Bacterial Genetics

Question 1

What is genetics?

Answer 1

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

Question 2

Describe the flow of genetic info when forming a protein

Answer 2

To form a protein, DNA transcribed into mRNA which is translated into a protein

Question 3

How does replication error occur during evolution?

Answer 3

DNA polymerase can make errors

e,g, single nucleotide polymorphisms (SNPs)

Question 4

What is a common plasmid transfer mechanism?

Answer 4

Bacterial Conjugation

Question 5

Why is the study of bacterial genetics so significant?

Answer 5

• bacteria cause infection & are key components of
  microbiome
• important industrial organisms
• small single-copy genomes that are relatively simple &
  easy to study
  –> manipulating tools for genomes originate from
  bacteria

Question 6

What is the function of proteins?

Answer 6

Perform most cell functions including catalysis and synthesis of other structures therefore genome sequence controls all cell functions

Question 7

How is bacterial transformation used in the lab?

Answer 7

Bacterial transformation is exploited in the lab to artificially move purified plasmids into bacteria

Question 8

What is the significance of next generation sequencing?

Answer 8

e.g. Illumine benchtop machines
can sequence a bacterial isolate for £50 a day
- routine research laboratory
- introduction into microbiology diagnostic labs

Question 9

Outline how plasmid transfer occurs

Answer 9

1. Donor cells attaches to recipient cell via pillus which
   draws cells together
2. Cells contact one another
3. One of strand of plasmid DNA transfers to recipient
4. The recipient synthesises a complementary strand to
   become F+ cell
   - the donor synthesises a complementary strand, restoring its complete plasmid

Question 10

What are MGEs?

Answer 10

Mobile gene element that move around within genomes

Question 11

How is the genome used in the lab?

Answer 11

New technologies leads to new understanding
whole genome sequencing + new computing power
Haemophillus influenza was the first bacterial genome sequenced

Question 12

What is the significance of prophages?

Answer 12

The prophage can encode important virulence genes
e.g. cholera, diphtheria, botulism toxins & panton-
valentine leukocidin

Question 13

What is CRISPR?

Answer 13

Clustered Regularly Interspaced Short Palindromic Repeats

- in 40% of bacteria

Question 14

How does bacterial genome replication occur?

Answer 14

Replication is the first step in bacterial cell division
DNA polymerase catalyses the reaction to synthesise new genome
Requires primers and DNA template strand

Question 15

How is genetic manipulation of eukaryotes carried out?

Answer 15

CRISPR is the most exciting new technology for eukaryotic cells gene editing and is a potential for treating genetic disorders e.g CF

Question 16

What are bacteriophages?

Answer 16

MGE Viruses of bacteria

• can lyse & kill bacteria
• or genome sits in bacterial chromosomes (prophage)

Question 17

What is the significance of bacterial gene regulation?

Answer 17

Bacteria are single celled organisms that are highly responsive to environmental triggers
e.g.
- nutrients, Oxygen, Iron
- Temperature, Bacterial pheromones, Mammalian cells &
hormones

Question 18

Describe the bacterial genome

Answer 18

All DNA in a bacterial cell includes

• chromosomes - circular, single copy
• plasmids - autonomously replicating circular DNA
• prophage - viruses integrated into chromosomes

Question 19

What is the consequence of genome replication errors?

Answer 19

Some errors can be fixed but errors can be accumulated

Errors can be advantageous, detrimental or neutral to the bacterial cell in a particular habitat

Question 20

What is the significance of MGEs?

Answer 20

Horizontal transfer of MGEs
Many MGEs encode virulence, antimicrobial resistance or host specific genes.
Acquisition of MGEs leads to new bacterial variants with enhanced virulence or resistance in their host ranges

Question 21

Describe the MRSA strain genome

Answer 21

2.9m base pairs in chromosomes
carries integrated prophage, transposons, pathogenicity islands and antimicrobial resistance elements
Encodes roughly 2800 genes

Question 22

Outline how knockout is constructed

Answer 22

1. Clone virulence gene into ‘suicide vector’ plasmid
2. Clone antibiotic resistance marker into gene to disrupt
   it
3. Transform it into bacterial cell
4. Recombination via RecA (rare)
5. Place onto agar with antibiotic and select for rare
   isolate that has resistance marker

Question 23

When are bacterial virulence factors expressed?

Answer 23

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

Question 24

What does the genome determine?

Answer 24

Bacterial capabilities - can predict function by identifying patterns homology of known genes & motifs

Question 25

What is the significance of studying bacterial gene functions?

