Mapping in Bacteria and Bacteriophages
Mapping in Bacteria and Bacteriophages BIO3010
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This 22 page Class Notes was uploaded by Samantha on Monday February 1, 2016. The Class Notes belongs to BIO3010 at University of Toledo taught by Dr Krishnamurthy in Winter 2016. Since its upload, it has received 37 views. For similar materials see Genetics in Biology at University of Toledo.
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Date Created: 02/01/16
Mapping in Bacteria and Bacteriophages Bacterial growth curve o Start culture with small inoculum of bacteria (a few thousand cells) o Lag phase-slow growth initially o Log phase- cells divide continuously Short interval between cell division May divide every 20 min Exponential growth period o Stationary phase 9 Culture reaches saturation (about 10 cells/ml Nutrients and Oxygen are depleted o Because bacteria grow so rapidly and produce so many offspring it is easy to identify spontaneous mutants Heritable variation How does the variation arise? Serial Dilution of Bacterial Culture o Cells grown in liquid medium can be quantified by transferring to a semisolid medium (agar plate) in a Petri dish. Each dilution varies by a factor of 10. Each colony is derived from a single bacterial cell. Mutation in Bacteria o Fluctuation test by Luria and Delbruck Spontaneous mutation is the primary source of genetic variation in bacteria o Phototrophs- Can synthesize all essential organic compounds for growth from a “minimal medium” Single carbon and nitrogen sources and inorganic ions Wild-type for all growth requirements grow on minimal medium o Auxotroph- mutation results in loss of the ability to make at least one essential organic component loss of ability to hake HIS amino acid must be added to minimal medium for growth do not grow on minimal medium, need supplements Genetic Recombination in bacteria o Genetic recombination in bacteria – process that leads to the formation of new gene combinations on chromosomes Replace one or more genes present in a strain with genes from a genetically distinct strain Produces an altered phenotype Led to investigations of the arrangement of genes on the chromosome Three processes that can lead to recombination in bacteria Conjugation - sources of genetic variation Transformation - genetic maps (order of Transduction genes on chromosome) Lederberg and Tatum (1946) Bacteria undergo a parasexual process Genetic material from one bacterium is transferred to and recombined with that of another o Call conjugation o Experiment: mixed tow multiple auxotroph’s and obtained prototroph’s o Transfer between the two strains led to genetic recombination Replacement of one or more genes present in one strain with those from a genetically distinct strain Requires thr, leu and thi to grow Requires met and bio to grow Very low probability that any of the cells underwent spontaneous mutation Prototrophs arose from some form of genetic exchange and recombination between the 2 mutants Recombination overview Techniques that allowed the identification and study of mutations let to investigations into the arrangement of genes on the bacterial chromosomes Lederberg and Tatum-described bacterial conjugation- allows the unidirectional flow of genetic information from one bacterial cell to another This work led to rapid advances in mapping on the E. coli (and other bacterial) chromosome Genetic recombination in bacteria-replaces one or more genes present in a strain with genes from a genetically distinct strain – different from eukaryotes where genetic material is exchanged in reciprocal crossover events The effect of recombination is that genetic information is transferred from one chromosome to another, resulting in an altered genotype Fertility factor – F Conjugation – a unidirectional transfer of genetic information. Two different strains are involved in the transfer + o Donor cells – F (provide genetic material DNA) o Recipient Cells – F (receive the donor genetic material) Physical contact between cells of the two strains is essential for genetic recombination Davis U-Tube Experiment When strain A and B auxotrophs are grown in a common medium but separated by a filter no genetic recombination occurs and no prototrophs are produced Showed that physical contact between the two cells is REQUIRED for genetic recombination Genetic recombination in bacteria – conjugation o F- factor is mobile (based on Davis U-tube experiment) Following conjugation and genetic recombination recipient cells always become F + F- factor is a large plasmid and is itself passed to ALL recipients Circular double stranded with about 100,000 base pairs 19 genes involved in formation of the pilus and conjugation o When f-factor is present the cell can form a pilus and has the potential to serve as a donor of genetic material o A single strand of the F factor plasmid is transferred during conjugation Both strands are replicated as the plasmid unwinds and the DNA is transferred to the recipient cells o In F x F conjugation none of the bacterial chromosome is transferred o Conjugation may be initiated by a bacterium housing a plasmid called F factor in its cytoplasm, making it a