RESUMEN
Ribonucleases (RNases) play critical roles in RNA metabolism and are collectively essential for cell viability. However, most knowledge about bacterial RNases comes from the studies on Escherichia coli; very little is known about the RNases in plant pathogens. The crucifer black rot pathogen Xanthomonas campestris pv. campestris (Xcc) encodes 15 RNases, but none of them has been functionally characterized. Here, we report the physiological function of the exoribonuclease RNase D in Xcc and provide evidence demonstrating that the Xcc RNase D is involved in 5S rRNA degradation and exopolysaccharide (EPS) production. Our work shows that the growth and virulence of Xcc were not affected by deletion of RNase D but were severely attenuated by RNase D overexpression. However, deletion of RNase D in Xcc resulted in a significant reduction in EPS production. In addition, either deletion or overexpression of RNase D in Xcc did not influence the tRNAs tested, inconsistent with the finding in E. coli that the primary function of RNase D is to participate in tRNA maturation and its overexpression degrades tRNAs. More importantly, deletion, overexpression, and in vitro enzymatic analyses revealed that the Xcc RNase D degrades 5S rRNA but not 16S and 23S rRNAs that share an operon with 5S rRNA. Our results suggest that Xcc employs RNase D to realize specific modulation of the cellular 5S rRNA content after transcription and maturation whenever necessary. The finding expands our knowledge about the function of RNase D in bacteria.
Asunto(s)
Xanthomonas campestris , Xanthomonas campestris/metabolismo , ARN Ribosómico 5S/metabolismo , Ribonucleasa III/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
The zinc uptake regulator (Zur) is a member of the Fur (ferric uptake regulator) family transcriptional regulators that plays important roles in zinc homeostasis and virulence of bacteria. Upon zinc perception, Zur binds to the promoters of zinc responsive genes and controls their transcription. However, the mechanism underlying zinc-mediated Zur activation remains unclear. Here we report a 2.2-Å crystal structure of apo Zur from the phytopathogen Xanthomonas campestris pv. campestris (XcZur), which reveals the molecular mechanism that XcZur exists in a closed inactive state before regulatory zinc binding. Subsequently, we present a 1.9-Å crystal structure of holo XcZur, which, by contrast, adopts an open state that has enough capacity to bind DNA. Structural comparison and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses uncover that binding of a zinc atom in the regulatory site, formed by the hinge region, the dimerization domain and the DNA binding domain, drives a closed-to-open conformational change that is essential for XcZur activation. Moreover, key residues responsible for DNA recognition are identified by site-directed mutagenesis. This work provides important insights into zinc-induced XcZur activation and valuable discussions on the mechanism of DNA recognition.
Asunto(s)
Proteínas Bacterianas/química , Zinc/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Transcripción Genética , Xanthomonas campestrisRESUMEN
Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in crucifers. Our previous findings revealed that Xcc can degrade 4-hydroxybenzoic acid (4-HBA) via the ß-ketoadipate pathway. This present study expands on this knowledge in several ways. First, we show that infective Xcc cells induce in situ biosynthesis of 4-HBA in host plants, and Xcc can efficiently degrade 4-HBA via the pobA/pobR locus, which encodes a 4-hydroxybenzoate hydroxylase and an AraC-family transcription factor respectively. Next, the transcription of pobA is specifically induced by 4-HBA and is positively regulated by PobR, which is constitutively expressed in Xcc. 4-HBA directly binds to PobR dimers, resulting in activation of pobA expression. Point mutation and subsequent isothermal titration calorimetry and size exclusion chromatography analysis identified nine key conserved residues required for 4-HBA binding and/or dimerization of PobR. Furthermore, overlapping promoters harboring fully overlapping -35 elements were identified between the divergently transcribed pobA and pobR. The 4-HBA/PobR dimer complex specifically binds to a 25-bp site, which encompasses the -35 elements shared by the overlapping promoters. Finally, GUS histochemical staining and subsequent quantitative assay showed that both pobA and pobR genes are transcribed during Xcc infection of Chinese radish, and the strain ΔpobR exhibited compromised virulence in Chinese radish. These findings suggest that the ability of Xcc to survive the 4-HBA stress might be important for its successful colonization of host plants.
