Abigail Ray
MIB200A Take Home
Professor Tsolis
4 December 2018
The following series of questions is related to the E. coli NarGHI respiratory nitrate reductase (also called Nitrate Reductase A), which is encoded by the operon narGHJI. Note, for some of the questions, the answer is not known, so it’s OK to generate a hypothesis based on what we discussed in lecture or on something you find in the literature—ie, there are multiple possible correct responses.
(1) Describe the ambient conditions under which E. coli would utilize NarGHI for nitrate respiration. How is expression of narGHIJ controlled at the transcriptional level and what transcriptional regulator(s) is/are involved? What is the molecular basis for this transcriptional regulation (ie what is being sensed)?
NarGHI is a membrane-bound enzyme that supports anaerobic respiration with nitrate as an electron receptor. Therefore, E. coli would use NarGHI in anaerobic conditions assuming that nitrate is present (Keseler, 2013). The transcriptional regulation of the narGHIJ operon is mediated by a two-component regulatory system. This involves NarQ and NarX (histidine kinases) and NarP and NarL (response regulators). The histidine kinases transmit signal to their cognate response regulators to adjust to dynamic conditions. The system is designed to sense nitrate and nitrite conditions. In low nitrate conditions, the system skews toward the production of nitrate reductase and Nrf nitrite reductase via NarXL and NarQP. However, in high nitrate conditions, the nitrase reductase is still synthesized, however, the nitrite reductase is repressed and NirB nitrate reductase is upregulated. Additional regulation comes from Fnr protein. Fnr (formate nitrate regulation) protein senses oxygen and under anaerobic conditions activates the narGHIJ operon (Moat, 2002).
Ingrid M. Keseler, Amanda Mackie, Martin Peralta-Gil, Alberto Santos-Zavaleta, Socorro Gama-Castro, César Bonavides-Martínez, Carol Fulcher, Araceli M. Huerta, Anamika Kothari, Markus Krummenacker, Mario Latendresse, Luis Muñiz-Rascado, Quang Ong, Suzanne Paley, Imke Schröder, Alexander G. Shearer, Pallavi Subhraveti, Mike Travers, Deepika Weerasinghe, Verena Weiss, Julio Collado-Vides, Robert P. Gunsalus, Ian Paulsen, Peter D. Karp; EcoCyc: fusing model organism databases with systems biology, Nucleic Acids Research, Volume 41, Issue D1, 1 January 2013, Pages D605–D612, https://doi.org/10.1093/nar/gks1027
Moat, Albert G., et al. Microbial Physiology. Wiley-Liss, 2002.
(2) NarGHI requires a cofactor for its activity, the molybdenum cofactor (Moco), whose biosynthesis requires the moaABCDE operon. This operon is regulated post-transcriptionally in response to the abundance of Moco. What would be some possible mechanisms for this regulation?
Work on moaA has showed that CsrA (carbon storage regulator) binds to moaA mRNA in vivo. CsrA represses stationary phase metabolism and activates carbon metabolism binds to moaA specifically and is able to activate moaA expression post-transcriptionally. They demonstrate that moaA forms an aptamer that is targeted by Moco and CsrA for post-transcriptional regulation (Patterson-Fortin, 2013).
Additionally, some work has indicated that Moco synthesis is a multi-step process. First, moaA and moaC convert GTP to cPMP. Then dithiolene is added to cPMP by moaD, moaE, and moeB. The last step, is to insert the molybdenum atom is added onto the dithiolene suphurs via a reaction with mogA. At any of these steps, post-transcriptional regulation could modify or alter the molybdenum thereby effecting the regulation of moaABCDE (Kozmin, 2013).
Kozmin SG, Schaaper RM. Genetic characterization of moaB mutants of Escherichia coli. Res Microbiol. 2013;164(7):689-94.
Patterson-Fortin LM, Vakulskas CA, Yakhnin H, Babitzke P, Romeo T. Dual posttranscriptional regulation via a cofactor-responsive mRNA leader. J Mol Biol. 2012;425(19):3662-77.