donor F cell. Following conjugation the recipient F cell receives + a copy of the F factor and is converted to the F status o Mating between F and F bacteria During conjugation, the DNA of the F factor replicates with one new copy entering the recipient The black bar added to the F factors follows their clockwise rotation during replication. F factor contains an originTransfer begins when one strand of the F t begins factor is nicked at the origin. Protein Once transfer is complete, encoded by a gene on F factor, endonuclease the F cell becomes F+, donor cell remains F+ A single strand of DNA is transferred to the recipient and the DNA is replicated through the rolling circle mechanism. As the DNA enters the F recipient it is o Hfr bacteria and chromosome mapping Treatment of an F strain of E. coli K12 with nitrogend mustard (chemical carcinogen, generates mutations) generated donor bacteria that recombined 1000 times + more frequently that the original F strain Designated Hfr, High Frequency Recombination, remain + donors F Two important features If the donor is from Hfr strain, the recipient cells NEVER becomes Hfr but remain F - + - + o F x F F (low rate of recombination) o Hfr x F F (high rate of recombination) Nature of recombination: In any given strain certain genes are more frequently recombined than others and some are not at all. Donor bacteria stains that undergo recombination at a + rate much higher than F cells Hfr cells are a special class of F cells (derivatives of F )+ Hfr cells originate by a rare crossover event in which the F factor integrates into the bacterial chromosome All genes on the F factor are now integrated into the host chromosome The F factor replicates with the hose chromosome instead of independently Because of the F factor genes Hfr cells can conjugate with - F cells F factor is integrated into the donor cell’s chromosome Chromosomal genes can be transferred during conjugation During conjugation the F factor is transferred last It rarely gets transferred because conjugation is broken apart before is can be transferred F cells almost never acquire the Hfr phenotype Interrupted mating technique Ellie Wollman and Francois Jacob; differences between Hfr and F and showed how Hfr strains allow genetic mapping of the E. coli chromosome - Hfr and F strains with suitable markers were mixed and recombination of specific genes was assayed at different times o Cultures of the two strains were mixed and samples were removed at various times and placed in a blender o The shear forces in the blender separated conjugating bacteria so that transfer of the chromosome was terminated o The cells were then assayed for recombination The progressive transfer during conjugation of various genes from a specific Hfr strain of E. coli to an F strain. Certain genes (azi and ton) transfer sooner than others and recombine more frequently. Others (lac and gal) take longer to transfer and recombine with a lower frequency. Demonstrates an Ordered Transfer of Genes Transfer correlated with the A time map of the genes studied in the experiment depicted in Figure 9–7. Different Hfr strains have the F factor integrated into the genome at different sites -Different point of origin for the start of Genetic mapping in Hfr strains overview transfer Interrupted mating technique o Certain genes were transferred before others o Genes always transferred in a certain order o Correlated with the length of time conjugation proceeded Chromosome of Hfr was transferred linearly and the gene order could be predicted Time between transfer of genes can be used as a measure of the distance of the genes between one another Used to create the first genetic map of E. coli with minutes corresponding to the map units in eukaryotes Different Hfr strains have F factor integrated at different sites o Argued that the chromosome is circular o F factor integrated int0 different regions of the chromosome o Serve as the origin of transfer of DNA from one strain to another Order of Transfer of genes in different Hfr strains Origin of transfer differs in different Hfr strains the order of the genes in the chromosome are the same, they just transfer at different times, depending on the location of the origin. (a) The order of gene transfer in four Hfr strains, suggesting that the E. coli chromosome is circular. (b) The point where transfer originates (O) is identified in each strain. Conversion of an F strain to Hfr Occurs spontaneously at a low frequency Occurs by integrating the F factor into the bacterial The pochromosome.gration of F factor determines the origin (O) of transfer. Genes adjacent to O are transferred first, and the F factor becomes the last part to be transferred. Conjugation does not last long enough to allow passage of entire chromosome across the conjugation tube Only A and B genes are transferred into an F- cell which may recombine with the host Chromosome. F factor remains in donor cell, so recipient cell remains F - + - Recombination of F x F mating At a low frequency the F factor spontaneously integrates from the cytoplasm to a random point in the chromosome converting F to the Hfr state - Newly formed Hfr can conjugate with F the genes that are transferred with