Answer 25

Genes are necessary for phenotype and pathogenesis especially for complex virulence pathways involving more than one gene

Question 26

Why are bacterial genomes manipulated?

Answer 26

To make tools for industrial production of proteins | To make tools for studying bacteria / gene function

Question 27

Can all DNA be transferred into bacteria?

Answer 27

No not all DNA can be transferred into bacteria as Bacterial immunity protects itself from foreign DNA

Question 28

Give examples of bacterial virulence factors being selectively expressed in vivo

Answer 28

Vibrio cholera expresses cholera toxin and pilin necessary for colonisation only in human intestinal tract Corynebacterium diphtheriae only produces diphtheriae toxin in low iron conditions e.g. in vivo

Question 29

Describe how gene expression is regulated via the Lac Operon

Answer 29

1. Absence of lactose - lac repressor binds to operator, blocking transcription 2. Presence of lactose - lactose binds to lac repressor, releasing it from operator, allowing RNA Pol. to proceed with transcription at a slow rate - cAMP and CAP complex increases RNA pol. activity - but have no effect in lactose absence

Question 30

What is general transduction?

Answer 30

Temperate bacteriophages 'accidentally' packages host bacterial DNA or plasmids into phage particles and delivers it to new bacteria

Question 31

What is the significance of RNA sequencing & transcriptional circuits?

Answer 31

Only genes important for survival & virulence are expressed Gene regulation pathways that respond to in vivo signals are targets for therapeutics

Question 32

How does restriction modification occur?

Answer 32

SRM molecule binds to DNA R (restriction) digests M (modification) methylates and protects DNA

Question 33

What is the consequence of the bacteriophage mechanism?

Answer 33

1. Genes encoded on phage begin forming proteins 2. Proteins formed assemble structures into phage particles 3. Phage particles formed suck up phage genome copies into heads 4. Bacteria killed and phage is released 5. Phage particles find a new host's outside to latch onto & infect DNA into new bacterial cell That DNA can now circuarise and integrate into chromosomes

Question 34

When is isogenic mutant pairs / knockouts used?

Answer 34

Used to study bacterial / gene function

Question 35

Give examples of homologous regions in bacterial genomes

Answer 35

Putative genes - encode proteins with predicted functions - toxins, virulence factors, metabolic pathways Gene regions with predicted functional regions - transmembrane regions, ATP binding region - start & stop codons - RNA pol gene regulatory binding site - Integrated mobile genetic elements

Question 36

What are Plasmids?

Answer 36

Type of MGE that replicate autonomously and have circular DNA Aren't essential for host Bacteria

Question 37

How do MGEs move between bacteria?

Answer 37

Bacterial Transformation - release of DNA and antibiotic resistant gene into recipient cell Bacterial Transduction - Release of phage from a phage-infected donor cell into recipient cell Bacterial conjugation - Transposin plasmid from donor cell into recipient cell

Question 38

What are the different sites present on plasmids?

Answer 38

- Origin of replication - Antibiotic resistant gene - selectable marker - promoter - 5' primer site - restriction sites - inserted gene - 3' primer site

Question 39

Outline how RNA sequencing is done in a lab

Answer 39

1. Grow bacteria in conditions of interest 2. Extract bacteria (extract bacterial mRNA & convert to DNA) 3. Sequence DNA and quantitate numbers of each genes transcript

Question 40

What is a gene?

Answer 40

Hereditary protein / DNA template / base sequence with potential to be translated into a specific protein

Question 41

Outline the bacteriophage mechanism

Answer 41

Induced by stress (UV light infection) 1. Phage genome pops out of chromosome & circularises 2. Circular phage pops back in chromosome 3. Replicates & makes many more copies => lysogenic phase

Question 42

Which bacteria are plasmids used as cloning vectors for?

Answer 42

E. coli ampR E. coli ori LacZ

Question 43

Outline the key features of DNA

Answer 43

- double helix of polynucleotide - DNA sequence composed of 4 nucleotide bases A,T,G,C - form complementary base pairs via hydrogen bonds AT and GC

Question 44

Give an example of how genomes are manipulated

Answer 44

Cloning genes - transfer of insulin genes via recombinant plasmid 1. Digest plasmid with restriction enzyme 2. Use same restriction enzyme to cut DNA strands ==> forms sticky ends 3. Seal gap with ligase enzyme

Question 45

Which region on the plasmid is highly targeted by multiple restriction enzymes?

Answer 45

The cloning region