The once inside its capsid dissolves in order

The use of bacteriophage P1 vir to transduce a drug-resistance marker from one strain of bacteria to another

Introduction

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There are two types of transduction: generalised and specialized, in this practical we demonstrated how generalised transduction works.

Transduction is a process which involves the transfer of DNA from one bacterial cell to another, by a virus called a bacteriophage. A bacteriophage is a virus which is capable of parasitizing a bacterium through infection and reproduction of itself. Transduction does not need physical contact between the cells  donating the DNA (donor)  and the one receiving the DNA (recipient) as commonly seen in bacterial conjugation. Transduction was first discovered by Zinder and Lederberg in 1952. 

There are two separate phases of transduction process: the lytic and the lysogenic cycle. The lytic cycle consists of multiple stages involving both bacteriophage and bacterial cell. With this experiment the virus component is a lysate of bacteriophage P1 grown on a tetracycline-resistant donor strain which is infecting a saturated culture of non-pathogenic prokaryote E.coli K-12 strain.

To begin the lytic cycle, the bacteriophage attaches itself onto the plasma membrane of host bacterial cell. This can be done by the tail of the bacteriophage that contains proteins which has an affinity to the cell wall. Attachment can also be through mechanical forces. Second is penetration where the bacteriophage punctures the cell membrane, once inside its capsid dissolves in order to release its genomic DNA into the bacterial cytoplasm. This is known as uncoating. The bacteriophage has now taken control of the host cells replicational, transcriptional and translation machinery. Next is biosynthesis of viral components such as viral DNA or RNA, viral nucleic acids and proteins. DNA is transcribed to messenger RNA (mRNA) that directs protein synthesis at ribosomes. One of the polypeptides made destroys the hosts DNA. At this stage capsid proteins and enzymes for new viruses are produced. After is assembly of viral components to produce complete viruses. Then is maturation phase where viral replication results in the accumulation of virions which is an ineffective form of a virus particle consisting of an RNA or DNA core surrounded by a protein coat. As virion numbers increase, specialised viral proteins start to dissolve the bacterial cell wall. Finally the cell lyses (bursts) because of high internal osmotic pressure caused by the entry of water which expands the cell. Also as a result of overcrowding from numerous virions surrounded by a viral capsid protein. Lysis releases all the contained virions which are able to infect other bacteria. The lytic cycle destroys the cell and the membrane which has been infected by bacteriophage.

In the lysogenic cycle the bacteriophage inserts its DNA into host cell bacteria, this viral DNA is then integrated into bacterial chromosome forming a prophage. The viral DNA is now part of the cells genetic material. Therefore the viral DNA is replicated along with the bacterial DNA though normal cell division, which doesn’t kill the cell as with the lytic cycle. The resulting daughter cells produced contain prophage and are called lysogens. These lysogens can remain part of the lysogenic cycle but are able to switch to the lytic cycle through a process known as induction.  Here viral DNA, capsids and proteins are produced until lysis occurs. The prophage can transfer genes which is one of the ways that bacteria are becoming increasingly resistant to antibiotics as well as the immune system. Transfer of genes also increases hosts cell virulence. The main difference between the lytic and lysogenic cycles is that viral DNA is distributed though normal prokaryotic reproduction whereas in the lytic cycle it is though the numerous phages from lysis of cell.

Generalised transduction involves the transfer of any genes part of the host chromosome whereas in specailised transduction only specific genes are transferred between host cell and recipient cell. However the main difference between the two types of transduction is that generalised transduction if carried out by virulent bacteriophages which cause cell lysis and release of newly replicated bacteriophages. Whereas specialised transduction is carried out by temperate bacteriophages which does not lyse the cell but does incorporate its viral DNA with bacterial chromosome which can survive for many generation within bacterial cell.

Generalised transduction occurs during the lytic cycle when a random part of the host cell DNA is injected into the capsid instead of viral DNA. When a bacteriophage infects another cell, it is not the viral DNA that is established but DNA from former bacterial cell. As there are no viral genes present they would not be able to replicate. The host chromosome is degraded and synthesis of phage DNA and protein begins. The bacteriophages which lack viral DNA but contains fragments of the bacterial genome are called transducing particles.

The phage coat protein is what determines the bacteriophages ability to attack and take control of a cell. Through injection of transducing phage contents into recipient cell’s cytoplasm, the bacterial genes can be incorporated through homologous recombination. Homologous recombination occurs between the DNA that has been introduced and the hosts genome. During crossing over homologous chromosomes break at one point in order to switch alleles. This exchange of genetic material results in a transductant in which the original bacterial DNA is replaced by fragments of  phage DNA. This results in two recombinant chromosomes with different genetic material.  . Homologous recombination is used to exchange genetic material between strains of bacteria and species of viruses which increases genetic diversity and helps pass on beneficial genes.

