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Bacterial Genetics. Bacteria are haploid identify loss-of-function mutations easier recessive mutations not masked 6-2 Copyright ©The McGraw-Hill Companies,

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Presentation on theme: "Bacterial Genetics. Bacteria are haploid identify loss-of-function mutations easier recessive mutations not masked 6-2 Copyright ©The McGraw-Hill Companies,"— Presentation transcript:

1 Bacterial Genetics

2 Bacteria are haploid identify loss-of-function mutations easier recessive mutations not masked 6-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3 Bacterial Genetics Bacteria reproduce asexually Crosses not used genetic transfer bacterial DNA segments transferred 6-3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

4 Enhances genetic diversity Types of transfer Conjugation direct physical contact & exchange Transduction phage Transformation uptake from environment 6-4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Genetic Transfer

5 Conjugation Many, but not all, species can conjugate Only certain strains can be donors Donor strain cells contain plasmid called F factor F + strains Plasmid circular, extra-chromosomal DNA molecule

6 Genes for conjugation F-factor Plasmid

7 Figure 6.4 Conjugation

8 Figure 6.4 Conjugation

9 Results of conjugation recipient cell acquires F factor converted from F – to F + cell F factor plasmid may carry additional genes called F’ factors F’ factor transfer can introduce genes & alter recipients genotype

10 1950s, Luca Cavalli-Sforza discovered E. coli strain very efficient at transferring chromosomal genes designated strain Hfr (high frequency of recombination) Hfr strains result from integration of F' factor into chromosome Hfr Strains Figure 6.5a

11 Hfr Conjugation Conjugation of Hfr & F – transfers portion of Hfr chromosome origin of transfer of integrated F factor starting point & direction of the transfer takes hrs for entire Hfr chromosome to be transfered Only a portion of the Hfr chromosome gets into the F – cell F – cells does not become F + or Hfr F – cell does acquire donor DNA recombines with homologous region on recipient chromosome

12 Figure 6.5b order of transfer is lac + – pro + F – now lac + pro – F – now lac + pro + Hfr Conjugation

13 Elie Wollman & François Jacob The rationale Hfr chromosome transferred linearly interruptions at different times  various lengths transferred order of genes on chromosome deduced by interrupting transfer at various time Interrupted Mating Technique

14 Wollman & Jacob started the experiment with two E. coli strains Hfr strain (donor) genotype thr + : Can synthesize threonine leu + : Can synthesize leucine azi s : Killed by azide ton s : Can be infected by T1 phage lac + : Can metabolize lactose gal + : Can metabolize galactose str s : Killed by streptomycin F – strain (recipient) genotype thr – leu – azi r ton r lac – gal – str r 6-21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

15 Figure 6.6

16 Interpreting the Data 6-26 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Minutes that Bacterial Cells were Allowed to Mate Before Blender Treatment Percent of Surviving Bacterial Colonies with the Following Genotypes thr + leu + azi s ton s lac + gal + 5–– After 10 minutes, the thr + leu + genotype was obtained The azi s gene is transferred first It is followed by the ton s gene The lac + gene enters between 15 & 20 minutes The gal + gene enters between 20 & 25 minutes There were no surviving colonies after 5 minutes of mating

17 6-27 From these data, Wollman & Jacob constructed the following genetic map: They also identified various Hfr strains in which the origin of transfer had been integrated at different places in the chromosome Comparison of the order of genes among these strains, demonstrated that the E. coli chromosome is circular Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

18 Conjugation experiments have been used to map genes on the E. coli chromosome The E. coli genetic map is 100 minutes long Approximately the time it takes to transfer the complete chromosome in an Hfr mating 6-28 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The E. coli Chromosome

19 6-29 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 6.7 Arbitrarily assigned the starting point Units are minutes Refer to the relative time it takes for genes to first enter an F – recipient during a conjugation experiment

20 6-30 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The distance between genes is determined by comparing their times of entry during an interrupted mating experiment The approximate time of entry is computed by extrapolating the time back to the origin Therefore these two genes are approximately 9 minutes apart along the E. coli chromosome Figure 6.7

21 Transduction is the transfer of DNA from one bacterium to another via a bacteriophage Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transduction A bacteriophage is a virus that specifically attacks bacterial cells It is composed of genetic material surrounded by a protein coat It can undergo two types of cycles Lytic Lysogenic Refer to Figure

