Unusual ß1-4-galactosidase activity of an α1-6-mannosidase from Xanthomonas manihotis in the processing of branched hybrid and complex glycans.

Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell-pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected ß1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel ß1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal ß1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.

A Glycoside Hydrolase Family 99-Like Domain-Containing Protein Modifies Outer Membrane Proteins to Maintain Xanthomonas Pathogenicity and Viability in Stressful Environments.

Protein glycosylation is an essential process that plays an important role in proteome stability, protein structure, and protein function modulation in eukaryotes. However, in bacteria, especially plant pathogenic bacteria, similar studies are lacking. Here, we investigated the relationship between protein glycosylation and pathogenicity by using Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight in rice, as a well-defined example. In our previous work, we identified a virulence-related hypothetical protein, PXO_03177, but how this protein regulates X. oryzae pv. oryzae virulence has remained unclear. BLAST analysis showed that most homologous proteins of PXO_03177 are glycoside hydrolase family 99-like domain-containing proteins. In the current study, we found that the outer membrane integrity of ΔPXO_03177 appeared to be disrupted. Extracting the outer membrane proteins (OMPs) and performing comparative proteomics and sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel staining analyses revealed that PXO_03177 deletion altered the protein levels of 13 OMPs. Western blot analyses showed that the protein level and glycosylation modification of PXO_02523, a related OmpA family-like protein, was changed in the ΔPXO_03177 mutant background strain. Additionally, the interaction between PXO_03177 and PXO_02523 was confirmed by coimmunoprecipitation. Both PXO_03177 and PXO_02523 play important roles in regulating pathogen virulence and viability in stressful environments. This work provides the first evidence that protein glycosylation is necessary for the virulence of plant pathogenic bacteria.

A Secreted Chorismate Mutase from Xanthomonas arboricola pv. juglandis Attenuates Virulence and Walnut Blight Symptoms.

Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.

Discovery of Novel Dihydrolipoamide S-Succinyltransferase Inhibitors Based on Fragment Virtual Screening.

A series of new oxadiazole sulfone derivatives containing an amide moiety was synthesized based on fragment virtual screening to screen high-efficiency antibacterial agents for rice bacterial diseases. All target compounds showed greater bactericidal activity than commercial bactericides. 3-(4-fluorophenyl)-N-((5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl)methyl)acrylamide (10) showed excellent antibacterial activity against Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola, with EC50 values of 0.36 and 0.53 mg/L, respectively, which were superior to thiodiazole copper (113.38 and 131.54 mg/L) and bismerthiazol (83.07 and 105.90 mg/L). The protective activity of compound 10 against rice bacterial leaf blight and rice bacterial leaf streak was 43.2% and 53.6%, respectively, which was superior to that of JHXJZ (34.1% and 26.4%) and thiodiazole copper (33.0% and 30.2%). The curative activity of compound 10 against rice bacterial leaf blight and rice bacterial leaf streak was 44.5% and 51.7%, respectively, which was superior to that of JHXJZ (32.6% and 24.4%) and thiodiazole copper (27.1% and 28.6%). Moreover, compound 10 might inhibit the growth of Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola by affecting the extracellular polysaccharides, destroying cell membranes, and inhibiting the enzyme activity of dihydrolipoamide S-succinyltransferase.

The Xanthomonas euvesicatoria type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling.

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.

Isolation and characterisation of carbapenem-resistant Xanthomonas citri pv. mangiferaeindicae-like strain gir from the faecal material of giraffes.

