Nien-Tai Hu

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 NH- 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(Δ29-428) or XpsD(Δ448-650) protein but not with XpsD(Δ74-303) or XpsD(Δ553-759) protein. We propose that the mutant XpsD(Δ29-428) protein caused secretion interference primarily by forming mixed nonfunctional multimers with the wild type XpsD protein in XC1701(pCD105), whereas the mutant XpsD(Δ74-303) did so by competing for unknown factor(s) in XC1701(pYL4).

XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL

GspE belongs to a secretion NTPase superfamily, members of which are involved in type II/IV secretion, type IV pilus biogenesis and DNA transport in conjugation or natural transformation. Predicted to be a cytoplasmic protein, GspE has nonetheless been shown to be membrane‐associated by interacting with the N‐terminal cytoplasmic domain of GspL. By taking biochemical and genetic approaches, we observed that ATP binding triggers oligomerization of Xanthomonas campestris XpsE (a GspE homolog) as well as its association with the N‐terminal domain of XpsL (a GspL homolog). While isolated XpsE exhibits very low intrinsic ATPase activity, association with XpsL appears to stimulate ATP hydrolysis. Mutation at a conserved lysine residue in the XpsE Walker A motif causes reduction in its ATPase activity without significantly influencing its interaction with XpsL, congruent with the notion that XpsE–XpsL association precedes ATP hydrolysis. For the first time, functional significance of ATP binding to GspE in type II secretion system is clearly demonstrated. The implications may also be applicable to type IV pilus biogenesis.

Association of the Cytoplasmic Membrane Protein XpsN with the Outer Membrane Protein XpsD in the Type II Protein Secretion Apparatus of Xanthomonas campestris pv. Campestris

An xps gene cluster composed of 11 open reading frames is required for the type II protein secretion in Xanthomonas campestris pv. campestris. Immediately upstream of the xpsD gene, which encodes an outer membrane protein that serves as the secretion channel by forming multimers, there exists an open reading frame (previously designated ORF2) that could encode a protein of 261 amino acid residues. Its N-terminal hydrophobic region is a likely membrane-anchoring sequence. Antibody raised against this protein could detect in the wild-type strain of X. campestris pv. campestris a protein band with an apparent molecular mass of 36 kDa by Western blotting. Its aberrant slow migration in sodium dodecyl sulfate-polyacrylamide gels might be due to its high proline content. We designated this protein XpsN. By constructing a mutant strain with an in-frame deletion of the chromosomal xpsN gene, we demonstrated that it is required for the secretion of extracellular enzyme by X. campestris pv. campestris. Subcellular fractionation studies indicated that the XpsN protein was tightly associated with the membrane. Sucrose gradient sedimentation followed by immunoblot analysis revealed that it primarily appeared in the cytoplasmic membrane fractions. Immune precipitation experiments indicated that the XpsN protein was coprecipitated with the XpsD protein. In addition, the XpsN protein was co-eluted with the (His)(6)-tagged XpsD protein from the metal affinity chromatography column. All observations suggested that the XpsN protein forms a stable complex with the XpsD protein. In addition, immune precipitation analysis of the XpsN protein with various truncated XpsD proteins revealed that the C-terminal region of the XpsD protein between residues 650 and 759 was likely to be involved in complex formation between the two.

Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system.

Secretion of fully folded extracellular proteins across the outer membrane of Gram-negative bacteria is mainly assisted by the ATP-dependent type II secretion system (T2SS). Depending on species, 12-15 proteins are usually required for the function of T2SS by forming a trans-envelope multiprotein secretion complex. Here we report crystal structures of an essential component of the Xanthomonas campestris T2SS, the 21-kDa N-terminal domain of cytosolic secretion ATPase XpsE (XpsEN), in two conformational states. By mediating interaction between XpsE and the cytoplasmic membrane protein XpsL, XpsEN anchors XpsE to the membrane-associated secretion complex to allow the coupling between ATP utilization and exoprotein secretion. The structure of XpsEN observed in crystal form P43212 is composed of a 90-residue α/β sandwich core domain capped by a 62-residue N-terminal helical region. The core domain exhibits structural similarity with the NifU-like domain, suggesting that XpsEN may be involved in the regulation of XpsE ATPase activity. Surprisingly, although a similar core domain structure was observed in crystal form I4122, the N-terminal 36 residues of the helical region undergo a large structural rearrangement. Deletion analysis indicates that these residues are required for exoprotein secretion by mediating the XpsE/XpsL interaction. Site-directed mutagenesis study further suggests the more compact conformation observed in the P43212 crystal likely represents the XpsL binding-competent state. Based on these findings, we speculate that XpsE might function in T2SS by cycling between two conformational states. As a closely related protein to XpsE, secretion ATPase PilB may function similarly in the type IV pilus assembly.

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

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, XpsD delta 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.

A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus.

