Bacterial Genetics

Question 1

why bacterial infection is a global challenge

Answer 1

• Bacterial infections cause diseases in all living organisms

• Infectious bacteria can be found in water, soil and air (secondary source)

Question 2

the role of antibiotics in combating bacterial infections

Answer 2

• Antibiotics inhibit the growth of or kills bacteria without harming the host
• Antibiotics are essential in treating infections and have saved countless lives
• The use of antibiotics resulted in the evolution of antibiotic resistance (anti microbial resistance, AMR)

Question 3

pre-antibiotic era

Answer 3

• 90% of children with bacterial meningitis died and survivors had severe disabilities
• Staphylococcus aureus was fatal in 80% of infected wounds
• Tuberculosis and pneumonia bacteria were famous killers
• In 1982 Alexander Fleming discovered penicillin- first antibiotic therapy
• Timelines for death due to infectious disease:
• Death rate started to decline after 1900, especially with introduction of vaccines

Question 4

• Mode of action of antibiotics:

Answer 4

• Based on mode of action, antibiotics are broadly classified into 4 classes
• Cell wall synthesis inhibitors:
• B-lactams (penicillin, cephalosporin, aztreonam, imipenem)
• Polypeptides (bacitracin, vancomycin)
• Protein synthesis inhibitors e.g. aminoglycosides, tetracyclins, macrolides, chloramphenicol, clindamycin
• Folate antagonists e.g. sulfonamides, trimethoprim
• Quinolones e.g. ciprofloxacin

Question 5

Resistance : decreased membrane permeability

Answer 5

• Beta-lactams, aminoglycosides
• Porins are proteins present in the outer membrane of gram-negative bacteria that form channels to allow the entry of antibiotics
• Resistant bacteria have reduced expression

Question 6

Resistance: efflux pump

Answer 6

• Tetracyclines, quinolones, macrolides
• Proteins that are localised and imbedded in the plasma membrane of the bacterium and whose function is to recognise noxious agents that have penetrated the protective cell wall of the organism and reached the periplasmic or cytoplasm and extrude them before they reach their intended targets
• Less accumulation of antibiotics, less effective

Question 7

Resistance: target alteration

Answer 7

• Beta-lactams, aminoglycosides, vancomycin, quinolones, macrolides
• Changes in the composition or structure of the target in the bacterium can stop the antibiotic from interacting with the target

Question 8

Resistance: enzymatic inactivation

Answer 8

• Beta-lactams, aminoglycosides, macrolides
• Antibiotic resistance mechanisms mediated by bacterial resistance enzymes:
• Resistance enzymes hydrolyse the antibiotics and confer resistance
• Resistance enzymes modify the structure of antibiotics or antibiotic targets, preventing the antibiotics from binding to the targets and conferring resistance
• Actively inactivates antibiotics

Question 9

how antibiotic resistance spreads

Answer 9

• 1: transmission of antibiotic resistant bacteria through the food chain and communities
• 2: transmission of antibiotic resistant genes to other bacteria: the same species and to different species

Question 10

Vertical gene transfer

Answer 10

spontaneous chromosomal mutation occurs that confers resistance, initially affecting a single bacterial cell which is able to multiply (parent to daughter transmission)

Question 11

Horizontal gene transfer

Answer 11

resistant bacterial gene is transferred to diverse species; to a new strain of the same species or to a different species
3 mechanisms:
Transformation
Transduction
Conjugation

Question 12

• Key factors driving spread of antibiotic resistance:

Answer 12

• Transmission of resistant organism between patients within hospitals
• Antibiotic over-prescription, misuse and self-medication
• Use of antibiotics in animals raised commercially for food
• Counterfeiting of drugs
• Limited testing capacity in hospital settings
• Poor hygiene
• Over prescribing antibiotics:
• Antibiotics given for conditions that are unlikely bacterial
• Antibiotics given for conditions that are usually self-limited
• Broad spectrum antibiotics given for conditions that can be successfully treated with narrow spectrum ones

