The Biochemical Analysis of Coexpressed and Copurified XpsE/MBP-XpsLN Complex

T2SS of Xanthomonas campestris pv campestris is assembled by 12 proteins. XpsE is the only cytoplasmic component and the likely energy supplier of the system, whereas XpsL is a bitopic membrane protein with a single transmembrane segment. The role of XpsL in T2SS is not so clear. It has been previously observed that the hexameric XpsE, whose formation is nucleotide-dependent, interacts in vitro directly with the cytoplasmic domain of XpsL as MBP-XpsLN. We thus speculated that XpsE may form complex with XpsLN in vivo. In this study, we attempted the complex isolation by coexpressing XpsE and MBP- XpsLN in E. coli. Copurification of MBP-XpsLN and Strep-tagged XpsE was observed on the SDS-PAGE when purified using double-affinity chromatography, indicating that a stable XpsE/MBP-XpsLN complex was formed as a consequence of their coexpression in E. coli. The molecular size of such a complex was estimated to be 800 kDa as revealed by size-exclusion chromatography. We thus postulated that the complex may be constituted of 6 molecules each component. The protein complex purified from size-exclusion chromatography exhibited an ATPase activity sixfold that of the singly expressed XpsE. In addition, the ATPase activity of the complex was stimulated by cardiolipin by threefold. The XpsE/MBP-XpsLN complex resulted from the coexpression strategy employed here might resemble an intermediate stage during secretion process in vivo, thus enabling us to study in the future the mechanistic events driven by the interaction between XpsE and XpsL.Introduction 1 Materials and Methods 7 Plasmids and bacterial strains 7 Media, reagents and buffers 7 Mini-preparation of plasmid DNA 7 Preparation of competent cell 7 Co-transformation 8 Protein analysis and immunological techniques 8 Small-scale induction of XpsE/MBP-XpsLN and XpsE/SUMO-XpsLN 9 Purification of XpsE/MBP-XpsLN complex using amylose affinity column 10 Purification of MBP-XpsLN using amylose affinity column 11 Purification of XpsE using Strep-Tactin column 11 Purification of XpsE/SUMO-XpsLN complex using two consecutive affinity columns 12 Purification of His-tagged SUMO-XpsLN complex using nickel column 13 Size-exclusion chromatography 13 Blue native gel electrophoresis 14 ATPase activity assay 14 Results 16 Expression and purification of XpsE/MBP-XpsLN 16 Stoichiometric analysis of XpsE and MBP-XpsLN interaction 18 In vitro ATPase activity of XpsE 19 In vitro ATPase activity of copurified XpsE/MBP-XpsLN complex 20 Expression and purification of XpsE/SUMO-XpsLN 21 Stoichiometric analysis of XpsE and SUMO-XpsLN interaction 22 Discussion 24 References 28 Figures 31 Tables 44 Appendix 4

Ancient co-option of an amino acid ABC transporter locus in Pseudomonas syringae for host signal-dependent virulence gene regulation.

Pathogenic bacteria frequently acquire virulence traits via horizontal gene transfer, yet additional evolutionary innovations may be necessary to integrate newly acquired genes into existing regulatory pathways. The plant bacterial pathogen Pseudomonas syringae relies on a horizontally acquired type III secretion system (T3SS) to cause disease. T3SS-encoding genes are induced by plant-derived metabolites, yet how this regulation occurs, and how it evolved, is poorly understood. Here we report that the two-component system AauS-AauR and substrate-binding protein AatJ, proteins encoded by an acidic amino acid-transport (aat) and -utilization (aau) locus in P. syringae, directly regulate T3SS-encoding genes in response to host aspartate and glutamate signals. Mutants of P. syringae strain DC3000 lacking aauS, aauR or aatJ expressed lower levels of T3SS genes in response to aspartate and glutamate, and had decreased T3SS deployment and virulence during infection of Arabidopsis. We identified an AauR-binding motif (Rbm) upstream of genes encoding T3SS regulators HrpR and HrpS, and demonstrated that this Rbm is required for maximal T3SS deployment and virulence of DC3000. The Rbm upstream of hrpRS is conserved in all P. syringae strains with a canonical T3SS, suggesting AauR regulation of hrpRS is ancient. Consistent with a model of conserved function, an aauR deletion mutant of P. syringae strain B728a, a bean pathogen, had decreased T3SS expression and growth in host plants. Together, our data suggest that, upon acquisition of T3SS-encoding genes, a strain ancestral to P. syringae co-opted an existing AatJ-AauS-AauR pathway to regulate T3SS deployment in response to specific host metabolite signals

