Wei-Ming Leu

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.

XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes

GspG, -H, -I, -J and -K proteins are members of the pseudopilin family. They are the components required for the type II secretion pathway, which translocates proteins across the outer membrane of Gram-negative bacteria to the extracellular milieu. They were predicted to form a pilus-like structure, and this has been shown for PulG of Klebsiella oxytoca by using electron microscopy. In the present study, we performed biochemical analyses of the XpsG protein of Xanthomonas campestris pv. campestris and observed that it is a pillar-like structure spanning the cytoplasmic and outer membranes. Subcellular fractionation revealed a soluble form (SF) of XpsG, in addition to the membrane form. Chromatographic analysis of SF XpsG in the absence of a detergent indicated that it is part of a large complex (>440 kDa). In vitro studies indicated that XpsG is prone to aggregate in the absence of a detergent. We isolated and characterized a non-functional mutant defective in forming the large complex. It did not interfere with the function of wild-type XpsG and was not detectable in the SF. Moreover, unlike wild-type XpsG, which was distributed in both the cytoplasmic and outer membranes, it appeared only in the cytoplasmic membrane. When wild-type XpsG was co-expressed with His6-tagged XpsH but not with untagged XpsH, SF XpsG bound to nickel and co-eluted with XpsH. This result suggests the presence of other pseudopilin components in the XpsG-containing large-sized molecules.

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.

Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation.

Membrane‐associated ATPase constitutes an essential element common to all secretion machineries in Gram‐negative bacteria. How ATP hydrolysis by these ATPases is coupled to secretion process remains unclear. Here we identified R286 as a key residue in the type II secretion system (T2SS) ATPase XpsE of Xanthomonas campestris that plays a pivotal role in coupling ATP hydrolysis to protein translocation. Mutation of R286 to alanine made XpsE hydrolyse ATP at a rate five times that of the wild‐type XpsE. Yet the mutant XpsE(R286A) is non‐functional in protein secretion via T2SS. Detailed analyses indicated that the mutant XpsE(R286A) lost the ability co‐ordinating the N‐ and C‐domain of XpsE. Without significantly influencing XpsE binding affinity with ATP or its oligomerization, R286A mutation however, caused XpsE lose the ability to associate with the cytoplasmic membrane via XpsLN. As a consequence, ATP hydrolysis by XpsE was uncoupled from protein secretion. Because R286 is highly conserved among members of the secretion NTPase superfamily, we speculate that its equivalent in other homologues may also play a critical energy coupling role for T2SS, type IV pilus assembly and type IV secretion system.

Increased Expression of 9-Cis-Epoxycarotenoid Dioxygenase, PtNCED1, Associated With Inhibited Seed Germination in a Terrestrial Orchid, Phaius tankervilliae

The phytohormone abscisic acid (ABA) is involved in regulating seed dormancy and germination. A crucial step of ABA biosynthesis in higher plants is the oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED). Seed development in orchids is unusual because the embryos are minute in size, without obvious histodifferentiation, and lack endosperm. To understand the regulation of ABA biosynthesis in orchid seeds, we isolated and characterized a full-length cDNA encoding an NCED homolog, PtNCED1, from developing seeds of an ornamental orchid, Phaius tankervilliae. Germination percentage was high at 90 days after pollination (DAP), when a globular embryo proper with a degenerating suspensor was evident. After 90 DAP, seed maturation was accompanied by a decrease in water content and a concomitant increase in ABA content and PtNCED1 mRNA level along with a marked decrease in germination percentage. Mature seeds pretreated with NaOCl solution lowered ABA content and improved seed germination. Moreover, after seed germination, developing protocorms could respond to dehydration stress. Dehydration of protocorms stimulated an increase in PtNCED1 level along with ABA content. Our results provide evidence of the involvement of PtNCED1 in regulating endogenous ABA content in developing seeds and protocorms. The accumulation of endogenous ABA content in orchid seeds may have a critical role in seed dormancy and the protocorm response to water stress after seed germination.