Question 13

What is transformation

Answer 13

In transformation, bacteria incorporates exogenous genetic material from its surroundings through the cell membrane into its genome
Bacterial cells that can undergo transformation are described as being competent
Unlike conjugation and transduction, the uptake of the genetic material is completely dependent on the recipient bacterium.
All required cellular machinery including proteins are encoded in the core genome
Most transformable bacteria do not permanently express the proteins that are involved but instead require specific conditions to develop competence for genetic transformation.
Competence is thus a transient ‘window of opportunity’ (transient process) for DNA internalization and thereby enables subsequent transformation.
The process is short lived as proteins are only produced when there is need, otherwise the genes are repressed

Question 14

How was transformation demonstrated

Answer 14

Bacterial transformation was first demonstrated by Griffith in 1928
Pneumonia was a serious cause of death after the Spanish influenza pandemic
Griffith was running experiments to create a vaccine for pneumonia
Griffith used 2 strains of streptococcus pneumoniae which infect mice
Type S (smooth) was virulent and type R (rough) was non-virululent
The virululence of type s was conferred by the presence of polysaccharide capsule which protected the bacteria from the host immune system
Hypothesis: material in dead bacterial cell can transform living bacterial cells
Conclusion: live type R strain bacteria had been transformed into type S by a transforming principle in the dead type s strain

Question 15

 Compare and contrast natural transformation in Gram +ve and –ve bacteria.

Answer 15

Both gram +ve bacteria and gram -ve are naturally transformable
In Griffith’s experiment, Streptococcus pneumoniae (gram +) spontaneously became competent to take up the genetic material
Approx 80 species are documented to be naturally transformable
An estimated 1% of bacteria are naturally transformable
Can be dangerous and cause mutation to the bacteria

Question 16

Benefits of bacteria becoming transformable:

Answer 16

Nutrition:
Degrade DNA as a source of nucleotides as de novo synthesis of nucleotides requires a lot of energy from the cell
Extracellular dna is broken down via nucleuses
DNA uptake is more efficient than breaking down extracellular DNA
Limits diffusion losses and avoids nucleoside re-phosphorylation
Genome repair:
Uptake of homologous DNA allows repair
DNA damage induces competent cells
“Self-DNA” uptake: high rates of transformation with self-DNA in Neisseria gonorrheoa leads to conservation of core regions of the genome
Genome diversification:
Uptake of exogenous DNA increases diversity
Could cause loss of beneficial alleles or replace deleterious alleles
Increase survival in future species, Darwinism, can help evade antibiotics
It is unclear what evolutionary factors enable transformation to persist in bacterial populations

Question 17

How bacteria become naturally transformable:

Answer 17

Transformation involves a series of molecular processes from the point of contact with exogenous dna to the point of insertion into the bacterial genome
Due to the differences in the structure of the cell envelopes of gram + and gram – bacteria there are some differences in dna uptake
Common mechanistic steps:
Bacteria develops competence
Cells bind double stranded DNA in the environment
Movement of DNA over cell membrane/cell wall
Conversion to ssDNA
Intracellular fate: Maintained in the genome as a plasmid or homologous recombination into genome or degradation

Question 18

Bacteria develop competence naturally:

Answer 18

Usually as a culture encounters stress e.g. nutrient limitation, high cell density, stationary phase of growth conditions
Cells coordinately express a new set of genes i.e. a regulon is activated
Cells synthesise new proteins
Construct a protein structure on the cell wall, the ‘com pilus’ and other surface proteins
Com pilus is related to type 4 pili
Diverse signals are involved and sometimes have opposing effects in different bacterial species
Only in Neisseria gonorrhoeae is competence constitutive, irrespective of stress it is competent. Consistently conserved.

Question 19

Binding of extracellular dsDNA (GRAM +VE):

Answer 19

Capture of exogenous dna by the transformation pilus (also called ComGC pilus,type 4 pseudo pilus, type 4 pilus(T4P,Tfp))
Binding of dsDNA by the DNA binding protein ComEA
Begins to move it through peptidoglycan
Recognition of the dsDNA by the nuclease EndA and conversion to ssDNA
(This happens in streptococcus pneumoniae but unidentified nucleuses (or strand separating proteins) generate ssDNA for uptake in other species)
Transport of the ssDNA strand by ComEC, driven by the ATP-dependent translocase ComFA
In firmicutes, ssDNA internalisation through ComEC is presumably driven by the ATP