Fha Interaction with Phosphothreonine of TssL Activates Type VI Secretion in <i>Agrobacterium tumefaciens</i>

<div><p>The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (<i>p-</i>TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, <i>Agrobacterium tumefaciens</i>. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif <i>in vivo</i> and <i>in vitro</i> and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in <i>A. tumefaciens</i>. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed.</p></div

Phos-tag SDS-PAGE for TssL phosphorylation analysis in various mutants.

<p>Detection of the phosphorylation status of TssL-His on Phos-tag gel. Western blot analysis of the same volumes of Ni-NTA resins (40 µl) associated with TssL-His from different strains treated with (+) or without (−) CIAP and examined with specific antibody against 6×His. Total proteins isolated from Δ<i>tssL</i> were a negative control. Phos-tag SDS-PAGE revealed the upper band indicating the phosphorylated TssL-His (<i>p</i>-TssL-His) and lower band indicating unphosphorylated TssL-His.</p

Hcp and Atu4347 secretion assays of various PpkA and TssL phosphorylation site mutations.

<p>(<b>A</b>) Hcp and Atu4347 secretion assay in <i>ppkA</i> mutants. The wild-type C58, Δ<i>ppkA</i>, and <i>ppkA</i> with D161AN166A substitutions strains (2 independent alleles) were analyzed for type VI secretion. (<b>B</b>) Type VI secretion and western blot analyses in various <i>tssL</i> mutants. Western blot analysis of total and secreted (Sup) proteins isolated from various <i>A. tumefaciens</i> strains with specific antibodies. ActC and RpoA were internal controls.</p

Phosphorylated TssL is a direct binding partner of Fha.

<p>Isothermal titration calorimetry (ITC) of specific binding between Fha and TssL peptides. Purified Fha7-267^WT (<b>A, B, C, E</b>) or Fha7-267^R30AS46A (<b>D</b>) was titrated with synthetic phosphorylated TssL (DLPpTVVEI, <i>p</i>-TssL^{8 mer} or DNPSSWQDLPpTVVEITEESR, <i>p</i>-TssL^{20 mer}), unphosphorylated TssL (DLPTVVEI, TssL^{8 mer}), or phosphorylated CHK2 (VSpTQEL, <i>p</i>-CHK2^{6 mer}) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003991#ppat.1003991-Wu3" target="_blank">[43]</a> peptides. The equilibrium dissociation constant (<i>K_d</i>) between truncated Fha7-267 proteins and synthetic peptides is shown at the bottom. Other combinations without any detectable binding ability are shown as undetectable. Top panel shows the raw calorimetric data for the interaction and bottom panel the integrated heat changes, corrected for heat of dilution, and fitted to a single-site binding model.</p

TssL-Strep pulldown and spheroplast protease susceptibility assays in <i>A. tumefaciens</i>.

<p>(<b>A</b>) Pulldown assay by TssL-Strep in various <i>A. tumefaciens</i> strains. Western blot analysis of the loaded Triton X-100 solublized protein fraction (L), wash (W), and elution (E) examined with specific antibodies. The soluble protein ActC and outer-membrane protein AopB <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003991#ppat.1003991-Jia1" target="_blank">[46]</a> were internal controls. (<b>B</b>) Spheroplasts from various <i>A. tumefaciens</i> strains were incubated with different protease concentrations as indicated. The degree of susceptibility or resistance to protease in various strains was analyzed by western blot analysis with specific antibodies. Cytoplasmic GroEL <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003991#ppat.1003991-Chang1" target="_blank">[47]</a> was an internal control for resistance to protease digestion. The amount of analyzed proteins was further quantified by use of UVP BioSpectrum 600 and normalized to the internal control GroEL. The relative intensity from TssM and TssL are shown at the bottom of analyzed strains by setting the wild-type C58 level to 100. The quantitative results shown with standard deviation were obtained and normalized from at least 2 independent experiments.</p

