Chapter 7: Bacterial and Viral Systems

Plaque

A clear patch of lysed cells in a lawn of cells on a plate.

Complete medium

A medium that contains all the substances, such as the amino acid leucine, required by bacteria for growth and reproduction.

minimal medium

A medium that contains only the nutrients required by prototrophic bacteria.

Concept Check 2: Which process of DNA transfer in bacteria requires a virus? a.) Conjugation b.) Transduction c.) Transformation d.) All the above

B.) Transduction

Transformants

Cells that receive genetic material through transformation

Competent

Cells that take up DNA through their cell membrane

Concept Check 4: A bacterial strain with genotype his- leu- thr- is transformed with DNA from a strain that is his+ leu+ thr+. A few leu+ thr+ cells and a few his+ th+ cells are found, but no his+ leu+ cells are observed. Which genes are farthest apart?

his and leu

oncogenes

stimulate cell division and cause formation of tumors

Conjugation

takes place when genetic material passes directly from one bacterium to another. Typically happens when two bacteria lie close together and a connection forms between them.

integrase

the enzyme responsible for integrating viral DNA into the host cell's DNA

Pili (pilus)

Extension of the surface of some bacteria that allows conjugation to take place. When a pilus on one cell makes contact with a receptor on another cell, the pilus contracts and pulls the two cells together.

Antigenic Shift

Major changes in the viral genome take place in which genetic material from different strains is combined in a process called reassortment.

Horizontal Gene transfer

Many bacteria have aquired genetic information from other species of bacteria, and sometimes even Eukaryotic organisms

21.)A geneticist isolates two mutations in a bacteriophage. One mutation causes clear plaques (c), and the other produces minute plaques( m). Previous mapping experiments have established that the genes responsible for these two mutations are 8 m.u. apart. The geneticist mixes phages with genotype c+m+ and genotype c-m- and uses the mixture to infect bacterial cells. She collects the progeny phages and cultures a sample of them on plated bacteria. A total of 1000 plaques are observed. What numbers of the different types of plaques (c+m+, c-m-,c+m-. c-m+) should she expect to see?

Number of plaques produced by c+m+=460; by c+m- = 40(recombinant); by c-m+= 40 (recombinant)

Concept Check 7: What enzyme is used by a retrovirus to make a DNA copy of its genome?

Reverse transcriptase

Retrovirus

viruses capable of integrating into the genomes of their hosts, much as temperate phages insert themselves into bacterial chromosomes

8.) Briefly explain how genes in phages are mapped.

To map genes in phages, bacterial cells are doubly infected with phage particles that differ in two or more genes. During the production of new phage progeny, the phage DNAs can undergo recombination, thus resulting in the formation of recombinant plaques. The rate of recombination is used to determine the linear order and relative distances between genes. The farther apart two genes are on the chromosome, the more frequently they will recombine.

3.) What types of matings are possible between F+, F-, Hfr and F' cells? What outcomes do these matings produce? What is the role of the F factor in conjugation?

Types of mating outcomesF+ X F- two F+ cellsHfr X F- one Hfr and one F- cellF' X F- two F' cellsF factor contains a number of genes that take part in the conjugation process, including genes necessary for the synthesis of the sex pilus. The F factor also has an origin of replication that enables the factor to be replicated in the conjugation process.

Specialized transduction

a highly specific part of the host genome is regularly incorporated into the virus

Transducing phages

a piece of the bacterial chromosome instead of phage DNA occasionally gets packaged into a phage coat

