and kill bacteria
OR
Genome sits in the bacterial
chromosome (prophage)
Prophage
Prophage can encode important virulence genes
e.g. Cholera toxin
Diphtheria toxin
Botulism toxin
Panton-Valentine leukocidin
Generalized Transduction
temperate bacteriophage
‘accidentally’ packages host bacterial DNA or plasmids into phage particles
and delivers it to new bacteria
Restriction Modification (RM)
specificity subunit
targets specific palindrome, eg. CGATCG
Can all DNA transfer into any bacteria?
NO.
Bacterial immunity to protect itself from foreign DNA, e.g. phage
Gene Expression
Not all genes are expressed all of the time
Example of DNA transfer
Importance of bacterial gene regulation
Bacteria are single-celled organisms that are highly responsive to environmental triggers.
Triggers include : nutrients oxygen iron temperature bacterial pheromones mammalian cells, hormones Etc.
Vibrio cholera
Vibrio cholera expresses
cholera toxin and pilin necessary for colonisation
only in the human intestinal tract
Corynebacterium diphtheriae
Corynebacterium diphtheriae
only produces diphtheria toxin in low iron conditions such as those found in vivo.
Many bacterial virulence factors are only
Many bacterial virulence factors are only expressed in vivo, or in conditions mimicking those found in vivo
Which genes are expressed in vivo?
Presumably, only genes important for survival and virulence are expressed, so it is a marker of their importance.
Gene regulation pathways that respond to in vivo signals are targets for therapeutics.
In vivo
Grow bac in conditions of interest
Extract bac
Extract bacterial DNA
Convert mRNA to DNA
Sequence the DNA and quantitate the numbers of transcripts of each gene
Manipulating genomes
Why?
To make tools for industrial productions of proteins
To make tools for studying bacteria or gene function
example - cloning genes by artificial ligation of DNA
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
Any DNA can be cloned
Isolate plasmid (vector) DNA and human DNA
Insert human DNA into plasmids
Cut both DNAs w/same restriction enzyme
Mix the DNAs; they join by base pairing (some plasmids; like this one, 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
Transform the cloned gene on a vector into bacteria
Put plasmids into lacZ^- bacteria by transformation
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
Is a gene necessary or essential for a phenotype or for pathogenesis?
Very powerful tool, especially for complex virulence pathways involving more than one gene.
Construction of a knockout
Clone virulence gene into “suicide vector” plasmid
(no ori for
replication)
Clone antibiotic resistance marker into the gene to disrupt it
Transform it into bacterial cell
Recombination via
RecA protein, rare
Plate onto agar with antibiotic and
select for the rare isolate that has the resistance marker
Genetic manipulation of complex eukaryotic cells
CRISPR is the most exciting new technology for gene editing of eurkaryotic cells, with potential for treating genetic disorders such as cystic fibrosis
CRISPR
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
