Insertion mutagenesis of XpsD, an outer-membrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris.
XpsD is an outer-membrane protein required for extracellular protein secretion in Xanthomonas campestris pv. campestris. Cross-linking and gelfiltration chromatography analyses have suggested that it forms a multimer. To determine its structure-function relationship, linker-insertion mutants were constructed in an xpsD gene carried on a plasmid. To assay for secretion function, each mutant gene was introduced into an xpsD::Tn5 mutant strain (XC1708) and assayed for alpha-amylase secretion on starch plates. To test whether the mutant genes exerted a dominant-negative effect, each was introduced into the parental strain XC1701 and examined for secretion interference. Nine functional, one semi-functional and eleven non-functional mutants were obtained. All the non-functional mutants, except two for which the mutant proteins were undetectable on immunoblots, showed interference of normal secretion. The insertion sites in the different mutant proteins are randomly distributed throughout the entire sequence of the XpsD protein. All the permissive insertion sites are located where beta-turn or coiled secondary structure is predicted. Over half of the non-permissive sites are located within predicted helical or beta-sheet regions. By pretreating total membranes of XC1701 in SDS at 50 degrees C, an immunoreactive band with high molecular mass (HMM) could be detected that remained in the stacking gel during SDS-PAGE. The semi-functional and all functional mutant proteins formed HMM complexes that were as SDS-resistant as those of the wild-type, whereas all except three of the non-functional mutant proteins formed HMM structures that were less resistant to SDS than the wild-type. By analysing the appearance of SDS-resistant HMM complexes, we were able to detect conformational alterations in XpsD that are too subtle to be detected by other assays.
Hu
Nien-Tai
NT
Agricultural Biotechnology Laboratories, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 40227.
Hung
Ming-Ni
MN
Agricultural Biotechnology Laboratories, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 40227.
Chen
David Chanhan
DC
Institute of Veterinary Microbiology, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 40227.
Tsai
Rong-Tzong
RT
Institute of Agricultural Biotechnology, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 40227.|Agricultural Biotechnology Laboratories, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 40227.
D000595:Amino Acid Sequence; D000681:Amylases / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D016959:Xanthomonas campestris / Q000201:enzymology* / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; C113125:XpsD protein, Xanthomonas campestris; D000681:Amylases