Structural and Functional Analyses of a Sterol Carrier Protein in Spodoptera litura

Backgrounds

In insects, cholesterol is one of the membrane components in cells and a precursor of ecdysteroid biosynthesis. Because insects lack two key enzymes, squalene synthase and lanosterol synthase, in the cholesterol biosynthesis pathway, they cannot autonomously synthesize cholesterol de novo from simple compounds and therefore have to obtain sterols from their diet. Sterol carrier protein (SCP) is a cholesterol-binding protein responsible for cholesterol absorption and transport.

Results

In this study, a model of the three-dimensional structure of SlSCPx-2 in Spodoptera litura, a destructive polyphagous agricultural pest insect in tropical and subtropical areas, was constructed. Docking of sterol and fatty acid ligands to SlSCPx-2 and ANS fluorescent replacement assay showed that SlSCPx-2 was able to bind with relatively high affinities to cholesterol, stearic acid, linoleic acid, stigmasterol, oleic acid, palmitic acid and arachidonate, implying that SlSCPx may play an important role in absorption and transport of these cholesterol and fatty acids from host plants. Site-directed mutation assay of SlSCPx-2 suggests that amino acid residues F53, W66, F89, F110, I115, T128 and Q131 are critical for the ligand-binding activity of the SlSCPx-2 protein. Virtual ligand screening resulted in identification of several lead compounds which are potential inhibitors of SlSCPx-2. Bioassay for inhibitory effect of five selected compounds showed that AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 inhibited the growth of S. litura larvae.

Conclusions

Compounds AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 selected based on structural modeling showed binding affinity to SlSCPx-2 protein and inhibitory effect on the growth of S. litura larvae.     

A Novel Two-Component Response Regulator Links rpf with Biofilm Formation and Virulence of Xanthomonas axonopodis pv. citri

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a serious disease that impacts citrus production worldwide, and X. axonopodis pv. citri is listed as a quarantine pest in certain countries. Biofilm formation is important for the successful development of a pathogenic relationship between various bacteria and their host(s). To understand the mechanisms of biofilm formation by X. axonopodis pv. citri strain XW19, the strain was subjected to transposon mutagenesis. One mutant with a mutation in a two-component response regulator gene that was deficient in biofilm formation on a polystyrene microplate was selected for further study. The protein was designated as BfdR for biofilm formation defective regulator. BfdR from strain XW19 shares 100% amino acid sequence identity with XAC1284 of X. axonopodis pv. citri strain 306 and 30–100% identity with two-component response regulators in various pathogens and environmental microorganisms. The bfdR mutant strain exhibited significantly decreased biofilm formation on the leaf surfaces of Mexican lime compared with the wild type strain. The bfdR mutant was also compromised in its ability to cause canker lesions. The wild-type phenotype was restored by providing pbfdR in trans in the bfdR mutant. Our data indicated that BfdR did not regulate the production of virulence-related extracellular enzymes including amylase, lipase, protease, and lecithinase or the expression of hrpG, rfbC, and katE; however, BfdR controlled the expression of rpfF in XVM2 medium, which mimics cytoplasmic fluids in planta. In conclusion, biofilm formation on leaf surfaces of citrus is important for canker development in X. axonopodis pv. citri XW19. The process is controlled by the two-component response regulator BfdR via regulation of rpfF, which is required for the biosynthesis of a diffusible signal factor.     

Correlation of Fragile Histidine Triad (Fhit) Protein Structural Features with Effector Interactions and Biological Functions

