Biochemical analysis of alginate biosynthesis protein AlgX from Pseudomonas aeruginosa: purification of an AlgX-MucD (AlgY) protein complex.
AlgX was found to be an essential protein for alginate biosynthesis, but its function is unknown. In this study, an isogenic, marker-free algX-knock out mutant was generated. In-frame fusions of algX with phoA and lacZ were analysed, respectively. No LacZ-activity was detected, but the PhoA fusion showed alkaline phosphatase activity. These data indicated that the C-terminus of AlgX is located in the periplasm, but is not required for protein function. Accordingly, AlgX with C-terminal fusion of strep tag II restored alginate production in the algX-negative mutant and was purified under native conditions from periplasmic and crude cell extracts, respectively. AlgX was identified by MALDI/TOF-MS analysis of tryptic peptides. TritonX-100 mediated solubilisation of cytoplasmic membrane and subsequent strep tag II affinity chromatography led to purification of an AlgX-MucD (AlgY) protein complex as identified by MALDI/TOF-MS analysis. This data suggested a protein-protein interaction between AlgX and MucD (AlgY) with a 1:1 stoichiometry. Thus AlgX might exert its function via interaction with MucD (AlgY). Immunoelectron microscopic localisation of AlgX-strep tag II suggested a localisation close to the cytoplasmic membrane.
Gutsche
J
J
Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Remminghorst
U
U
Rehm
B H A
BH
D000464:Alginates / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015415:Biomarkers; D002462:Cell Membrane / Q000378:metabolism; D007150:Immunohistochemistry; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C099517:Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; D000464:Alginates; D001426:Bacterial Proteins; D015415:Biomarkers; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C097649:MucD protein, Pseudomonas aeruginosa; D012697:Serine Endopeptidases