tal Would allow rdio to verity your answer. of researchers at the university vet
ID: 256617 • Letter: T
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tal Would allow rdio to verity your answer. of researchers at the university veterinary ratory isolated a new bacterium from tool of several horses that becam ill ta local farm. Prior to death, symptoms ded disorientation, loss ot motor function, and d paralysis, so the researchers suspected central ervous system involvement and possible production of neurotoxin. Based on 16S rRNA comparison, they found that the new bacterium was related to the gram-positive bacterium Clostridium botulinum, and they named it Clostridium equiniae. The researchers subsequently developed a mouse model of infection, in which bacteria are iniected into the bloodstreams of mice, and the mice are monitored for paralysis and death. Using this animal model, they have conducted oup resear 2. A group cs labo the bloo flacci nature-tagged mutagenesis to identify genes uhExplanation / Answer
A- Left Blot:
In the wild type bacterial cells, both the toxins (BltA and CptB) are produced- hence the bands at 50 kDa and 100 kDa respectively. In the cultured medium , we observe that both the toxins are released (but a little less in molecular weight)- it might be possible that they undergo some proteolytic activation by an important protease which is also released in the media by the bacterial cells. In the neuronal cells, the same bands are seen.
RIGHT BLOT:
In the CeMut1 mutant, it is very possible that the protease needed for activation of the toxins is not being released from the cells, but are produced in the cells. So in the cells, we observe both the variants of each toxin. In the culture medium, we observe lesser amount of the cleaved activated toxins. In the neuronal cells, the toxins (if they are activated, they might not form the A-B neurotoxin) cannot form the cativated A-B toxin, and hence no bands. it might be also possible that the protease in question might help in the entry of the toxins in the cells.
B- It is very likely that the protein deleted in CeMut1 is a protease secreted by the bacterial cells which act upon both the toxins, cleave them and activate them. this is the reason why in CeMut1 mutants, we observe all the variants of the toxins within the bacterial cells, but not outside. It might be a secretion defect which is not allowing the protease to be released.
If the CeMut1 mutants are grown and to that culutre, if we add the culture medium of any of the CeMut2 or CeMut3 (filtered through a bacterial filter), then we should observe copious amount of cleaved toxins of both type. This is because the culture medium of any of the CeMut2 or CeMut3 (filtered through a bacterial filter) will have the secreted bacterial protease.
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