Provirus

a viral genome incorporated into the host chromosome

18.) Rollin Hotchkiss and Julius Marmur studied transformation in the bacterium Streptococcus pneumoniae. They examine four mutations in this bacterium: penicillin resistance (P), streptomycin resistance (S), sulfanilamide resistance (F), and the ability to utilize mannitol (M). They extracted DNA from strains of bacteria with different combinations of different mutations and used this DNA to transform wild-type bacterial cells (P+S+F+M+). The results from one of their transformation experiments are shown below.' Donor DNA Recipient DNA Transformants %of all cells M S F M+ S+ F+ M+ S F+ 4.0 M+S+F 4.0 MS+F+ 2.6 M S F+ 0.41 M+ S F 0.22 M S+ F 0.0058 M S F 0.0071 a.) Hotchkiss and Marmur noted that the percentage of cotransformation was higher than would be expected on a random basis. For example, the results show that 2.6% of the cells were transformed into M and 4% were transformed into S. If the M and S traits were inherited independently, the expected probability of cotransformation of M and S (M S) would be 0.026 X 0.04= 0.001 or 0.1%. However, they observed 0.41% M S cotransformants, four times more than they expected. What accounts for the relatively high frequency of cotransformation of the traits they observed? b.) On the basis of the results, what conclusion can you draw about the order of the M, S, F genes on the bacterial chromosome? c,) WHy is the rate of cotransformations for all 3 genes almost the same as that of the cotransformation of M F alone?

a. M and S appear to be linked genes, i.e. the two are present at loci physically present very close to each other on the bacterial chromosome, which is why the two tend to move together and are transformed/ inherited at a higher frequency than expected. b. M and S appear to move together, whereas F moves separately. Also, the frequency of S and F moving together (0.22) is much higher than M and F moving together (0.0058). Hence, the correct order of genes is M-S---F. c. M and F are far apart and S lies in between them. Therefore, whenever M and F move together, S also moves along with them to transform the recipient. Hence, the rates of cotransformation with M F is almost the same as that for all three genes.

22.) E.coli cells are simultaneously infected with two strains of phage &. One strain has a mutant host range, is temperature sensitive, and produces clear plaques (genotype h st c); another strain carries wild-type alelles (genotype h+ st+ c+). Progeny phages are collected from the lysed cells and are plated on bacteria. The numbers of different progeny phages are as follows: Progeny Phage Genotype Number of plaques h+ c+ st+ 321 h c st 338 h+ c st 26 h c+ st+ 30 h+ c st+ 106 h c+ st 110 h+ c+ st 5 h c st+ 6 a.)Determine the order of the three genes on the phage chromosome. b.)Determine the map distances between the genes. c.)Determine the coefficient of coincidence and the interference.

a.) ---h+----------t+------------c+----- b.) --h+---------t+---------------------c+---- 7.1 m.u. 24.1 m.u. c.) Coefficient of coincidence= (6+5)/(0.071 X 942)= 0.68 interference= 1-0.68= 0.32.

Concept check 6: In gene mapping using generalized transduction, bacterial genes that are cotransduced are a.) Far apart on the bacterial chromosome b.) On different bacterial chromsomes c.) close together on the bacterial chromosome d.) on a plasmid

c.) close together on the bacterial chromosome

Temperate Phages

can undergo either the lytic or the lysogenic cycle

What are the advantages of using bacteria and viruses for genetic studies?

1. Reproduction is rapid 2. Many progeny are produced 3. The haploid genome allows all mutations to be expressed directly. 4. Asexual reproduction simplifies the isolation of genetically pure strains. 5. Growth in the laboratory is easy and requires little space. 6. Genomes are small. 7. Techniques are available for isolating and manipulating bacterial genes. 8. They have medical importance. 9. They can be genetically engineered to produce substances of commercial value.

Transductants

A recombinant bacteria in which bacterial genes can be moved from one bacterial strain to another.

Virus

A simple replicating structure made of nucleic acid surrounded by a protein coat.

Plasmid

A small, usually circular, supercoiled DNA molecules in bacteria. Generally carry genes that are nonessential to bacterial function.

6.) What types of genomes do viruses have?

A virus has either DNA or RNA genes and is called a DNA virus or a RNA virus, respectively. The vast majority of viruses have RNA genomes. Plant viruses tend to have single-stranded RNA genomes and bacteriophages tend to have double-stranded DNA genomes

colony

A visible clump of genetically identical cells when bacterium grow and divide.