22 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 6.9 Virulent phages only undergo a lytic cycle Temperate phages can follow both cycles 6-32 Prophage can exist in a dormant state for a long time It will undergo the lytic cycle

23 A plaque is a clear area on an otherwise opaque bacterial lawn on the agar surface of a petri dish It is caused by the lysis of bacterial cells as a result of the growth & reproduction of phages 6-54 Plaques Figure 6.14

24 Figure 6.10 Any piece of bacterial DNA can be incorporated into the phage This type of transduction is termed generalized transduction Transduction

25 Bacteria take up extracellular DNA Discovered by Frederick Griffith,1928, while working with strains of Streptococcus pneumoniae There are two types Natural transformation DNA uptake occurs without outside help Artificial transformation DNA uptake occurs with the help of special techniques Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transformation

26 Natural transformation occurs in a wide variety of bacteria Bacteria able to take up DNA = competent carry genes encoding competence factors proteins that uptake DNA into bacterium & incorporate it into the chromosome Transformation

27 6-47 Figure 6.12 A region of mismatch By DNA repair enzymes

28 Sometimes, the DNA that enters the cell is not homologous to any genes on the chromosome It may be incorporated at a random site on the chromosome This process is termed nonhomologous recombination Like cotransduction, transformation mapping is used for genes that are relatively close together Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-48 Transformation

29 Transfer of genes between different species vs Vertical gene transfer - transfer of genes from mother to daughter cell or from parents to offspring Sizable fraction of bacterial genes have moved by horizontal gene transfer Over 100 million years ~ 17% of E. coli & S. typhimurium genes have been shared by horizontal transfer Horizontal Gene Transfer

30 Genes acquired by horizontal transfer Genes that confer the ability to cause disease Genes that confer antibiotic resistance Horizontal transfer has contributed to acquired antibiotic resistance Horizontal Gene Transfer

31 Viruses are not living However, they have unique biological structures & functions, & therefore have traits We will focus our attention on bacteriophage T4 Its genetic material contains several dozen genes These genes encode a variety of proteins needed for the viral cycle Refer to Figure 6.13 for the T4 structure 6-51 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6.2 INTRAGENIC MAPPING IN BACTERIOPHAGES

32 6-52 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 6.13 Contains the genetic material Used for attachment to the bacterial surface

33 In the 1950s, Seymour Benzer embarked on a ten-year study focusing on the function of the T4 genes He conducted a detailed type of genetic mapping known as intragenic or fine structure mapping The difference between intragenic & intergenic mapping is: 6-53

34 A plaque is a clear area on an otherwise opaque bacterial lawn on the agar surface of a petri dish It is caused by the lysis of bacterial cells as a result of the growth & reproduction of phages 6-54 Plaques Figure 6.14

35 Some mutations in the phage’s genetic material can alter the ability of the phage to produce plaques Thus, plaques can be viewed as traits of bacteriophages Plaques are visible with the naked eye So mutations affecting them lend themselves to easier genetic analysis An example is a rapid-lysis mutant of bacteriophage T4, which forms unusually large plaques Refer to Figure 6.15 This mutant lyses bacterial cells more rapidly than do the wild-type phages Rapid-lysis mutant forms large, clearly defined plaques Wild-type phages produce smaller, fuzzy-edged plaques 6-55 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

36 Benzer studied one category of T4 phage mutant, designated rII (r stands for rapid lysis) It behaved differently in three different strains of E. coli In E. coli B rII phages produced unusually large plaques that had poor yields of bacteriophages The bacterium lyses so quickly that it does not have time to produce many new phages In E. coli K12S rII phages produced normal plaques that gave good yields of phages In E. coli K12( ) (has phage lambda DNA integrated into its chromosome) rII phages were not able to produce plaques at all As expected, the wild-type phage could infect all three strains 6-56 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

37 Benzer collected many rII mutant strains that can form large plaques in E. coli B & none in E. coli K12( ) But, are the mutations in the same gene or in different genes? To answer this question, he conducted complementation experiments Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-57 Complementation Tests

38 6-58 Figure 6.16 shows the possible outcomes of complementation experiments involving plaque formation mutants Figure 6.16 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

