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Chapter 7 C Recombination in Bacteria and Their Viruses

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Chapter 7 C Recombination in Bacteria and Their Viruses Powered By Docstoc
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Chapter 7 – Recombination in Bacteria and Their Viruses


Questions to be addressed:
1) By what mechanisms is genetic information recombined in bacteria?
2) How are the distances between genes determined in bacteria?


Terminology


conjugation: the union of bacterial cells during which genetic information is transferred
from donor (F+) to recipient(F-)
plasmid: extrachromosomal, circular DNA
F (Fertility) factor: bacterial episome (present on plasmid or chromosome) which allows
a bacterial cell to be the donor during conjugation
Hfr (high frequency of recombination): a bacterial cell in which the F factor is
integrated into the chromosome; during conjugation, the F factor acts as the origin of
chromosomal transfer
exconjugate: a bacterium that has undergone conjugation
endogenote: the endogenous recipient chromosome
exogenote: the exogenous donor chromosome
merozygote: a cell which is a partial diploid containing both an endogenote and an
exogenote
transformation: introduction of foreign DNA material through external application
bacteriophage: "bacteria eater" - a virus that infects bacteria
lytic cycle: mode of infection in which the bacteriophage genome enters the bacterium,
replicates, lyses the cell and progeny are released
virulent bacteriophage - undergo the lytic cycle
prophage: bacteriophage genome which is integrated into the bacterial chromosome
lysogenic: "lysis causing" - prophage is replicated with bacterial chromosome; if
prophage is excised from the chromosome, lytic cycle is initiated
temperate bacteriophage - phage can enter the lysogenic cycle
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transduction: movement of genetic material from donor to recipient through a
bacteriophage vector




Characteristics of Bacteria:
• single-celled
• prokaryotic (i.e. no nucleus or membrane-bound organelles)
• DO NOT exhibit meiosis


Typical phenotypes in bacteria (see Table 7-1)
-often alternative phenotypes of protrophy (self-feeding) and auxotrophy (outside-
feeding) are used as markers for genetic analysis
CLASS 1: pairs of alleles that confer ability to synthesize amino acids, nucleotides, or
other essential macromolecules
ad+ - wild type allele, able to synthesize amino acid adenine - prototrophic
ad- - mutant allele, unable to synthesize amino acid adenine - auxootrophic
CLASS 2: pairs of alleles that confer ability to utilize energy sources
gal + - wild type allele, able to utilize galactose as a carbon source, prototrophic
gal - - mutant allele, unable to utilize galactose as a carbon source, auxotrophic
CLASS 3: pairs of alleles that confer resistance to compounds that normally inhibit
bacterial growth (e.g. antibiotics)
Strs - wild type allele, sensitive to streptomycin, unable to form colonies in presence of
streptomycin
Strr - mutant allele, resistant to streptomycin, able to form colonies in presence of
streptomycin




Scoring phenotypes (Figure 7-4):
-   a selection system is used
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-   e.g. to select antibiotic resistant bacteria, cells are grown on medium containing
    antibiotic
    -   wild type cells die, resistant cells form colonies which can be scored and selected
-   e.g. to select revertants, auxotrophic bacteria are grown on minimal medium
    -   cells of original (auxotrophic) phenotype die, cells which have reverted to wild
        type allele form colonies
-   e.g to select for auxotrophic mutants, bacteria are grown in a low concentration of
    penicillin
    -   rapidly dividing cells (prototrophs) die, auxotrophs which cannot divide survive
    -   if cells are washed of penicillin and replated on various supplemented media,
        auxotrophs can be selected


Question 1: How are new combinations of genetic information achieved?


Genetic material enters the bacterial cell by one of three mechanisms, and then undergoes
RECOMBINATION with homologous regions of the original bacterial chromosome.