Question 20

Binding of extracellular dsDNA (GRAM -VE):

Answer 20

Capture and transport of DNA across outer membrane: PilQ secretore channel enables pilus to traverse outer membrane, bind dsDNA (at specific sequences) and transport it into periplasm
Binding of dsDNA by the DNA binding protein (ComE)
Transport of the DNA strand across inner membrane by a homologue of the ComFA translocase, currently unclear
Endonuclease probably exists but not found yet

Question 21

DNA uptake by transformation is highly conserved

Answer 21

Gram + and gram – bacteria rely on highly similar DNA uptake systems
The only major difference is the requirement for transport across the outer membrane in gram -, which involves the PilQ secretin channel

Question 22

Homologous recombination:

Answer 22

In the cytosol, transformation-dedicated DNA processing A (DprA) protein binds the internalised ssDNA
DprA is activated by ssDNA being in the cytosol
DprA aims to stabilise ssDNA
DprA also recruits and loads the recombinant RecA onto ssDNA
RecA polymerises on the ssDNA and promotes a homology search along chromosomal DNA, followed by strand exchange
Homologous recombination is initiated in all organisms by ssDNA
DNA processing (DprA) is also conserved

Question 23

Artificial transformation:

Answer 23

Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature
Many bacterial species can be transformed artificially e.g. E.coli, salmonella, staphylococcus
Uses circular double stranded dna (natural transformation prefers ds linear dna)
Artificial transformation is relatively inefficient but is a key tool in gene cloning to introduce recombinant dna into the host

Question 24

Transformation efficiency determinants:

Answer 24

Genetic background of transformed bacteria: restriction system
Preparation of artificially competent bacteria : manipulations that alter membrane permeability
Quality of the transforming dna : ion chealators, salt contaminants, concentration, damaged dna
Size of transforming dna: large plasmids are taken up less efficiently
Artificial transformation is still fairly inefficient: e.g 10^8 transformants per mg DNA, most bacteria in a sample are not transformed e.g. 1 in 1000
Selection/ screen required to identify transformants

Question 25

 Understand how transformation applies to gene cloning

Answer 25

Transformation is one of the steps in gene cloning
Gene cloning is the isolation and amplification of a specific DNA fragment such as a gene
The key steps include:
Isolation of dna of interest (restriction enzymes required)
Ligation (dna ligase required)
Transformation
Selection and screening

Question 26

Why introduce a dna fragment into bacteria?

Answer 26

Propagate recombinant dna:
To allow sequencing of DNA and further analysis/ manipulation (but not when undertaking high throughput genome sequencing)
Protein expression:
To allow high-level expression of a protein of interest for subsequent purification and structural or biochemical studies
DNA function:
Protomter studies (and other regulatory regions)
Gene function studies

Question 27

What is conjugation

Answer 27

• In conjugation (bacterial sex) there is a direct transmission of DNA from on bacterial cell to another. Similar to sexual reproduction
• Unlike transformation and transduction, there is a physical contact between the recipient and the donor
• There is unidirectional dna transfer from the donor to the recipient
• DNA is exchanged via pilus
• In most cases, conjugation involves transfer of plasmid dna, although chromosomal dna transfer can also occur in some organisms
• Conjugation occurs predominantly in gram -ve bacteria
• Conjugation is not confined to members of the same species

Question 28

How was conjugation demonstrated

Answer 28

• Conjugation was demonstrated by lederberg and Tatum in 1947
• Mixed cultures of nutritional (auxotrophic) mutants of E.coli has strains which differ from their parent wild type, strain K-12, in lacking the ability to synthesise growth factors
• As a result of these deficiencies, they will only grow in media supplemented with their specific nutritional requirements
• Two mutant strains were used: strain A, requiring methionine and biotin, strain B required threonine, leucine and thiamin

Question 29

how do we know physical contact is required for conjugation

Answer 29

• This was demonstrated by Bernard Davies
• In the experiment, a U tube was used, with such filter that the pores were too small to allow the bacteria to pass, but large enough to allow passage of fluid with dissolved substances
• Strain A was kept in one arm and strain B in the other
• After they had been incubated for a few hours, the contents of each arm were separately tested on minimal medium
• None of them were found to grow on a minimal medium, suggesting that physical contact between strain A and strain B was necessary for gerneration of wild type strains or prototrophs
• Genetic recombination involving physical contact was responsible for the appearance of prototrophs

