Structural insights into Xanthomonas campestris pv. campestris NAD+ biosynthesis via the NAM salvage pathway.

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.

RNase D Is Involved in 5S rRNA Degradation and Exopolysaccharide Production in Xanthomonas campestris pv. campestris.

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.

Structural basis for zinc-induced activation of a zinc uptake transcriptional regulator.

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.

Extracellular Amylase Is Required for Full Virulence and Regulated by the Global Posttranscriptional Regulator RsmA in Xanthomonas campestris Pathovar campestris.

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.

Genome-wide screen and functional analysis in Xanthomonas reveal a large number of mRNA-derived sRNAs, including the novel RsmA-sequester RsmU.

Although bacterial small noncoding RNAs (sRNAs) are known to play a critical role in various cellular processes, including pathogenesis, the identity and action of such sRNAs are still poorly understood in many organisms. Here we have performed a genome-wide screen and functional analysis of the sRNAs in Xanthomonas campestris pv. campestris (Xcc), an important phytopathogen. The 50-500-nt RNA fragments isolated from the wild-type strain grown in a virulence gene-inducing condition were sequenced and a total of 612 sRNA candidates (SRCs) were identified. The majority (82%) of the SRCs were derived from mRNA, rather than specific sRNA genes. A representative panel of 121 SRCs were analysed by northern blotting; 117 SRCs were detected, supporting the contention that the overwhelming majority of the 612 SRCs identified are indeed sRNAs. Phenotypic analysis of strains overexpressing different candidates showed that a particular sRNA, RsmU, acts as a negative regulator of virulence, the hypersensitive response, and cell motility in Xcc. In vitro electrophoretic mobility shift assay and in vivo coimmunoprecipitation analyses indicated that RsmU interacted with the global posttranscriptional regulator RsmA, although sequence analysis displayed that RsmU is not a member of the sRNAs families known to antagonize RsmA. Northern blotting analyses demonstrated that RsmU has two isoforms that are processed from the 3'-untranslated region of the mRNA of XC1332 predicted to encode ComEA, a periplasmic protein required for DNA uptake in bacteria. This work uncovers an unexpected major sRNA biogenesis strategy in bacteria and a hidden layer of sRNA-mediated virulence regulation in Xcc.

A SAM-I riboswitch with the ability to sense and respond to uncharged initiator tRNA.

All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-IXcc) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-IXcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-IXcc expression platform not only can repress gene expression in response to SAM binding to SAM-IXcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.

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.

The Gram-negative phytopathogen Xanthomonas campestris pv. campestris employs a 5'UTR as a feedback controller to regulate methionine biosynthesis.

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.

The RNA chaperone Hfq is important for the virulence, motility and stress tolerance in the phytopathogen Xanthomonas campestris.

The RNA chaperone, Hfq, is known to play extensive roles in bacterial growth and development. More recently, it has been shown to be required for virulence in many human and animal bacterial pathogens. Despite these studies little is known about the role Hfq plays in phytopathogenic bacteria. In this study, we show Hfq is required for full virulence of the crucifer black rot pathogen Xanthomonas campestris pv. campestris (Xcc). We demonstrate that an Xcc hfq deletion strain is highly attenuated for virulence in Chinese radish and shows a severe defect in the production of virulence factors including extracellular enzymes and extracellular polysaccharide. Furthermore, the Xcc strain lacking Hfq had significantly reduced cell motility and stress tolerance. These findings suggest that Hfq is a key regulator of important aspects of virulence and adaptation of Xcc. Taken together, our findings are suggestive of a regulatory network placing Hfq at the centre of virulence gene expression control in Xcc.

Identification of a putative cognate sensor kinase for the two-component response regulator HrpG, a key regulator controlling the expression of the hrp genes in Xanthomonas campestris pv. campestris.

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 HrpG 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.

Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris

It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.

Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris

It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.

High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signalling reveals novel genes that contribute to Xanthomonas phytopathogenesis.

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.

Transcriptome profiling of Xanthomonas campestris pv. campestris grown in minimal medium MMX and rich medium NYG.

Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease in cruciferous plants worldwide. Although the complete genomes of several Xcc strains have been determined, the gene expression and regulation mechanisms in this pathogen are far from clear. In this work, transcriptome profiling of Xcc 8004 grown in MMX medium (minimal medium for Xanthomonas campestris) and NYG medium (peptone yeast glycerol medium) were investigated by RNA-Seq. Using the Illumina HiSeq 2000 platform, a total of 26,514,630 reads (90 nt in average) were generated, of which 15,708,478 reads mapped uniquely to coding regions of Xcc 8004 genome. Of the 4273 annotated protein-coding genes of Xcc 8004, 629 were found differentially expressed in Xcc grown in MMX and NYG. Of the differentially expressed genes, 495 were up-regulated and 134 were down-regulated in MMX. The MMX-induced genes are mainly involved in amino acid metabolism, transport systems, atypical condition adaptation and pathogenicity, especially the type III secretion system, while the MMX-repressed genes are mainly involved in chemotaxis and degradation of small molecules. The global transcriptome analyzes of Xcc 8004 grown in MMX and NYG might facilitate the gene functional characterization of this phytopathogenic bacterium.

Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris.

It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.

RsmA regulates biofilm formation in Xanthomonas campestris through a regulatory network involving cyclic di-GMP and the Clp transcription factor.