Three mechanisms by which genetic information enters a bacterial cell:
1) CONJUGATION - exchange of genetic material between bacteria involving cell to
cell contact


2) TRANSFORMATION - exchange of genetic material between bacteria and their
environment


3) TRANSDUCTION - bacteriophage (virus) mediated transfer of DNA between
bacteria


CONJUGATION
- one member of the conjugating pair carries a fertility factor (F) within the F PLASMID
F plasmid - a small, circular, extrachromosomal piece of DNA
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F+ strains - bacteria contain the F factor
F- strains - bacteria lack the F factor


Properties of the F Factor (See figure 7-5):
1) enables the production of PILI (proteinaceous attachment tube between bacteria
facilitating cell to cell contact)
2) replication of F plasmid permits F to be maintained (F factor moves into F- cells during
conjugation)
3) prevents conjugation between two F+ cells


Lederberg and Tatum (1952) studied bacterial gene transfer and conjugation in
Escherichia coli (see box 7-1)


STRAIN A = -bio- cys leu+ phe thi thr+
STRAIN B = bio+ cys+ leu phe+ thi thr


- both strains are AUXOTROPHIC
- if strains A and B are combined, a low frequency (1 x 10-7) of the resulting bacteria are
PROTOTROPHIC


Question: How does recombination of genetic information occur?
Hypothesis 1: physical contact between the cells is required.
Experiment: place bacterial strains on either side of a filter, and determine if gene
transfer occur
Results:
• unable to transfer genes through filter


Hypothesis 2: the direction of gene transfer is directed, with one strain acting as donor,
the other as recipient.
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Experiment: expose one bacterial strain to streptomycin (an antibiotic; allows transfer of
DNA by bacterium, but inability to produce progeny)


     STUDY
Strain A       1) treat with streptomycin
               2) incubate with Strain B                 RESULT = prototrophs

Strain B       1) treat with streptomycin
               2) incubate with Strain A                 RESULT = no prototrophs

    CONCLUSION: transfer is directed; only Strain A may act as the donor
       and only Strain B acts as the recipient


Observation: Some F+ strains spontaneously become strains with a high frequency of
recombination (Hfr) (1000x more frequent recombination) of chromosomal genes


Question: Are some genes more likely to be recombined than others?


Experiment (see figure 7-9):



                         Hfr strain: strs tonr lac+ gal + azir
                         F- strain: strr tons lac- gal- azis


NOTE: To control the experiment, the time of contact between the two cells must be
constant.
•    combine the two strains
•    interrupt matings (disrupt conjugation) at specific times and sample cells resulting
     from mating
•    plate the samples on medium containing streptomycin
                                                                                       -
•    streptomycin kills the original Hfr cells (since strs) but permits the growth of F cells

     (strr)
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•   determine the genotype (test for tonr lac+ gal+ azir genetic markers) of the surviving
    bacteria by evaluating their ability to carry out metabolic pathway (e.g. a lac+
    bacterium can use lactose as a carbon source)


Results: the genetic markers are present with different frequencies at the different
sampling (mating) times (see figure 7-9)


                         genetic characters among strr exconjugates
                                                                      100
                                                                                                          azir
                                                                       80                                 tonr


                                                                       60

                                                                       40                                 lac+
                             Frequency of Hfr




                                                                                                          gal+
                                                                       20

                                                                       0
                                                                            0   10   20   30   40   50   60   70
                                                                                 Mating Time (minutes)




azir - high frequency at short matings
gal+ - greatest frequency only in long matings


Conclusions:
1) In Hfr strains, F factor is integrated into the bacterial chromosome promoting transfer
of chromosomal genes (Figure 7-6)
2) there is a fixed point at which transfer begins (origin) and a linear order to the transfer
process of the genes (Figure 7-8)
3) the time taken to transfer a gene is related to the distance from the origin to that gene
(Figure 7-9)


Additional observation: if the mating is permitted to continue (i.e. it is not interrupted),
the recipient cells become Hfr
Conclusions (Figure 7-7):
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F-factor consists of two parts
    • the first part to enter = origin
    • the last part to enter = terminus


- a cell must receive both the origin and the terminus to become Hfr


Question: Is the position of genes, the position of the origin, and the direction of transfer
constant in different Hfr strains? (see figure 7-11)


experiment: Consider 5 different Hfr strains (all derived from the same F+ strain) and
determine the sequence of gene transfer




                           = origin      a - e = genetic marker   F = F factor

                      strain 1 =         a     b      c       d     e      F
                      strain 2 =         c     d      e       a     b      F
                      strain 3 =         b     c      d       e     a      F
                      strain 4 =         c     b      a       e     d      F
                      strain 5 =         e     d      c       b     a      F



Results:
1) in each strain, the gene which enters first is different
2) the relative position of each gene is constant, (e.g. gene a in each strain is always
flanked by genes e and b


conclusion: the difference between Hfr strains is the position and orientation of the
origin within the circular chromosome