The purpose of this study was to determine if giraffes (Giraffa camelopardalis) living in captivity at the Jacksonville Zoo and Gardens, Jacksonville, FL were colonised with carbapenem-resistant bacteria and, if found, to identify underlying genetic mechanisms contributing to a carbapenem-resistant phenotype. Faecal samples from seven giraffes were examined for carbapenem-resistant bacteria. Only one isolate (a Xanthomondaceae) was found to be carbapenem-resistant by antibiotic susceptibility testing. This isolate was selected for additional characterization, including whole genome sequencing (WGS). Based on average nucleotide identity, the bacterium was identified as Xanthomonas citri pv. mangiferaeindicae-like strain gir. Phenotypic carbapenemase tests and PCR for the most common carbapenemase genes produced negative results, suggesting that carbapenem resistance was mediated by another mechanism. Resistance gene profile analysis of WGS results confirmed these results. Among identified resistance genes, a chromosomal class A beta-lactamase with 71% identity to the penP beta-lactamase gene from Xanthomonas citri ssp. citri was identified, which could contribute to a carbapenem-resistant phenotype.

The crystal structure of the phytopathogenic bacterial sensor PcrK reveals different cytokinin recognition mechanism from the plant sensor AHK4.

Plant cytokinins (CKs) are essential for many central cellular processes and play important roles in the interaction between bacteria and plants. Perception of CK is executed by the CHASE domain in the histidine kinase sensors of a class of two-component regulatory systems. Despite advances in understanding the structural basis for CK perception by the sensor AHK4 in Arabidopsis, the molecular mechanism of CK binding by other sensors is unclear. Here, we report the crystal structure of the CHASE domain in the histidine kinase PcrK of the bacterial plant pathogen Xanthomonas campestris pathovar campestris, which senses plant CK, determined at 2.55 Šresolution. The structure reveals that the PcrK has an AHK4-like overall topology and assembles into a homodimer. Strikingly, detailed structural analysis unveils two unique features of the PcrK ligand binding pocket: the size of the pocket is restricted for CK binding, and the PcrK applies a positively charged arginine but not a negatively charged aspartate to recognize the ligand. We propose a model to explain how the PcrK accommodates CK-sized compounds through conformational changes, providing a potential mechanistic framework for understanding ligand recognition by the PcrK.

Structural basis of exo-ß-mannanase activity in the GH2 family.

The classical microbial strategy for depolymerization of ß-mannan polysaccharides involves the synergistic action of at least two enzymes, endo-1,4-ß-mannanases and ß-mannosidases. In this work, we describe the first exo-ß-mannanase from the GH2 family, isolated from Xanthomonas axonopodis pv. citri (XacMan2A), which can efficiently hydrolyze both manno-oligosaccharides and ß-mannan into mannose. It represents a valuable process simplification in the microbial carbon uptake that could be of potential industrial interest. Biochemical assays revealed a progressive increase in the hydrolysis rates from mannobiose to mannohexaose, which distinguishes XacMan2A from the known GH2 ß-mannosidases. Crystallographic analysis indicates that the active-site topology of XacMan2A underwent profound structural changes at the positive-subsite region, by the removal of the physical barrier canonically observed in GH2 ß-mannosidases, generating a more open and accessible active site with additional productive positive subsites. Besides that, XacMan2A contains two residue substitutions in relation to typical GH2 ß-mannosidases, Gly439 and Gly556, which alter the active site volume and are essential to its mode of action. Interestingly, the only other mechanistically characterized mannose-releasing exo-ß-mannanase so far is from the GH5 family, and its mode of action was attributed to the emergence of a blocking loop at the negative-subsite region of a cleft-like active site, whereas in XacMan2A, the same activity can be explained by the removal of steric barriers at the positive-subsite region in an originally pocket-like active site. Therefore, the GH2 exo-ß-mannanase represents a distinct molecular route to this rare activity, expanding our knowledge about functional convergence mechanisms in carbohydrate-active enzymes.

A bipartite periplasmic receptor-diguanylate cyclase pair (XAC2383-XAC2382) in the bacterium Xanthomonas citri.