Question 30

Conjugation step • 1) tra gene expression and formation of mating pair

Answer 30

• The initiation of conjugation requires the expression of the tra genes
• Some of the produced Tra proteins form the T4SS and the conjugative pilus that will recruit the recipient cell and mediate mating pair stabilisation
• The conjugative pilus contacts receptors on the surface of the recipient cell, forming a mating pair
• The pili contract to bring the cells into intimate contact and a channel or pore is made
• The dna passes through the pore or channel from the donor to the recipient
• Tra proteins constitute the relaxosome (Tral, TraM, TraY)
• Tra proteins form a pore complex, mating bridge, between 2 cells
• Tral (DNA relaxase) nicks one strand of F plasmid at oriT site and attaches to the 5’P

Question 31

Steps in conjugation

Answer 31

• 1) tra gene expression and formation of mating pair

• 2) transfer of DNA into recipient bacteria
• 3) stabilisation and circularisation of ssDNA

• 4) conversion of ssDNA to dsDNA

• 5) cell separation and phenotypic expression in recipient cell

Question 32

Conjugation step 2 transfer of DNA into recipient bacteria

Answer 32

• Interaction between the relaxosome and the type IV coupling protein (T4CP) initiates the transfer of the T-strand by the T4SS
• DNA relaxase and ssDNA migrates to the Tra pore complex and transfers to recipient cell
• Conversion of donor ssDNA to dsDNA by DNA polymerase begins

Question 33

conjugation step 3 stabilisation and circularisation of ssDNA

Answer 33

Upon entry into the recipient, the ssDNA T-strand is coated by the host chromosomal SSB, and the single stranded promoter Frpo adopts a stem-loop structure recognised by the host RNA polymerase to initiate the synthesis of rna primers

• Tral performs circularisation of the fully internalised T-strand

Question 34

Conjugation step 4 conversion of ssDNA to dsDNA

Answer 34

• The rna-dna duplex is recognised by the host DNA polymerase to initiate the complementary strand synthesis reaction
• Re-synthesis of the F plasmid is completed in donor cell
• DNA transfer completed and ssDNA converted to ds circular DNA in recipient cell
• Conversion of ssDNA to dsDNA occurs by rolling circle replication (RCR) in the donor

Question 35

Conjugation step 5 cell separation and phenotypic expression in recipient cell

Answer 35

• Plasmid gene expression results in the phenotypic conversion of the recipient cell into a transconjugant cell
• Cells separate
• Both cells now have F plasmid and can make f pilus
• Both can act as donors to new recipient cells
• Neither cell can be a recipient because of surface exclusion

Question 36

• in conjugation Which cells can be recipient?

Answer 36

• The host range is defined by several factors:
• Recognition of recipient by donor
• Successful completion of DNA transfer
• Replication of transferred plasmid
• Host range of some conjugal plasmids is very broad
• Includes transfer to bacteria, yeast, plant cells and mammalian cells
• However, plasmid replication is often limited

Question 37

Resistance (R) factors in conjugation

Answer 37

• Plasmids that encode multiple antibiotic resistances
• Not necessarily conjugative plasmids
• But many are conjugative and can rapidly spread between diverse bacterial species

Question 38

• CONJUGATION IN GRAM +VE

Answer 38

• Many gram +ve bacteria also possess plasmids that are transmissible by conjugation, and in many cases the mechanism of DNA transfer is similar to that described above
• However, there are substantial differences in other respects
• Fewer genes required for conjugation (less than 5 genes in some cases)
• No requirement for production of pilus because they don’t have outer membrane
• Absence of pilus means its harder for human immune system to detect them

Question 39

• CONJUGATION IN GRAM -VE

Answer 39

• In the absence of a recipient, the transfer genes are switched off by TraA
• The recipient produces a peptide, cAD1, which inactivates TraA, allowing conjugation to proceed
• One of the products, aggregation substance (Asa) coats the outside of the donor cell, resulting in the formation of a mating aggregate with donor and recipient cells bound together

