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1.
Int J Mol Sci ; 22(19)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34638715

RESUMO

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.


Assuntos
Proteínas de Bactérias/metabolismo , Corismato Mutase/metabolismo , Juglans/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas , Xanthomonas/enzimologia , Xanthomonas/patogenicidade
2.
Nat Commun ; 12(1): 5479, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531388

RESUMO

The Xanthomonas outer protein C2 (XopC2) family of bacterial effectors is widely found in plant pathogens and Legionella species. However, the biochemical activity and host targets of these effectors remain enigmatic. Here we show that ectopic expression of XopC2 promotes jasmonate signaling and stomatal opening in transgenic rice plants, which are more susceptible to Xanthomonas oryzae pv. oryzicola infection. Guided by these phenotypes, we discover that XopC2 represents a family of atypical kinases that specifically phosphorylate OSK1, a universal adaptor protein of the Skp1-Cullin-F-box ubiquitin ligase complexes. Intriguingly, OSK1 phosphorylation at Ser53 by XopC2 exclusively increases the binding affinity of OSK1 to the jasmonate receptor OsCOI1b, and specifically enhances the ubiquitination and degradation of JAZ transcription repressors and plant disease susceptibility through inhibiting stomatal immunity. These results define XopC2 as a prototypic member of a family of pathogenic effector kinases and highlight a smart molecular mechanism to activate jasmonate signaling.


Assuntos
Proteínas de Bactérias/metabolismo , Oryza/metabolismo , Fosfotransferases/metabolismo , Proteínas de Plantas/metabolismo , Estômatos de Plantas/metabolismo , Xanthomonas/enzimologia , Proteínas de Bactérias/genética , Resistência à Doença/genética , Interações Hospedeiro-Patógeno , Oryza/genética , Fosforilação , Fosfotransferases/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Estômatos de Plantas/genética , Estômatos de Plantas/microbiologia , Plantas Geneticamente Modificadas , Xanthomonas/genética , Xanthomonas/fisiologia
3.
Nat Commun ; 12(1): 367, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446650

RESUMO

Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Xanthomonas/enzimologia , Proteínas de Bactérias/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Cinética , Modelos Moleculares , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Xanthomonas/química , Xanthomonas/genética , Xilose/química , Xilose/metabolismo
4.
Int J Biol Macromol ; 166: 190-199, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33164774

RESUMO

Cold-adapted endo-ß-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-ß-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal ß-1,4-linkages of cellulose and mixed-linkage ß-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10-50 °C), which are distinguishing features from conventional thermophilic ß-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-ß-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.


Assuntos
Proteínas de Bactérias/metabolismo , Cafeína/química , Temperatura Baixa , Glucana 1,4-beta-Glucosidase/metabolismo , Sementes/química , Proteínas de Bactérias/química , Biocatálise , Cafeína/análise , Cobalto/química , Estabilidade Enzimática , Glucana 1,4-beta-Glucosidase/química , Paullinia/química , Xanthomonas/enzimologia
5.
PLoS One ; 15(12): e0243867, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33338036

RESUMO

The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase ß subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity.


Assuntos
Proteínas de Bactérias/metabolismo , Periplasma/metabolismo , Proteômica , Xanthomonas/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carbono/metabolismo , Genes Bacterianos , Anotação de Sequência Molecular , Fosfoglucomutase/metabolismo , Reprodutibilidade dos Testes , Xanthomonas/enzimologia , Xanthomonas/genética , Xanthomonas/crescimento & desenvolvimento
6.
Acta Crystallogr D Struct Biol ; 76(Pt 8): 778-789, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32744260