2.) Briefly explain the differences between F+, F-, Hfr, and F' cells.

An F+ cell will contain a circular plasmid seperate from the chromosome. the Hfr cell has the f factor integrated into its chromosome. in F' strains the f factor exists as a seperate circular plasmid but the plasmid carries bacterial genes that were originally part of the bacterial chromosome. The F- strain does not contain the f factor and can recieve DNA from cells that contain the F factor. (F+, Hfr, and F' )

reverse transcriptase

An enzyme encoded by some certain viruses (retroviruses) that uses RNA as a template for DNA synthesis.

1.) What is the difference between complete medium and minimal medium? How are complete media and minimal media to which one or more nutrients have been added (selective media) used to isolate auxotrophic mutants of bacteria?

Auxotrophic mutants lack one or more enzymes necessary for synthesizing some essential molecule, such as an amino acid. Complete medium contains all the substances required by bacteria for growth and reproduction. Minimal medium contains only nutrients necessary for prototrophic (wild-type) bacteria. To isolate auxotrophic mutants that require leucine, bacteria are first grown on complete medium. Bacterial colonies that grow are transferred to plates with minimal medium to which has been added leucine and plates with minimal medium that lacks leucine. Any colony that grows on the plate containing leucine but fails to grow on the plate without leucine contains is a leu- mutant that cannot synthesize leucine.

15.) Austin Taylor and Edward Adelberg isolated some new strains of Hfr cells that they then used to map several genes in E. coli by using interrupted conjugation. In one experiment, they mixed cells of Hfr strain AB-312, which were xyl+ mtl+ mal+ met+ and sensitive to phage T6, with F- strain AB-531, which was xly- mtl- mal- met+ and resistant to phage T6. The cells were allowed to undergo conjugation. At regular intervals, the researchers removed a sample of cells and interrupted the conjugation by killing the Hfr cells with the phage T6. The F- cells, which were resistant to phage T6, survived and were then tested for the presence of genes transferred from the Hfr strain. The results of this experiment are shown in the accompanying graph. On the basis of these data, give the order of the xyl, mtl, mal, met genes on the bacterial chromosome and indicate the minimum distances between them.

By using different Hfr strains in which F plasmid has been integrated into different places and different orientations, one can practically map genes in E. coli simply by measuring and comparing the time of transfer. Determine the map position of a gene by : - Time of entry mapping - Hfr mapping genes/mutations relative to known markers The no. of recombinants for each marker increases with the time of mating. Screen for recombinant markers: As the cross was in between HfrH ( xyl+ mtl+ mal+ met+) X F - ( xyl- mtl- mal- met-) From the above figure we can see that the direction of gene transfer is clockwise which means the ordering of gene is mal->xyl->mtl->met. Note: Lesser the time taken for gene transfer means the faster it will move from hfr strain to F- strain. So, from the graph, the order of gene is mal xyl mtlmet Map, based on mating results from the graph mal gene transfer in 23 minutes [(from the arrow/start (0) to mal(23)] xyl gene transfer in 30 minutes [(from the arrow/start (0) to xyl(30). Distance between mal and xyl is 7 minute (23+7=30) ] mtl gene transfer in 32 minutes [(from the arrow/start (0) to mtl(32). Distance between xyl and mtl is 2 minute (30+2=32) ] met gene tansfer in 41 minutes [(from the arrow/start (0) to met (41). Distance between mtl and met is 32+9=41) ] The distance between met and mal is 41-23=18 minutes So the minimum distance between mal and xyl is 7 minutes xyl and mtl is 2 minutes mtl and met is 9 minutes.

Concept Check 1: Which of the following statements is true of plasmids? a.) They are composed of RNA b.) They normally exist outside of bacterial cells. c.) The possess only a single strand of DNA d.) They contain an origin of replication

D.)They contain an origin of replication

23.) A donor strain with alleles a+ b+ c+ is infected with phages to map the donor chromosome using generalized transduction. The phage lysate from the bacterial cells is collected and used to infect a second strain of bacteria that are a-b-c-. Bacteria with the a+ gene are selected, and the percentages of cells with cotransduced b+ and c+ genes are recorded Donor Recipient Selected Gene Cells w/ cotransd (%) a+b+c+ a-b-c- a+ 25 b+ a+ 3 c+ Is the b or c gene closer to a? Explain your reasoning.