Question 40

F factor

Answer 40

• Bacterial strains that can transfer genes by conjugation were designated as possessing a fertility (F) factor
• The F factor (also called conjugative plasmid) include genes that codes for the proteins that make up the sex pilus and other cellular machinery for conjugation
• The F factor also contains a special site where DNA transfer during conjugation begins
• Confers ability to donate dna
• Can be lost and regained easily
• Strains carrying F are donors and designated F+
• Strains lacking are recipients and designated F-

Question 41

• The F pilus:

Answer 41

• The F pilus:
• In e.coli and other gram -ve bacteria, the donor cell carries appendages on the cell surface known as pilli
• These vary considerably in structure, e.g. the pilus specified by the F plasmid is long, thin and flexible while the RP4 pilus is short, thicker and rigid

Question 42

F plasmid

Answer 42

• The F plasmid is a small, circular, double stranded DNA molecule that is distinct from a cells chromosomal dna
• Can exist independently of the host chromosome
• Can intergrate reversibly into the host chromosome (episome)
• Plasmid contains genes that specify:
• Type 4 secretion system (T4SS) a nano machine that synthesises the F pilus
• “Surface exclusion”
• Stabilisation of mating pairs
• DNA transfer
• Regulation

Question 43

Hfr strains

Answer 43

• Hfr strains arise by integration of the F plasmid into the bacterial chromosome
• Also, chromosomal transfer starts from a defined point and proceeds in a specific direction and the origin of transfer is determined by the site of insertion
• Hfr occurs due to homologous recombination between insertional sequences (IS)
• Multiple IS sites present throughout bacterial genomes
• The combination of the partial transfer of chromosomal dna with the ordered transfer of genes made conjugation an important tool in the mapping of bacterial chromosomes
• Duration of transfer is no more than 100 mins
• The dna strand transferred is very long and often breaks
• The F plasmid would be the last piece transferred, therefore recipients do nt become F+ as it can’t be incorporated into the genome
• Only fragment of F factor is transferred into recipient cell so recipient doesn’t have all the properties
• Linear transport of Hfr DNA:
• Directional
• Can use this to map gene order

Question 44

F’ plasmid:

Answer 44

• F plasmid can both integrate and excise from the genome
• Improper excision of integrated F can occur, resulting in F’
• F’ usually contains chromosomal genes
• F’ plasmids:
• Behave like F-plasmid, autonomously replicating, conjugation-competent
• Can incorporate host genomic fragments (replacing some plasmid sequences)
• Are stable plasmids, with integration fairly rare because of the altered IS sites
• In F’ x F- cross, F’ remains F’ and F- becomes F’
• Recipient bacterium are now known as merodiploid or partial diploid
• Because of presence of 2 copies of a particular gene (chromosomal and F’)

Question 45

Appreciate the value of conjugation to genetic mapping

Answer 45

• Interrupted mating experiments use the process of conjugation to map the order of genes on a bacterial chromosome
• Gene mapping can be done by interrupting the conjugation at multiple times and taking account of which genes the recipient cell has
• Genes that take longer to enter the recipient cell are further from the origin
• The percentage of recipient cells with a given gene also provides information on the order of genes on a chromosome
• The sex pilus can randomly break at any time
• When a gene takes a longer amount of time to enter a recipient cell (bc further from origin) there will be more chances that the sex pilus could break before it is donated
• The fertility factor Hfr is the last gene to enter a recipient cell so very few recipient cells have it

Question 46

What are bacteriophages

Answer 46

• Bacteriophages are viruses that infect and kill bacteria
• Most phages are specific to one form of bacteria, some phases are polyvalent
• Believed to be the most abundant life form in nature
• They can be virululent (lytic) or temperate (lysogenic)
• Structure:
• Head: nucleic acid (DNA or RNA, ds or ss) and capsid (protective protein coat)
• Tail: facilitates attachment
• Bacteriophage means eater of bacteria

