please be very descriptive and clear thank you 9 Mutations c

please be very descriptive and clear. thank you

9. Mutations can lead to altered phenotypes, subjecting the mutated strain to selective pressure.
a. Aside from a mutation in the nleA coding sequence itself, describe two other mutations that could result in constant repression in NleA production. In terms of mechanism, how would your mutations impact expression? (4pts each; 8pts total)

b. Describe two mutations that would result in constant production of NleA. Again, describe their mechanism (4pts each; 8pts total)

c. Of the two: constant repression (always off, the mutations in part A) or constant production (always on, the mutations in part B), which would you imagine to be the most disadvantageous? Why (4pts)?

Solution

Non LEE encoded effectors, nleA or Espl was observed to be essential for the virulence of Attaching/Effacing pathogens.

Ler is encoded by the first gene of LEE1 operon and acts as a master regulator of all the LEE operons by stopping the repression shown by H-NS. LEE also encodes two other regulators called GrlA which is the stimulator of ler transcriptional activation. GrlR is the negative regulator of LEE gene expression.

A)

Ler regulates nleA expression. GrlA and Pch increased nleA expression and secretion. It is also studied that Ler requires the upstream position to -123 to activate nleA expression. It was also found that the sequence between -123 and -85 act as a negative control in the expression of nleA. The -35 and -10 hexamer promoters were also seen activating nleA expression.

The expression of nleA was measured in ler, grlA, grlR and hns mutants. Its expression was observed to be drastically reduced in the ler and grlA mutants.

B)

Deletion of the region between -149 and -123 upstream of nleA promoter reduced the expression of nleA genes. Deletion of positions between -149 and -123, between -87, 65 and -56 could reduce the promoter activity while deletion between -123 and -87 could not show reduction in promoter activity. This shows that region between -123 and -87 has negative regulation on the transcriptional activity of nleA gene. It was also shown that there are certain cis-acting regulatory elements between -56 and -47 that help in the activation of nleA expression.

C)

Repression of nleA genes reduces the virulence of the A/E pathogens that cause infection. So, obviously it is disadvantageous for having the positive regulators of nleA genes. Hence, mutations in the regions that enhance the expression of nleA gene are advantageous than the mutations that occur in the regions that are involved in the negative regulation of the nleA gene expression.