Asunto(s)
Parabenos/metabolismo , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismo , Factor de Transcripción de AraC/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Parabenos/química , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Virulencia/genética , Xanthomonas campestris/patogenicidadRESUMEN
As with many phytopathogenic bacteria, the virulence of Xanthomonas campestris pv. campestris, the causal agent of black rot disease in cruciferous plants, relies on secretion of a suite of extracellular enzymes that includes cellulase (endoglucanase), pectinase, protease, and amylase. Although the role in virulence of a number of these enzymes has been assessed, the contribution of amylase to X. campestris pv. campestris virulence has yet to be established. In this work, we investigated both the role of extracellular amylase in X. campestris pv. campestris virulence and the control of its expression. Deletion of XC3487 (here renamed amyAXcc), a putative amylase-encoding gene from the genome of X. campestris pv. campestris strain 8004, resulted in a complete loss of extracellular amylase activity and significant reduction in virulence. The extracellular amylase activity and virulence of the amyAXcc mutant could be restored to the wild-type level by expressing amyAXcc in trans. These results demonstrated that amyAXcc is responsible for the extracellular amylase activity of X. campestris pv. campestris and indicated that extracellular amylase plays an important role in X. campestris pv. campestris virulence. We also found that the expression of amyAXcc is strongly induced by starch and requires activation by the global posttranscriptional regulator RsmA. RsmA binds specifically to the 5'-untranslated region of amyAXcc transcripts, suggesting that RsmA regulates amyAXcc directly at the posttranscriptional level. Unexpectedly, in addition to posttranscriptional regulation, the use of a transcriptional reporter demonstrated that RsmA also regulates amyAXcc expression at the transcriptional level, possibly by an indirect mechanism.
Asunto(s)
Xanthomonas campestris , Amilasas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Enfermedades de las Plantas , Virulencia , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismoRESUMEN
Xanthomonas campestris pv. campestris is the causative agent of black rot disease in crucifer plants. This Gram-negative bacterium utilizes the type III secretion system (T3SS), encoded by the hrp gene cluster, to aid in its resistance to host defenses and the ability to cause disease. The T3SS injects a set of proteins known as effectors into host cells that come into contact with the bacterium. The T3SS is essential for the virulence and hypersensitive response (HR) of X. campestris pv. campestris, making it a potential target for disease control strategies. Using a unique and straightforward high-throughput screening method, we examined a large collection of diverse small molecules for their potential to modulate the T3SS without affecting the growth of X. campestris pv. campestris. Screening of 13,129 different compounds identified 10 small molecules that had a significant inhibitory influence on T3SS. Moreover, reverse transcription-quantitative PCR (qRT-PCR) assays demonstrated that all 10 compounds repress the expression of the hrp genes. Interestingly, the effect of these small molecules on hrp genes may be through the HpaS and ColS sensor kinase proteins that are key to the regulation of the T3SS in planta Five of the compounds were also capable of inhibiting X. campestris pv. campestris virulence in a Chinese radish leaf-clipping assay. Furthermore, seven of the small molecules significantly weakened the HR in nonhost pepper plants challenged with X. campestris pv. campestris. Taken together, these small molecules may provide potential tool compounds for the further development of antivirulence agents that could be used in disease control of the plant pathogen X. campestris pv. campestris.IMPORTANCE The bacterium Xanthomonas campestris pv. campestris is known to cause black rot disease in many socioeconomically important vegetable crops worldwide. The management and control of black rot disease have been tackled with chemical and host resistance methods with variable success. This has motivated the development of alternative methods for preventing this disease. Here, we identify a set of novel small molecules capable of inhibiting X. campestris pv. campestris virulence, which may represent leading compounds for the further development of antivirulence agents that could be used in the control of black rot disease.