PpkA- and Fha-dependent post-translational regulation of Hcp secretion from <i>A. tumefaciens.</i>

<p>(<b>A</b>) The <i>imp</i> operon encoding 14 genes (<i>atu4343</i> to <i>atu4330</i> or <i>impA-N</i>) and the <i>hcp</i> operon encoding 9 genes (<i>atu4344</i> to <i>atu4352</i>) and <i>atu3642</i> (<i>vgrG-2</i>) encoded by <i>A. tumefaciens</i> strain C58 was designated <i>tss</i> or <i>tag</i> based on nomenclature proposed by Shalom et al. (2007) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003991#ppat.1003991-Shalom1" target="_blank">[18]</a> and common names <i>ppkA</i>, <i>tagF-pppA</i>, and <i>fha</i>. This schematic was from Lin et al. (2013) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003991#ppat.1003991-Lin1" target="_blank">[11]</a>. (<b>B</b>) Hcp secretion analysis. Western blot analysis of total (T) and secreted (S) proteins isolated from wild-type C58, Δ<i>ppkA</i>, and Δ<i>tagF-pppA</i> mutants harboring the vector pRL662 (V) or <i>ppkA</i> complemented plasmid (pPpkA) or <i>tagF-pppA</i> complemented plasmid (pTagF-PppA) with specific antibodies. (<b>C</b>) The amino acid sequence alignment of the FHA domain of Fha (Atu4335) and selected Fha-family proteins indicating the conserved pThr binding motif. Conserved amino acid residues are highlighted in black and marked below, and R30 and S46 used for mutagenesis are indicated with an asterisk. Sequences were aligned and highlighted by use of ClustalW2 (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). (<b>D</b>) Hcp secretion assay for chromosomally encoded <i>fha</i> variants, including <i>fha</i> deletion (Δ<i>fha</i>), <i>fha</i> wild-type revertant with double crossover (<i>fha</i> R), <i>fha</i> with deletion of the entire FHA domain from 25 to 76 aa (<i>fha</i>^ΔFHA), and <i>fha</i> with substitutions of pThr binding motif (<i>fha</i>^R30A, <i>fha</i>^S46A, and <i>fha</i>^R30AS46A). For secretion assays in (B) and (D), the non-secreted protein ActC was an internal control. The proteins analyzed and sizes of molecular weight standards are on the left and right, respectively.</p

Mass spectrometry identification of TssL phosphorylation at Thr 14.

<p>(<b>A</b>) TssL domain organization presented according to the topology analysis by Ma et al. (2009) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003991#ppat.1003991-Ma1" target="_blank">[20]</a> and information from the NCBI database (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>). TssL is an integral inner membrane protein (1–501 aa) with an N-terminal conserved IcmH/DotU VasF-like domain (35–296 aa) harboring one transmembrane domain (TM, 256–278 aa) and C-terminal peptidoglycan binding domain (367–470 aa) located in periplasm. (<b>B</b>) TssL-His purified from Δ<i>tssL</i>(pTssL-His) was separated by 12% SDS-PAGE, and Coomassie blue-stained TssL-His protein band was excised for in-gel digestion followed by mass spectrometry (MS) analysis. MS/MS ion spectrum with the matched b and y ions of the pT14-containing tryptic peptide STDNPSSWQDLPpTVVEITEESR in TssL-His is shown.</p

TssL phosphorylation analysis by Phos-tag.

<p>(<b>A</b>) Western blot analysis with regular SDS-PAGE of total proteins isolated from various <i>A. tumefaciens</i> strains examined with specific antibodies. RNA polymerase α subunit RpoA was an internal control. The TssL protein band with faster migration in Δ<i>ppkA</i> mutant is marked with an asterisk. (<b>B</b>) Phos-tag SDS-PAGE analysis. Different volumes of Ni-NTA resins containing TssL-His were treated with (+) or without (−) calf intestinal alkaline phosphatase (CIAP). Western blot analysis of proteins were separated by 7% Phos-tag SDS-PAGE and examined by specific antibody against 6×His. Total proteins from Δ<i>tssL</i> mutant were a negative control. Phos-tag SDS-PAGE revealed the upper band indicating phosphorylated TssL-His (<i>p</i>-TssL-His) and lower band indicating unphosphorylated TssL-His.</p