Lecture 3: Bacterial Genetics Flashcards
1) DNA in bacterial cells 2) Natural transformation, plasmid transformation, and artificially induced transformation 3) Generalized and specialized transduction 4) Bacterial conjugation and mechanism of DNA transfer in conjugation 5) Transposons and their function
Genetics
The manipulation of DNA to study cellular and organismal function
Bacterial Chromosome
- shape
- arrangement
- size
- single large convalently closed molecule of ds DNA
- some species of Borrelia and Streptomyces have linear chromosomes
- no major histones and no nuclear membrane
- contains all essential bacterial genes
- size varies from 0.4x10^9-8.6x10^9 daltons
Plasmid DNA
- supercoiled, circular, ds extrachromosomal pieces of DNA
- replicate independently of chromosome
Plasmid DNA
-Functions
- may be important to pathogenicity or to survive under special conditions
- production of toxins
- synthesis of cell structure req’d for adherence or colonization
Plasmid DNA
-Types
- Fertility factors (episomes) -F plasmid
- Drug resistance factors -R plasmid
- Conjugative plasmids
- Col plasmids
- Stable phage DNA - Phage DNA arises through infection with a temperate phage whose viral production is repressed
Transformation
uptake of extracellular DNA by bacteria from the environment
Transduction
Transfer of bacterial genes (DNA) via a bacterial virus (bacteriophage) vector
*Generalized or Specialized
Conjugation
Transfer of DNA by direct donor-recipient contact
Natural Transformation
- naturally transformable bacteria can take up DNA from environment
- natural competence
- both gram (+) and (-) bacteria such as Bacillus subtilis, Haemophilus influenzae, Neisseria gonorrheae, Streptococcus pneumoniae, Synechococcus
Determinants of Transformation
- In B.subtilis and S.pneumoniae, a complex of 3-5 proteins including:
- a labile competence factor
- a specific endonuclease
- a specific endonuclease
- the DNA-binding polypeptides
- an autolysin to increase cell permeability
Plasmid transformation
- neither plasmids nor phage DNA’s can be efficiently introduced into naturally competent cells
- ss plasmids do not recyclize or make complementary strand
- plasmid or phage DNA transformation occurs only with DNA’s that are dimerized
Plasmid or phage DNA transformation occurs only with DNA’s that are ___________
Dimerized
Artificially Induced Competence
-Chemical transformation
- bacteria are exposed to solutions (calcium ions) which alter their cell membranes enough to make the DNA molecules pass through and into the cell
- heat shock treatment is used
- after transformation, the cells are plated on selection media to select the transformants
Artificially Induced Competence
- Electroporation
- optimal/high efficiency achieved how?
- E.coli cells are small -require very high field strengths (12.5-15kV/cm)
- optimal efficiency achieved using small volumes with dense slurry of bacteria (2x10^10 cells/ml)
- best carried out at 0-4C
- high efficiency when conc. of input DNA is high (1-10ug/ml)
Generalized Transduction
essentially any region of the bacterial DNA can be transferred
Specialized Transduction
certain genes close to attachment site of a lysogenic phage in the chromosome can be transferred
Transducing phages
phages capable of transduction
Donor strain
original bacterial strain in which the transducing phage had multiplied
Recipient strain
infected bacterial strain
Transductants
cells that received DNA from another bacterium by transduction
What makes a transducing phage?
a) phage must not degrade the host DNA completely after infection
b) packaging sites or pac sites of the phage must not be specific to the sequences of host DNA (e.g. phage P1 is a good transducer as it has less pac site specificity)
c) non-transducing phages can be converted by special treatments
Generalized transduction
-how does it differ from specialized?
1) any gene of donor bacterium can be transduced into recipient
2) transducing phage carries only the bacterial genes
Specialized transduction
-how does it differ from generalized?
1) only bacterial genes close to the attachment site of the prophage can be transduced
2) transducing phage carries both bacterial and phage genes
Bacterial Conjugation
- def
- directionality
- 2 ways it differs from transformation
-the transfer of DNA directly from one living bacterium to another
-transfer is unidirectional
Differs from transformation in 2 ways:
1) requires direct cell to cell contact
2) cells must be of opposite mating types
Transconjugant
recipient cell that receives the DNA
Most naturally occuring plasmids are either _____ _____ or __________.
self transmissable; mobilizable
Bacterium harboring a self transmissable plasmid is a potential _________
donor
Donor cells must have ______
fertility factors
F+ cell
a cell with a free fertility factor
Hfr cell
has a fertility factor inserted into the bacterial chromosome
Recipient cells (aka ____ cells) lack _______
F- cells; fertility factors
Fertility factor -F plasmid
- size/shape
- ds or ss?
- what controls replication?
- 2 origins of replication
- self _______
- transfer originates where?
- Large plasmid 94.5kb, circular, dsDNA
- controls its own replication
- 2 origins of replication
1) oriV -origin for bidirectional replication
2) oriS- origin for unidirectional replication - self mobilizable
- transfer originates at a special origin of transfer oriT, and proceeds via a rolling circle mechanism
OriV
Origin of vegetative replication
-bidirectional replication
OriT
Origin of transfer
-ss break occurs
Insertion sequences
where the bacterial chromosome integrates
The most important genes for transfer are:
1) Sex Pili genes (protein and assembly)
2) Genes for conjugal DNA metabolism
When an F factor (a plasmid) is transferred from a donor (F+) to a recipient (F-), the F- cell is converted to a ______ cell.
F+
Mechanism of DNA transfer during conjugation
1) one strand of plasmid DNA nicked at oriT by endonuclease encoded by tra genes
2) a specific helicase (encoded by tra) displaces the broken strand and other tra gene products transfer it to recipient cell
3) once inside the recipient cell the two ends are rejoined to make a circular molecule
4) new complimentary strand synthesized in the donor and recipient
Transfer (tra) genes
- def
- products are _____ acting
- genes required for transfer
- products of tra genes are trans acting and can act on another plasmid in the same cell
(e.g. pKM101 has 11 tra genes as well as eex gene (entry exclusion) which prevents the entry of other plasmids with same Tra functions)
Sex Pilus
- an appendage which serves to bring 2 cells together for mating
- only one protein pilin, product of tra genes makes up the pilus
- tra genes transport pilin through cell membrane & assemble pilus on the cell surface
- structure of sex pilus depends on the type of self transmissible plasmid
Regulation of tra genes
- naturally occuring plasmids transfer with a high efficiency for only a short time
- tra genes are repressed without synthesis of pilin & other tra gene products
- repression of tra genes is to prevent infection by some types of phages
- when plasmid containing population encounters non plasmid containing population, tra genes are expressed and plasmid is transferred
When an F factor becomes integrated into the chromosome of an F+ cell, it makes the cell a ________________(___) cell
high frequency of recombination (Hfr)
When an Hfr donor passes a portion of its chromosome into an F- recipient, a ______________ results
recombinant F- cell
Transposons
- def
- aka
- no need for DNA _______
- insert into DNA of?
- how are they replicated?
- frequently cause what?
- can carry genes for what?
- mobile genetic elements
- aka “jumping genes”
- short pieces of DNA that can insert in a donor chromosome without having DNA homology
- insert into DNA of phages, plasmids, and bacterial chromosomes
- do not replicate independently but are copied during their host’s DNA transcription
- frequently cause aberrant excision and carry DNA from the original location to a new site
- can carry genes for antibiotic resistance and virulence factors
A transposon can move from place to place in the chromosome, leaving a ___________ behind at the previous site
copy of itself
What 2 genes control the movement of the transposon?
Transposase & Resolvase