Asunto(s)
Enfermedades de las Plantas/prevención & control , Sistemas de Secreción Tipo III/genética , Xanthomonas campestris/fisiología , Proteínas Bacterianas/genética , Productos Agrícolas/microbiología , Regulación Bacteriana de la Expresión Génica , Enfermedades de las Plantas/microbiología , Factores de Transcripción/genética , Sistemas de Secreción Tipo III/metabolismo , Virulencia , Xanthomonas campestris/química , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidadRESUMEN
A characteristic feature of phytopathogenic Xanthomonas bacteria is the production of yellow membrane-bound pigments called xanthomonadins. Previous studies showed that 3-hydroxybenzoic acid (3-HBA) was a xanthomonadin biosynthetic intermediate and also, that it had a signaling role. The question of whether the structural isomers 4-HBA and 2-HBA (salicylic acid) have any role in xanthomonadin biosynthesis remained unclear. In this study, we have selectively eliminated 3-HBA, 4-HBA, or the production of both by expression of the mhb, pobA, and pchAB gene clusters in the Xanthomonas campestris pv. campestris strain XC1. The resulting strains were different in pigmentation, virulence factor production, and virulence. These results suggest that both 3-HBA and 4-HBA are involved in xanthomonadin biosynthesis. When both 3-HBA and 4-HBA are present, X. campestris pv. campestris prefers 3-HBA for Xanthomonadin-A biosynthesis; the 3-HBA-derived Xanthomonadin-A was predominant over the 4-HBA-derived xanthomonadin in the wild-type strain XC1. If 3-HBA is not present, then 4-HBA is used for biosynthesis of a structurally uncharacterized Xanthomonadin-B. Salicylic acid had no effect on xanthomonadin biosynthesis. Interference with 3-HBA and 4-HBA biosynthesis also affected X. campestris pv. campestris virulence factor production and reduced virulence in cabbage and Chinese radish. These findings add to our understanding of xanthomonadin biosynthetic mechanisms and further help to elucidate the biological roles of xanthomonadins in X. campestris pv. campestris adaptation and virulence in host plants.
Asunto(s)
Hidroxibenzoatos , Parabenos , Pigmentos Biológicos , Xanthomonas campestris , Hidroxibenzoatos/metabolismo , Parabenos/metabolismo , Pigmentos Biológicos/biosíntesis , Pigmentos Biológicos/genética , Enfermedades de las Plantas/microbiología , Factores de Virulencia/genética , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismo , Xanthomonas campestris/patogenicidadRESUMEN
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 2.55â¯Å resolution. 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.
Asunto(s)
Proteínas Bacterianas/química , Citocininas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Xanthomonas/enzimología , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Unión Proteica , Conformación ProteicaRESUMEN
The HprK serine kinase is a component of the phosphoenolpyruvate phosphotransferase system (PTS) of bacteria that generally regulates catabolite repression through phosphorylation/dephosphorylation of the PTS protein PtsH at a conserved serine residue. However, many bacteria do not encode a complete PTS or even have an HprK homologue. Xanthomonas campestris pv. campestris (Xcc) is a pathogen that cause black rot disease in crucifer plants and one of the few Gram-negative bacteria that encodes a homologue of HprK protein (herein HprKXcc ). To gain insight into the role of HprKXcc and other PTS-related components in Xcc we individually mutated and phenotypically assessed the resulting strains. Deletion of hprK Xcc demonstrated its requirement for virulence and other diverse cellular processes associated including extracellular enzyme activity, extracellular-polysaccharide production and cell motility. Global transcriptome analyses revealed the HprKXcc had a broad regulatory role in Xcc. Additionally, through overexpression, double gene deletion and transcriptome analysis we demonstrated that hprK Xcc shares an epistatic relationship with ptsH. Furthermore, we demonstrate that HprKXcc is a functional serine kinase, which has the ability to phosphorylate PtsH. Taken together, the data illustrates the previously unappreciated global regulatory role of HprKXcc and previously uncharacterized PTS components that control virulence in this pathogen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Xanthomonas campestris/enzimología , Xanthomonas campestris/patogenicidad , Proteínas Serina-Treonina Quinasas/genética , Virulencia/genéticaRESUMEN