RESUMO

The genus Xanthomonas comprises several cosmopolitan plant-pathogenic bacteria that affect more than 400 plant species, most of which are of economic interest. Citrus canker is a bacterial disease that affects citrus species, reducing fruit yield and quality, and is caused by the bacterium Xanthomonas citri subsp. citri (Xac). The Xac3819 gene, which has previously been reported to be important for citrus canker infection, encodes an uncharacterized glutathione S-transferase (GST) of 207 amino-acid residues in length (XacGST). Bacterial GSTs are implicated in a variety of metabolic processes such as protection against chemical and oxidative stresses. XacGST shares high sequence identity (45%) with the GstB dehalogenase from Escherichia coli O6:H1 strain CFT073 (EcGstB). Here, XacGST is reported to be able to conjugate glutathione (GSH) with bromoacetate with a Km of 6.67 ± 0.77 mM, a kcat of 42.69 ± 0.32 s-1 and a kcat/Km of 6.40 ± 0.72 mM-1 s-1 under a saturated GSH concentration (3.6 mM). These values are comparable to those previously reported for EcGstB. In addition, crystal structures of XacGST were determined in the apo form (PDB entry 6nxv) and in a GSH-bound complex (PDB entry 6nv6). XacGST has a canonical GST-like fold with a conserved serine residue (Ser12) at the GSH-binding site near the N-terminus, indicating XacGST to be a serine-type GST that probably belongs to the theta-class GSTs. GSH binding stabilizes a loop of about 20 residues containing a helix that is disordered in the apo XacGST structure.


Assuntos
Proteínas de Bactérias , Glutationa Transferase , Glutationa/metabolismo , Doenças das Plantas/microbiologia , Xanthomonas , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citrus/metabolismo , Citrus/microbiologia , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Interações entre Hospedeiro e Microrganismos , Ligação Proteica , Conformação Proteica , Xanthomonas/enzimologia , Xanthomonas/patogenicidade
7.
Virology ; 548: 160-167, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838937

RESUMO

Filamentous Inoviridae phages integrate into the chromosome of plant pathogens Xanthomonas as prophages, but their diversity and integrative mechanism are not completely understood. A proviral Cf2 sequence of 6454 bases from Xanthomonas citri genome was revived as infectious virions able to lysogenize its host. Unlike other Xanthomonas phages (Cf1c, φLf, Xf109, XacF1), Cf2 phage has RstA/RstB replication protein, and its attP has XerD binding arm and dif central region but lacks XerC binding arm. XerC+/Xf109 and XerD+/Cf2 attPs are in the opposite direction in phage genomes. Moreover, XerCD binding and XerD catalysis for strand exchange are necessary for site-specific integration of XerD+/Cf2 and XerC+/Xf109 attPs. Taken together, these results provide a new insight into the mechanism of XerCD-mediated recombination at XerD + attP.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Inovirus/fisiologia , Integrases/metabolismo , Xanthomonas/enzimologia , Xanthomonas/virologia , Sítios de Ligação Microbiológicos , Proteínas de Bactérias/genética , Bacteriófagos/genética , Genoma Bacteriano , Inovirus/genética , Integrases/genética , Lisogenia , Integração Viral , Xanthomonas/genética
8.
Biochim Biophys Acta Gen Subj ; 1864(3): 129514, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31911239

RESUMO

BACKGROUND: Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker is maintained as an epiphyte on citrus leaves until entering the plant tissue. During epiphytic survival, bacteria may encounter low water availability that challenges the infection process. Proteomics analyses of Xcc under saline stress, mimicking the conditions found during epiphytic survival, showed increased abundance of a putative NAD(P)H dehydrogenase encoded by XAC2229. METHODS: Expression levels of XAC2229 and a Xcc mutant in XAC2229 were analyzed in salt and oxidative stress and during plant-pathogen interaction. An Escherichia coli expressing XAC2229 was obtained, and the role of this protein in oxidative stress resistance and in reactive oxygen species production was studied. Finally, Xac2229 protein was purified, spectrophotometric and cofactor analyses were done and enzymatic activities determined. RESULTS: XAC2229 was expressed under salt stress and during plant-pathogen interaction. ΔXAC2229 mutant showed less number of cankers and impaired epiphytic survival than the wild type strain. ΔXAC2229 survived less in the presence of H2O2 and produced more reactive oxygen species and thiobarbituric acid-reactive substances than the wild type strain. Similar results were observed for E. coli expressing XAC2229. Xac2229 is a FAD containing flavoprotein, displays diaphorase activity with an optimum at pH 6.0 and has quinone reductase activity using NADPH as an electron donor. CONCLUSIONS: A FAD containing flavoprotein from Xcc is a new NADPH quinone reductase required for bacterial virulence, particularly in Xcc epiphytic survival on citrus leaves. GENERAL SIGNIFICANCE: A novel protein involved in the worldwide disease citrus canker was characterized.