Useful Terminology:
EXCONJUGATE: a bacterium that has undergone conjugation
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    - select for recipient cells
ENDOGENOTE: the endogenous recipient chromosome
EXOGENOTE: the chromosome from the donor
MEROZYGOTE: partial diploid formed through conjugation


Hypothesis: using the sequence and time of transfer, a map of the chromosome can be
obtained (Figure 7-10)
1) based on the order of transfer it is possible to deduce the linear order of genes
2) based on the first gene transferred it is possible to determine the position and
orientation of the F-factor
3) the time it takes to transfer the genes from the donor to the recipient is equal to the
distance between the genes


EXAMPLE
Five Hfr strains derived from the same E. coli original strain are allowed to conjugate
with an F- strain. The following table shows entry times (minutes) of the first four genes.


              Strain 1        Strain 2      Strain 3       Strain 4       Strain 5
               a (10)          c (5)         b (3)          c (6)          e (25)
               b (18)              d (20)    c (13)         b (16)          d (45)
               c (28)              e (40)    d (28)         a (24)          c (60)
               d (43)              a (70)    3 (48)         e (54)          b (70)



question: derive a map of the original E. coli strain


MAPPING BASED ON RECOMBINATION FREQUENCY (Figure 7-12)
hypothesis: once genes are transferred into the F- strain, they are able to recombine with
genes in the host chromosome and the frequency of recombination between two genes
will depend upon the distance between those genes
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    gal+            arg+           met +
                                                    transferred DNA

    gal- -
    gal             arg--
                    arg            met- -
                                   met
                                                     host chromosome




Solving the problem of gradient of transfer - gene transfer is based on the distance
from the origin, with genes nearest the origin being transferred at the highest frequency,
and genes farthest from the origin transferred at the lowest frequency = gradient of
transfer


INTEGRATION = transfer and recombination
•   a gene must be transferred before it can be integrated into the chromosome.
•   gradient of transfer means that genes closer to the origin will be transferred and
    therefore integrated more frequently than genes further from the origin
•   distorts the recombination frequency as a measure of distance


How to remove the gradient of transfer distortion?


EXAMPLE
             Hfr strain gal+ arg+ met+         X    F- strain gal- arg- met-

           RESULT
             gene            frequency of                implication
                             inheritance
             met+              100%                  first gene to be transferred
             arg+              70%
             gal+              30%                   last gene to be transferred


• since met+ is the most frequently integrated gene, it is the first gene transferred
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• since gal+ is the least frequently integrated gene, it is the last gene transferred


recall that:
integration = transfer and recombination
•    if transfer is equal for all genes then integration = recombination


therefore, frequency of integration
                        = frequency of recombination
                        = distance between two genes
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              PARENTALS
                          endogenote = gal- arg- met-      (F-)
                          exogenote = gal + arg+ met+      (Hfr)

              RESULTS
                   • all exconjugates (i.e. 100%) selected are gal+

                      •       5%    arg- met-
                              10%   arg+ met-
                              0%    arg- met+
                              85%   arg+ met+

                     order of transfer: gal+ arg+ met+


gal+ arg- met- = recombination between gal and arg

     gal+         arg+          met +           % recombinants = map distance gal - arg
                                                                   = 5 m.u.
     gal-         arg-          met-


gal+ arg+ met- = recombination between arg and met

     gal+         arg+          met +           % recombinants = map distance arg - met
                                                               = 10 m.u.
     gal-         arg-          met-

gal+ arg- met+ = recombination 1 + 2

     gal+         arg+          met +           4 x crossover class = least frequent


     gal-         arg-          met-

gal+ arg+ met+ = recombination past met

     gal+         arg+          met +


     gal-         arg-          met-
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TRANSFORMATION
- introduction of DNA fragments from the environment through the bacterial cell wall
- recombination leads to integration


- transformation can also be induced in plant and animal cells
- the frequency of bacterial transformation can be increased by manipulating [Ca+2] and
electric shock
     (a treated cell is said to be COMPETENT to take up DNA)


Linkage Information and Transformation
-DNA is introduced as fragments, no direction of transfer
- the closer together two genes are, the more likely that they will be on the same fragment
and integrate into the chromosome together
-    can deduce the relative distance between pairs of genes based on
     COTRANSFORMATION frequency (the introduction of more than one gene
     simultaneously)
-    if two genes are unlinked, the frequency of cotransformation will be equal to the
     product of their individual transformation frequencies