The ability of the bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc) to cause disease is dependent on the type III secretion system (T3SS). Proteins of the Xcc T3SS are encoded by hrp (hypersensitive response and pathogenicity) genes and whose expression is mainly controlled by the regulators HrpG and HrpX. Here, we describe the identification and characterization of a previously unknown regulatory protein (named HpaP), which plays important role in hrp gene expression and virulence in Xcc. Clean deletion of hpaP demonstrated reduced virulence and HR (hypersensitive response) induction of Xcc and alterations in cell motility and stress tolerance. Global transcriptome analyses revealed that most hrp genes were down regulated in the hpaP mutant, suggesting HpaP positively regulates hrp genes. GUS activity assays implied that HpaP regulates the expression of hrp genes via controlling the expression of hrpX. Biochemical analyses revealed that HpaP protein had both ATPase and phosphatase activity. While further site-directed mutagenesis of conserved residues in the PTP loop (a protein tyrosine phosphatase signature) of HpaP resulted in the loss of both phosphatase activity and regulatory activity in virulence and HR. Taken together, the findings identify a new regulatory protein that controls hrp gene expression and virulence in Xcc.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Sistemas de Secreción Tipo III/genética , Xanthomonas campestris/metabolismo , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Monoéster Fosfórico Hidrolasas/genética , Enfermedades de las Plantas/microbiología , Factores de Transcripción/genética , Virulencia , Xanthomonas campestris/genéticaRESUMEN
The synthesis of methionine is critical for most bacteria. It is known that cellular methionine has a feedback effect on the expression of met genes involved in de novo methionine biosynthesis. Previous studies revealed that Gram-negative bacteria control met gene expression at the transcriptional level by regulator proteins, while most Gram-positive bacteria regulate met genes at post-transcriptional level by RNA regulators (riboregulators) located in the 5'UTR of met genes. However, despite its importance, the methionine biosynthesis pathway in the Gram-negative Xanthomonas genus that includes many important plant pathogens is completely uncharacterized. Here, we address this issue using the crucifer black rot pathogen Xanthomonas campestris pv. campestris (Xcc), a model bacterium in microbe-plant interaction studies. The work identified an operon (met) involved in de novo methionine biosynthesis in Xcc. Disruption of the operon resulted in defective growth in methionine-limited media and in planta. Western blot analysis revealed that the expression of the operon is dependent on methionine levels. Further molecular analyses demonstrated that the 5'UTR, but not the promoter of the operon, is involved in feedback regulation on operon expression in response to methionine availability, providing an example of a Gram-negative bacterium utilizing a 5'UTR region to control the expression of the genes involved in methionine biosynthesis.
Asunto(s)
Regiones no Traducidas 5' , Retroalimentación Fisiológica , Regulación Bacteriana de la Expresión Génica , Metionina/biosíntesis , Xanthomonas campestris/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Operón , Xanthomonas campestris/genética , Xanthomonas campestris/crecimiento & desarrolloRESUMEN
BACKGROUND: The Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris recruits the hrp/T3SS system to inject pathogenicity effector proteins into host cells and uses the rpf/DSF cell-cell signaling system to regulate the expression of virulence factors such as extracellular enzymes and polysaccharide. Whether these two systems have any connection is unknown. METHODS: Positive regulator candidates affecting hrpX expression were identified by sacB strategy. The transcriptional expression was determined by qRT-PCR and GUS activity analysis. Transcriptome analysis was performed by RNA deep-sequencing. The hypersensitive response (HR) was determined in the nonhost plant pepper ECW-10R and electrolyte leakage assay. RESULTS: Mutation of the gene encoding the sensor RpfC of the rpf/DSF system significantly reduced the expression of hrpX, the key regulator of the hrp/T3SS system, all of the genes in the hrp cluster and most reported type III effector genes. Mutation of rpfG did not affect the expression of hrpX. The rpfC mutant showed a delayed and weakened HR induction. CONCLUSIONS: RpfC positively regulates the expression of hrpX independent of RpfG, showing a complex regulatory network linking the rpf/DSF and hrp/T3SS systems.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Enfermedades de las Plantas/microbiología , Factores de Transcripción/metabolismo , Xanthomonas campestris/metabolismo , Proteínas Bacterianas/genética , Capsicum/microbiología , Mutación , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Xanthomonas campestris/genéticaRESUMEN
Stenotrophomonas maltophilia is an antibiotic-resistant Gram-negative pathogen, which is associated with hospital-acquired infection. The genome encodes a protein highly related to the Ax21 protein of Xanthomonas oryzae that is implicated in interactions of this plant pathogen with rice. Here, we report on the pleiotropic nature of ax21 mutation in S. maltophilia and the effects of addition of the Ax21 protein on the restoration of the wild-type phenotype. We show that loss by mutation of Ax21 leads to reduced motility, reduced biofilm formation, reduced tolerance to the antibiotic tobramycin and reduced virulence to larvae of Galleria mellonella, as well as alteration in the expression of specific genes associated with virulence or antibiotic resistance. Addition of the Ax21protein restored motility and the level of gene expression towards wild type. These findings are consistent with the notion that the Ax21 protein is involved in intraspecies communication, although other interpretations cannot be discounted.
