Genetics chapter 7 - Bacteria and Viruses

Reasons for bacteria being a good organism 

• Rapid reproduction

• Large progeny

• Haploid genome allows all mutations to be expressed

• Asexual reproduction

• Small genome

• techniques available to isolate and manipulate genes

• medical importance

Type of bacteria

• Prototrophic: wild type bacteria that can synthesize inorganic materials and survive on its own

• Auxotrophic: mutant type bacteria that lacks enzyme responsible for synthesizing an essential molecule, requiring a complete medium

Techniques for studying bacteria

• Minimum medium: only contains essential nutrients for prototrophic bacteria

• Complete medium: Contains nutrients essential for all bacteria including auxotrophic 

Difference between single colony and multiple colonies (in terms of genes)

• Single colony: All progeny in a colony are genetically identical

• Multiple colonies: One colonies genome may be different than another’s 

Lab test for mutation steps

• grow bacteria in test tube by placing into inoculate medium

• After division, pipette onto growth medium

• Spread and after 1-2 days colonies form

• use velvet to stick and transfer colonies onto two mediums, one lacking amino acid, and other with it

• Compare results (see if any colonies are missing to identify a mutation)

Bacterial genome

• Bacterial DNA: mostly singular and circular chromosome

• Plasmid: Extra, small circular DNA. Contain epistomes (f factors)

Plasmid conflict

A single plasmid can carry many genes. They can carry multiple different types of antibiotic resistant genes. 

Use of antibiotics promoting natural selection

Bacteria containing a plasmid that has a gene for resistance against an antibiotic will survive when antibiotics are used. These bacteria will produce and more bacteria with the plasmid containing antibiotic resistant gene will spread.

Replication process for plasmid

• Replication begins at ori site

• strands separate and replication begins in both directions for each strand

• Two daughter cells will be produced, possibility of new strand inside or outside

F factor

Fertility factor, DNA segment that allows for exchange of genes through conjugation

3 types of gene function for f factor

• Genes for regulating plasmid replication

• genes that regulate plasmid transfer to other cells

• Genes for regulating insertion of plasmid into bacterial chromosome 

Experiment to identify if bacteria exchange genes and result

• separate bacteria that are auxotrophic for opposite strains

• Place both in a minimum medium where they cannot grow individually

• Formation of colonies indicates that bacteria exchanged info to make completely prototrophic bacteria

Result: Bacteria do exchange genetic info

Experiment to see if direct contact is needed and result

• Two bacteria, auxotrophic for differing segments, are placed in the same medium

• A filter is present to allow medium to cross but not the bacteria

• After, the bacteria are pipetted onto minimal medium and none survive

Result: Bacteria need direct contact to exchange genetic info

Three types of bacteria genetic exchange

• Conjugation: direct contact between bacteria in order to exchange genetic info

• transformation: Cell lysis and a new bacteria picks up free gene fragment

• Transduction: Virus lysis cell and takes gene fragment, then virus infects another bacterial cell and the gene fragment is passed on

Different F factors

• F+: plasmid is separate DNA molecule

• F-: plasmid is absent

• Hfr: Present and integrated into bacterial chromosome

• F’: Plasmid is separate DNA molecule containing some bacterial genes 

F factors roles in conjugation

• F+: donate plasmid

• F-: receive plasmid

• Hfr: high frequency plasmid donor

• F’: donate plasmid

Conjugation

Direct transfer of one plasmid strand from F+ cell, through conjugation pilus (sex pilus), into receiving F- cell. (Not reciprocal) 

Formation of Hfr cell

crossing over between F factor on plasmid and bacterial chromosome results in integration into bacterial chromosome

Merozygote

Partial diploid bacterial cell containing F plasmid carrying some bacterial genes

How does merozygote form

F’ transfers f plasmid containing some chromosomal gene to F- and then variations of same gene can arise

Result of conjugation between cells with different f type

• F+ x F- : Two F+ cells (F- becomes F+)

• Hfr x F- : One Hfr and one F- (F- might have genetic change as crossing over can occur from loose Hfr fragment and bacterial chromosome

• F’ x F- : Two F’ cells (F- becomes F’ and partial diploid/merozygote) 

gene mapping with conjugation

Distance between genes is measure using time it takes to transfer DNA from Hfr to F-

Interrupted mating 

Experimental way to measure gene location using conjugation

Transformation

A bacterium takes a DNA fragment from medium. Recombination then takes place to integrate into the bacterial chromosome

Component cell

Cells that prepares to take up DNA from medium during transformation when under stress

Types of transformation cells

• transformant: Cell that receives genes and integrates

• cotransformed: Cells transformed by two or more genes

transformation steps

• Cell takes one strand of DNA from medium and other strand is hydrolyzed

• the strand attaches to bacterial chromosome and recombination occurs

• When the cell replicates, original strand will produce a daughter cell with original bacterial genome and other will contain two transformed strands

Laboratory use of transformation

heat shocking cell produces component cell which is more readily able to take up foreign DNA

Transformation efficiency

Probability that cell will take extracellular DNA and express genes encoded in it

factors affecting transformation efficiency

• Plasmid size

• DNA type

• cell genotype

• transformation method

• cell growth rate

gene mapping using co transformation

Transformants that take up two or more genes from cell fragments will exhibit the trend:

Closer two genes are = higher rate of co-transformation

Bacterial defense mechanisms

• reduce expression of receptors viruses need to attach to

• Secretion of polysaccharides to limit infection

• Block viral replication

• CRISPR-Cas systems (main focus)

CRISPR-Cas system

Recognizes and remembers DNA of specific pathogens. Acts as bacteria’s immune system

CRISPR - Clustered regularly interspaced short palindromic repeats.

(repeated sequences separated by spacers that are the same forward or backward)

CRISPR-Cas explained

Viral DNA is copied by CRISPR and copied viral sequences are made so that when viral DNA comes back, Cas can recognize it and cut it out.

How are double stranded DNA breaks repaired?

NHEJ (non homologous end joining) - Brings two ends of a break together 

HDR (Homologous directed repair) - Adds donor DNA to break

Problem with NHEJ

Often results in nucleotide insertions or deletions 

Virus 

replicating structure of DNA/RNA and a protein coat

Virulent phage

will always kill host cell and reproduce through lytic cycle

temperate phage

inactive prophage whose DNA integrates into bacterial chromosome

Lytic cycle (6 steps)

• 1) attachment - virus locks onto bacterial cell

• 2) injection - virus injects DNA into bacterial cell

• 3) Biosynthesis - bacterial cell stops its own work and instead follows genome of virus

• 4) Assembly - Viral parts begin to assemble themselves into many new viruses inside cell

• 5) Lysis - Cell breaks open

• 6) Release - Viruses are released after cell lysis

Lysogenic cycle

Instead of degrading host cell chromosome, viral DNA is incorporated into it. Temperate prophages enter this state