Bacteria Genetics Flashcards

1
Q

Define genetics

A

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

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2
Q

Why study bacterial genetics?

A
  • 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
  • Many exciting tools for manipulating genomes also come from bacteria.
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3
Q

What does the bacteria DNA include?

A

Chromosome (single copy, circular, essential for life)

▪ Mobile genetic elements (MGE), such as

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4
Q

What are the two types of mobile genetic elements?

A

● Plasmids (autonomously replicating circular DNA)

● Prophage (viruses integrated into the chromosome)

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5
Q

Describe the genome of MRSA

A

Chromosome is 2.9 million bp
o Carries integrated prophage, transposons, pathogenicity islands, antimicrobial resistance elements
o Encodes roughly 2800 genes

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6
Q

Putative genes:

A

encoding proteins with predicted functions (e.g. toxins, virulence factors, metabolic pathways)

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7
Q

What is SNP?

A

single nucleotide polymorphism- DNA polymerase error

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8
Q

What can errors be?

A

advantageous, detrimental or neutral

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9
Q

What is mobile genetics elements?

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 enhances virulence or resistance or host range.
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10
Q

How MGEs move between bacteria?

A

Conjugation- The process that exchanges genetic information between two bacteria through a direct cell-to-cell contact is known as conjugation.

Transduction- The process of transferring bacterial DNA from one cell to another cell by means of bacteria-infecting viruses is known as transduction.

Transformation- The process of taking the free fragments of DNA by bacteria from the surrounding medium is known as transformation.

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11
Q

Recall the parts the structure of plasmids

A

Selective marker
promotor
5’primer site

inserted gene
restriction site

3’ primer site

origin of replication

antibiotic resistance gene

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12
Q

Where are antimicrobial resistance genes normally found in the bacteria

A

plasmid

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13
Q

Which type of exchange is most used by plasmids?

A

conjugation

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14
Q

How is transformation used in the liver?

A

artificially move purified plasmids into bacteria

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15
Q

How do bacteriophage work?

A

The phage genome pops out of the chromosome and circularises
▪ The circular phage can pop back in to the chromosome and it can go on to replicate and make many copes
Lysogenic phage

integrated into the bacterial chromosome. Phage in this state is called prophage
Viral DNA being the part of bacterial chromosome passes to each daughter cell in all succeeding generations.
Sometimes, nevertheless, the viral DNA gets detached from the host’s chromosome and the lytic cycle starts.

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16
Q

What can alternatively happen after the phage circularises?

A

the phage itself replicates like a plasmid and makes lots of copies of itself
▪ Then the genes encoded on the phage start to make proteins
▪ Those proteins start to make all the structures to make a phage particle
▪ The phage particles then get made and many copies of the phage genome get sucked up into those phage particle heads
▪ The bacteria is then killed and the phage is released
▪ The phage particles find new hosts and latch on the outside and they inject the DNA into the new bacterial cell.
● That DNA can circularise and then integrate into the chromosome

17
Q

What is generalised transduction?

A
  • Temperate bacteriophage

- “Accidentally” packages host bacterial DNA or plasmids into phage particles and delivers it to new bacteria

18
Q

Can all DNA transfer into any bacteria?

A

No. o Bacterial immunity to protect itself from foreign DNA, e.g. phage
- Restriction modification (RM) o S – specificity subunit o R – restriction (digests/cuts)

o M – modification (methylates and protects)

19
Q

Where will vibrio cholera express cholera toxin and pilin?

A

human intestinal tract

20
Q

Where will corynebacterium diptheriae produce diptheria toxin

A

low iron conditions

21
Q

Which genes are expressed in vivo?

A

only genes important for survival and virulence are expressed

extract bacteria
sequence the DNA and quantitate the number of transcript of each gene

22
Q

Manipulating genomes - Why?

A
  • To make tools for industrial production of proteins

- To make tool for studying bacteria or gene function o E.g. cloning gene

23
Q

Cloning, How?

A
  • Digest the plasmid with a restriction enzyme
    o It cuts one strand on one side of the specific sequence
    o Cuts the other strand at a lightly different location
    ▪ End up with a “sticky end”
    ▪ We cut the DNA we want to implement using the same restriction enzyme so that we end up with a complementary “sticky end”
    ▪ Seal the gap with ligase
24
Q

Plasmids as cloning vectors

A
  1. selection for plasmid presence in E. coli ampR
    2, Cloning region= target sites for multiple restriction enzymes
  2. lacZ for selection of plasmids with insert
25
Q

Describe CRISPR?

A

gene editing of eukaryotic cells, with potential for treating genetic disorders such as cystic fibrosis
- CRISPR – clustered regularly interspaced short palindromic repeats o Found in 40% of bacteria o Adaptive immunity or protection
from foreign DNA or MGE previously encountered o CRISPR technology has now been
harnessed to genetically modify eukaryotic DNA at specific target sequences

26
Q

Example of lac operon gene expression

A

Lactose absent:

  1. repressor bound to operator, operon
  2. RNA polymerase cannot move down the lac operon

Lactose present:

  1. repressor is converted to inactive form which unbinds from operator
  2. RNA can move along and transcribe lac Z, Y, A
27
Q

Construction of a knowckout

A

. Clone virulence gene into “suicide vector” plasmid (no ori for replication)
2. Clone antibiotic resistance marker into the gene to disrupt it
3. Transform it into bacterial cell
4. Recombination via
RecA protein, rare
5. . Plate onto agar with antibiotic and
select for the rare isolate that has the resistance marker

28
Q

DNA cloning

A
  1. Isolate plasmid DNA and human DNA
  2. Insert human DNA into plasmid by cutting both DNA with same restriction enzyme. DNAs join by base pairing. DNA ligase bonds them covalently
  3. Put plasmids into lacZ- bacteria by transformation
  4. Cone cells: plate cells onto medium with ampicillin and X-gal
  5. Identify clones of cells containing recombinant plasmids by their ability to grow in ampiciin and their colour
  6. Identify clone carrying gene of interest
29
Q

What is X-gal?

A

X-gal is a sugar that is added to the agar. If the bacteria carry an intact lacZ gene, the LacZ protein will break down the X-gal and turn the colonies blue.