Download 2054, Chap. 13, page 1 I. Microbial Recombination and Plasmids

2054, Chap. 13, page 1
I. Microbial Recombination and Plasmids (Chapter 13)
A. recombination = process of combining genetic material from 2 organisms to produce a
genotype different from either parent (exchange of DNA between different genes)
1. occurs during meiosis as crossing over between homologous chromosomes
2. genetic recombination (homologous recombination) is the most common form,
involving reciprocal exchange between a pair of homologous DNA sequences
a. involves exchange between nearly identical DNA sequences
b. can occur anywhere on the chromosome
c. DNA strands break and reunite after crossing over
d. involves products of rec genes
3. site-specific recombination (nonhomologous recombination) = nonreciprocal
recombination = exchange of DNA with different sequences
a. allows joining of DNA from different sources (e.g., phages, other bacteria,
plasmids)
b. most important for integration of viral genomes into bacterial chromosomes
c. transposons use a type of site-specific recombination called replicative
recombination
4. horizontal gene transfer = transfer of genetic material from one mature individual to
another
a. common in bacteria (think antibiotic resistance genes)
b. all living systems are capable of vertical gene transfer or the transfer of genetic
information to progeny
5. recombination is important in bacterial populations as a means of increasing genetic
diversity
6. recombination is also an important tool for creation of new strains useful in
biotechnology
B. Plasmids = small, circular DNA molecules that can exist independently of host
chromosomes (extrachromosomal)
1. have their own replication origins
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2. contain few genes (usually < 30)
3. not essential to host
4. curing = elimination of plasmid from host cell
5. plasmids with the fertility or F factor contain the information to allow cell
attachment and plasmid transfer between specific bacterial strains (conjugation)
a. F factor is about 100 kb long
b. F factor is an episome (= plasmid that can exist either with or without being
integrated into the host’s chromosome) by virtue of insertion sequences
6. R factors are plasmids that contain genes for resistance to antibiotics
a. do not integrate with the chromosome
b. may contain several different resistance genes
c. can be conjugative or nonconjugative, although nonconjugative plasmids may
transfer during plasmid promoted conjugation
d. can transfer between different genera, becoming a much greater public health
problem
7. Col plasmids code for bacteriocins (proteins that kill other bacteria)
a. bacteriocins open channels in the membrane
b. provides a competitive advantage
c. can be conjugative
8. other plasmids may carry genes that increase the virulence or provide additional
metabolic capabilities to the host
C. transposons = transposable genetic elements = jumping genes = genes that move
(transposition) around the chromosome
1. small segments of DNA that can move (be transposed) from one region of DNA
molecule to another
a. 700 - 40,000 bp
b. Barbara McClintock studied them in maize, but they occur in all organisms
2. all transposons carry the information for their own transfer
a. simplest are insertion sequences (IS elements)
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(1) short DNA sequence that contains only the genes required for transposition
(a) both ends have inverted repeat sequence that identifies integration site
(b) each type of IS has its own characteristic inverted repeats
(2) contain the gene for transposase (catalyzes the cutting and ligating of DNA)
and recognition sites
b. recognition sites are short regions of DNA in inverted repeats that the enzyme
recognizes as recombination sites between the transposon and the chromosome
3. composite transposons carry other genes, like toxins or antibiotic resistance
4. usually the original transposon remains at the parental site, while the replicate inserts
elsewhere
5. target sites are cut, transposon (ds) inserted, and ss gaps filled in
6. some transposons are conjugative
7. transposons have been observed in eukaryotes, bacteria, Archaea, yeast, maize,
Drosophila, and humans
8. transposons contribute to genetic diversity
D. conjugation = transfer of DNA between bacteria in direct contact
1. depends on plasmids (free or integrated)
2. Lederberg and Tatum mixed multiple auxotrophs and plated on minimal medium to
demonstrate genetic transfer
3. Davis demonstrated the need for contact by separating strains with a glass filter
4. F factor = fertility plasmid; plays a major role in conjugation
a. donors have F (F+), produce sex pilus, transfer the plasmid to recipients (F-),
which become F+
b. Hfr (high frequency of recombination) cell has the F factor integrated into the
chromosome
c. Hfr strains transfer part of their chromosome during conjugation
(1) origin of transfer lies within the F gene, so recipient does not contain complete
F unless entire chromosome is transferred
(2) through recombination, transferred DNA incorporated into chromosome at
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homologous site
(3) linear transfer, useful for mapping genes by using interrupted mating
(location related to time before transfer)
d. F’ plasmid is F plus chromosomal genes (deintegration of F plasmid)
(1) functions similar to F+
(2) provides diploid, important to determining dominant gene and mapping
E. transformation = transfer of genes as "naked" DNA in solution
1. DNA in environment (released from lysed cells) can be taken up by related bacteria
and incorporated into the chromosome by recombination
2. occurs naturally in a few genera of bacteria and can be induced in others
a. works best when donor DNA from closely related species
b. when recipient can take up DNA, it is said to be competent
c. increased concentrations of calcium chloride make cells more permeable
3. can be very useful for genetic engineering
4. classic case involve Streptococcus pneumoniae
a. capsule formation is required for virulence
b. dead virulent + live avirulent = live virulent + live avirulent
F. transduction = transfer of DNA between bacteria via a bacteriophage
1. in the lytic cycle, a phage infects a host, takes over machinery, produces new phages,
and lyses the host
a. 4 stages:
(1) attachment (adsorption and penetration)
(2) synthesis
(3) assemby
(4) lysis
b. usually called virulent bacteriophages
2. temperate phages are lysogenic
a. genome integrates with chromosome and is replicated as part of cell
b. under appropriate conditions, phages are reproduced
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c. new phages are released when the cell lysis
d. synthesis of lysogenic phage particles = induction
3. generalized transduction
a. phage attaches to bacterium and injects its DNA
b. DNA directs synthesis of new phages
c. during infection, bacterial chromosome breaks apart and some fragments can be
packaged inside the phage
d. later infections transfer bacterial genes to new cells
e. normal replication results in lysis but if the phage carries bacterial instead of viral
DNA, lysis does not occur (defective phage)
4. specialized (restricted) transduction = specific regions of bacterial DNA are
transferred
a. due to an error in the lysogenic life style
b. chromosomal DNA is excised with phage DNA when the prophage deintegrates
c. bacterial genes may get incorporated in an infected host
G. Mapping the genome
1. a genetic map shows the relative positions of different genes on a genome
2. interrupted mating of Hfr strains can determine relative locations through frequency
of transfer
3. frequency of gene transfer can also be used to map genes using transformation or
transduction
4. genetic maps provide information on gene locations, useful for genetic engineering,
and may provide physiological information
a. is the 33 minute region, which has few recognized genes, involved in attachment
of the chromosome to the cell membrane during replication and cell division?
b. genes can now be compared with DNA sequence information