We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing mutants that do not bind substrate or cannot be phosphorylated showed decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization. Expression of Fhit or mutants that bind interactor proteins results in oxidative damage and accumulation of cells in G₂/M or sub-G₁ fractions after peroxide treatment; noninteracting mutants are defective in these biological effects. Gastric cancer clones expressing noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity, confirming that substrate binding, interaction with heat shock proteins, mitochondrial localization, and interaction with Fdxr are important for Fhit tumor suppressor function.Fhit protein is a powerful tumor suppressor that is frequently lost or reduced in cancer cells because of rearrangement of the exquisitely DNA damage-sensitive fragile FHIT gene. Restoration of Fhit expression suppresses tumorigenicity of cancer cells of various types, and the ability to induce apoptosis in cancer cells in vitro is reduced by specific Fhit mutations (1, 2).Through studies of signal pathways affected by Fhit expression, by searches for Fhit protein effectors, and by in vitro analyses of Fhit activity, we and others have defined Fhit enzymatic activity in vitro (3), apoptotic activity in cells and tumors (4–6), and most recently identification of a Fhit protein complex that affects Fhit stability, mitochondrial localization, and interaction with ferredoxin reductase (Fdxr)⁵ (7). The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where Fhit interacts with Fdxr, which is responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Virally mediated Fhit restoration in Fhit-deficient cancer cells increases production of intracellular reactive oxygen species (ROS), followed by increased apoptosis of cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, likely carrying oxidative DNA damage that contributes to accumulation of mutations.The Fhit protein sequence, showing high homology to the histidine triad (HIT) family of proteins, suggested that the protein product would hydrolyze diadenosine tetraphosphate or diadenosine triphosphate (Ap₃A) (8), and in vitro studies showed that Ap₃A was cleaved into ADP and AMP by Fhit. The catalytic histidine triad within Fhit was essential for catalytic activity (3), and a Fhit mutant that substituted Asn for His at the central histidine (H96N mutant) was catalytically inactive, although it bound substrate well (3). Early tumor suppression studies showed that cancer cells stably transfected with wild type (WT) or H96N mutant Fhit were suppressed for tumor growth in nude mice. This suggested the hypothesis that the Fhit-substrate complex sends the tumor suppression signal (9, 10). To test this hypothesis, a series of FHIT alleles was designed to reduce substrate-binding and/or hydrolytic rates and was characterized by quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in k_cat and increases as large as 30-fold in K_m. Mutants with 2–7-fold increases in K_m had significantly reduced apoptotic indices and the mutant with a 30-fold increase in K_m retained little apoptotic function. Thus, the proapoptotic function of Fhit, which is likely associated with tumor suppressor function, is limited by substrate binding and is unrelated to substrate hydrolysis (11).Fhit, a homodimeric protein of 147 amino acids, is a target of tyrosine phosphorylation by the Src family protein kinases, which can phosphorylate Tyr-114 of Fhit in vitro and in vivo (12). After co-expression of Fhit with the Elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit, unphosphorylated, mono-, and diphosphorylated Fhit were purified, and enzyme kinetics studies showed that monophosphorylated Fhit exhibited monophasic kinetics with K_m and k_cat values ∼2- and ∼7-fold lower, respectively, than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics; one site had K_m and k_cat values ∼2- and ∼140-fold lower, respectively, than for unphosphorylated Fhit; the second site had a K_m ∼60-fold higher and a k_cat ∼6-fold lower than for unphosphorylated Fhit (13). Thus, it was possible that the alterations in K_m and k_cat values for phosphorylated forms of Fhit might favor formation and lifetime of the Fhit-Ap₃A complex and enhance tumor suppressor activity (see

Functional and Structural Properties of a Novel Protein and Virulence Factor (Protein sHIP) in Streptococcus pyogenes

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.     

Defining the Predicted Protein Secretome of the Fungal Wheat Leaf Pathogen Mycosphaerella graminicola

The Dothideomycete fungus Mycosphaerella graminicola is the causal agent of Septoria tritici blotch, a devastating disease of wheat leaves that causes dramatic decreases in yield. Infection involves an initial extended period of symptomless intercellular colonisation prior to the development of visible necrotic disease lesions. Previous functional genomics and gene expression profiling studies have implicated the production of secreted virulence effector proteins as key facilitators of the initial symptomless growth phase. In order to identify additional candidate virulence effectors, we re-analysed and catalogued the predicted protein secretome of M. graminicola isolate IPO323, which is currently regarded as the reference strain for this species. We combined several bioinformatic approaches in order to increase the probability of identifying truly secreted proteins with either a predicted enzymatic function or an as yet unknown function. An initial secretome of 970 proteins was predicted, whilst further stringent selection criteria predicted 492 proteins. Of these, 321 possess some functional annotation, the composition of which may reflect the strictly intercellular growth habit of this pathogen, leaving 171 with no functional annotation. This analysis identified a protein family encoding secreted peroxidases/chloroperoxidases (PF01328) which is expanded within all members of the family Mycosphaerellaceae. Further analyses were done on the non-annotated proteins for size and cysteine content (effector protein hallmarks), and then by studying the distribution of homologues in 17 other sequenced Dothideomycete fungi within an overall total of 91 predicted proteomes from fungal, oomycete and nematode species. This detailed M. graminicola secretome analysis provides the basis for further functional and comparative genomics studies.     