also appear to be random + - within the F / F population Recipient bacterium will appear as a recombinant - but will remain F Subsequent conjugation with an F cell will convert + it to F Genetic recombination in bacteria + A Hfr strain has an integrated F factor (plasmid) Occasionally this will recombine back out of the chromosome and revert to new type F state (F’) + o Excision is imprecise and the new F factor will carry adjacent genes – this is called F’ In this case bacterial genes are now part of the F factor plasmid and not part of the bacterial chromosome When an F’ factor is transferred to another cell (F’) by conjugation there will be two copies of the genes in the recipient cell + o The F recipient becomes partial diploid or merozygote Conversion of Hfr cells to F’ cell - The F recipient becomes partial diploid or Merozygote Recombination and Rec Proteins How does recombination occur in bacteria o Rec genes o Mutation bacterial strains isolated that had reduced/absent ability to undergo recombination recA, recB, recC, and recD – reduced recombination frequencies o recA - ~1000 fold decrease in recombination o others ~ 100 fold decrese recA protein – when deDNA enters a recipient cell one strand is degraded, complementary strand finds its homologous region on the chromosome facilitated by recA recBCD – 3 subunits – complex has enzymatic activity unwinding the helix other types of F factor plasmids o R plasmids – contain antibiotic resistance genes-makes it easy for bacterial cells to pass on resistance to other strains (or species) Genetic recombination in bacteria Mutations in the rec genes decrease the frequency of recombination of F plasmid o recA, recB, recC, and recD diminish recombination from 100-1000 fold F factors and R plasmids o Small circular double-stranded DNA molecules o Distinct from the genomic DNA R plasmids contain one or more resistance factors o Such as antibiotic resistance genes o Very important in hospitals o A BIG problem when they carry multiple drug resistance Transposons often allow movement of the R genes R-plasmids resistance transfer factor and r-determinants A Plasmid of E. coli (a) Electron micrograph of a plasmid isolated from E. coli. (b) An R plasmid containing (b) transfer factors (RTFs) and multiple r- Bacterial Transformation determinants (Tc, tetracycline; Kan, Mechanism for uptake of DNA into bacterial cellnamycin; o Can result in recombination (c) Sm, streptomycin; Su, sulfonamide; The DNA recombines with a homologous region iand Hg, mercury). the recipients chromosome o Leads to stable exchange of genetic information Another technique for genetic mapping o Only if the recombined DA produces a different phenotype than the original phenotype of the recipient strain Transformation o Transformation Bacterial transformation – another mechanism for transfer of genetic information form one bacterial strain to another Different process than conjugation Griffith and Avery, MacLeod, McCarty used transformation in their experiments o Mechanism by which small pieces of DNA (10-20kb) are taken up by bacteria through a receptor-mediated mechanism o DNA that is taken up recombines with the homologous region of the recipient’s chromosome o DNA fragments many be large enough to contain several genes-allows for this to be used as a technique for genetic mapping o Genes that are close to one another on the bacterial chromosome (linked) will be more likely to transform together than genes that are far apart o Linkage in bacteria – genes that are close together on the chromosome have a greater likelihood of transferring together Co-transformation Proposed steps for transforming a bacterial cell by exogenous DNA o Entry of DNA into the cell o Recombination of the donor DNA with its homologous region in the recipient chromosome Bacteria must be competent to take up the DNA o Occurs through a receptor-mediated process (requires energy) Only one of the two entering DNA strands is involved in the transformation event o One strand is digested away by nucleases o The other strand aligns with its complementary region of the chromosome o Recombination enzymes excise the counterpart and replace it with the new strand o Heteroduplex – two complementary strands may have a different DNA sequence Co-transformation and Linkage-transformation between 10-20,000 bp o May include several genes o Co-transformed genes are linked Mechanism of bacterial transformation o Genetic analysis of bacteriophage Transduction- bacterial recombination mediated by bacteriophage Lederberg- Zinder experiment Used a Davis-U tub for this experiment Found that recombination between two strains of salmonella occurred without the cells touching A ‘filterable agent’ as cause o Determined to be a phage Bacteriophage structure Placed two auxotrophic strains on opposite sides of a Davis U-tube Cells share the same media but are NOT in physical contact because the cells are not physically touching the prototrophs could nto have arisen due to conjugation Recovered prototrophs from the side containing the LA-22 strain but not from the side containing the LA-2. FA ws produced by the LA-2 cells only when grown in association with LA-22. Culture medium from LA-2 cells