EXAMPLE
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                    Original Cell:                 met- gal- leu-
                    Transforming DNA:              met+ gal+ leu+

Method: select for 1 marker and determine the frequency of the other
     two markers

Results:
      Experiment                Selected Marker                  Unselected Marker
                1                      met+                       20% gal+, 2% leu +
                2                      leu+                       8% gal+, 1% met+

Experiment 1 Conculsion:
• met is closer to gal than to leu
        Option A       met gal                             leu
     OR

          Option B       gal     met                       leu

Experiment 2 Conclusion
• leu is closer to gal than to met

          therefore, Option A is correct (met           gal                    leu)



BACTERIOPHAGES (refer to Figures 7-16, 7-17, 7-18)
- viruses that infect bacteria (e.g. T2, T4, l = lambda)
- consist of nucleic acid surrounded by a protein coat (= capsid)


Lytic Cycle (refer to Figure 7-17, 7-18)
1) phage recognizes and attaches to bacterium
2) viral genome enters bacterium
3) bacterium makes copies of the viral genome and capsid
4) new phages assemble within host bacterium
5) bacterial cell is lysed and viral particles are released
- if bacteria are infected and then grown on an agar plate, the lysis of the bacterial cell
results in the formation of a clear area of dead bacteria known as a PLAQUE
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Mapping in Bacteriophages
-    requires that two genetically distinct phages come together and have the opportunity
     to recombine
     -   the Phage Cross (Figure 7-20, 7-21, Table 7-2)
-    coinfection of a bacterium with two distinct phage genotypes by incubating bacteria
     with a very high phage concentration (Figure 7-20)
     -   e.g. phage 1 = h+ r-
         phage 2 = h- r+
         -   h = "host range" - can only affect one strain of E. coli
         -   h - can affect two strains of E. coli
         -   r - "rapid lysis" - rapidly lyses E.coli
         -   r - slowly lyses E.coli
-    progeny of infection is incubated with a combination of both E.coli strains, and 4
     genotypes (2 parental , 2 recombinant) scored
-    recombination frequency = #recombinant plaques/total plaques


Lysogenic Cycle
•    the viral genome is integrated into the bacterial chromosome (PROPHAGE)
•    integration of the prophage occurs at a particular site within the bacterial chromosome
•    example:
     •   lambda and E. coli (both have circular genomes)
     •   integration is accomplished through a recombination event at specific
         ATTACHMENT SITES (att sites) in the phage and bacterial genomes (see figure
         7-23)
     •   the incorporated viral genome is replicated each time the bacterial chromosome is
         duplicated
     •   adverse conditions may trigger the viral genome to become released from the
         bacterial chromosome and subsequently induce lysis
         •   excision (entry into the lytic cycle) is through reversal of the recombination
             event
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       •   if a lysogenic Hfr bacterium conjugates with a nonlysogenic F-bacterium, the
           transferred prophage immediately enters the lytic cycle = zygotic induction
           (Figure 7-22)
       •   repressors present in cytoplasm of lysogenic cell maintain prophage in
           lysogenic state
       •   nonlysogenic cell has no repressors, therefore lysis is induced


TRANSDUCTION: the bacteriophage mediated transfer of genetic material
- information is transferred between two bacteria via a bacteriophage
Hypothesis: physical contact is necessary for transfer of genetic material
experiment: assess if genetic material can be transferred between bacteria when they are
separated by a filter
results:
• the exclusion limit (point at which genetic material cannot be transferred) is determined
by the size of the phage particle
• the phage is the VECTOR of genetic material transfer


- there are two types of transduction
1) Generalized Transduction (Figure 7-24) - random sample of genes are transduced
- the bacterial chromosome disintegrates when the cell is lysed
- genomic fragments may be incorporated into the phage particle (FAULTY HEAD
STUFFING)
- subsequent phage infection of a new bacterial cell introduces the old disintegrated
fragment into new bacterium
- recombination leads to integration of the fragment


- each gene is equally likely to be transduced
- proximity of genes is implied by …? (figure 7-26)


2) Specialized Transduction (see Figure 7-25)
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•    excision of prophage initiates the lytic cycle
•    if recombination is not exact, bacterial genes close to the attachment site may be
     incorporated
•    in lambda (l), the gal+ genes is adjacent to the attachment site
•    if the phage leaves it genes it may be incapable of integrating (i.e. becomes defective)
        •   (lambda d gal)
•    the gene most frequently transduced is nearest the attachment site

				
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