Structural and Functional Similarities between a Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO)-like Protein from Bacillus subtilis and Photosynthetic RuBisCO

The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO₂ fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg²⁺ for catalysis and, like RuBisCO, was stimulated by CO₂. Four residues that are essential for the enolization reaction of RuBisCO, Lys¹⁷⁵, Lys²⁰¹, Asp²⁰³, and Glu²⁰⁴, were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys¹²³, the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a K_i value of 103 μm. RuBP and d-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)⁴ catalyzes the carboxylation and oxygenation reactions of ribulose 1,5-bisphosphate (RuBP) in photosynthesis (1-4). This enzyme is the sole CO₂-fixing enzyme in plants; however, it has certain inefficiencies. It has a very low turnover rate, a low affinity for the substrate, CO₂, and low specificity between the carboxylation and oxygenation reactions (5-7). Thus, the intrinsic enzymatic properties of RuBisCO are inadequate for efficient incorporation of CO₂ into organic matter in photosynthesis (7). However, plants have overcome these disadvantages by investing a huge amount of leaf nitrogen in RuBisCO synthesis (8).In nature, there are wide variations in the properties and primary sequences of RuBisCO among different photosynthetic organisms (9-12). The primary sequences vary as much as 73% without loss of activity. The relative specificity ranges from ∼0.5 in a small subunitless RuBisCO to 238 in a red algal, hexadecameric RuBisCO (13, 14). The affinity for CO₂ varies some 100-fold (15). Comparisons between these kinetic parameters and the primary sequences are expected to reveal promising strategies for improving the enzyme, and many studies have been conducted on this topic (7, 16-18).A RuBisCO-like protein (RLP) with no CO₂-fixing activity was first demonstrated in Chlorobium tepidum (19), and a similar protein in Bacillus subtilis was found to be involved in the methionine salvage pathway (20). These findings have pointed to a new direction in RuBisCO research (17, 21). The phylogenetic tree of the catalytic subunits of RuBisCOs and their homologs shows four major clusters, forms I-III, and form IV (Fig. 1A). Form I and II RuBisCOs are involved in photosynthetic or chemosynthetic CO₂ fixation, whereas the metabolic function of form III RuBisCOs remains unclear, although they can fix CO₂ on RuBP (9, 22). Forms I-III conserve almost all 19 amino acid residues that are essential for CO₂ fixation in RuBisCO (Fig. 1B). The form IV cluster in the phylogenetic tree consists of RLPs that show ∼20% homology to plant form I or bacterial form II RuBisCOs (12, 20, 21, 23-25). There are 8-18 RuBisCO-essential residues that are conserved in RLPs (Fig. 1B). Form IV RLPs are further subdivided into four groups; α1, α2, β, and γ (21). The RLP of B. subtilis is classified in α1 and catalyzes the enolization reaction of 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) but not the carboxylation of RuBP (Fig. 2A) (20, 21, 23). The absence of CO₂-fixing activity in the B. subtilis RLP may be ascribed to changes in 8 of the 19 amino acid residues essential for CO₂ fixation in RuBisCO (Fig. 1B). Several of these residues are located at the C-terminal domains of B. subtilis RLP and RuBisCO. The dimeric RuBisCO from Rhodospirillum rubrum catalyzes the DK-MTP-1-P enolase reaction with very low activity (20). These findings, together with the similarity in the chemical structures of substrates for B. subtilis RLP and RuBisCO (Fig. 2A), suggest that they may have a close evolutionary relationship (12, 21, 23-25).Open in a separate windowFIGURE 1.Homology between RLPs and RuBisCOs. A, phylogenetic tree of RLPs and RuBisCOs. Deduced amino acid sequence of B. subtilis subsp. subtilis str. 168 RLP ({"type":"entrez-protein","attrs":{"text":"NP_389242","term_id":"16078423","term_text":"NP_389242"}}NP_389242) was compared with sequences of RLPs of Thermotoga lettingae TMO ({"type":"entrez-protein","attrs":{"text":"YP_001471302","term_id":"157364535","term_text":"YP_001471302"}}YP_001471302), Beggiatoa sp. SS ({"type":"entrez-protein","attrs":{"text":"ZP_01997270","term_id":"153864333","term_text":"ZP_01997270"}}ZP_01997270), Ostreococcus tauri (Ostreococcus tauri IV, {"type":"entrez-protein","attrs":{"text":"CAL54998","term_id":"116059291","term_text":"CAL54998"}}CAL54998), Alkalilimnicola ehrlichei MLHE-1 ({"type":"entrez-protein","attrs":{"text":"YP_742007","term_id":"114320324","term_text":"YP_742007"}}YP_742007), R. rubrum ATCC 11170 (R. rubrum IV, {"type":"entrez-protein","attrs":{"text":"YP_427085","term_id":"83593333","term_text":"YP_427085"}}YP_427085), R. palustris CGA009 (R. palustris IV-1, {"type":"entrez-protein","attrs":{"text":"NP_947514","term_id":"39935238","term_text":"NP_947514"}}NP_947514), Archaeoglobus fulgidus DSM 4304 (A. fulgidus IV, {"type":"entrez-protein","attrs":{"text":"NP_070416","term_id":"11499182","term_text":"NP_070416"}}NP_070416), M. aeruginosa PCC 7806 (M. aeruginosa IV, {"type":"entrez-protein","attrs":{"text":"CAJ43366","term_id":"112821115","term_text":"CAJ43366"}}CAJ43366), G. kaustophilus HTA426 ({"type":"entrez-protein","attrs":{"text":"YP_146806","term_id":"56419488","term_text":"YP_146806"}}YP_146806), Bacillus cereus ATCC 14579 ({"type":"entrez-protein","attrs":{"text":"NP_833754","term_id":"30022123","term_text":"NP_833754"}}NP_833754), B. bronchiseptica RB50 ({"type":"entrez-protein","attrs":{"text":"NP_887583","term_id":"33600023","term_text":"NP_887583"}}NP_887583), Polaromonas sp. JS666 ({"type":"entrez-protein","attrs":{"text":"YP_546958","term_id":"91786006","term_text":"YP_546958"}}YP_546958), C. tepidum TLS ({"type":"entrez-protein","attrs":{"text":"NP_662651","term_id":"21674586","term_text":"NP_662651"}}NP_662651), and R. palustris CGA009 (R. palustris IV-2, {"type":"entrez-protein","attrs":{"text":"NP_945615","term_id":"39933339","term_text":"NP_945615"}}NP_945615) and of RuBisCOs of R. palustris CGA009 (R. palustris II, {"type":"entrez-protein","attrs":{"text":"NP_949975","term_id":"39937699","term_text":"NP_949975"}}NP_949975), R. rubrum ATCC 11170 (R. rubrum II, {"type":"entrez-protein","attrs":{"text":"YP_427487","term_id":"83593735","term_text":"YP_427487"}}YP_427487), M. jannaschii DSM 2661 ({"type":"entrez-protein","attrs":{"text":"NP_248230","term_id":"15669420","term_text":"NP_248230"}}NP_248230), A. fulgidus DSM 4304 (A. fulgidus III, {"type":"entrez-protein","attrs":{"text":"NP_070466","term_id":"11499228","term_text":"NP_070466"}}NP_070466), Thermococcus kodakaraensis KOD1 ({"type":"entrez-protein","attrs":{"text":"YP_184703","term_id":"57642225","term_text":"YP_184703"}}YP_184703), Galdieria partita ({"type":"entrez-protein","attrs":{"text":"BAA75796","term_id":"4519903","term_text":"BAA75796"}}BAA75796), R. palustris CGA009 (R. palustris I, {"type":"entrez-protein","attrs":{"text":"NP_946905","term_id":"39934629","term_text":"NP_946905"}}NP_946905), M. aeruginosa PCC 7806 (M. aeruginosa I, {"type":"entrez-protein","attrs":{"text":"CAJ43363","term_id":"112821111","term_text":"CAJ43363"}}CAJ43363), O. tauri (O. tauri IV, {"type":"entrez-protein","attrs":{"text":"YP_717262","term_id":"113170470","term_text":"YP_717262"}}YP_717262), and S. oleracea ({"type":"entrez-protein","attrs":{"text":"NP_054944","term_id":"11497536","term_text":"NP_054944"}}NP_054944). When an organism has more than one RuBisCO and/or RLP sequence, the form number of each sequence in the RuBisCO family follows the name of the organism. ClustalW and TreeView programs (available on the World Wide Web) were used to construct the phylogenetic tree. B, multiple alignments of sequences underlined in A. Identical amino acid residues are indicated by black shading, and similar amino acid residues are indicated by gray shading. Sequences are numbered according to the S. oleracea sequence. Catalytic and RuBP-binding residues deduced for RuBisCO are indicated by open triangles and filled triangles, respectively. Alignment was visualized with the BOXSHADE program (available on the World Wide Web).Open in a separate windowFIGURE 2.Catalytic