39 6-59 Benzer carefully considered the pattern of complementation & noncomplementation He determined that the rII mutations occurred in two different genes, which were termed rIIA & rIIB Benzer coined the term cistron to refer to the smallest genetic unit that gives a negative complementation test So, if two mutations occur in the same cistron, they cannot complement each other A cistron is equivalent to a gene However, it is not as commonly used Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

40 At an extremely low rate, two noncomplementing strains of viruses can produce an occasional viral plaque, if intragenic recombination has occurred Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-60 Coinfection rII mutations Viruses cannot form plaques in E. coli K12( ) Function of protein A will be restored Therefore new phages can be made in E. coli K12( ) Viral plaques will now be formed Figure 6.17

41 Figure 6.18 describes the general strategy for intragenic mapping of rII phage mutations 6-61

42 6-62 r103 r104 Take some of the phage preparation, dilute it greatly (10 -8 ) & infect E. coli B Take some of the phage preparation, dilute it somewhat (10 -6 ) & infect E. coli K12( ) 66 plaques 11 plaques Total number of phages Number of wild-type phages produced by intragenic recombination Both rII mutants & wild-type phages can infect this strain rII mutants cannot infect this strain

43 6-63 The data from Figure 6.18 can be used to estimate the distance between the two mutations in the same gene Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The phage preparation used to infect E. coli B was diluted by 10 8 (1:100,000,000) 1 ml of this dilution was used & 66 plaques were produced Therefore, the total number of phages in the original preparation is 66 X 10 8 = 6.6 X 10 9 or 6.6 billion phages per milliliter The phage preparation used to infect E. coli k12( ) was diluted by 10 6 (1:1,000,000) 1 ml of this dilution was used & 11 plaques were produced Therefore, the total number of wild-type phages is 11 X 10 6 or 11 million phages per milliliter

44 6-64 In this experiment, the intragenic recombination produces an equal number of recombinants Wild-type phages & double mutant phages However, only the wild-type phages are detected in the infection of E. coli k12( ) Therefore, the total number of recombinants is the number of wild- type phages multiplied by two Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 2 [wild-type plaques obtained in E. coli k12( )] Frequency of recombinants = Total number of plaques obtained in E. coli B 2(11 X 10 6 ) 6.6 X 10 9 Frequency of recombinants = = 3.3 X 10 –3 = In this example, there was approximately 3.3 recombinants per 1,000 phages

45 6-65 As in eukaryotic mapping, the frequency of recombinants can provide a measure of map distance along the bacteriophage chromosome In this case the map distance is between two mutations in the same gene The frequency of intragenic recombinants is correlated with the distance between the two mutations The farther apart they are the higher the frequency of recombinants Homoallelic mutations Mutations that happen to be located at exactly the same site in a gene They are not able to produce any wild-type recombinants So the map distance would be zero Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

46 Benzer used deletion mapping to localize many rII mutations to a fairly short region in gene A or gene B He utilized deletion strains of phage T4 Each is missing a known segment of the rIIA and/or rIIB genes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-66 Deletion Mapping

47 Let’s suppose that the goal is to know the approximate location of an rII mutation, such as r103 E. coli k12( ) is coinfected with r103 & a deletion strain If the deleted region includes the same region that contains the r103 mutation No intragenic wild-type recombinants are produced Therefore, plaques will not be formed If the deleted region does not overlap with the r103 mutation Intragenic wild-type recombinants can be produced And plaques will be formed Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-67

48 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-68 Figure 6.19

49 As described in Figure 6.19, the first step in the deletion mapping strategy localized rII mutations to seven regions Six in rIIA & one in rIIB Other strains were used to eventually localize each rII mutation to one of 47 regions 36 in rIIA & 11 in rIIB At this point, pairwise coinfections were made between mutant strains that had been localized to the same region This would precisely map their location relative to each other This resulted in a fine structure map with depicting the locations of hundreds of different rII mutations Refer to Figure 6.20 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-69

50 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-70 Figure 6.20 Contain many mutations at exactly the same site within the gene

51 Intragenic mapping studies were a pivotal achievement in our early understanding of gene structure Some scientists had envisioned a gene as being a particle-like entity that could not be further subdivided However, intragenic mapping revealed convincingly that this is not the case It showed that Mutations can occur at different parts within a single gene Intragenic crossing over can recombine these mutations, resulting in wild-type genes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6-71


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