added to LA-22, no recombination. LA-22 play some role in FA production only when they share common growth medium Filterable agent from LA-22 strain is responsible for the recombination DNAse digestion did not affect recombination. FA is nto naked DNA ruling out transformation Reduced pore size in Davis U-tube blocked recombination These initial observations led to the discovery of the phenomenon called transduction The Structure of Bacteriophage T4 -an icosahedral head filled with DNA (about 150 genes) -a tail consisting of a collar, tube, sheath, base plate, and tail fibers. -During assembly, the tail components are added to Life cycle of bacteriophage T4 the head and then tail Life cycle: fibers are added. o Bacteriophage binds (adsorbs) to the bacterial host cell During infection the tail o Tail fibers recognize/bind to regions of the cell fibers recognize binding wall sites on the surface of the o Central core contracts and penetrates the cell bacterial cell wall wall (ATP dependent process) o DNA in the head is extruded into the bacterial cell Infection by the bacteriophage blocks bacterial DNA, RNA, and protein synthesis o Host cell DNA s degraded Synthesis of viral molecules begins o Phage DNA replication begins first o Protein components are then synthesized Head, tail, and tail fibers Mature viruses are assembled o DNA packaging into viral head o Tail assembly o Tail fiber assembly Lysozyme-enzyme produced by the phage o Digests bacterial cell wall, results in lysis and release of phage Plaque Assay for bacteriophage infection A Plaque Assay for Bacteriophage Analysis -Bacteriophage are an important tool used in molecular biology/ molecular genetic labs -Phage are used to infect a lawn of bacteria growing on an agar plate -Perform 10-fold serial dilutions and add phage to melted agar -Pour agar over lawn of bacteria -Because the phae cBacteriophage Infection – Lysogenic or Lytic clear spot will Lytic- bacteriophage infects the cell, replicates, and lyses the cell form in the lawn Lysogenic- bacteriophage infects the cell, but the -Caused by a single phage tphage DNA integrates into the host cell genome bact. o No new phage are produced -Phage in the plaque are clones of the originalcates with the hose cell DNA infecting phage o No cell lysis Cell stress conditions like chemical or UV light – -The phage can then be isolviral DNA loses its integrated status and initiates further studies replication, phage reproduction and lysis of the bacterium to release new phage Lysogeny vs Lytic Prophage- viral DNA that is integrated into the bacterial chromosome Temperate phage- viruses that can either lyse the cell or integrate into the bacterial chromosome to become a prophage Virulent phage- viruses that only lyse the cell Lysogenic bacteria- a bacteria that harbors a prophage o This cell is capable of being lysed as a result of induced viral reproduction Episome- viral DNA which can either replicate in the bacterial cytoplasm or become integrated into the chromosome Bacteriophages can be: o Lytic – infect the host cell, reproduce and then lyse it o Lysogenic – infect, integrate into the chromosome, do not reproduce Transduction Bacterial recombination mediated by bacteriophage Bacteriophage P22 is present as a prophage in the chromosome of LA-22 Rarely enter vegetative or lytic phase, reproduce and are released by LA-22 cells P22 phages being much smaller than bacterium pass through the filter and infect and lyse some of LA-2 cells In the process P22 occasionally package a region of the LA-2 chromosome in their heads If this region carries phe+ and trp+ genes and the phages pass back across the filter and infect LA-22 cells these newly lysogenized cells behave like prototrophs Mechanism for bacteriophage – mediated transduction Generalized Transduction -Host cell DNA is degraded by the phage -DNA fragments can be packaged into virus head instead of the viral DNA Lytic Phage – Induces degradation of host cell Transduction by bacteriophage DNA upon infection Excision of prophage DNA from bacterial genome ise – Reactivation of prophage imprecise and can take bacterial DNA as well induces degradation of host cell (similar to Hfr conversion to F’) DNA This DNA is packaged into the phage head and can then integrate into the chromosome of a newly infected bacteria The initial transduction was discovered in a temperate phage and lysogenized bacterium also applies to lytic cycle Phage lysis of bacteria causes the host bacteria DNA to be degraded. This DNA can then be packaged into viral head o Abortive transduction- a phage with bacterial DNA infects a cell but the DNA does not recombine into the new hosts chromosome transmitted to one progeny cell following each division o Complete Transduction- when the bacterial DNA recombine with its homologous region of the bacterial chromosome transduced genes are replicated and passed to all daughter cells Co-transduction – genes that are closely linked on the bacterial chromosome can be simultaneously transduced Transduction mapping- used to determine gene order o Similar to mapping using the interrupted mating technique o Genes that co-translate are closely linked
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