[micro] bacterial genetics (6 jan)

Bacteria use three main mechanisms to adapt to

changing environments

– Mutation

– Gene transfer

– Regulation of gene expression

Mutation

Spontaneous mutations occur infrequently

and randomly in the natural environment

Rate of spontaneous mutation

Probability that a mutation will be observed in a given

gene each time the cell divides

Rate is generally between 1 in 10,000 and 1 in a trillion

Mutations are stable heritable changes inthe base sequence of DNA

Mutations can be caused by:

Base substitutions

Removal or addition of nucleotides

Transposable elements

Base substitutionsMost common type of mutation

Results from mistakes during DNA

replication

Point mutations: when one base pair is

changed

Missense mutation

Nonsense mutation: Mutation that

changes an amino acid codon to a stop

codon

Removal and addition of nucleotidesShifts the translational reading frame

Called frameshift mutation because it affects all amino acids downstream from addition or deletion (frequently result in premature stops)

Transposable elements

Special segments (transposons) of DNA that move

spontaneously from one gene to a different gene

Transposons may disrupt the integrity of the gene and

render its protein product nonfunctional

Read about Barbara McClintock (A Glimpse of History) on pg. 191

Mutations are essential for understanding genetics

Mutations can be intentionally produced (induced

mutations) to demonstrate function of particular gene or

set of genes

Mutations can be induced via:

Chemical mutagens

Transposition

Radiation

Chemical mutagens

Nitrous acidChanges cytosine to uracil

Alkylating agents

Alter hydrogen bonding of bases

Nitrosoguanine is common alkylating agentUsed as antineoplastic drugs

Base analogs

Chemicals that are structurally similar to the nitrogenous bases buthave slightly altered base pairing properties

Base analogs include:2-aminopurine which incorporates in the place of adenine butbinds with cytosine

5-bromouracil which incorporates in the place of thymine but binds with guanine

Intercalating agents

Molecules that insert themselves between adjacent

bases and create space between bases

Ethidium bromide is common intercalating agent

Potential carcinogen

Used extensively in biochemical/molecular biological

research

Transposition

Common procedure used to induce mutation in thelaboratory

A transposon inserts into a gene (insertion mutation)

Radiation

Ultraviolet light

Causes covalent bonding between adjacent thymine bases forming thymine dimers which distort DNA

X rays

Cause breaks and alterations in DNA. Breaks that occur on both strands are often lethal

Mutations and Their Consequences

Mutations provide the organism with a way to respond to environmental stresses

Environment selects for cells suited to survive

Environment does not cause mutation

WESTERN BLOT

WESTERN BLOT

WESTERN BLOT

WESTERN BLOT

NORTHERN BLOT

NORTHERN BLOT

NORTHERN BLOT

NORTHERN BLOT

SOUTHERN BLOT

Southern blot

Mechanisms of Gene Transfer

Genes are naturally transferred between bacteria

DNA-mediated transformation

Transduction

Conjugation

Gene exchange in bacteria

1. Donor DNA is transferred and accepted by the recipient cell

Three mechanisms

DNA-mediated transformation

Transduction

Conjugation

2. Donor DNA is integrated into the recipient cell’s chromosome

No integration

Integration

DNA-mediated transformation (transformation)

The transfer of naked DNA from one bacterium toanother

Discovered by Fredrick Griffith in 1928 while working with Streptococcus pneumoniae

Griffith realized S. pneumoniae existed in two formsEncapsulated, virulent form (smooth in appearance)Nonencapsulated, avirulent form (Rough in appearance)

Griffith hypothesized that injections with the smoothstrain could protect mice from pneumonia

Griffith injected mice with the two different strains

Griffith’s Results

Natural transformation occurs when bacterial

cells are “competent”

Competence is a condition in which bacterial cells

are capable of taking up and integrating large

fragments of DNA into their chromosome

Competence usually occurs naturally during the late log,

early stationary phase

Dying cells rupture during the stationary and death phases. The chromosome breaks into small pieces and explodes through the ruptured

cell wall

Recipient cells absorb pieces of “naked” DNA

The naked DNA is integrated into the recipient

cell’s chromosome

Naked DNA integrates at a homologous site on the recipient’s

chromosome

Entry of the DNAOnly single strands enter

Integration of the donor DNADonor DNA is integrated at ahomologous site

Enzymes cleave recipient DNA

Donor DNA replaces recipient DNAvia recombination

Cell multiplicationTransformed cells multiply under selectiveconditions in which non transformed cellswill not grow

Transduction

Bacterial DNA that is transferred from donor to

recipient via a bacterial virus (bacteriophage)

Two types of transductionGeneralized

Any gene from the donor can be transferred

SpecializedOnly specific genes can be transferred

Transduction

Conjugation

Conjugation is mediated by a plasmid R plasmids

F plasmids

Conjugation requires direct contact between cells

Cells must be of opposite mating types

Donor cells carry a plasmid that codes for fertility factor or “Ffactor”

This cell is designated F+

Recipient cell does not carry a plasmidThis cell is designated F-

Conjugation

High frequency of recombination – Hfr strains