Genetic Transfer in Bacteria
Lecture Outline:
1. Mechanisms of Transfer and Features in Common.
- survival- for bacteria to survive they must change, evolve,
and this can be accomplished by the accumulation of mutations
and/or the acquisition of "new" genetic information.
- acquisition of "new" genetic information- The 3 mechanisms
that allow DNA to be transferred from one cell, called the donor
cell, to another cell, called the recipient cell, differ in how
the DNA is transferred, how much genetic information is
transferred, and the types of genes transferred, i.e., chromosomal
versus plasmid genes.
- mechanisms of transfer-
- DNA-mediated transformation- free or naked DNA (isolated
from or released from a donor cell) is taken up by a recipient
cell.
- transduction- donor DNA is transferred from donor to
recipient cell by a bacteriophage.
- conjugation- cell to cell contact mediated by the sex-pilus
is required for DNA transfer.
- features in common between mechanisms of transfer-
- there is always a unidirectional transfer of DNA, not a
reciprocal exchange.
- only a portion of donor chromosomal DNA is transferred or
in the case of a plasmid the entire plasmid is
transferred.
- for donor chromosomal DNA to become an inheritable property
of recipient, DNA must integrate, insert itself, into the
recipient genome. Insertion occurs by the breakage and reunion
model (homologous recombination). This is not required of
plasmids. Why?
- all have potential to change genotype/phenotype of
recipient cell. To demonstrate that transfer has occurred there
must be a difference(s) between the donor, recipient, and the
resulting recombinant cell(s). In most cases looking for
phenotypic difference, or a selection procedure that will
select for a particular type of recombinant.
2. Mechanism of Transfer:
- DNA-mediated transformation- many bacteria, both Gram-positive
and negatives, are naturally transformable, while others including
yeast and mammalian cells can be artificially transformed. Will
discuss transformation that occurs in the Gram-positive bacterium
Streptococcus pneumoniae.
- source of DNA- lysed cell or extracted DNA from a donor
cell.
- competence state- in order for recipient cells to take up
the DNA, recipient must be in a state of competence. In S.
pneumoniae competence is established late in exponential
phase of growth, and is the result of having a few cells
synthesize and release a small protein called competence
factor, which induces cells in the population to synthesize
8-14 new proteins that establish the competence state (two of
these proteins are a DNA binding protein and a nuclease).
- binding/entry of DNA- recipient cell binds fragments of DNA
(~400 genes), which are cut into small fragments of ds-DNA (~40
genes). Entry of DNA is accompanied by the degradation of one
of the two strands.
- integration of DNA into recipient cell- if homology exists
between transferred DNA and recipients genome, have homologous
recombination that leads to the formation of a heterogenote
(two strands are not completely complimentary).
- DNA replication- resolves heteroduplex, and gives a
recombinant cell.
- Transduction:
- Generalized transduction- Can involve a lytic or temperate
bacteriophage. In generalized transduction can have transfer of
any group of genes of the infected host, but the maximum amount
of host DNA that can be transferred is equivalent to the size
of the phage genome (~100 genes).
- during phage gene expression, host chromosome is
fragmented.
- during maturation, by mistake, host DNA is packaged into
phage heads (no phage DNA in these heads). These particles
are called generalized transducing viruses.
- upon infection of new host, previous host DNA is
injected into cell, and if this DNA is homologous with
recipients DNA, will recombine.
- Specialized transduction- occurs with temperate
bacteriophage that are in the prophage state, and transfer only
a few chromosomal genes, and only those bacterial genes to one
side or the other of where prophage existed.
- during the excision of the prophage from host
chromosome, an error occurs in that DNA to one side or the
other of where prophage existed is cut out with phage genes
(a complete genomes worth of DNA is cut out). Excised DNA
contains chromosomal genes plus most of phage genome (some
of phage genome left in chromosome!).
- This excised DNA is replicated and packaged into phage
heads. These viruses are call specialized transducing
particles.
- upon infection of new host, DNA is injected, and if
homology exists with recipient DNA recombination occurs.
This type of infection never leads to a productive phage
infection. Why?
- Conjugation:
- F-plasmid- donor cell, designated the male cell,
synthesizes a sex-pilus which is encoded by genes on the F
(fertility) plasmid. The F-plasmid can replicate independent of
chromosome (cell called an F+ cell) or can integrate
into host chromosome (called an Hfr cell). Unlike a prophage,
F-plasmid genes are expressed. F-plasmid can intergrate into
several, but specific sites on chromosome. Cell lacking
F-plasmid is recipient cell, also designated the F-
cell or female cell.
- F'- occasionally when F-plasmid excises itself from
chromosome can take chromosomal genes to either side of
plasmid, but unlike prophage does not leave any plasmid genes
behind.
- role of sex-pilus- sex-pilus allows contact with an F-
cell. Sex-pilus retracts, bring mating cell together and
allowing DNA transfer to occur.
- types of mating-
- F+ X F-- in this type of mating
the entire F-plasmid is transferred to F- cell,
and no chromosomal genes are transferred. Donor retains copy
of F-plasmid. No integration of F-plasmid required.
- Hfr X F-- in mating of this type, chromosomal
genes to one side or the other of where F-plasmid is
integrated are transferred in a sequential manner. Since
conjugation bridge fragile, rarely, if ever, is the entire
donor chromosome transferred (F-plasmid genes would be last
to be transferred, and time required would be 100 minutes).
By interrupting mating, can map location of genes on
chromosome (~30-40 genes transferred per minutes, and 8 to
10 minute conjugations experiments are very realistic).
Integration of transferred DNA required. Why?
- F' X F-- mating is similar to that of an
F+ cell, but recipient cell may know have two
copies for certain genes (one copy on plasmid and another on
chromosome of recipient).
3. R-plasmids and their importance: Contain gene(s) for resistance
to antibiotics and/or heavy (toxic) metals. Some R-plasmids behave
life F-plasmid, and thus allow transfer, say, of R-plasmid from a
nonpathogenic bacteria to a pathogenic bacterium.
Learning Objectives:
- What is meant by homologous recombination?
- What are the basic features of DNA-mediated
transformation?
- How are bacterial genes transferred in transduction, and what
is the deference between generalized and specialized
transduction.
- How is gene transfer accomplished in conjugation, and how do
the different matings that can occur influence the number and
types of genes that are transferred.
- You should be familiar with the following words: competence,
plasmid, R-factors.
7/22/09