The second messenger cyclic diguanylate monophosphate (c-di-GMP) is a central regulator of bacterial lifestyle, controlling several behaviors, including the switch between sessile and motile states. The c-di-GMP levels are controlled by the interplay between diguanylate cyclases (DGCs) and phosphodiesterases, which synthesize and hydrolyze this second messenger, respectively. These enzymes often contain additional domains that regulate activity via binding of small molecules, covalent modification, or protein-protein interactions. A major challenge remains to understand how DGC activity is regulated by these additional domains or interaction partners in specific signaling pathways. Here, we identified a pair of co-transcribed genes (xac2382 and xac2383) in the phytopathogenic, Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), whose mutations resulted in opposing motility phenotypes. We show that the periplasmic cache domain of XAC2382, a membrane-associated DGC, interacts with XAC2383, a periplasmic binding protein, and we provide evidence that this interaction regulates XAC2382 DGC activity. Moreover, we solved the crystal structure of XAC2383 with different ligands, indicating a preference for negatively charged phosphate-containing compounds. We propose that XAC2383 acts as a periplasmic sensor that, upon binding its ligand, inhibits the DGC activity of XAC2382. Of note, we also found that this previously uncharacterized signal transduction system is present in several other bacterial phyla, including Gram-positive bacteria. Phylogenetic analysis of homologs of the XAC2382-XAC2383 pair supports several independent origins that created new combinations of XAC2382 homologs with a conserved periplasmic cache domain with different cytoplasmic output module architectures.

CRISPR RNA and anti-CRISPR protein binding to the Xanthomonas albilineans Csy1-Csy2 heterodimer in the type I-F CRISPR-Cas system.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide microbial adaptive immunity against bacteriophages. In type I-F CRISPR-Cas systems, multiple Cas proteins (Csy1-4) compose a surveillance complex (Csy complex) with CRISPR RNA (crRNA) for target recognition. Here, we report the biochemical characterization of the Csy1-Csy2 subcomplex from Xanthomonas albilineans, including the analysis of its interaction with crRNA and AcrF2, an anti-CRISPR (Acr) protein from a phage that infects Pseudomonas aeruginosa The X. albilineans Csy1 and Csy2 proteins (XaCsy1 and XaCsy2, respectively) formed a stable heterodimeric complex that specifically bound the 8-nucleotide (nt) 5'-handle of the crRNA. In contrast, the XaCsy1-XaCsy2 heterodimer exhibited reduced affinity for the 28-nt X. albilineans CRISPR repeat RNA containing the 5'-handle sequence. Chromatographic and calorimetric analyses revealed tight binding between the Acr protein from the P. aeruginosa phage and the heterodimeric subunit of the X. albilineans Csy complex, suggesting that AcrF2 recognizes conserved features of Csy1-Csy2 heterodimers. We found that neither XaCsy1 nor XaCsy2 alone forms a stable complex with AcrF2 and the 5'-handle RNA, indicating that XaCsy1-XaCsy2 heterodimerization is required for binding them. We also solved the crystal structure of AcrF2 to a resolution of 1.34 Å, enabling a more detailed structural analysis of the residues involved in the interactions with the Csy1-Csy2 heterodimer. Our results provide information about the order of events during the formation of the multisubunit crRNA-guided surveillance complex and suggest that the Acr protein inactivating type I-F CRISPR-Cas systems has broad specificity.

Xanthomonas oryzae pv. oryzae Response Regulator TriP Regulates Virulence and Exopolysaccharide Production Via Interacting With c-di-GMP Phosphodiesterase PdeR.

PdeR, a response regulator of the two-component system (TCS) with the cognate histidine kinase PdeK, has been shown to be an active phosphodiesterase (PDE) for intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) turnover and positively regulates the virulence of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight of rice. To further reveal the key components and pathways involved in the PdeR-mediated c-di-GMP regulation of virulence, 16 PdeR-interacting proteins were identified, using the yeast two-hybrid (Y2H) assay. Among them, PXO_04421 (named as TriP, a putative transcriptional regulator interacting with PdeR) was verified via Y2H and glutathione-S-transferase pull-down assays, and its regulatory functions in bacterial virulence and exopolysaccharide (EPS) production were assessed by biochemical and genetic analysis. The REC domain of TriP specifically interacted with the EAL domain of PdeR. TriP promoted the PDE activity of PdeR to degrade c-di-GMP in the presence of PdeK. In-frame deletion in triP abolished the polar localization of PdeR in the cell. Notably, the ∆triP mutant showed significantly reduced virulence on susceptible rice leaves and impaired EPS production compared with wild type, whereas the double mutant ∆triP∆pdeR, like ∆pdeR, caused shorter lesion lengths and produced less EPS than ∆triP. In addition, cross-complementation showed in trans expression of pdeR in ∆triP restored its EPS production to near wild-type levels but not vice versa. Taken together, our results suggest that TriP is a novel regulator that is epistatic to PdeR in positively regulating virulence expression in X. oryzae pv. oryzae.

