< Back to previous page


Cell surface display of quorum quenching enzymes in Pseudomonas stutzeri.

Autotransporters (ATs) represent a large family of cell surface and extracellularly secreted proteins in Gram-negative bacteria. They are expressed as a single polypeptide consisting of a Sec-specific cleavableN-terminal signal peptide, a surface exposed or extracellularly secretedpassenger domain and a C-terminal outer membrane-anchored translocation unit, made up of a C-terminal ß-barrel and an upstream a-helical linker.ATs are often regarded as the most simple secretion mechanism in Gram-negative bacteria. Their modular structure makes ATs suited for surface display of heterologous passenger proteins in various biotechnological applications including whole-cell biocatalysis. In thisregard, the versatility and universality of the AT-based surface displaytechnology has often been advocated.
In this doctoral research, the esterase A (EstA) AT of the rice root-colonizing beneficial bacterium Pseudomonas stutzeri A15 was studied. The EstA AT of P. stutzeri A15 is a member of clade II of the GDSL esterase family. Based on the crystal structure of the homologous EstA AT of Pseudomonas aeruginosa, a three-dimensional model was constructed. This model exhibits the typical characteristics of ATs withan a-helix running through a ß-barrel domain thereby connecting it to the passenger domain. The overall picture of the passenger domain shows a globular fold with a-helices and loops.
Until now, no phenotypic differences have been observed for an estA-negative mutant compared to the wild type P. stutzeri A15. In combination with the low expression level of EstA under standard laboratory conditions, a dedicated role for EstA in particular environmental conditions is likely. Autologous overexpression of the EstA AT in P. stutzeri A15 was needed for its biochemical characterization. Cellular fractionation of P. stutzeri A15 cells expressing EstA revealed its presence in the total membrane fraction. The observation of a heat modifiability shift of EstA in this membrane fraction indicated the correct folding of the ß-barrel domain of EstA. The expression of EstA at the cell surface was demonstrated by fluorescent labeling with EstA specific antibodies and its accessibilityfor degradation by externally added proteinase K. The EstA passenger domain, which remained covalently attached to the cell surface after outer membrane translocation, exhibited hydrolytic activity towards the ester substrate p-nitrophenyl butyrate. Using site-directed mutagenesis the residues S37, D304 and H307 were experimentally shown to be essential for the catalytic activity of EstA. Mutations in these catalytically important residues did not change the cell surface displayof the mutant proteins in P. stutzeri A15 compared to the wild type EstA. In contrast, the biogenesis of EstA variants with non-conservativemutations in conserved hydrophobic amino acid residues of the N-terminalpart of the a-helix was affected as no EstA mutant proteins could be detected in P. stutzeri A15 cells expressing these variants. Based on the three-dimensional model of EstA and a multiple sequence alignment ofATs with a GDSL esterase passenger domain, conserved hydrophobic amino acid residues in the ß-barrel domain were identified as candidate interacting partners of the amino acid residues in the a-helix. Again, no EstA mutant proteins with non-conservative mutations in these ß-barrel residues could be detected in cells expressing these EstA variants. The unaffected cell surface exposure of most EstA variants with conservative mutations underlined the importance of the hydrophobiccharacter of the amino acid residues in both the a-helix and the ß-barrel for AT assembly.
As many P. stutzeri strains are involved in environmental processes, alltools for developing an AT-based cell surface display system for environmental use were available. An expression module for the surface display of heterologous passenger proteins was developed based on the P.stutzeri A15 EstA AT translocation unit. The correct surface display andesterase activity of the native EstA passenger domain validated the application of this expression module. Furthermore, the expression module was successfully applied for the functional display of an Escherichia coli ß-lactamase protein at the cell surface of P. stutzeri A15. In contrast, the expression module could not be successfully used for the cell surface display of three fluorescent proteins and three lactonases. The incorporation of the native EstA passenger in the expression module did not improve the surface display of these heterologous passenger proteins. In a subsequent study, only a minority of diverse proteins of P. aeruginosa PAO1 were successfully displayed atthe cell surface of P. stutzeri A15 using the above mentioned expressionmodule. Therefore the EstA AT cannot be considered as a broad spectrum cell surface display system in P. stutzeri A15. As all factors were invariant in our set-up, this finding indicates that the nature of the heterologous passenger domain determines the ability of a passenger to be displayed at the cell surface. Although successful surface display could not be correlated to a single characteristic of the passenger domain, the results suggest that compatibility with the Sec-mediated translocation process is an important issue.
In conclusion, the P. stutzeri A15 EstA AT cannot be regarded as an universal cell surface display system of heterologous passenger proteinsand its application still remains a black box. The analysis of more passenger proteins and the detailed examination of specific passenger proteins is necessary to determine what factors influence the ability ofa passenger domain to be displayed at the cell surface using ATs.
Date:1 Oct 2009  →  30 Sep 2013
Keywords:Autotransporter, Pseudomonas, Quorum quenching, Cell surface display, Biofilm
Disciplines:Systems biology, Medical biochemistry and metabolism, Biochemistry and metabolism, Microbiology, Laboratory medicine
Project type:PhD project