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Fundamentals of Pathology > W12 Bacterial genetics > Flashcards

W12 Bacterial genetics Flashcards

1
Q

Genetics

A

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

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2
Q

. Bacterial Genome :

A

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)

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3
Q

Bacterial Genome example

A

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

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4
Q

The genome determines

A

what the bacteria is capable of.

Bacteria can only do what their genome allows them to do

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5
Q

Predicting cell function examples

A

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

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6
Q

Predicting cell function - theory

A

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

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7
Q

The first bacterial genome sequence

A

Haemophilus influenza

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8
Q

next generation sequencing technology

A

(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

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9
Q

How do bacteria vary?

A

Comparison of the first 5 S. aureus whole genome sequences

chromosomes are represented linearly in grey, and homology between genomes is red

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10
Q

DNA polymerase can make errors.

A

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.

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11
Q

The tree is based on

A

The tree is based on all of the SNP differences in each sequenced isolate (1000s in total)

Colours are geography of isolates

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12
Q

WGS to identify

A

identify specific mutations for evolutionary success
HA-MRSA CC22 clone causes >75% of UK MRSA

Geography/location by colour

Estimate year of evolutionary change

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13
Q

HA-MRSA CC22

A

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…

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14
Q

Mobile Genetic Elements (MGE)

A

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

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15
Q

How MGEs move between bacteria

A

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

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16
Q

Plasmids are a type of MGE

A

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

17
Q

MGEs: Bacteriophage – viruses of bacteria

A

Can lyse and kill bacteria
OR
Genome sits in the bacterial
chromosome (prophage)

18
Q

Prophage

A

Prophage can encode important virulence genes

e.g. Cholera toxin
Diphtheria toxin
Botulism toxin
Panton-Valentine leukocidin

19
Q

Generalized Transduction

A

temperate bacteriophage
‘accidentally’ packages host bacterial DNA or plasmids into phage particles
and delivers it to new bacteria

20
Q

Restriction Modification (RM)

A

specificity subunit

targets specific palindrome, eg. CGATCG

21
Q

Can all DNA transfer into any bacteria?

A

NO.

Bacterial immunity to protect itself from foreign DNA, e.g. phage

22
Q

Gene Expression

A

Not all genes are expressed all of the time

Example of DNA transfer

23
Q

Importance of bacterial gene regulation

A

Bacteria are single-celled organisms that are highly responsive to environmental triggers.

Triggers include :
nutrients
oxygen
iron
temperature
bacterial pheromones
mammalian cells, hormones
Etc.
24
Q

Vibrio cholera

A

Vibrio cholera expresses
cholera toxin and pilin necessary for colonisation
only in the human intestinal tract

25
Q

Corynebacterium diphtheriae

A

Corynebacterium diphtheriae

only produces diphtheria toxin in low iron conditions such as those found in vivo.

26
Q

Many bacterial virulence factors are only

A

Many bacterial virulence factors are only expressed in vivo, or in conditions mimicking those found in vivo

27
Q

Which genes are expressed in vivo?

A

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.

28
Q

In vivo

A

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

29
Q

Manipulating genomes

A

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

30
Q

Plasmids as cloning vectors

A

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

31
Q

Any DNA can be cloned

A

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

32
Q

X-gal

A

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

33
Q

Transform the cloned gene on a vector into bacteria

A

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

34
Q

Is a gene necessary or essential for a phenotype or for pathogenesis?

A

Very powerful tool, especially for complex virulence pathways involving more than one gene.

35
Q

Construction of a knockout

A

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

36
Q

Genetic manipulation of complex eukaryotic cells

A

CRISPR is the most exciting new technology for gene editing of eurkaryotic cells, with potential for treating genetic disorders such as cystic fibrosis

37
Q

CRISPR

A

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

Fundamentals of Pathology (23 decks)

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