Asunto(s)
Biopelículas , Infección Hospitalaria/microbiología , Infecciones por Bacterias Gramnegativas/microbiología , Stenotrophomonas maltophilia/fisiología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/aislamiento & purificación , Stenotrophomonas maltophilia/patogenicidad , VirulenciaRESUMEN
A number of species of bacteria from the genus Burkholderia have been shown to be causal agents of diseases of rice. These diseases, caused by Burkholderia glumae, B. gladioli and B. plantarii, are becoming increasingly common across the globe. This is particularly so for B. glumae, whose ability to grow at elevated temperatures suggests that it may become a prevalent problem in an era of global warming. Despite the increasing threat to rice, relatively little is known about the virulence mechanisms employed by these pathogens. Work over the last 5 years has provided an increasing insight into these factors and their control by environmental and other cues. In addition, the determination of a number of genome sequences has allowed bioinformatic predictions of further possible mechanisms, which can now be investigated experimentally. Here, we review recent advances in the understanding of virulence of Burkholderia to rice, to include discussion of the roles of toxins, type II secreted enzymes, type III secreted effectors and motility as well as their regulation by quorum sensing, two-component systems and cyclic di-GMP signalling. Finally, we consider a number of approaches for the control of bacterial virulence through the modulation of quorum sensing and toxin degradation.
Asunto(s)
Burkholderia/patogenicidad , Oryza/microbiología , Burkholderia/genética , Genoma Bacteriano , Enfermedades de las Plantas/microbiología , Percepción de Quorum , Virulencia/genéticaRESUMEN
BACKGROUND: Bacterial plasmids have a major impact on metabolic function and adaptation of their hosts. An indigenous plasmid was identified in a Chinese isolate (GX01) of the invasive phytopathogen Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of rice bacterial leaf streak (BLS). To elucidate the biological functions of the plasmid, we have sequenced and comprehensively annotated the plasmid. METHODS: The plasmid DNA was extracted from Xoc strain GX01 by alkaline lysis and digested with restriction enzymes. The cloned and subcloned DNA fragments in pUC19 were sequenced by Sanger sequencing. Sequences were assembled by using Sequencher software. Gaps were closed by primer walking and sequencing, and multi-PCRs were conducted through the whole plasmid sequence for verification. BLAST, phylogenetic analysis and dinucleotide calculation were performed for gene annotation and DNA structure analysis. Transformation, transconjugation and stress tolerance tests were carried out for plasmid function assays. RESULTS: The indigenous plasmid from Xoc strain GX01, designated pXOCgx01, is 53,206-bp long and has been annotated to possess 64 open reading frames (ORFs), including genes encoding type IV secretion system, heavy metal exporter, plasmid stability factors, and DNA mobile factors, i.e., the Tn3-like transposon. Bioinformatics analysis showed that pXOCgx01 has a mosaic structure containing different genome contexts with distinct genomic heterogeneities. Phylogenetic analysis indicated that the closest relative of pXOCgx01 is pXAC64 from Xanthomonas axonopodis pv. citri str. 306. It was estimated that there are four copies of pXOCgx01 per cell of Xoc GX01 by PCR assay and the calculation of whole genome shotgun sequencing data. We demonstrate that pXOCgx01 is a self-transmissible plasmid and can replicate in some Xanthomonas spp. strains, but not in Escherichia coli DH5α. It could significantly enhance the tolerance of Xanthomonas oryzae pv. oryzae PXO99A to the stresses of heavy metal ions. The plasmid survey indicated that nine out of 257 Xoc Chinese isolates contain plasmids. CONCLUSIONS: pXOCgx01 is the first report of indigenous plasmid from Xanthomonas oryzae pv. oryzicola, and the first completely sequenced plasmid from Xanthomonas oryzae species. It is a self-transmissible plasmid and has a mosaic structure, containing genes for macromolecule secretion, heavy metal exportation, and DNA mobile factors, especially the Tn3-like transposon which may provide transposition function for mobile insertion cassette and play a major role in the spread of pathogenicity determinants. The results will be helpful to elucidate the biological significance of this cryptic plasmid and the adaptive evolution of Xoc.
