Characterization of Esterase A, a Pseudomonas stutzeri A15 Autotransporter.
Autotransporters are a widespread family of proteins, generally known as virulence factors produced by Gram-negative bacteria. In this study, the esterase A (EstA) autotransporter of the rice root-colonizing beneficial bacterium Pseudomonas stutzeri A15 was characterized. A multiple sequence alignment identified EstA as belonging to clade II of the GDSL esterase family. Autologous overexpression allowed the investigation of several features of both autotransporter proteins and GDSL esterases. First, the correctly folded autotransporter was shown to be present in the membrane fraction. Unexpectedly, after separation of the membrane fraction, EstA was detected in the N-laurylsarcosine soluble fraction. However, evidence is presented for the surface exposure of EstA based on fluorescent labeling with EstA specific antibodies. Another remarkable feature is the occurrence of a C-terminal leucine residue instead of the canonical phenylalanine or tryptophan residue. Replacement of this residue with a phenylalanine residue reduced the stability of the β-barrel. Regarding the esterase passenger domain, we show the importance of the catalytic triad residues, with the serine and histidine residues being more critical than the aspartate residue. Furthermore, the growth of an estA-negative mutant was not impaired and cell mobility was not disabled compared to the wild type. No specific phenotype was detected for an estA-negative mutant. Overall, P. stutzeri A15 EstA is a new candidate for the surface display of proteins in environmentally relevant biotechnological applications.
Centre of Microbial and Plant Genetics, KU Leuven, Heverlee, Belgium. stijn.spaepen@biw.kuleuven.be
Toon
T
Nicolay
Ken
K
Devleeschouwer
Jos
J
Vanderleyden
Stijn
S
Spaepen
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D016000:Cluster Analysis; D026901:Membrane Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D044223:Pseudomonas stutzeri / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D002265:Carboxylic Ester Hydrolases; C098953:EstA protein, bacteria