Investigation of the biogenesis of Type V protein secretion in Gram-negative bacteria
Biochemistry
Metabolism, Biochemistry and Genetics of Microorganisms
Final Report Abstract
The general objective of this project was to define the exact function of BamA-E in the biogenesis of ATs in order to understand the molecular mechanism that underlie the biogenesis of BAM related events. To this end, we purified each BAM component then prepared various combinations of proteoliposomes. Based on our results we propose that E. coli BamA and BamD forms a basic translocation module to mediate the membrane translocation of ATs, while BamB, BamC and BamE forms a regulation module to regulate the release of successfully translocated ATs from BamAD into the outer membranes. Further detailed investigations to elucidate how BamA and BamD forms the translocation module and how BamBCE regulates the release of ATs await future investigations. In attempts to use photo-crosslinking strategy to define the interaction map between the essential component BamA and ATs, however, were not successful with the whole BAM complex (i.e. BamA-E) probably because the translocation and release is too fast, which in this case requires further efforts to obtain any kinds of translocation intermediate(s) first. We currently over-expressed these BamA stop-codon mutants and planned to purify them then make either BamAmtantsBamD proteoliposomes first to define the molecular details of the translocation process, which will be followed by the analysis of the regulation function of BamBCE using the same strategy. Another development of the project is the identification of the representative function of TtOmp85, which will be definitely followed up to define in general the molecular details of the function of BAM complex. The chaperone function during the biogenesis of two-partner-secretion (TPS) was analyzed using knock-out and photo-crosslinking strategy. The results indicate that single knock out has no obvious effect, instead double knock out of yfgM and ppiD negatively affects the outer membrane translocation of FhaB. In particular, photo-crosslinking strategy suggests that various potential interaction partners interact with FhaB, which even depends on if it was followed by outer membrane translocation or not as it shows different interaction patterns. Overall, we should apologize that because of the extensive efforts to set-up the purification protocol for BAMs, the whole project was significant delayed. Despite the encountered technical difficulties prevented us from achieving several goals, we believe what we had tried lead to a significant basis development for future investigations. Taken together, our project brought new insights into the biogenesis of ATs, function of BAM as well as roles of chaperones in TPS. Of course, it should be noted that despite these progresses, the molecular mechanism of the BAM and chaperone function are far from being understood and should be addressed in future studies.
Publications
- An In Vitro Assay for Substrate Translocation by FhaC in Liposomes. Methods in Molecular Biology, 2015, 1329:111-125
Enguo Fan, Derrick Norell, Matthias Müller
(See online at https://doi.org/10.1007/978-1-4939-2871-2_8)
