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1 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 2 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 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 3 Strr - mutant allele, resistant to streptomycin, able to form colonies in presence of streptomycin Scoring phenotypes (Figure 7-4): - a selection system is used - 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 4 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 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 5 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. 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): 6 s tonr lac+ gal+ azir Hfr strain: str r tons lac- gal- azis F- strain: str 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) • 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 Frequency of Hfr geneticcharacters among strr exconjugates the different sampling (mating) times (see figure 7-9) 100 azir tonr 80 60 lac+ 40 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 7 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): 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 Results: 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 8 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 - 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. 9 Strain 1 a (10) Strain 2 c (5) Strain 3 b (3) Strain 4 c (6) Strain 5 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 gal+ arg+ met+ gal gal- - arg-- met met- - transferred DNA 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 10 • distorts the recombination frequency as a measure of distance How to remove the gradient of transfer distortion? EXAMPLE Hfr strain gal+ arg+ met+ X -Fstrain gal- arg- met- RESULT gene met+ arg+ gal+ frequency of inheritance 100% 70% 30% implication first gene to be transferred last gene to be transferred • since met+ is the most frequently integrated gene, it is the first gene transferred • 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 11 12 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 13 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 14 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 720) - e.g. phage 1 = h+ rphage 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 15 • excision (entry into the lytic cycle) is through reversal of the recombination event • if a lysogenic Hfr bacterium conjugates with a nonlysogenic Fbacterium, 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 16 - proximity of genes is implied by …? (figure 7-26) 2) Specialized Transduction (see Figure 7-25) • 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