(3) The NarGHI-Moco complex functions in the periplasm of E. coli. However, it is not known how the assembled enzyme gets there. Propose a hypothesis on the mechanism and two experiments to test it (with controls, of course). How will you interpret the results of your experiments to support or refute your hypothesis?
The NarGHI-Moco complex functions in the cytoplasm, not the periplasm (Bertero, 2003). If it were actually doing nitrate reduction in the periplasm, NarGHI would be consuming the protons, rather than pumping them out of the cell, which would diminish the proton motive force as well as acidifying the cell.
If it were possible for the NarGHI-Moco complex to function in the periplasm, two possible pathways that would allow the complex to get to the periplasm. Proteins can be transported by either the twin arginine translocation (TAT) pathway or the general secretory (Sec) pathway. The TAT pathway secretes folded proteins, however it has a 70 angstrom (+ or – 10) size limit. However, the NarGHI protein is 143 angstroms, making it too large for the TAT pathway to successfully secrete and thereby preventing it from entering the periplasm. However, the Sec pathway secretes unfolded proteins only. Therefore, it would be able to secrete the protein to the periplasm. Therefore, I hypothesize that if the NarGHI-Moco complex is functioning in the periplasm, it would only be able to be transported there by the Sec pathway. To determine if the Sec pathway is in fact transporting the NarGHI-Moco complex you could approach it in two ways. First, the Sec pathway requires a signal sequence for transport. The signal sequence is a motif found in the N terminus of the secreted protein. To test this, I would use a protease that cleaves the N terminus of NarG. Without this signal, the complex would not be able to transport it to the periplasm. A second experiment I would propose would be to knockdown the Sec pathway. Then you could culture the knockdown and wildtype E. coli in a nitrate rich and molybdenum supplemented media in an anaerobic environment. To quantify this mutation, you could measure ATP production as an indicator of NarGHI functionality.
Bertero, Michela G, et al. “Insights into the Respiratory Electron Transfer Pathway from the Structure of Nitrate Reductase A.” Nature Structural & Molecular Biology, vol. 10, no. 9, 2003, pp. 681–687., doi:10.1038/nsb969.
(4) It’s Thanksgiving, and time to think about diarrhea!
The CDC has just issued two food safety advisories: Contamination of Romaine Lettuce by Shiga toxin-producing E. coli https://www.cdc.gov/ecoli/2018/o157h7-11-18/index.html and contamination of raw turkey by Salmonella https://www.cdc.gov/salmonella/reading-07-18/index.html#_blank . Both of these pathogens cause diarrhea and are use type III secretion systems to inject effector proteins into epithelial cells lining the intestine.
How do diarrheal pathogens sense their proximity to the epithelium to regulate expression of their secretion systems? Read one of these papers to find out and explain what you figured out (one paragraph and/or draw a diagram). What are the main lines of evidence presented?
Paper #1: Enterotoxigenic E. coli virulence gene regulation in human infections
http://www.pnas.org/content/115/38/E8968.long
Paper #2: Modulation of Shigella virulence in response to available oxygen in vivo
https://www.nature.com/articles/nature08970
Based on the Marteyn et al. paper, Shigella flexneri, modulates its virulence based on the concentration gradient of oxygen in various microenvironments of the gut. This study demonstrates the modulation of virulence is directly due to changes in S. flexneri’s type three secretion system (T3SS). They conclude that in anaerobic environments, the T3SS expresses the T3SS needles and reduces Ipa (invasion plasmid antigen) expression. At the same time, FNR (fumarate and nitrate reduction) is mediating this process. The virulence of S. flexneri is based entirely on its ability to deliver Ipa effector via T3SS. Taken together with the information about T3SS expression in anaerobic environments it can be concluded that virulence depends on oxygen levels in the gut. However, oxygen alone is not able to induce virulence via T3SS and the secreted Ipa is also required. Interestingly, this paper also demonstrates that there is a slim zone of oxygenation that has a protective effect immediately proximal to the gut mucosa.