Which membrane transport proteins are responsible for the uptake of proline in E-Coli?

Week One: Introduction to the PUT Project

October 8th, 2021

Introduction

The truth is, this is still unknown, and work was put in during this past week to get closer to the answer. E-Coli is a simple bacterium, yet the different physiological processes it performs have long been the interest of laboratory scientists. It could be the topic of investigation to look for cytoplasmic cellular structures responsible for a certain metabolic function, or the different genes responsible for a certain phenotypic allele; nevertheless, the function under question for the current project is which transport proteins E-Coli utilizes to transport proline inside the cell and use it as a sole nitrogen source to grow.

Discussion

During the week, multiple procedures were completed to further the knowledge needed to conclude the results of the experiment, including growing different E-Coli strains on different growth media, isolating bacterial DNA and quantifying its concentration, as well as carrying out Polymerase Chain Reaction (PCR) for each strain. Most important of these experiments is the PCR portion which confirmed that the right strains were being experimented on. Briefly, a triple mutant strain namely CDC11-1 engineered by Dr. Deutch et al 2014 was utilized, a strain without the three main proline transport proteins, ProP, PutP, ProW, nor a kanamycin cassette. This inhibited the translation of these membrane proteins. Also, two more strains were ordered by Dr. Deutch which are missing the genes to one of the two membrane transporters newly in question, ProY and BrnQ. The gist of the experiment is finding out whether either or both of the two new membrane transporters, ProY and BrnQ, could uptake proline, and to do so, a quadruple mutant is under construction. 

Conclusion

To dive deeper, primers specifically chosen from the ProY and BrnQ genes were searched for in the whole genome of the four strains. The results of the PCR prove that the parent strain BW2513 and the triple mutant strain CDC11-1 have the genes for both of the transporters of interest, ProY and BrnQ.  However, the strain JW5055 which is delta ProY (missing the ProY gene) only has the gene for BrnQ while JW0391 is delta BrnQ (delta noting an absent gene). These new strains have a kanamycin cassette which inserts in the middle of the genes of interest and defects it. All in all, the experiments that have already been executed along with future experiments will provide the answer as to which transport proteins, ProY, BrnQ, or both, are responsible for the growth of E. coli on proline as the only nitrogen source. Results from the PCR of all four strains are shown below. 

References

Deutch, C.E., Spahija, I., and Wagner, C.E. (2014) Susceptibility of Echerichia coli to the toxic L-proline analogue L-selenaproline is dependent on two L-cystine transport systems. Journal of Applied Microbiology 114, 1487-1499.