Biofilm formation and dispersal in the black rot pathogen Xanthomonas campestris pathovar campestris (Xcc) is influenced by a number of factors. The extracellular mannanase ManA has been implicated in biofilm dispersal whereas biofilm formation requires a putative glycosyl transferase encoded by the xag gene cluster. Previously we demonstrated that the post-transcriptional regulator RsmA exerts a negative regulatory influence on biofilm formation in Xcc. Here we address the mechanisms by which RsmA exerts this action. We show that RsmA binds to the transcripts of three genes encoding GGDEF domain diguanylate cyclases to influence their expression. Accordingly, mutation of rsmA leads to an increase in cellular levels of cyclic di-GMP. This effect is associated with a down-regulation of transcription of manA, but an upregulation of xag gene transcription. Mutation of clp, which encodes a cyclic di-GMP-responsive transcriptional regulator of the CRP-FNR family, has similar divergent effects on the expression of manA and xag. Nevertheless Clp binding to manA and xag promoters is inhibited by cyclic di-GMP. The data support the contention that, in common with other CRP-FNR family members, Clp can act as both an activator and repressor of transcription of different genes to influence biofilm formation as a response to cyclic di-GMP.

Effect of interactions between Mip and PrtA on the full extracellular protease activity of Xanthomonas campestris pathovar campestris.

Mip (macrophage infectivity potentiator) and Mip-like proteins have been demonstrated to be involved in virulence of several animal pathogens, but as yet none of their native bacterial targets has been identified. Our previous work demonstrated that the Mip-like protein found in the plant pathogen Xanthomonas campestris pv. campestris (Xcc) (hereafter called Mip(Xcc)) is also involved in virulence. Inactivation of the mip(Xcc) gene leads to a significant reduction in exopolysaccharide production and extracellular protease activity via an unknown mechanism. The Xcc genome encodes six extracellular proteases, all of which are secreted via the type II secretion system. The serine protease PrtA makes the largest contribution to Xcc's total extracellular proteolytic activity. In this study, Western blotting analysis demonstrated that Mip(Xcc) was located in the periplasm. Bacterial two-hybrid and far-Western analysis indicated that Mip(Xcc) interacted with PrtA directly. Purified Mip(Xcc) was found to be able to rescue the protease activity of periplasmic proteins extracted from the mip(Xcc) mutant. These findings show that Mip(Xcc) plays a role in the maturation of PrtA, which is the novel native target for at least one Mip or Mip-like protein.

sRNA-Xcc1, an integron-encoded transposon- and plasmid-transferred trans-acting sRNA, is under the positive control of the key virulence regulators HrpG and HrpX of Xanthomonas campestris pathovar campestris.

sRNA-Xcc1 is a trans-acting sRNA recently identified from the plant pathogenic bacterium Xanthomonas campestris pathovar campestris (Xcc). Here, the phylogenetic distribution, predicted secondary structure and regulation of expression of sRNA-Xcc1 were analyzed. The analysis showed (1) a total 81 sRNA-Xcc1 homologs that are found in some bacterial strains that are taxonomically unrelated, belonging to the α-, ß-, γ- and δ-proteobacteria (2) that some sRNA-Xcc1 homologs are located in a plasmid-borne transposon or near a transposase coding gene, (3) that sRNA-Xcc1 is encoded by a integron gene cassette in Xcc and sRNA-Xcc1 homologs occur in integron gene cassettes of some uncultured bacteria and (4) that sRNA-Xcc1 homologs have a highly conserved sequence motif and a stable consensus secondary structure. These findings strongly support the idea that sRNA-Xcc1 represents a novel family of sRNAs which may be originally captured by integrons from natural environments and then spread among different bacterial species via horizontal gene transfer, possibly by means of transposons and plasmids. The expression analysis results demonstrated that the transcription of sRNA-Xcc1 is under the positive control of the key virulence regulators HrpG and HrpX, indicating that sRNA-Xcc1 may be involved in the virulence regulation of Xcc.

Systematic mutagenesis of all predicted gntR genes in Xanthomonas campestris pv. campestris reveals a GntR family transcriptional regulator controlling hypersensitive response and virulence.

The GntR family is one of the most abundant and widely distributed groups of helix-turn-helix transcriptional regulators in bacteria. Six open reading frames in the genome of the plant pathogen Xanthomonas campestris pv. campestris were predicted to encode GntR regulators. All six of the predicted GntR-encoding genes were individually mutagenized and mutants from five of them were successfully obtained. Plant disease response assays revealed that one, whose product belongs to the YtrA subfamily and has been named HpaR1, is involved in the hypersensitive response (HR) and virulence. Electrophoretic mobility shift assays and in vitro transcription assays revealed that HpaR1 could repress its own transcription level through binding to its promoter sequence, indicating an autoregulatory feedback inhibition mechanism for HpaR1 expression. Promoter-gusA reporter and reverse-transcription polymerase chain reaction analyses revealed that HpaR1 positively and negatively affects the expression of HR and pathogenicity (hrp) genes in host plant and standard media, respectively. Constitutive expression of the key hrp regulator, hrpG, in the hpaR1 mutant could bypass the requirement of HpaR1 for the induction of wild-type HR, suggesting that HpaR1 regulates the expression of hrp genes that encode the type III secretion system via hrpG.