Asunto(s)
Plásmidos/aislamiento & purificación , Xanthomonas/genética , China , Biología Computacional , Conjugación Genética , Replicación del ADN , Farmacorresistencia Bacteriana , Escherichia coli/genética , Transferencia de Gen Horizontal , Metales Pesados/toxicidad , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Oryza/microbiología , Filogenia , Enfermedades de las Plantas/microbiología , Análisis de Secuencia de ADN , Homología de Secuencia , Xanthomonas/aislamiento & purificaciónRESUMEN
The bacterium Xanthomonas campestris is an economically important pathogen of many crop species and a model for the study of bacterial phytopathogenesis. In X. campestris, a regulatory system mediated by the signal molecule DSF controls virulence to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon a system comprising the sensor RpfC and regulator RpfG. Here we have addressed the action and role of Rpf/DSF signalling in phytopathogenesis by high-resolution transcriptional analysis coupled to functional genomics. We detected transcripts for many genes that were unidentified by previous computational analysis of the genome sequence. Novel transcribed regions included intergenic transcripts predicted as coding or non-coding as well as those that were antisense to coding sequences. In total, mutation of rpfF, rpfG and rpfC led to alteration in transcript levels (more than fourfold) of approximately 480 genes. The regulatory influence of RpfF and RpfC demonstrated considerable overlap. Contrary to expectation, the regulatory influence of RpfC and RpfG had limited overlap, indicating complexities of the Rpf signalling system. Importantly, functional analysis revealed over 160 new virulence factors within the group of Rpf-regulated genes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/microbiología , Transducción de Señal , Xanthomonas campestris/patogenicidad , Proteínas Bacterianas/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Bacterianos , Factores de Transcripción/metabolismo , Factores de Virulencia/biosíntesis , Xanthomonas campestris/genéticaRESUMEN
The bacterial phytopathogen Xanthomonas campestris pv. campestris (Xcc) relies on the hrp (hypersensitive response and pathogenicity) genes to cause disease and induce hypersensitive response (HR). The hrp genes of bacterial phytopathogens are divided into two groups. Xcc hrp genes belong to group II. It has long been known that the group II hrp genes are activated by an AraC-type transcriptional regulator whose expression is controlled by a two-component system (TCS) response regulator (named HrpG in Xcc). However, no cognate sensor kinase has yet been identified. Here, we present evidence showing that the Xcc open-reading frame XC_3670 encodes a TCS sensor kinase (named HpaS). Mutation of hpaS almost completely abolished the HR induction and virulence. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacted with HrpG. Phos-tag™ SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of HrpGâ in vivo. These data suggest that HpaS and HrpG are most likely to form a TCS. We also showed that XC_3669 (named hpaR2), which is adjacent to hpaS and encodes a putative TCS response regulator, is required for full virulence but not HR induction. HpaR2 also physically interacted with HpaS, suggesting that HpaS may also form another TCS with HpaR2.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Genes Reguladores , Proteínas Quinasas/genética , Factores de Transcripción/genética , Xanthomonas campestris/patogenicidad , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Brassicaceae/microbiología , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Fosforilación , Enfermedades de las Plantas/microbiología , Unión Proteica , Proteínas Quinasas/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción Genética , Virulencia , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismoRESUMEN
Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) via the nicotinamide (NAM) salvage pathway. While the structural biochemistry of eukaryote NAMPT has been well studied, the catalysis mechanism of prokaryote NAMPT at the molecular level remains largely unclear. Here, we demonstrated the NAMPT-mediated salvage pathway is functional in the Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) for the synthesis of NAD+, and the enzyme activity of NAMPT in this bacterium is significantly higher than that of human NAMPT in vitro. Our structural analyses of Xcc NAMPT, both in isolation and in complex with either the substrate NAM or the product nicotinamide mononucleotide (NMN), uncovered significant details of substrate recognition. Specifically, we revealed the presence of a NAM binding tunnel that connects the active site, and this tunnel is essential for both catalysis and inhibitor binding. We further demonstrated that NAM binding in the tunnel has a positive cooperative effect with NAM binding in the catalytic site. Additionally, we discovered that phosphorylation of the His residue at position 229 enhances the substrate binding affinity of Xcc NAMPT and is important for its catalytic activity. This work reveals the importance of NAMPT in bacterial NAD+ synthesis and provides insights into the substrate recognition and the catalytic mechanism of bacterial type II phosphoribosyltransferases.