Assuntos
NAD(P)H Desidrogenase (Quinona)/metabolismo , Xanthomonas/enzimologia , Benzoquinonas/metabolismo , Citrus/metabolismo , Citrus/microbiologia , Peróxido de Hidrogênio/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , NADP/metabolismo , Estresse Oxidativo , Folhas de Planta/metabolismo , Estresse Salino/genética , Estresse Salino/fisiologia , Virulência , Xanthomonas/metabolismo , Xanthomonas/patogenicidade , Xanthomonas/fisiologia
9.
J Antibiot (Tokyo) ; 73(2): 125-127, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31654037

RESUMO

Peptidoglycan is an indispensable component of bacterial cell walls. We recently discovered an alternative peptidoglycan biosynthetic pathway, which involves two enzymes, MurD2 and MurL, catalyzing the ligation of L-Glu to UDP-MurNAc-L-Ala and epimerization of the terminal L-Glu of the MurD2 product, respectively. Because the pathway operates in Xanthomonas oryze, a pathogen causing bacterial blight of rice, we searched for specific inhibitors from metabolites produced by actinomycetes to obtain a lead compound to function as an agrochemical. Actinomycin D was isolated from Streptomyces parvulus NBRC 13193 as a specific inhibitor of the pathway. In vitro analysis indicated that actinomycin D inhibited the MurD2 reaction.


Assuntos
Antibacterianos/farmacologia , Dactinomicina/farmacologia , Peptidoglicano/efeitos dos fármacos , Streptomyces/metabolismo , Xanthomonas/efeitos dos fármacos , Antibacterianos/isolamento & purificação , Dactinomicina/isolamento & purificação , Peptidoglicano/biossíntese , Xanthomonas/enzimologia
10.
Antonie Van Leeuwenhoek ; 113(1): 137-145, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31485840

RESUMO

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.


Assuntos
Carbapenêmicos/farmacologia , Fezes/microbiologia , Xanthomonas/enzimologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Girafas , Sequenciamento Completo do Genoma , Xanthomonas/efeitos dos fármacos , Xanthomonas/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo
11.
Biochimie ; 170: 57-64, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31883957

RESUMO

Bacteriophage-encoded transcription antiterminators play essential roles in the regulation of gene expression during infection. Here, we characterize the effects of the antiterminator protein P7 of bacteriophage Xp10 on transcriptional pausing by Xanthomonas oryzae RNA polymerase (RNAP) at different types of pause-inducing signals. When acting alone, P7 inhibits only hairpin-stabilized pauses, likely by preventing hairpin formation. In the presence of NusA, P7 also suppresses backtracking-stabilized pauses and the his elemental pause, but not the consensus elemental pause, suggesting that these pause signals may be mechanistically different. Thus, P7 and other bacteriophage proteins that bind near the RNA exit channel of RNAP have evolved to regulate transcription by suppressing RNAP pausing at a subset of regulatory signals, and to co-opt NusA in doing so.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Bacteriano/genética , Transcrição Genética , Proteínas Virais/metabolismo , Xanthomonas/enzimologia , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Bacteriano/metabolismo , Proteínas Virais/genética
12.
BMC Plant Biol ; 19(1): 530, 2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31783788

RESUMO

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.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/imunologia , Oryza/genética , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Ubiquitina-Proteína Ligases/genética , Xanthomonas/fisiologia , Arabidopsis/imunologia , Parede Celular/imunologia , Oryza/imunologia , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Proteínas de Plantas/imunologia , Ubiquitina-Proteína Ligases/imunologia , Xanthomonas/enzimologia
13.
Int J Mol Sci ; 20(20)2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31615004

RESUMO

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.


