XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv campestris, forms a multimer

XpsD is an outer membrane lipoprotein, required for the secretion of extracellular enzymes by Xanthomonas campestris pv. campestris. Our previous studies indicated that when the xpsD gene was interrupted by transposon Tn5, extracellular enzymes were accumulated in the periplasm (Hu, N.-T., Hung, M.-N., Chiou, S.-J., Tang, F., Chiang, D.-C., Huang, H.-Y. and Wu, C.-Y. (1992) J. Bacteriol. 174, 2679-2687). In this study, we constructed a series of substitutions and deletion mutant xpsD genes to investigate the roles of NH2- and COOH-terminal halves of XpsD in protein secretory function. Among these secretion defective xpsD mutations, one group (encoded by pCD105, pYL4, pKdA6, and pKD2) caused secretion interference when co-expressed with wild type xpsD, but the other (encoded by pMH7, pKdPs, and pKDT) did not. Cross-linking studies and gel filtration chromatography analysis indicated that the wild type XpsD protein forms a multimer in its native state. Similar gel filtration analysis of xpsD mutants revealed positive correlations between multimer formation and secretion interfering properties exerted by the mutant XpsD proteins in the parental strain XC1701. Those mutant XpsD proteins (encoded by pCD105, pYL4, pKdA6, and pKD2) that caused secretion interference formed multimers that are similar to the wild type XpsD multimers and those (encoded by pMH7, pKdPs, and pKDT) that did not formed smaller ones. Furthermore, gel filtration and anion exchange chromatography analyses indicated that the wild type XpsD protein co-fractionated with XpsD(Delta 29-428) or XpsD(Delta 448-650) protein but not with XpsD(Delta 74-303) or XpsD(Delta 553-759) protein. We propose that the mutant XpsD(Delta 29-428) protein caused secretion interference primarily by forming mixed nonfunctional multimers with the wild type XpsD protein in XC1701(pCD105), whereas the mutant XpsD(Delta 74-303) did so by competing for unknown factor(s) in XC1701(pYL4).