Transduction results in genetic diversity among a population as there is crossing over which is the interchanging of genetic material between donor and recipient that forms a recombinant cell. Despite viruses having a smaller genome size they have high mutation rates so can, in particular RNA viruses as they use RNA polymerases which don’t proof read so increases risk of mistakes made in RNA strand. Along with this natural selection allows viruses to conform to changes in the environment of the host. Combination of high mutation rates and natural selection, viruses are able to replicate extremely rapidly Thus the transfer of genes allows viruses to pass on any beneficial genes such as those conferring antibiotic resistance for example to their progenies.

 

 

 

Results

Plate Number

Volume of Lysate Added (?l)

Number of Bacterial Colonies

Control 1

0

0

Control 2

0

0

Plate 1

10

2

Plate 2

10

3

Plate 3

50

20

Plate 4

50

25

Lab Practical Results

 

Figure 1a: Table of result obtained from lab practical showing the different volumes of lysate and the number of bacterial colonies found after incubation

 

Figure 1b: Graph for volume of lysate added against average number of transductants found according to date in Fig.1 with trend line

 

 

 

 

 

Volume of Lysate ((ml)

Total Number of Colonies (transduction) Counted

Total Number of Plates Counted

Average Number of Colonies Per Plate

0

0

85

0

10

106

100

1.06

20

183

103

1.78

30

248

97

2.56

40

213

103

2.07

50

112

106

1.06

P1 Transduction Class Data

Figure 2b: Graph for results of the class data shown in Fig.2a of the number of colonies per plate  at different lysate volumes.
 

 

 

 

Discussion

The data obtained from the in lab session (Fig 1a) shows that as the volume of lysate increases, the number of bacterial colonies found on the Petri dishes also increases. For instance, when the lysate volume was 0 µl the number of colonies identified was also 0. As the volume of the lysate increases to 10 µl accordingly the number of bacterial colonies found on medium increased to 2 and 3 on plates 1 and 2 respectively. The highest volume of lysate used in the lab session was 50 µl and as anticipated, gave the highest number of bacterial colonies. On plate 3 there were 20 colonies whilst on plate 4 there were 25. This giving the average of 22.5, thus following the trend of the other plates. When plotted as a graph in Fig 1b the trendline shows a positive correlation supported by an R2 value of 0.9922. The positive correlation illustrates a strong relationship between the two variables, as the volume of lysate increase so do the number of colonies found on agar after incubation.

Although the graph along with the R2 value shows a positive correlation, the results are quite unreliable as it was conducted only once. In comparison to the class data graph there are few data point meaning that more data needed to be collected to give results that are more reliable. Repeating the experiment more than once is vital in scientific experiments as it can help calculate the average especially if you see a pattern.  Additionally repetition of the method can help identify any anomalous results that would have been missed if the experiment was only conducted once or twice.

On the other hand with results of the class data, exhibited in graph form in Fig 2b shows that as the volume of lysate added increased, the number of colonies found on each plate also increased however only to a certain point. After this peak has been reached the number of colonies decreases despite the volume of lysate added increases. This is an example of polynomial distribution, as at first both values increase but after the peak has been reached one of the variable decreases even though the other continues to increase. This is because the bacteriophages have a certain capacity and once it becomes filled with phage copies, lysis occurs and the cell bursts releasing its contents. Therefore this is why after the peak which is approximately at 30 µl which gave a 2.5 bacterial colony average, the number of colonies decreased because there are no more to infect with a virus and they would have been destroyed during cell lysis.

Comparing the two graphs it is clear that as volume of lysate increase the number of colonies increases exponentially, however with the class data we see that this doesn’t continue for long. With the lab data it appears that the bacterial colonies will continue to increase as the volume of lysate increases however there wasn’t a variation in the volumes to see the decrease in bacterial colonies. With the class data there were 6 different volumes used whereas with the lab data there were only 3 and this may be the reason for the differences seen in the two results.

There are many factors involved for the differences seen between the class data and own lab data. One of them include errors made in plate counting, for example the size of colonies vary and when small colonies develop they may not be completely visible to the naked eye and possibly be missed during counting. A more accurate way to count bacterial colonies is by using a microscope which has the magnification and resolution to help identify any missed colonies that were too small to see which gives a better interpretation of the actual number of bacterial colonies present on the surface. However since the bacterial count here was less than 30 it was easier to just directly count by looking at the plate.