Assuntos
ADP Ribose Transferases/química , Arginina/química , Peptídeos/química , Xanthomonas/química , ADP Ribose Transferases/genética , Sequência de Aminoácidos/genética , Arginina/genética , Domínio Catalítico/genética , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Oxigênio/química , Peptídeos/genética , Plantas/genética , Plantas/microbiologia , Ligação Proteica , Conformação Proteica , Transdução de Sinais/genética , Xanthomonas/enzimologia , Xanthomonas/patogenicidade
14.
J Struct Biol ; 208(1): 69-76, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31419523

RESUMO

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.


Assuntos
Proteínas de Bactérias/química , Citocininas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Xanthomonas/enzimologia , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica
15.
Phytopathology ; 109(11): 1869-1877, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31290730

RESUMO

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.


Assuntos
Arginase , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Oryza , Xanthomonas , África , Arginase/metabolismo , Ásia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/genética , Oryza/microbiologia , Virulência , Xanthomonas/enzimologia , Xanthomonas/patogenicidade
16.
J Agric Food Chem ; 67(25): 6962-6969, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31150235

RESUMO

Target validation of current drugs remains the major challenge for target-based drug discovery, especially for agrochemical discovery. The bactericide 0 represents a novel lead structure and has shown potent efficacy against those diseases that are extremely difficult to control, such as rice bacterial leaf blight. However, no detailed target analysis of this bactericide has been reported. Here, we developed a panel of 0-derived probes 1-6, in which a conservative modification (alkyne tag) was introduced to keep the antibacterial activity of 0 and provide functionality for target identification via click chemistry. With these cell-permeable probes, we were able to discover dihydrolipoamide S-succinyltransferase (DLST) as an unprecedented target in living cells. The probes showed good preference for DLST, especially probe 1, which demonstrated distinct selectivity and reactivity. Also, we reported 0 as the first covalent DLST inhibitor, which has been used to confirm the involvement of DLST in the regulation of energy production.


Assuntos
Aciltransferases/química , Antibacterianos/química , Proteínas de Bactérias/química , Oryza/microbiologia , Doenças das Plantas/microbiologia , Sulfonas/química , Xanthomonas/enzimologia , Aciltransferases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Xanthomonas/química , Xanthomonas/efeitos dos fármacos
17.
ACS Chem Biol ; 14(5): 975-978, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30977993

RESUMO

We recently revealed that a previously unknown pathway for peptidoglycan biosynthesis operates in some microorganisms, including Xanthomonas oryzae. It involves two enzymes, MurD2 and MurL, which catalyze the ligation of l-glutamate (l-Glu) to UDP- N-acetylmuramic acid-l-alanine and the epimerization of the terminal l-Glu of the product, respectively. MurD2 of X. oryzae possesses a 26% identity with MurD of Escherichia coli (MurDec), which ligates d-Glu to UDP- N-acetylmuramic acid-l-alanine. To understand how X. oryzae MurD2 recognizes the isomer substrate, we estimated its structure based on that of MurDec during docking simulations. Several amino acid residues, which may be responsible for l-Glu recognition, were replaced with their corresponding amino acid residues in MurDec. Consequently, we obtained a mutated MurD2 enzyme that contained two amino acid substitutions and accepted only d-Glu as the substrate. We next tried to convert the substrate specificity of MurDec using the same strategy, but the mutant enzyme still accepted only d-Glu. Then, MurD of Streptococcus mutans (MurDsm), which possesses the key amino acid residue for l-Glu recognition identified in MurD2, was used for random screenings of mutant enzymes accepting l-Glu. We obtained a mutated MurDsm that had one amino acid substitution and slightly accepted l-Glu. A mutated MurDec possessing the corresponding one amino acid substitution also accepted l-Glu. Thus, we revealed that a few amino acid residues in MurD/MurD2 might control the acceptability of substrates with different stereochemistries.


