W12 Bacterial genetics Flashcards by Sharjeel Ahmad

Genetics

The study of genomes and DNA/RNA, genome replication, gene expression,
genetic variation and distribution

How well did you know this?

Not at all

Perfectly

. 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)

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

The genome determines

what the bacteria is capable of.

Bacteria can only do what their genome allows them to do

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

The first bacterial genome sequence

Haemophilus influenza

How well did you know this?

Not at all

Perfectly

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 well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

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.

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

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…

How well did you know this?

Not at all

Perfectly

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 well did you know this?

Not at all

Perfectly

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

How well did you know this?

Not at all

Perfectly

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 ```