MICB 211 Unit 2 Case Studies

Chapter 8 Case Study 1

Objective: Study the role of cAMP in repressing lactose metabolism in the presence of glucose in E. coli

Background:

• β-galactosidase is an enzyme encoded in lac operon (lacZ)

• presence of β-galactosidase levels indicate activation/expression of lac operon

• glycerol is a poor, alternative carbon source for E. coli

• assume all levels of the sugars are considered high levels and that they grew bacteria for 100 minutes

Hypothesis: cAMP is involved in promoting lactose metabolism when there is little to no glucose because it helps activate the CAP protein

Control: glycerol used as negative control — no glucose, no lactose control. Glycerol treatment used to demonstrate that glucose/lactose has an effect (negative control).

Data
Fig. 1: β-galactosidase levels as an indication of activation/expression of the lac operon and total cAMP levels under 4 conditions. Glycerol is a poor carbon source for E. coli. cAMP levels don’t match low glucose levels → low glucose should have high cAMP levels but it is not displayed in this figure. Figure shows that cAMP grows in the absence of both glucose and lactose.
Fig. 2: Growth measured in OD600 and β-galactosidase activity under a condition where cAMP is added to the media at high levels and a strain that in unable to produce cAMP. Both cAMP grown in 0.4% glucose and 0.2% lactose (assume high levels).
Expected results doesn’t match curve of β-galactosidase when cAMP added — amount of β-galactosidase resulted matches expected results but the curve doesn’t match: There is a lag when cAMP added → not expected → there should be no lag (β-galactosidase should be highly expressed from the start)
Expected results doesn’t match curve of β-galactosidase and OD curve: no cAMP produced (CAP can’t bind to CAP site without cAMP) → low levels of β-galactosidase expressed not high levels of β-galactosidase shown in the curve

Conclusion: Refute hypothesis; cAMP is always present and the CAP is always bound to the CAP site, regardless of glucose levels, but cAMP always binds to CAP.

Chapter 8 Case Study 2

Characteristics of bacteriophage isolates and expression of Shiga toxin genes transferred to non Shiga toxin producing E.coli by transduction

Background: Shiga toxin is a type of bacterial toxin produced by pathogenic E. coli and S.dysenteriae

Objectives: Not all strains of E.coli can produce this toxin, but there is a concern that environmental bacteriophages are helping to transfer this toxin gene to non-toxin producing E.coli → bacteriophage transferring toxin

Hypothesis: Strains of phage carrying Shiga toxin that were most effective at infecting E.coli would also result in the highest rate of gene transfer

Data:
Table 1: Infection detection of 9 different Stx-encoding phage (each row) against 5 different non-toxic strains of E. coli (each column). + infection detected, - infection not detected. E.coli ATCC 9637 and E. coli ATCC 10536 strains infected by the highest number of phages (6) → strains that are most susceptible to phage infection.
Table 2: Detection of two types of Shiga toxin genes chromosomally in originally non-toxic E. coli strains (decreasing susceptibility). Stx1 gene has a higher rate of transfer → detected in a greater proportion of bacteria infected with different phages.
There is no correlation between E.coli strain susceptibility and presence of toxin gene → E.coli ATCC 9637 most susceptible but doesn’t have stx2 expression &&& E.coli ATCC 25922 least susceptible (only infected by 1 phage), but expressed both stx1 and stx2 → presence of toxin gene and susceptibility to infection aren’t correlated. E.coli ATCC 9637 should have both stx1 & stx2 IF there was a correlation since they are most susceptible but since they only have stx1 → no correlation between strain and susceptibility.

Table 3: Shiga toxin levels (stx1 and stx2) and bacterial cell viability with increasing salt concentration. Cell viability decreases with higher salt concentration (negative correlation). Maximum stx1 concentration at 6% NaCl concentration. Increasing NaCl induces stx1 production.

Conclusion: Bacteriophages can mediate the transfer of Shiga toxin through transduction (Shiga toxin (stx1) transferred more than stx2). More bacterial cell death observed under high osmotic pressure (higher salt concentration). Shiga toxin more highly expressed under high osmotic pressure (increased expression in high salt could correlate to gut conditions).