Assuntos
Ácido Glutâmico/química , Peptídeo Sintases/química , Peptidoglicano/química , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Mutação , Peptídeo Sintases/genética , Estereoisomerismo , Streptococcus mutans/enzimologia , Especificidade por Substrato , Xanthomonas/enzimologia
18.
Mol Plant Microbe Interact ; 32(6): 729-739, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30589364

RESUMO

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.


Assuntos
Oryza , Virulência , Xanthomonas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Oryza/microbiologia , Diester Fosfórico Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Virulência/genética , Xanthomonas/enzimologia , Xanthomonas/genética , Xanthomonas/patogenicidade
19.
Microbiologyopen ; 8(5): e00706, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30085414

RESUMO

Xanthomonas citri subsp. citri (Xac) is the causative agent of citrus canker, a plant disease that significantly impacts citriculture. In earlier work, we showed that alkylated derivatives of gallic acid have antibacterial action against Xac and target both the cell division protein FtsZ and membrane integrity in Bacillus subtilis. Here, we have purified native XacFtsZ and characterized its GTP hydrolysis and polymerization properties. In a surprising manner, inhibition of XacFtsZ activity by alkyl gallates is not as strong as observed earlier with B. subtilis FtsZ. As the alkyl gallates efficiently permeabilize Xac membranes, we propose that this is the primary mode of antibacterial action of these compounds.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/isolamento & purificação , Proteínas do Citoesqueleto/metabolismo , Xanthomonas/enzimologia , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Citrus/microbiologia , Inibidores Enzimáticos/metabolismo , Ácido Gálico/farmacologia , Guanosina Trifosfato/metabolismo , Hidrólise , Doenças das Plantas/microbiologia , Multimerização Proteica , Xanthomonas/efeitos dos fármacos
20.
PLoS One ; 13(10): e0206187, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30335828

RESUMO

The rare branched-chain sugar apiose, once thought to only be present in the plant kingdom, was found in two bacterial species: Geminicoccus roseus and Xanthomonas pisi. Glycans with apiose residues were detected in aqueous methanol-soluble fractions as well as in the insoluble pellet fraction of X. pisi. Genes encoding bacterial uridine diphosphate apiose (UDP-apiose) synthases (bUASs) were characterized in these bacterial species, but the enzyme(s) involved in the incorporation of the apiose into glycans remained unknown. In the X. pisi genome two genes flanking the XpUAS were annotated as hypothetical glycosyltransferase (GT) proteins. The first GT (here on named XpApiT) belongs to GT family 90 and has a Leloir type B fold and a putative lipopolysaccharide-modifying (LPS) domain. The second GT (here on XpXylT) belongs to GT family 2 and has a type A fold. The XpXylT and XpApiT genes were cloned and heterologously expressed in E. coli. Analysis of nucleotide sugar extracts from E. coli expressing XpXylT or XpApiT with UAS showed that recombinant XpApiT utilized UDP-apiose and XpXylT utilized UDP-xylose as substrate. Indirect activity assay (UDP-Glo) revealed that XpApiT is an apiosyltransferase (ApiT) able to specifically use UDP-apiose. Further support for the apiosyltransferase activity was demonstrated by in microbe co-expression of UAS and XpApiT in E. coli showing the utilization of UDP-apiose to generate an apioside detectable in the pellet fraction. This work provides evidence that X. pisi developed the ability to synthesize an apioside of indeterminate function; however, the evolution of the bacterial ApiT remains to be determined. From genetic and evolutionary perspectives, the apiose operon may provide a unique opportunity to examine how genomic changes reflect ecological adaptation during the divergence of a bacterial group.


Assuntos
Pentoses/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Xanthomonas/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Óperon , Pentosiltransferases/química , Filogenia , Plantas/microbiologia , Domínios Proteicos , Xanthomonas/genética
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