First to answer this question you meed to understand what co transduced means. It means that if another gene is transduced along with the gene of interest. Let there be a gene of interest x and a gene b and c .when x gene is transduced then along with x ,b also gets transduced this means b is co transduced with x gene. The more cells we get with co transduced genes it means that those genes are very closer hence they are transferred together. If the genes were far away than they would bot have been co transduced. In this case we can see that cells with gene b co transduced with a are more than gene c co transduced with a. Since b is cotransduced much more than c we can clearly say that b gene is closer to a than c then only they are transduced together. Hence b is closer to gene a.

19.) Two mutations that affect plaque morphology in phages (a- and b-) have been isolated. Phages carrying both mutations (a-b-) are mixed with wild-type phages (a+b+) and added to a culture of bacterial cells. Subsequent to infection and lysis, samples of the phage lysate are collected and cultured on bacterial cells. The following numbers of plaques are observed: Plaque Phenotype Number a+b+ 2043 a+b- 320 a-b+ 357 a-b- 2134 What is the frequency of recombination between the a and b genes?

Hint: Recombinant progeny number is lesser always than the parental combinations. So that Recombinant progeny are a+b- and a- b+. Recombination frequency =( no. of Recombinants / Total progeny)*100 = (677/ 4854)*100 = 13.95%

Progeny Phages produced from h- r+ X h+ r-

Phenotype Genotype Clear and small h- r+ cloudy and large h+ r- Cloudy and small h+ r+ Clear and large h- r-

Transformation

Takes place when a bacterium takes up DNA from the medium in which it is growing. After transformation, recombination may take place between the introduced genes and those of the bacterial chromosome.

Transduction

Takes place when bacterial viruses (bacteriophages, or phages) carry DNA from one bacterium to another.

4.) Explain how interrupted conjugation, transformation, and transduction can be used to map bacterial genes. How are these methods similar and how are they different?

To map genes by conjugation, an Hfr strain is mixed with F- strain. the conjugation process is interrupted at regular intervals. The chromosomal transfer from the Hfr strain always begins with a part of the integrated F factor and proceeds in a linear fashion. the time required for individual genes to be transferred is relative to their position on the chromosome. Gene distances are mapped in minutes of conjugation.

Cotransduced

When genes located close together on the bacterial chromosome get transferred together

Antigenic Drift

When new mutations are continually introduced into the viral genome

prophage

When the lysogenic cycle begins like the lytic cycle, but inside the cell, the phage DNA integrates into the bacterial chromosome, where it remains as an inactive prophage. The prophage is replicated along with the bacterial DNA and is passed on when the bacterium divides.

Cotransformed

When two or more genes are transferred together.

Generalized Transduction

Where any gene may be transferred

20.) T. Myake and M. Demerec examined proline-requiring mutations in the bacterium Salmonella typhimurium. On the basis of complementation studies, they found four proline autotrophs: proA, proB, proC, proD. To determine whether these loci were located close together on the bacterial chromsome, they conducted a transduction experiment. Bacterial strains that were proC + and had mutations at proA, proB, and proD were used as donors. The donors were infected with bacteriophages, and progeny phages were allowed to infect recipient bacteria with genotype proC- proA+ proB+ and proD+. The recipient bacteria were then plated on a selective medium that allowed only proC+ bacteria to grow.. After this, the proC+ transductants were plated on selective media to reveal their genotypes at the other three loci. The following results were obtained: Donor Genotype Transductant Genotype Number proC+proA-ProB+proD+ proC+proA+proB+proD+ 2765 proC+proA-proB+proD+ 3 proC+proA+proB-proD+ proC+proA+proB+proD+ 1838 proC+proA+proB-proD+ 2 proC+proA+proB+proD- proC+proA+proB+proD+ 1166 proC+proA+ProB+proD- 0 a.)Why are there no proC- genotypes among the transductants? b.) Which genotypes represent single transductants and which represent cotransductants? c.) Is there evidence that proA, proB, and proD are located close to proC? Explain your answer.

