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

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 [3H]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, XpsDd414-759, 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.

HxcQ Liposecretin Is Self-piloted to the Outer Membrane by Its N-terminal Lipid Anchor

“…Previously, BfpB, TcpC and XpsD were experimentally demonstrated to be lipoproteins (10 -11, 24). The involvement of lipidation in secretin biogenesis was only tested for XpsD where this post translational fatty acylation turned out to be dispensable for secretin function (24). For BfpB and TcpC two small nonlipidated periplasmic proteins have been shown to be required for their stabilization and multimerization respectively (25,11).…”

“…One, XpsD of Xanthomonas campestris pv. campestris, belongs to a T2SS (24), and two others, BfpB of enteropathogenic E. coli (25) and TcpC of Vibrio cholerae (11) are members of type IV pili systems.…”

XpsD, an Outer Membrane Protein Required for Protein Secretion by Xanthomonas campestris pv. campestris, Forms a Multimer

“…Subcellular Fractionation-The supernatant collected from centrifuging cultured cells was saved as extracellular fraction and concentrated by cold trichloroacetic acid precipitation (3). The other subcellular fractions, i.e.…”

“…The xpsD⌬29 -428 and xpsD⌬414 -759 mutations were constructed in our previous work (3). For the construction of the xpsD⌬545-553 mutation, the following two primers were used: 5Ј-TTTTTCCAAGAAATTCTAGAGCGACGC-CATTGC-3Ј for the upstream XbaI site and 5Ј-CTAGCGCACTA-ATATCTAGAGCTGCATTTTTTCC-3Ј for the downstream XbaI site.…”

“…campestris required for the secretion of extracellular proteins with a cleavable NH 2 -terminal signal peptide (1)(2)(3). It is likely to be involved in the second step of a two-step secretory pathway, the type II pathway (4 -6).…”