Since the spread plate method was used to place the bacteria in the tetracycline-agar plates they may have been spread out unevenly across the surface causing more colonies to develop on one side more than the other. Which may lead to overpacking is and when these colonies combine it may be difficult to distinguish between them. A metal loop was used to streak plate and if a swirling motion was used to distribute bacteria, more bacteria are likely to found near the rim of the Petri dish, increasing the likeliness for miss counting of colonies. Additionally if the heated loop is not allowed to cool properly it may damage the tetracycline on the agar plate which may cause the development of undesirable bacteria.

Another likely error may occur if the sterility of all the equipment used to distribute the bacteria is not maintained. For instance not flaming the loop under the sterile region of the Bunsen burner may cause contamination of the entire culture. Or not holding the loop under the blue flame long enough to destroy any contaminating bacteria and not re-sterilising the loop after it has been used can also lead to contamination of Petri dish.

Inaccuracies in pipetting the liquid sample onto agar plate can also contribute to the difference seen in results. In some cases air bubbles may have formed when picking up and releasing the liquid leading to there been less on the surface. Furthermore not setting the pipette to its correct volume and picking up less liquid that required can lead to inaccurate results as there may be less sample on one plate than the other.

Insufficient mixing of recipient strain and the CaCl2/MgSO4  buffer may lead to their being more E.coli in one plate than the other, which can obscure the results. In addition its likely that some on the mixture remained in the Eppendorf tubes after pipetting

The lab was conducted over the course of two days, Monday and Tuesday and the colony counts happened on the Wednesday morning. Therefore the Monday groups plates were in incubation for a whole day longer than those plates which were only placed in incubation on the Tuesday. As a result it is likely that there may have been more distinct bacterial colonies present on the plates form Mondays lab than those from Tuesdays lab.

Questions

(1)   The E.coli genome is 4.6 x 106 base pairs in length. What is the maximum separation between two genes that is possible for them to be co-transduced (simultaneously transduced) into a donor strain. Please explain your answer. (5%).

(2) Suppose that you obtain the following results in the experiment described above:-

      Plate number of colonies

If the culture that you used in the experiment contained 3 x107 bacteria per millilitre, calculate the average frequency of transduction. Explain carefully how you arrived at your answer. Do not forget to include units. (5%)

 

Mean  = 8 + 5 + 7 + 4 + 10 = 6.8
                            5
6.8/3 x 107 = 2.27 x 10-6      0.05 x 3.0 x 107
 
     6.8        x 100 =  2.26 x 10-5 x 0.1 = 2.27 x 10-6
  3 x 107
 

 

 

 

 

 

(3)   Why is it essential to be able to select for transductant in a transduction experiment? (5%)

 

Its vital to be able to select for transductant as we are testing for antibiotic resistance within a culture of bacteria. On the agar plates the bacterial colonies are resistant to tetracycline. If tetracycline wasn’t present, then the non-resistant cultures would be present on the medium.  The bacteria could have been destroyed by the virulent strain and gone through transduction with an agent such as a bacteriophage.

 

(4)   Do you think that it would be possible to transfer a drug resistance marker from a recA mutant (defective in generalised or homologous recombination) donor strain into a recombination proficient recipient strain? Please explain your answer. (10 %)

 

Yes it would be possible to transfer a drug resistance marker from a recA mutant into a recombinant proficient recipient strain . This is because the recipient cell is most defective in generalised or homologous recombination and therefore the infected linear bacterial DNA can undergo the recombination events has no effect of recombination in the second cell. When DNA enters the recipient, homologous recombination can happen.

 

(5)   It has been observed that as the amount of bacteriophage added to a culture increases, the number of transduction subsequently isolated rises to a maximum level, and then declines. Provide a hypothesis to explain these results. (10%)

 

The more bacteriophage you add the more transduction happen until reaches a critical level where bacteriophages begin to lysate cells bursting them and releasing all its contents. With no more cell left therefore, no transductions occur. A hypothesis is that the more bacteriophage is added to a culture the more likely lysis will occur after the critical level has been reached.

(6) It has been noticed that the difference in frequency of transduction between two genes can vary by almost 100-fold. Offer two hypotheses to explain this observation. Please explain your answers carefully (15 %)

The pac sequence in DNA determines the position of the enzyme that will make the initial cleavage, so the closer the gene to the pac sequence the more likely it is to be transduced.

If a gene doesn’t get swapped over in recombination there is a chance that is can get lost because it hasn’t been moved from one chromosomes to the other which means during cleaving it won’t be transduced as it can be lost.

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