The cytoplasmic membrane proteins XpsL, XpsM and XpsN are components required for type II secretion in Xanthomonas campestris pv. campestris. We performed metal-chelating chromatography to partially purify the His(6)-tagged XpsM (XpsMh)-containing complex. Immunoblot analysis revealed that both XpsL and XpsN co-eluted with XpsMh. The co-fractionated XpsL and XpsN proteins co-immune precipitated with each other, suggesting the existence of an XpsL-XpsM-XpsN complex. Ternary complex formation does not require other Xps protein components of the type II secretion apparatus. Further purification upon size-exclusion chromatography revealed that XpsN is prone to dissociate from the complex. Reassociation of XpsN with the XpsL-XpsMh complex immobilized on a nickel column is more effective than with XpsMh alone. Membrane-mixing experiments suggested that the XpsL-XpsMh complex and XpsN probably dissociate and reassociate in the membrane vesicles. Comparison of the half-lives of the XpsL-XpsMh-XpsN and XpsL-XpsMh complexes revealed that XpsL dissociates from the latter at a faster rate than from the former. Dissociation and reassociation between XpsL and XpsM were also demonstrated with membrane-mixing experiments. A dynamic model is proposed for the XpsL-XpsM-XpsN complex.

Involvement of the XpsN Protein in Formation of the XpsL-XpsM Complex in Xanthomonas campestris pv. campestris Type II Secretion Apparatus

The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestris pv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from the xpsM mutant strain, and vice versa. Introduction of the wild-type xpsM gene carried on a plasmid into the xpsM mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the xpsN mutant strain. They were recovered either by reintroducing the wild-type xpsN gene or by introducing extra copies of wild-type xpsL or xpsM individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of X. campestris pv. campestris caused inhibition of secretion. Complementation of an xpsL or xpsM mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the xpsN gene on the plasmid along with the xpsL and the xpsM genes caused more severe inhibition in both cases. Furthermore, complementation of the xpsN mutant strain was also inhibited. In both the wild-type strain and a strain with the xps gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.

Functional Dissection of the XpsN (GspC) Protein of the Xanthomonas campestris pv. campestris Type II Secretion Machinery

Type II secretion machinery is composed of 12 to 15 proteins for translocating extracellular proteins across the outer membrane. XpsL, XpsM, and XpsN are components of such machinery in the plant pathogen Xanthomonas campestris pv. campestris. All are bitopic cytoplasmic-membrane proteins, each with a large C-terminal periplasmic domain. They have been demonstrated to form a dissociable ternary complex. By analyzing the C-terminally truncated XpsN and PhoA fusions, we discovered that truncation of the C-terminal 103 residues produced a functional protein, albeit present below detectable levels. Furthermore, just the first 46 residues, encompassing the membrane-spanning sequence (residues 10 to 32), are sufficient to keep XpsL and XpsM at normal abundance. XpsN46(His6), synthesized in Escherichia coli, is able to associate in a membrane-mixing experiment with the XpsL-XpsM complex preassembled in X. campestris pv. campestris. The XpsN N-terminal 46 residues are apparently sufficient not only for maintaining XpsL and XpsM at normal levels but also for their stable association. The membrane-spanning sequence of XpsN was not replaceable by that of TetA. However, coimmunoprecipitation with XpsL and XpsM was observed for XpsN97::PhoA, but not XpsN46::PhoA. Only XpsN97::PhoA is dominant negative. Single alanine substitutions for three charged residues within the region between residues 47 and 97 made the protein nonfunctional. In addition, the R78A mutant XpsN protein was pulled down by XpsL-XpsM(His6) immobilized on an Ni-nitrilotriacetic acid column to a lesser extent than the wild-type XpsN. Therefore, in addition to the N-terminal 46 residues, the region between residues 47 and 97 of XpsN probably also plays an important role in interaction with XpsL-XpsM.

The type IV pre‐pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues

Type IV pre‐pilin leader peptidase was demonstrated to be required for protein secretion, in addition to its involvement in biogenesis of type IV pili. The type IV pre‐pilin leader peptidase gene of Xanthomonas campestris pv. campestris was located on a 3 kb Accl fragment on account of its hybridization with the DNA fragment containing the type IV pre‐pilin leader‐peptidase gene pilD/xcpA of Pseudomonas aeruginosa. Sequencing of the cloned fragment revealed an open reading frame (ORF) (designated xpsO) of 287 amino acid residues. A protein with an apparent molecular mass of approximately 32.5 kDa was synthesized in vitro from a DNA fragment containing the xpsO gene. The amino acid sequence shares 50% identity with that of PilD throughout the entire sequence. Among other type IV pre‐pilin leader peptidases, XpsO is unique in not having the two conserved ‐CXXC‐ motifs in a cytoplasmic domain. Instead, new motifs were noted when the protein was compared with XpsE, which is another member of the extracellular protein‐secretion machinery. When the xpsO gene was introduced into the pilD mutant of P. aeruginosa, both the sensitivity against infection with the pilus‐specific phage PO4 and the ability to secrete extracellular protein were recovered. Furthermore, immunoblot analysis indicated that the P. aeruginosa pilin was apparently processed in vivo by the xpsO gene product.

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.