a.) Transductants were initially screened for the presence of proC+. Thus, only proC+ transductants were identified. b.) The wild-type genotypes (proC+, proA+, proB+, proD+) represent single transductants of proC+. Both the proC+ proA- proB+ proD+ and proC+ proA+ proB- proD+ genotypes represent cotransductants of proC+, proA_ and proC+, proB-. c.) Both proA and proB were cotransduced with proC at about the same rate, which suggests that they are similar in distance to proC. The proD gene was never cotransduced with proC, suggesting that proD is more distant from proC than proA and proB. However, there were a small number of all types of cotransductants, and the absence of cotransduction between proD and proC might be due to chance.

17.) DNA from a bacterial strain that is his+ leu+ lac+ is used to transform a strain that is his- leu-lac-. The following percentages of cells are transformed: Donor strain Rec. Strain Genotype of Trans cells %of all cells his+leu+lac+ his-leu-lac- his+leu+lac+ 0.02 his+leu+lac- 0.00 his+leu-lac+ 2.00 his+leu-lac- 4.00 his-leu+lac+ 0.10 his-leu-lac+ 3.00 his-leu+lac- 1.50 a.) What conclusions can you draw about the order of these three genes on the chromosome? b.)Which two genes are the closest?

a.) he answer must be Lac gene in the middle , His and lac are closest. If His in the middle then you should get some amount of recombinants for HIs and leu because leu is far away. But what you get is 0.10% of lac+ and leu+ so lac+ should be in the middle . Leu and lac are farther apart and that is why you get 0.10 % recombinants. His and lac are close so that you get 2% recombinants. b.) His and lac

Concept Check 2: Conjugation between an F+ and an F- cell usually results in a.) two F+ cells b.) two F- cells c.) an F+ and an F- cell d.) an Hfr cell and an F+ cell

a.) two F+ cells

F Factor of E. coli

an episome that controls mating and gene exchange between E. coli cells

16.) DNA from a strain of Bacillus subtilis with genotype a+b+c+d+e+ is used to transform a strain with the genotype a-b-c-d-e-. Pairs of genes are checked for cotransformation, and the following results were obtained: Pair of Genes Cotransformation Pair of Genes Cotransfor a+ and b+ NO b+ and d+ NO a+ and c+ NO b+ and e+ YES a+ and d+ YES c+ and d+ NO a+ and e+ YES c+ and e+ YES b+ and c+ YES d+ and e+ NO On the basis of these results, what is the order of the genes on the bacterial chromosome?

b+c+e+a+d+ Explanation Genes that transform together /close together: a --- d, a --- e, b --- c, b --- e ,c --- e. Genes that do not transform together /far away : a --- b ,a --- c, b --- d ,c --- d, d --- e . To find the order of gene, consider which ones transform together or close together: b --- c, b --- e , c --- e Therefore, a possible order is - b --- c --- e Seeing that , a --- d transform together and they are close and as a --- e transform together and they are also close. Therefore , a possible order is: b --- c --- e --- a It has seen that, d doesn't transform with b, c or e and d is far from b, c and e and as a doesn't transform with b or c. Therefore, a possible order is: b --- c --- e --- a --- d

Concept Check 5: In which bacteriophage life cycle does the phage DNA become incorporated into the bacterial chromosome? a.) Lytic b.)Lysogenic c.) Both lytic and lysogenic d.) Neither lytic or lysogenic

b.)Lysogenic

Episomes

plasmids that are capable of replicating freely and are able to integrate into the bacterial chromosomes

Virulent phages

reproduce strictly through the lytic cycle and kill their host cells