and structural similarity of RLPs and RuBisCOs. A, catalytic reactions of RuBisCO and RLP. B, comparison of active sites between S. oleracea RuBisCO binding CABP (8RUC) and G. kaustophilus RLP (2OEM) modeled to bind DK-MTP-1-P. DK-MTP-1-P in G. kaustophilus RLP was depicted by substituting the methyl group of DK-H-1-P in 2OEM with the thiomethyl group of MTRu-1-P bound to MtnA (28). Side chains of active site residues and ligands are shown as sticks. These five residues of B. subtilis were substituted with other amino acids in this study. CABP and DK-MTP-1-P are shown in white, and their phosphate groups are shown in red and orange, respectively. Mg²⁺ atoms are shown in yellow. Protein structures were drawn with PyMOL (available on the World Wide Web).The RuBisCO reaction starts with the abstraction of the C3 proton from RuBP to form the cis-enediol(ate) of RuBP (Fig. 2A) (26). Using the spinach numbering format to identify RuBisCO and RLP residues, the carbamate formed on the ε-amino group of Lys²⁰¹ may be the general base to abstract the proton, and the cis-enediol(ate) form of RuBP is stabilized in the combination of side chains from Lys¹⁷⁵ and His²⁹⁴ (27). Asp²⁰³, Glu²⁰⁴, and the carbamate Lys²⁰¹ of the enzyme active site stabilize the cis-enediol(ate) and CO₂ through the Mg²⁺ ion (26). The B. subtilis RLP abstracts the C1 proton of its substrate DK-MTP-1-P to start the DK-MTP-1-P enolization reaction (12, 21, 23). The ε-amino group of Lys¹²³ is thought to be required for the abstraction of the 1-proS proton in the Geobacillus kaustophilus RLP, which belongs to group α1, together with the B. subtilis RLP (Fig. 2B) (25). Lys¹²³ is conserved among DK-MTP-1-P enolases and resides very near the C1 of 2,3-diketohexane 1-phosphate (DK-H-1-P), a structural analogue of DK-MTP-1-P. As is the case in RuBisCO, the enolate intermediate is stabilized by Mg²⁺ and several amino acid residues: Lys¹⁷⁵, Asp²⁰³, Glu²⁰⁴, His²⁹⁴, and the carbamylated Lys²⁰¹.The results of these studies suggest that the DK-MTP-1-P enolase is structurally and functionally related to photosynthetic RuBisCO. However, research on the G. kaustophilus RLP revealed that the proton-abstracting, reaction-starting residues differed between the DK-MTP-1-P enolase and RuBisCO (25). It has been reported that when lysine at 201 is substituted with an alanine in the G. kaustophilus RLP, the enzyme is still capable of catalyzing enolization of DK-MTP-1-P (25). This result raises a question about the above hypothesis on the close evolutionary relationship between the RLP and RuBisCO, because a carbamylated lysine residue would be required at this position to form the Mg²⁺-chelating triad linkage together with Asp²⁰³ and Glu²⁰⁴ and to stabilize the reaction intermediate in the RuBP enolization reaction of RuBisCO.Evolutionary relationships of genes with similar sequences are deduced by comparing gene sequence homology of the genes and amino acid sequence homology of the predicted proteins and by analyzing conservation of functional motifs of the predicted proteins in silico. Comparison of protein structures at the active sites also provides important information. However, it may difficult to predict their mutual evolutionary relationship more precisely when they catalyze different reactions in individual metabolic pathways. The present research adopted a new method to resolve such an issue.We studied the structural and functional interrelationships of RLP and RuBisCO after enzymological characterization of B. subtilis RLP as the DK-MTP-1-P enolase enzyme. The results showed that DK-MTP-1-P enolase activity was limited to some RLPs in the cluster, including B. subtilis in form IV RLPs. All of the catalytic residues for the RuBisCO reaction were also indispensable for DK-MTP-1-P enolase activity. The architecture of the B. subtilis RLP substrate-binding residues stereospecifically stabilized the transition state analog in CO₂ fixation of RuBisCO. The fact that the transition state analog of RuBisCO interacts with the active site of Bacillus RLP strongly supports their evolutionary proximity.     