Chapter 9 Case Study 3

Aim: Study how S.enterica senses different metal ions using two two-component regulatory systems, PhoPQ & PmrAB

Background:
PhoPQ
-senses/responds to low Mg²⁺
-induces expression of pmrD

PmrAB
-senses/responds to high Fe²⁺
-induces expression of pbgP

-pmrA, pmrB, phoP or phoQ knock-out mutants

• all 4 genes of the 2 two-component systems

-low Mg, high Fe: signals detected by sensors

• what low and high levels are doesn’t matter

-lacZ activity = pbG expression/PmrA activity

• easily measured

-PhoQ and PmrB are the sensors of 2 two-component systems

-PhoP and PmrA are the response regulators of the 2 two-component systems

Steps:

1. determine what signals can activate PmrA → cause pbgP expression

   • signals that can activate PmrA: low Mg (via PhoPQ response PmrD), high Fe (via PmrB)

     • crosstalk between PhoPQ and PmrAB

   • PmrA

2. create a reporter plasmid using promoters from genes that they suspect are regulated by PmrA

   • test reporter gene expression for each reporter in PhoQ mutant

     • need to eliminate chances of genes getting activated because of PhoPq (low Mg) instead of PmrAB (high Fe)

     • isolate impact of PmrA gene regulation

   • promoter: pbgP promoter

   • reporter gene: beta-galactosidase (lacZ)

   • create mutants for phoPq and pmrAB

3. Determine if two-component systems are involved in antibiotic resistance and test these strains to see if they are resistant to antibiotic polymyxin B

   • create strains where response regulators have been deleted

   • ΔPmrA or ΔPhoP

Data:
Fig. 1: A reporter plasmid created using promoter of pbgP in front of lacZ (b-galactosidase). B-galactosidase activity as an indicator of pbgP expression was measured in low Mg or low Mg and high Fe in knock-out mutants of pmrA, pmrB, phoP and phoQ. The no plasmid control are WT S.enterica cells not transformed with the reporter plasmid. Either low Mg or high Fe are minimally sufficient to activate PmrA.
Bacterial cells that are wild-type

• low Mg alone is inducing expression (B-galactosidase)

Bacterial cells that are ΔpmrA

• no expression with any levels of Mg or Fe

Bacterial cells that are ΔpmrB

• no expression with any levels of Mg or Fe

Bacterial cells that are ΔpmrP

• high Fe, low Mg results in expression

Bacterial cells that are ΔpmrQ

• high Fe, low Mg results in expression

Fig. 2: ugd, pbgP, pmrC genes are regulated by PmrA

Fig. 3: both PhoPq and PmrAB play a role in resistance against polymyxin B. PmrAB confers more resistance against polymyxin B. Deleting pmrAB has more of an impact than phoPQ.

Conclusion:
PmrAB can be activated by either low MG (via PhoPQ) or high Fe (independent). PmrA regulates pbgP, ugd, pmrC found to be involved in antibiotic resistance.

Tutorial Case Study 1

Aim: Determine how gene expression of the arabinose operon is regulated by arabinose and glucose through AraR

Hypothesis: The ara operon is only activated when arabinose is present and glucose is absent; and AraR is a repressor.

Background: The ara operon is a series of genes that encode for proteins involved in metabolizing the sugar, arabinose. It has 4 promoter binding sites that regulate 6 genes (3 of the genes are contained as part of an operon, one gene is regulator AraR). Assume ara operon works like the lac operon (arabinose instead of lactose). AraR is repressor, CAP is activator.

Based on the arabinose operon, the promoter is controlling an “operon” is promoter B. The promoter(s) controlling single genes are promoter A, D, and C.

Conclusion:
Accept hypothesis; ara operon only gets activated when arabinose is present. Promoters for araA and araE are controlled in the same way in response to glucose and/or arabinose.
No glucose, no arabinose

• araR bound

• CAP bound

• low levels of transcription

Glucose & arabinose

• araR not bound

• CAP not bound

• basal levels of transcription

Glucose, no arabinose

• araR bound

• CAP not bound

• low levels of transcription

No glucose, Arabinose

• araR not bound

• CAP bound

• high levels of transcription

Tutorial Case Study 2

Aim: Create a genetic variant of E. coli that can sequester and remove copper particles from the environment.

Hypothesis: The introduction of a plasmid that contains copper-sequestering proteins with the CusC promoter controlled by a signal transduction system that senses copper can produce a strain of E. coli that remove environmental copper.

Fig. 1: The signal transduction system used is two-component. The sensor is CusS and the response regulator is CusR. The tOmpC-CBP gene is the signal transduction system is acting on.

Fig. 2: The type of plasmid being used is expression plasmid (could possibly also be reporter plasmid). The selection marker is AmpR. Plasmids are self-replicating. In order to replicate, the DNA polymerase binds to PMB1 ori.

Fig. 3:
Control:
-pUC19 (empty plasmid control)
-pCC1056 with zinc
-uninduced cells in pCC1056 (a)

Treatment:
-cells induced with 0.5 mM & 1.0 mM CuSO₄ (B, C) of pCC1056

Under 0.5 mM & 1.0 mM CuSO₄ with copper ions will have expression of the copper binding protein. Under negative control conditions there would not have expression of copper binding protein. A lower concentration of CuSO₄ promoted better copper adsorption.

Conclusion:
Authors reached their aim and conclusion was accepted because they the E.coli genetic variant that was created was able to remove copper particles from the environment. The plasmid introduced into E.coli was able to sense copper and remove it from the environment.