Asunto(s)
Niacinamida , Xanthomonas campestris , Humanos , Niacinamida/metabolismo , NAD/metabolismo , Mononucleótido de Nicotinamida/metabolismo , Mononucleótido de Nicotinamida/farmacología , Xanthomonas campestris/metabolismo , Nicotinamida Fosforribosiltransferasa/química , Nicotinamida Fosforribosiltransferasa/metabolismo , FosforilaciónRESUMEN
The black rot pathogen Xanthomonas campestris utilizes molecules of the diffusible signal factor (DSF) family as signals to regulate diverse processes contributing to virulence. DSF signal synthesis and transduction requires proteins encoded by the rpf gene cluster. RpfF catalyzes DSF synthesis, whereas the RpfCG two-component system links the perception of DSF to alteration in the level of the second messenger cyclic di-GMP. As this nucleotide can exert a regulatory influence at the post-transcriptional and post-translational levels, we have used comparative proteomics to identify Rpf-regulated processes in X. campestris that may not be revealed by transcriptomics. The abundance of a number of proteins was altered in rpfF, rpfC, or rpfG mutants compared with the wild type. These proteins belonged to several functional categories, including biosynthesis and intermediary metabolism, regulation, oxidative stress or antibiotic resistance, and DNA replication. For many of these proteins, the alteration in abundance was not associated with alteration in transcript level. A directed mutational analysis allowed us to describe a number of new virulence factors among these proteins, including elongation factor P and a putative outer membrane protein, which are both widely conserved in bacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enfermedades de las Plantas/microbiología , Proteómica , Raphanus/microbiología , Transducción de Señal , Xanthomonas campestris/metabolismo , Proteínas Bacterianas/genética , Comunicación Celular , Análisis Mutacional de ADN , Electroforesis en Gel Bidimensional , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Mutación , Hojas de la Planta/microbiología , ARN Bacteriano/genética , ARN Mensajero/genética , Proteínas Recombinantes de Fusión , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidad , Xanthomonas campestris/fisiologíaRESUMEN
Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial leaf blight, one of the most important rice bacterial diseases in China and many other countries. The upstream portions of 41 candidate genes encoding non-transcription activator-like effectors of Xoo Chinese strain 13751 were fused with the coding sequence of AvrBs159-445 in a broad host-range vector. The constructed plasmids were respectively introduced into Xoo strain 13751 and avrBs1 deletion mutant of X. campestris pv. campestris strain 8004 by tri-parental conjugation. The resultant transconjugants were respectively tested for hypersensitive response (HR) elicitation on pepper ECW-10R. Nine strains were able to elicit HR on pepper, indicating that the nine genes (XOO0037, XOO0103, XOO0110, XOO0315, XOO1488, XOO2875, XOO3150, XOO3222 and XOO4134) encoded effectors. Among them, xopAE 13751 (XOO0110), expressed in Xoo strain 13751 growing in rice leaves, was a new experimentally confirmed effector gene. XopAE13751 contains 11 leucine rich repeats. Furthermore, mutants for the nine effector genes were created in Xoo strain 13751 and subsequently tested for virulence in rice. As a result, only the xopR 13751 (XOO4134) deletion mutant GXMxopR showed a significant reduction in virulence in hybrid rice cv. Teyou63 compared to the wild type. However, the growth of GXMxopR in host plant rice was not affected. These results indicated that xopR 13751 was required for full virulence of Xoo strain 13751 by inducing rice disease tolerance.