Overexpression of OsPUB41, a Rice E3 ubiquitin ligase induced by cell wall degrading enzymes, enhances immune responses in Rice and Arabidopsis.

BACKGROUND: Cell wall degrading enzymes (CWDEs) induce plant immune responses and E3 ubiquitin ligases are known to play important roles in regulating plant defenses. Expression of the rice E3 ubiquitin ligase, OsPUB41, is enhanced upon treatment of leaves with Xanthomonas oryzae pv. oryzae (Xoo) secreted CWDEs such as Cellulase and Lipase/Esterase. However, it is not reported to have a role in elicitation of immune responses. RESULTS: Expression of the rice E3 ubiquitin ligase, OsPUB41, is induced when rice leaves are treated with either CWDEs, pathogen associated molecular patterns (PAMPs), damage associated molecular patterns (DAMPs) or pathogens. Overexpression of OsPUB41 leads to induction of callose deposition, enhanced tolerance to Xoo and Rhizoctonia solani infection in rice and Arabidopsis respectively. In rice, transient overexpression of OsPUB41 leads to enhanced expression of PR genes and SA as well as JA biosynthetic and response genes. However, in Arabidopsis, ectopic expression of OsPUB41 results in upregulation of only JA biosynthetic and response genes. Transient overexpression of either of the two biochemically inactive mutants (OsPUB41C40A and OsPUB41V51R) of OsPUB41 in rice and stable transgenics in Arabidopsis ectopically expressing OsPUB41C40A failed to elicit immune responses. This indicates that the E3 ligase activity of OsPUB41 protein is essential for induction of plant defense responses. CONCLUSION: The results presented here suggest that OsPUB41 is possibly involved in elicitation of CWDE triggered immune responses in rice.

The effector AvrRxo1 phosphorylates NAD in planta.

Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3'-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3'-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3'-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst.

The Role of a Host-Induced Arginase of Xanthomonas oryzae pv. oryzae in Promoting Virulence on Rice.

The plant bacterial pathogen Xanthomonas oryzae pv. oryzae causes bacterial blight of rice, which is one of the most destructive rice diseases prevalent in Asia and parts of Africa. Despite many years of research, how X. oryzae pv. oryzae causes bacterial blight of rice is still not completely understood. Here, we show that the loss of the rocF gene caused a significant decrease in the virulence of X. oryzae pv. oryzae in the susceptible rice cultivar IR24. Bioinformatics analysis demonstrated that rocF encodes arginase. Quantitative real-time PCR and Western blot assays revealed that rocF expression was significantly induced by rice and arginine. The rocF deletion mutant strain showed elevated sensitivity to hydrogen peroxide, reduced extracellular polysaccharide (EPS) production, and reduced biofilm formation, all of which are important determinants for the full virulence of X. oryzae pv. oryzae, compared with the wild-type strain. Taken together, the results of this study revealed a mechanism by which a bacterial arginase is required for the full virulence of X. oryzae pv. oryzae on rice because of its contribution to tolerance to reactive oxygen species, EPS production, and biofilm formation.

Crystal Structure-Based Exploration of Arginine-Containing Peptide Binding in the ADP-Ribosyltransferase Domain of the Type III Effector XopAI Protein.

