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Grant Bubrig

Professor Gregg Pettis

BIOL 4200

October 26, 2014

Written Assignment 2: Fujita and Losick

1. The first strain used in Fig. 1 is strain MF237, thePspoIIG-gfp fusion. This strain attaches

the green fluorescent protein to the SpoIIG protein. The strain is induced to sporulate,

and after three hours of sporulation, tagged with the membrane stain FM4-64. The cell is

then ready for viewing with fluorescence microscopy. The second strain is MF277,which contains thegfpgene fused with thePspoIIEgene. This fusion is also forced to

sporulate, treated with the membrane stain three hours later, and then observed under

fluorescence microscopy. The last strain shown in Fig. 1 is MF339, a strain containing aPspacc-gfp fusion. Other than the fusion itself, this strain was not manipulated, and was

simply observed after engulfment.

2. Fig. 1 proves that Spo0A remains present in the cell and is active primarily in the mother

cell after polar division. In Fig. 1, it is observed that the strain containing the PspoIIG-gfpfusion has GFP accumulating in the mother cell once the cell reaches engulfment. GFP is

completely absent in the forespore of the cell. This result is fairly identical in the second

column of the figure; the strain containing PspoIIE-gfp has heavy fluorescence in themother cell, but none in the forespore of the cell. Because both SpoIIG and SpoIIE are

controlled by Spo0A, these results show that Spo0A remains active in the mother cell.

The final column of the figure showed the Pspacc-gfp strain, which was used as a control.

Cells with this fusion strain exhibit equal fluorescence throughout the cell, both in themother cell and forespore.

3. In the top panels of Fig. 3, cells were taken from the third hour of sporulation and put

through SDS-PAGE, and then an immunoblot analysis using the anti-Spo0A antibodies.This was done with not only the wild type cell, but four other fusion strains. In the

bottom panels, the figures show the fluorescence after four hours of sporulation. The first

non-wild type strain used was MF828, a fusion protein containing PspoIIQ-spo0A. Thisstrain places the normal, wild-type Spo0A protein under the control of the promoter for

the sporulation genespoIIQ, which is transcribed in the forespore. A similar strain,

MF826, contains PspoIIQ-sad67. Sad67 is a constantly-active mutant of Spo0A. Strain

MF825 contains thesad67gene under the control of the promoter forspoIID, a celltranscribed in the mother cell. The final strain contains PspoIID-sad67.

4.

Fig. 3 ultimately shows that Spo0A reduces sporulation efficiency when it is active in the

forespore of the cell. This is shown by the reading at the bottom of Fig. 3. Because the

sad67mutant is forcibly expressed at all times, when it is placed in the forespore bySpoIIQ, the sporulation efficiency is lowered significantly. The strain containing PspoIIQ-

spo0A proves that the constant activation ofsad67in the forespore is what inhibits

sporulation, as this strain has much, much higher sporulation efficiency. The PspoIID-spo0A and PspoIID-sad67 containing strains both show that the localization of the protein

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Spo0A is important, as these strains have the protein located in the mother cell. Neither

of these two strains is very inhibited.

5. The top portion of Fig. 4-A takes cells from four different strains, including the wild type,after one hour intervals after the beginning of sporulation. Just like the experiment in

Fig. 3, these cells are put through SDS-PAGE, then immunoblot analysis using anti-

Spo0A antibodies. The bottom portion of Fig. 4-A shows these cells after hour 4 ofsporulation, when they have been treated with the membrane stain, FM4-64. Thesporulation efficiencies are listed below, similar to in Fig. 3. Aside from the wild type

(PY79), this figure exhibits three strains. The first strain is MF972, which uses a

truncated form of Spo0A, Spo0A-N, placed under the control of Spo0A. The secondstrain, MF973, places Spo0A-N under the control of SpoIID. The last fusion strain is

MF974, where Spo0A-N is controlled by SpoIIQ.

6. Fig. 4-A shows that inhibiting the activation of Spo0A in the mother cell prevents the cell

from sporulating effectively. This is shown primarily in strain MF972, the Pspo0A-spo0A-N fusion strain. Spo0A-N, a shortened version of Spo0A, is able to bind phosphates, but

is not able to actually bind DNA to signal anything. This means that this form is simply a

phosphate sponge. When Spo0A-N and Spo0A are present together, they compete forphosphates, leading to reduced Spo0A activation, which ultimately inhibits sporulation.

This can be seen in the second lane of Fig. 4-A. MF973 also had low sporulation

efficiency. This is because MF973 put Spo0A-N in the promoter of SpoIID, meaning

that Spo0A-N was forcibly placed into the mother cell only. Again, this low sporulationefficiency further proves that Spo0A inactivation in the mother cell is what inhibits

sporulation. Lastly, the strain MF974 used SpoIIQ to put Spo0A-N in the forespore of

the cell. However, because there is no Spo0A present in the forespore of the cell, therewas no competition for phosphates, and sporulation was left unaffected.