A Comparison of Pathogen Isolation in Culture and Injection–infiltration Bioassay of Citrus Leaves for Detecting Xanthomonas citri subsp. citri

Citrus canker [caused by Xanthomonas citri subsp. citri (Xcc)] can cause yield loss of susceptible citrus and result in trade restrictions of fresh fruit. For both regulatory purposes and epidemiological studies, accurate detection and quantification of viable inoculum are critical. Two accepted methods used to detect and quantify Xcc are injection–infiltration bioassay and culture, but these two methods have not been directly compared using field‐obtained samples. The two methods were compared using washates of lesions taken from fruit, leaves and shoots in a commercial orchard in Florida in 2009–2010 and 2010–2011, with bioassay being the assumed standard. Despite some misclassifications, true positives (sensitivity) and true negatives (specificity) were the dominant classes using culture. False positives for lesions from shoots ranged from 13.1 to 21.4% in 2009–2010 and 2010–2011, respectively, and false positives for lesions from fruit and leaves ranged from 4.3 to 15.7%, in the two seasons, respectively. The false positive rate for culture compared with injection–infiltration bioassay was highest (0.16–0.55), due to more frequent recovery of Xcc by culture at ≤10³ colony‐forming units (CFU) Xcc per ml. The false negative rate was consistently lower (0.02–0.21), confirming that in only a few cases did culture fail to detect Xcc when it was present. The area under the curve for receiver operator characteristic analysis ranged from 0.80 to 0.97, confirming that culture provided an accurate diagnosis in most cases. There was a higher frequency of lesions from shoots with a CFU ≤10³ Xcc compared with lesions from fruit or leaves, making culture more effective at detecting these. The data demonstrate that culture is a reliable way to detect and quantify Xcc compared with injection–infiltration bioassay, particularly when the CFU is ≤10³ Xcc per ml.     

Characterization of Citrus Rootstock Responses to Tylenchulus semipenetrans (Cobb)

Citrus rootstocks which significantly limited the reproduction of Tylenchulus semipenetrans (Cobb) "Citrus" and "Poncirus" biotypes responded to infection by producing a hypersensitive-type response in the root hypodermis, wound periderm and/or cavities in the root cortex, and/or abnormal vacuoles in nurse cell cytoplasm. Rootstocks which limited nematode reproduction also had significantly fewer nematodes in the rhizoplane within 8 d of inoculation than did rootstocks which did not limit reproduction. Germplasm sources of the cellular responses which limited citrus nematode reproduction were identified.     

Identification of the Protein Target of Myelin-Binding Ligands by Immunohistochemistry and Biochemical Analyses

The ability to visualize myelin is important in the diagnosis of demyelinating disorders and the detection of myelin-containing nerves during surgery. The development of myelin-selective imaging agents requires that a defined target for these agents be identified and that a robust assay against the target be developed to allow for assessment of structure-activity relationships. We describe an immunohistochemical analysis and a fluorescence polarization binding assay using purified myelin basic protein (MBP) that provides quantitative evidence that MBP is the molecular binding partner of previously described myelin-selective fluorescent dyes such as BMB, GE3082, and GE3111.     

BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC.     