Question 47

Discovery of phage mediated transfer of genetic material

Answer 47

• Salmonella typhimurium
• Mixed 2 auxotrophic mutants together
• Obtained prototrophic strain
• Repeated with a U-tube with filter (large pore size) (bacteriophage can pass through)
• Obtained prototrophic recombinants
• Repeated with a U tube with filter (small pore size) (bacteriophage can’t pass through)
• Did not obtain prototrophic recombinants
• Concluded genetic transfer was mediated by bacteriophage

Question 48

Lytic phage lifecycle

Answer 48

• Key stages:
• Adsorption to bacterium
• Production of phage specific mRNA, translation to produce enzymes, incl. nucleases that degrade the host chromosome
• Digestion of bacterial dna
• Replication of viral dna
• Coat proteins encoded by virus appear in cell
• Coat proteins condense around phage DNA; phage produces lysozyme
• Lysis of bacterial cell, 100s of progeny viruses released
• Infectious virus, contains the genetic material (usually DNA) surrounded by a complex protein head; protein tail required for adsorption to host cell

Question 49

Lysogenic phage lifecycle

Answer 49

• Temperate/ quiescent phages
• Prophage can be activated into lytic cycle:
• DNA is excised from genome
• Replicates
• Produces progeny
• And lyses host cell
• In a lysogen:
• Bacteria grows (doesn’t damage bacteria)
• Most phage genes repressed (exception is the repressor)
• No phage particles constructed
• Phage nucleic acid replicates as part of the host chromosome

Question 50

What is transduction

Answer 50

• In transduction, foreign DNA is introduced into a cell by a virus or viral vector
• No physical contact between the cells is required
• Bacteriophage serves as the medium of gene transfer between 2 bacteria
• Phases infect bacteria, use host cell machinery to multiply and while assembling their components, they mistakenly pack host dna fragments
• When these viruses infect new bacterial cells, the bacterial genome that they carry may get inserted into the host genome

Question 51

Generalised transduction

Answer 51

• Any part of the bacterial chromosome
• Occurs during lytic cycle
• Random packing of host dna into phage particle
• Bacteriophage must digest host dna (nuclease)
• Approx 1 in 10^4 phage particles carry host DNA
• Known as transducing particle when host dna is present
• Bacteria with donor dna recombined into chromosome ‘transduction’
• DNA fragment integrates into chromosome
• Homologous recombination using host RecA enzyme

Question 52

Specialised transduction

Answer 52

• Only transfers dna adjacent to Prophage insertion
• Occurs during lysogenic to lytic conversion
• Inaccurate excision of Prophage from host chromosome
• Repression relieved:
• Lytic cycle kicks off
• Prophage excision
• Excision of phage from chromosome:
• Via a site-specific recombination process
• ‘Integrase’ and ‘excisionase’
• 1 excision per 10^6 is incorrect and will take with it some host dna
• Result is ‘specialised transducing phage’
• Can still infect
• May have a defective genome
• Might need a wild type helper to infect
• Carries genes adjacent to original site of integration
Only specific portions of bacterial chromosome are transduced

Question 53

 Know the difference between abortive and stable transduction

Answer 53

• Stabilised transduction uses linear dna
• Transduced dna fragment is replicated along with chromosome
• Propogated to daughter cells
• Abortive transduction uses circularised DNA fragment
• Circularisation introduced by proteins and prevents nuclease incorporating it into chromosome
• Prevents synthesis of ds dna
• Doesn’t integrate into chromosome
• Unable to replicate autonomously
• Bacterial cells continue to divide
• Thus diluting out single bacterium with transduced dna fragment

Question 54

Appreciate bacteriophage can be used for gene mapping

Answer 54

• Generalised transduction for gene mapping
• Infect bacterial strain (strain 1) that is met+ arg+ with phage
• Strain 1 is lysed and the phages that are formed are now used to infect strain 2 which is met- arg-
• After the infection, count how many cells of strain 2 were met+ (growth on medium unsupplemented by met)
• Then count number of met+ that were also arg+ (growth on medium unsupplemented by met and arg)
• The higher the frequency of met+ arg+ to met+ arg- cells, the closer the two loci
• The met+ arg+ strains are cotransductants (because the two loci have been transferred via transduction together)
• The principle: genes close togther are more likely to be packaged together, hence a higher co-transduction frequency