Plant pathogens secrete proteins called effectors into the cells of their host to modulate the host immune response against colonization. Effectors can either modify or arrest host target proteins to sabotage the signaling pathway, and therefore are considered potential drug targets for crop disease control. In earlier research, the Xanthomonas type III effector XopAI was predicted to be a member of the arginine-specific mono-ADP-ribosyltransferase family. However, the crystal structure of XopAI revealed an altered active site that is unsuitable to bind the cofactor NAD+, but with the capability to capture an arginine-containing peptide from XopAI itself. The arginine peptide consists of residues 60 through 69 of XopAI, and residue 62 (R62) is key to determining the protein-peptide interaction. The crystal structure and the molecular dynamics simulation results indicate that specific arginine recognition is mediated by hydrogen bonds provided by the backbone oxygen atoms from residues W154, T155, and T156, and a salt bridge provided by the E265 sidechain. In addition, a protruding loop of XopAI adopts dynamic conformations in response to arginine peptide binding and is probably involved in target protein recognition. These data suggest that XopAI binds to its target protein by the peptide-binding ability, and therefore, it promotes disease progression. Our findings reveal an unexpected and intriguing function of XopAI and pave the way for further investigation on the role of XopAI in pathogen invasion.

Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution.

Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. We mutated distinguishing active-site residues to generate enzymes that had a common Zn2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of these pruned enzymes with a series of substrates. A substantial rate enhancement of ∼1011-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 107-108-fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.

Identification of a repressor and an activator of azoreductase gene expression in Pseudomonas putida and Xanthomonas oryzae.

Genes responsible for the production of azoreductase enzymes in 2 gram-negative bacteria, the soil bacterium Pseudomonas putida (AzoP) and the plant pathogen Xanthomonas oryzae (AzoX), were identified. The deduced amino acid sequences of AzoP and AzoX, share 46% amino acid identity to each other. Two different bacterial transcription factors, a repressor (AzoPR) and an activator (AzoXR), in P. putida and X. oryzae, respectively, were found to be divergently oriented to their respective azoreductase genes. Both regulators are LysR-type transcriptional regulators (LTTR) that respond to the azo dye inducer, methyl red (MR). AzoPR represses transcription of azoP in P. putida, which is reversed when cells are exposed to MR. Interestingly, in X. oryzae, AzoXR positively regulates azoX transcription upon MR induction. Moreover, despite their similarity, with 51% amino acid sequence identity, azoPR and azoXR are expressed differently in response to MR. The transcription of azoPR is increased in a dye concentration-dependent manner, while azoXR transcription is constitutive and relatively higher than azoPR. Both regulators are autoregulatory. Gel mobility shift assays (EMSA) verified the binding between the regulators and their corresponding promoter regions. Additionally, binding only occurred under reduced conditions in the presence of 0.5 mM dithiothreitol (DTT), indicating that the proteins are active in their reduced form.

Phosphodiesterase EdpX1 Promotes Xanthomonas oryzae pv. oryzae Virulence, Exopolysaccharide Production, and Biofilm Formation.

In Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, there are over 20 genes encoding GGDEF, EAL, and HD-GYP domains, which are potentially involved in the metabolism of second messenger c-di-GMP. In this study, we focused on the characterization of an EAL domain protein, EdpX1. Deletion of the edpX1 gene resulted in a 2-fold increase in the intracellular c-di-GMP levels, which were restored to the wild-type levels in the complemented ΔedpX1(pB-edpX1) strain, demonstrating that EdpX1 is an active phosphodiesterase (PDE) in X. oryzae pv. oryzae. In addition, colorimetric assays further confirmed the PDE activity of EdpX1 by showing that the E153A mutation at the EAL motif strongly reduced its activity. Virulence assays on the leaves of susceptible rice showed that the ΔedpX1 mutant was severely impaired in causing disease symptoms. In trans expression of wild-type edpX1, but not edpX1E153A, was able to complement the weakened virulence phenotype. These results indicated that an active EAL domain is required for EdpX1 to regulate the virulence of X. oryzae pv. oryzae. We then demonstrated that the ΔedpX1 mutant was defective in secreting exopolysaccharide (EPS) and forming biofilms. The expression of edpX1 in the ΔedpX1 mutant, but not edpX1E153A, restored the defective phenotypes to near-wild-type levels. In addition, we observed that EdpX1-green fluorescent protein (EdpX1-GFP) exhibited multiple subcellular localization foci, and this pattern was dependent on its transmembrane (TM) region, which did not seem to directly contribute to the regulatory function of EdpX1. Thus, we concluded that EdpX1 exhibits PDE activity to control c-di-GMP levels, and its EAL domain is necessary and sufficient for its regulation of virulence in X. oryzae pv. oryzae.IMPORTANCE Bacteria utilize c-di-GMP as a second messenger to regulate various biological functions. The synthesis and degradation of c-di-GMP are catalyzed by GGDEF domains and an EAL or HD-GYP domain, respectively. Multiple genes encoding these domains are often found in one bacterial strain. For example, in the genome of X. oryzae pv. oryzae PXO99A, 26 genes encoding proteins containing these domains were identified. Therefore, to fully appreciate the complexity and specificity of c-di-GMP signaling in X. oryzae pv. oryzae, the enzymatic activities and regulatory functions of each GGDEF, EAL, and HD-GYP domain protein need to be elucidated. In this study, we showed that the EAL domain protein EdpX1 is a major PDE to regulate diverse virulence phenotypes through the c-di-GMP signaling pathway.

The Xanthomonas effector XopK harbours E3 ubiquitin-ligase activity that is required for virulence.

Xanthomonas oryzae pv. oryzae is the causative agent of rice bacterial leaf blight. While the type III secretion system of X. oryzae pv. oryzae is essential for virulence, the biochemical activities and virulence mechanisms of non-transcription activator-like (non-TAL) effectors delivered by this system are largely unknown. Here, by screening for non-TAL effectors that contribute to X. oryzae pv. oryzae virulence, we revealed that Xanthomonas outer protein K (XopK) inhibits pathogen-associated molecular pattern-triggered immunity upstream of mitogen-activated protein kinase cascades. Specifically, XopK interacted with and directly ubiquitinated rice somatic embryogenic receptor kinase 2 (OsSERK2), resulting in its degradation. Accordingly, mutation of a putative ubiquitin-conjugation enzyme (E2) binding site abolished XopK-induced degradation of OsSERK2 and compromised XopK-dependent virulence. As crucial immune regulators associated with a multitude of immune receptors, SERKs have been shown to be perturbed by Pseudomonas effectors via different mechanisms. Our study revealed a distinct perturbation mechanism of SERK activity via ubiquitination achieved by Xanthomonas non-TAL effector.

Structural and functional characterization of the phosphoglucomutase from Xanthomonas citri subsp. citri.

Citrus canker, caused by bacteria Xanthomonas citri subsp. citri, can affect all economically important varieties of citrus. Studying Xanthomonas genes related to the invasive capacity may improve the knowledge on how this works and ultimately use the information to avoid the disease. Some annotated genes from Xanthomonas citri subsp. citri published genome are addressed to an interesting class of genes named "pathogenicity, virulence and adaptation". One of them is xanA, which encodes a predicted phosphoglucomutase. Phosphoglucomutases are ubiquitous enzymes among the living kingdoms that play roles in carbohydrate metabolism, catalyzing the reversible conversion of 1- to 6-phosphoglucose. In Xanthomonas, phosphoglucomutase activity is required to synthesize precursors of the pathogenesis-related polysaccharide xanthan. In this work, a characterization of this gene product is presented by structural and functional studies. Molecular cloning was used for heterologous expression and deletion of xanA. A Michaelis-Menten kinetics model was obtained using the recombinant protein. The protein structure was also determined by X-ray diffraction on the recombinant enzyme substrate-free, bound to glucose-1,6-biphosphate and to glucose-1-phosphate. Deletion of xanA was done with a suicide plasmid construct and the obtained mutant was tested for pathogenic capacity. This study is the first describing the properties of the Xanthomonas citri subsp. citri phosphoglucomutase.