Structural and Functional Characterization of the Protein Kinase Mps1 in Arabidopsis thaliana

In eukaryotes, protein kinases catalyze the transfer of a gamma-phosphate from ATP (or GTP) to specific amino acids in protein targets. In plants, protein kinases have been shown to participate in signaling cascades driving responses to environmental stimuli and developmental processes. Plant meristems are undifferentiated tissues that provide the major source of cells that will form organs throughout development. However, non-dividing specialized cells can also dedifferentiate and re-initiate cell division if exposed to appropriate conditions. Mps1 (Monopolar spindle) is a dual-specificity protein kinase that plays a critical role in monitoring the accuracy of chromosome segregation in the mitotic checkpoint mechanism. Although Mps1 functions have been clearly demonstrated in animals and fungi, its role in plants is so far unclear. Here, using structural and biochemical analyses here we show that Mps1 has highly similar homologs in many plant genomes across distinct lineages (e.g. AtMps1 in Arabidopsis thaliana). Several structural features (i.e. catalytic site, DFG motif and threonine triad) are clearly conserved in plant Mps1 kinases. Structural and sequence analysis also suggest that AtMps1 interact with other cell cycle proteins, such as Mad2 and MAPK1. By using a very specific Mps1 inhibitor (SP600125) we show that compromised AtMps1 activity hampers the development of A. thaliana seedlings in a dose-dependent manner, especially in secondary roots. Moreover, concomitant administration of the auxin IAA neutralizes the AtMps1 inhibition phenotype, allowing secondary root development. These observations let us to hypothesize that AtMps1 might be a downstream regulator of IAA signaling in the formation of secondary roots. Our results indicate that Mps1 might be a universal component of the Spindle Assembly Checkpoint machinery across very distant lineages of eukaryotes.     

Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis

Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its “pupylome”. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtb response regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation.     

Single-molecule Analyses of the Dynamics of Heat Shock Protein 104 (Hsp104) and Protein Aggregates

Hsp104 solubilizes protein aggregates in cooperation with Hsp70/40. Although the framework of the disaggregase function has been elucidated, the actual process of aggregate solubilization by Hsp104-Hsp70/40 remains poorly understood. Here we developed several methods to investigate the functions of Hsp104 and Hsp70/40 from Saccharomyces cerevisiae, at single-molecule levels. The single-molecule methods, which provide the size distribution of the aggregates, revealed that Hsp70/40 prevented the formation of large aggregates from small aggregates and that the solubilization of the small aggregates required both Hsp104 and Hsp70/40. We directly visualized the individual association-dissociation dynamics of Hsp104 on immobilized aggregates and found that the lifetimes of the Hsp104-aggregate complex are divided into two groups: short (∼4 s) and long (∼30 s). Hsp70/40 stimulated the association of Hsp104 with aggregates and increased the duration of this association. The single-molecule data provide novel insights into the functional mechanism of the Hsp104 disaggregation machine.     

The Role of Mitogen-Activated Protein (MAP) Kinase Signaling Components in the Fungal Development,Stress Response and Virulence of the Fungal Cereal Pathogen Bipolaris sorokiniana

Mitogen-activated protein kinases (MAPKs) have been demonstrated to be involved in fungal development, sexual reproduction, pathogenicity and/or virulence in many filamentous plant pathogenic fungi, but genes for MAPKs in the fungal cereal pathogen Bipolaris sorokiniana have not been characterized. In this study, orthologues of three MAPK genes (CsSLT2, CsHOG1 and CsFUS3) and one MAPK kinase kinase (MAPKKK) gene (CsSTE11) were identified in the whole genome sequence of the B. sorokiniana isolate ND90Pr, and knockout mutants were generated for each of them. The ∆Csfus3 and ∆Csste11 mutants were defective in conidiation and formation of appressoria-like structures, showed hypersensitivity to oxidative stress and lost pathogenicity on non-wounded leaves of barley cv. Bowman. When inoculated on wounded leaves of Bowman, the ∆Csfus3 and ∆Csste11 mutants were reduced in virulence compared to the wild type. No morphological changes were observed in the ∆Cshog1 mutants in comparison with the wild type; however, they were slightly reduced in growth under oxidative stress and were hypersensitive to hyperosmotic stress. The ∆Cshog1 mutants formed normal appressoria-like structures but were reduced in virulence when inoculated on Bowman leaves. The ∆Csslt2 mutants produced more vegetative hyphae, had lighter pigmentation, were more sensitive to cell wall degrading enzymes, and were reduced in virulence on Bowman leaves, although they formed normal appressoria like the wild type. Root infection assays indicated that the ∆Cshog1 and ∆Csslt2 mutants were able to infect barley roots while the ∆Csfus3 and ∆Csste11 failed to cause any symptoms. However, no significant difference in virulence was observed for ∆Cshog1 mutants while ∆Csslt2 mutants showed significantly reduced virulence on barley roots in comparison with the wild type. Our results indicated that all of these MAPK and MAPKKK genes are involved in the regulation of fungal development under normal and stress conditions and required for full virulence on barley plants.     

