Characterization of the lipopolysaccharide transport machinery using an Escherichia coli/Pseudomonas aeruginosa hybrid system and Escherichia coli lptC mutants The lipopolysaccharide (LPS) transport (Lpt) is an essential process for the biogenesis of the outer membrane (OM) in Gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. OM, the first bacterial defense against harsh environment and antimicrobial compounds, is composed by an inner leaflet of phospholipids, an outer leaflet of LPS, and outer membrane proteins. Lpt system, originally identified in E. coli, is a protein machine responsible for LPS delivery from the inner membrane, where it is synthesized, to the outer membrane. It is composed of seven proteins, LptA through LptG, which form a complex spanning the IM and OM. LptBCFG are located at the IM, LptA in the periplasm, and LptDE at the OM. The lpt genes are evolutionarily conserved and appear to play an essential role in most Proteobacteria. To understand how LPS is delivered to the OM crossing the hydrophilic periplasmic space, we focused on the role of the periplasmic components LptC and LptA by two main approaches: i) comparison between E. coli and P. aeruginosa Lpt systems to search for conserved features shared by the Lpt machines of different Gram negative species. By plasmid shuffling technique we showed that P. aeruginosa lptCAB genes can complement E. coli ΔlptCAB mutants, thus indicating that the Pseudomonas proteins can interact with both the other proteins of the Lpt machine and the LPS of E. coli. Although E. coli and P. aeruginosa LptC and LptA proteins exhibit limited sequence identity and similarity, their 3D conformation is conserved. This suggests that LptA and LptC overall structure rather than their amino acid sequence may play a major role in Lpt assembly and LPS recognition and transport; ii) analysis of E. coli lptC mutants in order to elucidate the role of LptC in the system. E. coli appears to tolerate several mutations in lptC, including deletion of the trans membrane domain; moreover the lethality of the lptC C-terminal region deletion can be suppressed by an appropriate expression of lptB. We thus tested whether E. coli could tolerate the lack of LptC, which was thought to be essential. By plasmid shuffling we obtained viable mutants missing lptC. Genome sequencing of such mutants revealed single amino acid substitutions at the R212 residue of the IM component LptF (lptFSupmutants); in complementation tests, lptFSup mutants suppress lethality of LptC conditional expression mutants. These data show that a specific mutation in LptF can compensate the lack of LptC and suggest that LptC may serve as a chaperon of the Lpt machine assembly and/or activity rather than an essential structural component.
|Titolo:||CHARACTERIZATION OF THE LIPOPOLYSACCHARIDE TRANSPORT MACHINERY USING AN ESCHERICHIA COLI/PSEUDOMONAS AERUGINOSA HYBRID SYSTEMAND ESCHERICHIA COLI LPTC MUTANTS.|
|Data di pubblicazione:||28-mag-2015|
|Settore Scientifico Disciplinare:||Settore BIO/19 - Microbiologia Generale|
|Citazione:||CHARACTERIZATION OF THE LIPOPOLYSACCHARIDE TRANSPORT MACHINERY USING AN ESCHERICHIA COLI/PSEUDOMONAS AERUGINOSA HYBRID SYSTEMAND ESCHERICHIA COLI LPTC MUTANTS. / M. Benedet ; tutor: G. Dehò, A. Polissi, G. Bertoni. - Milano : Università degli studi di Milano. DIPARTIMENTO DI BIOSCIENZE, 2015 May 28. ((27. ciclo, Anno Accademico 2014.|
|Digital Object Identifier (DOI):||10.13130/m-benedet_phd2015-05-28|
|Appare nelle tipologie:||Tesi di dottorato|