Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris

1 Agricultural Biotechnology Laboratories National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 40227, Republic of China 2 Graduate Institute of Molecular Biology National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 40227, Republic of China 3 Graduate Institute of Botany, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan 40227, Republic of China *Tel: +886 4 2874754. Fax: +886 4 2861905. e-mail: 711nthu@vax9K.nchu.edu.tw ABSTRACT The last ORF of an xps gene cluster, designated xpsD , is required for the secretion of extracellular enzymes across the outer membrane in Xanthomonas campestris pv. campestris. It could encode a protein of 759 amino acid residues. A consensus N-terminal lipoprotein signal peptide was revealed from its deduced amino acid sequence. A [ 3 H]palmitate labelling experiment indicated that XpsD was fatty-acylated. Differential extraction with Triton X-100 disclosed that XpsD was fractionated with the outer membrane. Sucrose gradient sedimentation analysis of total membranes also indicated that XpsD was mainly located in the outer membrane. At least part of XpsD is exposed to the cell surface as suggested by trypsin experiment results. Intact cells pretreated with antibody against XpsD could indirectly be labelled with fluorescent agent. When the N-terminal lipoprotein signal peptide was replaced with a nonlipoprotein signal peptide cleavable by signal peptidase I, non-fatty-acylated XpsD was synthesized. Its subcellular location was indistinguishable from that of the fatty-acylated XpsD. Complementation of an xpsD ::Tn5 mutant of X. campestris pv. campestris indicated that this non-fatty-acylated XpsD remains functional in extracellular protein secretion. A stable, C-terminal truncated protein, XpsD 414-759, was synthesized from a mutated xpsD gene. Although it stayed associated with the outer membrane and exposed to the cell surface, it no longer could complement the xpsD ::Tn5 mutant of X. campestris pv. campestris. Keywords: Xanthomonas campestris pv. campestris, general secretion pathway, outer-membrane protein, lipoprotein, main terminal branch