Physiological and Biochemical Characteristics of Iranian Strains of Xanthomonas axonopodis pv. citri, the Causal Agent of Citrus bacterial Canker Disease

Twenty-four strains of Xanthomonas axonopodis pv. citri ( Xac ), the causal agent of bacterial canker of citrus, isolated from Mexican lime ( Citrus aurantifolia ) and lemon ( Citrus limon ) in southern Iran, were characterized phenotypically. Strains were all pathogenic on C. aurantifolia . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed slight differences in soluble protein profiles among the strains. Based on host range specificity and phenotypic characteristics, representative strains were differentiated into two groups of Asiatic (A) and atypical Asiatic (aA) forms. DNA fingerprinting analysis using Eco RI as the restriction endonuclease showed a negligible difference in restriction pattern between the two groups. On the basis of isozymic analysis, the two groups were distinct with respect to superoxide dismutase (SOD) and esterase (EST) banding patterns. Plasmid DNA profile analysis showed that the bacterial strains were different from each other in terms of plasmid number and molecular weight. Phage typing study revealed that most of group A strains were susceptible to Cp1 and/or Cp2 and some were resistant to both phage types including the strain in aA group. Bacteriocin production test indicated that there was a variation among Xac strains using different indicators for each bacteriocin producer. It is concluded that the Iranian strains of Xac are heterogeneous and constitute a subgroup(s) within the pathotype A.     

Adaptability and Persistence of the Emerging Pathogen Bordetella petrii

The first described, environmentally isolated, Bordetella petrii was shown to undergo massive genomic rearrangements in vitro. More recently, B. petrii was isolated from clinical samples associated with jaw, ear bone, cystic fibrosis and chronic pulmonary disease. However, the in vivo consequences of B. petrii genome plasticity and its pathogenicity remain obscure. B. petrii was identified from four sequential respiratory samples and a post-mortem spleen sample of a woman presenting with bronchiectasis and cavitary lung disease associated with nontuberculous mycobacterial infection. Strains were compared genetically, phenotypically and by antibody recognition from the patient and from inoculated mice. The successive B. petrii strains exhibited differences in growth, antibiotic susceptibility and recognition by the patient’s antibodies. Antibodies from mice inoculated with these strains recapitulated the specificity and strain dependent response that was seen with the patient’s serum. Finally, we characterize one strain that was poorly recognized by the patient’s antibodies, due to a defect in the lipopolysaccharide O-antigen, and identify a mutation associated with this phenotype. We propose that B. petrii is remarkably adaptable in vivo, providing a possible connection between immune response and bacterial evasion and supporting infection persistence.     

Pathogen and Circadian Controlled 1 (PCC1) Protein Is Anchored to the Plasma Membrane and Interacts with Subunit 5 of COP9 Signalosome in Arabidopsis

The Pathogen and Circadian Controlled 1 (PCC1) gene, previously identified and further characterized as involved in defense to pathogens and stress-induced flowering, codes for an 81-amino acid protein with a cysteine-rich C-terminal domain. This domain is essential for homodimerization and anchoring to the plasma membrane. Transgenic plants with the ß-glucuronidase (GUS) reporter gene under the control of 1.1 kb promoter sequence of PCC1 gene display a dual pattern of expression. At early post-germination, PCC1 is expressed only in the root vasculature and in the stomata guard cells of cotyledons. During the transition from vegetative to reproductive development, PCC1 is strongly expressed in the vascular tissue of petioles and basal part of the leaf, and it further spreads to the whole limb in fully expanded leaves. This developmental pattern of expression together with the late flowering phenotype of long-day grown RNA interference (iPCC1) plants with reduced PCC1 expression pointed to a regulatory role of PCC1 in the photoperiod-dependent flowering pathway. iPCC1 plants are defective in light perception and signaling but are not impaired in the function of the core CO-FT module of the photoperiod-dependent pathway. The regulatory effect exerted by PCC1 on the transition to flowering as well as on other reported phenotypes might be explained by a mechanism involving the interaction with the subunit 5 of the COP9 signalosome (CSN).