Bacteria Genetics

Question 1

Define genetics

Answer 1

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

Question 2

Why study bacterial genetics?

Answer 2

• 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.

Question 3

What does the bacteria DNA include?

Answer 3

Chromosome (single copy, circular, essential for life)

▪ Mobile genetic elements (MGE), such as

Question 4

What are the two types of mobile genetic elements?

Answer 4

● Plasmids (autonomously replicating circular DNA)

● Prophage (viruses integrated into the chromosome)

Question 5

Describe the genome of MRSA

Answer 5

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

Question 6

Putative genes:

Answer 6

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

Question 7

What is SNP?

Answer 7

single nucleotide polymorphism- DNA polymerase error

Question 8

What can errors be?

Answer 8

advantageous, detrimental or neutral

Question 9

What is mobile genetics elements?

Answer 9

• 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.

Question 10

How MGEs move between bacteria?

Answer 10

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.

Question 11

Recall the parts the structure of plasmids

Answer 11

Selective marker
promotor
5’primer site

inserted gene
restriction site

3’ primer site

origin of replication

antibiotic resistance gene

Question 12

Where are antimicrobial resistance genes normally found in the bacteria

Answer 12

plasmid

Question 13

Which type of exchange is most used by plasmids?

Answer 13

conjugation

Question 14

How is transformation used in the liver?

Answer 14

artificially move purified plasmids into bacteria

Question 15

How do bacteriophage work?

Answer 15

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.

Question 16

What can alternatively happen after the phage circularises?

Answer 16

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

Question 17

What is generalised transduction?

Answer 17

• Temperate bacteriophage

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

Question 18

Can all DNA transfer into any bacteria?

Answer 18

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)

Question 19

Where will vibrio cholera express cholera toxin and pilin?

Answer 19

human intestinal tract

Question 20

Where will corynebacterium diptheriae produce diptheria toxin

Answer 20

low iron conditions

Question 21

Which genes are expressed in vivo?

Answer 21

only genes important for survival and virulence are expressed

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

Question 22

Manipulating genomes - Why?

Answer 22

• To make tools for industrial production of proteins

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

Question 23

Cloning, How?

Answer 23

• 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

Question 24

Plasmids as cloning vectors

Answer 24

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

Question 25

Describe CRISPR?

Answer 25

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

Question 26

Example of lac operon gene expression

Answer 26

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

Question 27

Construction of a knowckout

Answer 27

. 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

Question 28

DNA cloning

Answer 28

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 5. Identify clone carrying gene of interest

Question 29

What is X-gal?

Answer 29

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.