Table of Contents:
Proline is a very important compound and serves several functions for the gram+ bacterium Bacillus subtilis. Prolin is an amino acid and therefore essential for protein synthesis, it can be used by B. subtilis as a single source of carbon and nitrogen and furthermore proline acts as an osmostress protectant.
B. subtilis possess two different ways for the de novo synthesis of proline: the anabolic and the osmoadaptive pathway. Both pathways are connected via the shared ProA protein. The deletion of the proA gene leads to a perturbation in both the anabolic and osmoadaptive proline biosynthesis. Suppressor mutations within the rocR-rocDEF region recruit the arginine degradation pathway for the synthesis of proline. The suppressor mutations were of two types: (i) single amino acid substitutions in the activator protein RocR resulting in partial inducer-independent RocR* variants; (ii) mutants in the promoter region of rocDEF activates a cryptic SigA-type promoter. Both types of mutants enhance the transcription of rocDEF leading to increased amounts of RocD. RocD as part of the arginine degradation pathway synthesizes the same reaction product as the ProA enzyme and thereby bypassing the ProA mediated enzyme reaction. Furthermore, the suppressor mutants also developed a new regulatory mechanism that allows the enhanced rocDEF transcription in response to proline in the presence of ammonium. This demonstrates how effective bacteria can adapt to limitations on their essential biosynthetic pathways.
In his natural habitat B. subtilis is frequently exposed to osmotic fluctuations. B. subtilis can adapt to high osmolarity growth conditions through the osmotically induced de novo synthesis or the uptake of proline. But B. subtilis can also use proline containing peptides as osmostress protectants. Osmoprotection by peptides depends on their import via peptide uptake systems (App, Dpp, Opp, DtpT) and the subsequent hydrolysis to release proline. The liberated proline is then accumulated to protect the cell against the disadvantageous effects of high osmolarity. The peptidases responsible for the hydrolysis of various types of Xaa-Pro and Xaa-Pro-Xaa peptides could be identified: PapA (YqhT) and PapB (YkvY). This adds a new aspect to the use of proline as an osmostress protectant by B. subtilis and demonstrates how gainful available resources can be used by bacteria to adapt to changes in environmental conditions.
In addition to PutP and OpuE, B. subtilis possess a so far uncharacterized proline transport system. To identify the unknown proline transporter a genetic screening approach was developed. In a B. subtilis mutant strain unable to transport proline, the transcription of the proline utilization genes (putBCP) did no longer occur in the presence of proline. This demonstrates the necessity of the proline import to induce putBCP expression.
In conclusion, the knowledge acquired in this dissertation revealed novel facets of the physiological role of proline in B. subtilis and furthermore it illustrates impressively how effective bacteria can adapt on a variety of changing.