Analysis of transcriptional regulators HpaR1 and Clp regulating the expression of glycoside hydrolase-encoding gene in the Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that causes black rot in host. It is an important model strain for studying the interaction between the phytopathogen and plants. In Xcc, global transcription regulator HpaR1 that belongs to the GntR family regulates many cellular processes such as the movement and synthesis of extracellular polysaccharides and extracellular enzymes, and is associated with hypersensitive response (HR) and pathogenicity. On the other hand, the global transcriptional regulator Clp regulates the secretion and synthesis of extracellular enzymes and extracellular polysaccharides, and is associated with the pathogenicity of Xanthomonas. Previous studies have shown that both HpaR1 and Clp bind to the promoter region of the glycoside hydrolase encoding gene (named ghy gene). This study investigates the molecular mechanism of the co-regulation of HpaR1 and Clp on the expression of ghy gene. Through electrophoresis mobility shift assay (EMSA), we found that both HpaR1 and Clp bind to the promoter regions of gene ghy in vitro. Both HpaR1 and Clp also bind to the promoter regions of gene ghy in vivo by chromatin immunoprecipitation (ChIP) assays. DNase I footprinting and 5'-RACE assays showed that both HpaR1 and Clp bind to the -35 region upstream of the ghy promoter. The HpaR1 binding site was located upstream of the Clp binding site. RT-qPCR and in vitro transcription assays showed that HpaR1 negatively while Clp positively regulates the transcription of gene ghy. Furthermore, HpaR1 inhibits the activation of Clp on the transcription of gene ghy in vitro. Our findings indicate that HpaR1 and Clp exhibit opposite effect on the transcription of gene ghy. It is speculated that HpaR1 may regulate the expression of gene ghy by inhibiting the activity of RNA polymerase.

Progress on the GntR family transcription regulators in bacteria.

In bacteria, GntR family transcription regulators are the widespread family of transcription factors. Members of this family consist of two functional domains, a conserved N-terminal DNA-binding domain that contains a typical helix-turn-helix (HTH) motif and a C-terminal effector-binding or oligomerization domain. Usually, the amino acid sequences of N-terminal DNA-binding domains are highly conserved, but differ in the C-terminal effector-binding or oligomerization domains. In the past several decades, many GntR family transcription regulators have been characterized in a number of bacteria. These regulators control a variety of cellular processes such as cell motility, glucose metabolism, bacterial resistance, pathogenesis and virulence. In this review, we summarized the discovery, C-terminal domains, biological function and regulation mode of GntR family transcription regulators. This review will help researchers to obtain more knowledge about the functions and mechanisms of the GntR family transcriptional regulatory factors.

PilG and PilH antagonistically control flagellum-dependent and pili-dependent motility in the phytopathogen Xanthomonas campestris pv. campestris.

BACKGROUND: The virulence of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) involves the coordinate expression of many virulence factors, including surface appendages flagellum and type IV pili, which are required for pathogenesis and the colonization of host tissues. Despite many insights gained on the structure and functions played by flagellum and pili in motility, biofilm formation, surface attachment and interactions with bacteriophages, we know little about how these appendages are regulated in Xcc. RESULTS: Here we present evidence demonstrating the role of two single domain response regulators PilG and PilH in the antagonistic control of flagellum-dependent (swimming) and pili-dependent (swarming) motility. Using informative mutagenesis, we reveal PilG positively regulates swimming motility while and negatively regulating swarming motility. Conversely, PilH negatively regulates swimming behaviour while and positively regulating swarming motility. By transcriptome analyses (RNA-seq and RT-PCR) we confirm these observations as PilG is shown to upregulate many genes involved chemotaxis and flagellar biosynthesis but these similar genes were downregulated by PilH. Co-immunoprecipitation, bacterial two-hybrid and pull-down analyses showed that PilH and PilG were able to interact with district subsets of proteins that potentially account for their regulatory impact. Additionally, we present evidence, using mutagenesis that PilG and PilH are involved in other cellular processes, including chemotaxis and virulence. CONCLUSIONS: Taken together, we demonstrate that for the conditions tested PilG and PilH have inverse regulatory effects on flagellum-dependent and pili-dependent motility in Xcc and that this regulatory impact depends on these proteins influences on genes/proteins involved in flagellar biosynthesis and pilus assembly.

HprKXcc is a serine kinase that regulates virulence in the Gram-negative phytopathogen Xanthomonas campestris.

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.

HpaP, a novel regulatory protein with ATPase and phosphatase activity, contributes to full virulence in Xanthomonas campestris pv. campestris.

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.

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.

Xanthomonas campestris VemR enhances the transcription of the T3SS key regulator HrpX via physical interaction with HrpG.

VemR is a response regulator of the two-component signalling systems (TCSs). It consists solely of a receiver domain. Previous studies have shown that VemR plays an important role in influencing the production of exopolysaccharides and exoenzymes, cell motility, and virulence of Xanthomonas campestris pv. campestris (Xcc). However, whether VemR is involved in the essential pathogenicity determinant type III secretion system (T3SS) is unclear. In this work, we found by transcriptome analysis that VemR modulates about 10% of Xcc genes, which are involved in various cellular processes including the T3SS. Further experiments revealed that VemR physically interacts with numerous proteins, including the TCS sensor kinases HpaS and RavA, and the TCS response regulator HrpG, which directly activates the transcription of HrpX, a key regulator controlling T3SS expression. It has been demonstrated previously that HpaS composes a TCS with HrpG or VemR to control the expression of T3SS or swimming motility, while RavA and VemR form a TCS to control the expression of flagellar genes. Mutation analysis and in vitro transcription assay revealed that phosphorylation might be essential for the function of VemR and phosphorylated VemR could significantly enhance the activation of hrpX transcription by HrpG. We infer that the binding of VemR to HrpG can modulate the activity of HrpG to the hrpX promoter, thereby enhancing hrpX transcription. Although further studies are required to validate this inference and explore the detailed functional mechanism of VemR, our findings provide some insights into the complex regulatory cascade of the HpaS/RavA-VemR/HrpG-HrpX signal transduction system in the control of T3SS.

HpaP divergently regulates the expression of hrp genes in Xanthomonas oryzae pathovars oryzae and oryzicola.

The bacterial pathogens Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc) cause leaf blight and leaf streak diseases on rice, respectively. Pathogenesis is largely defined by the virulence genes harboured in the pathogen genome. Recently, we demonstrated that the protein HpaP of the crucifer pathogen Xanthomonas campestris pv. campestris is an enzyme with both ATPase and phosphatase activities, and is involved in regulating the synthesis of virulence factors and the induction of the hypersensitive response (HR). In this study, we investigated the role of HpaP homologues in Xoo and Xoc. We showed that HpaP is required for full virulence of Xoo and Xoc. Deletion of hpaP in Xoo and Xoc led to a reduction in virulence and alteration in the production of virulence factors, including extracellular polysaccharide and cell motility. Comparative transcriptomics and reverse transcription-quantitative PCR assays revealed that in XVM2 medium, a mimic medium of the plant environment, the expression levels of hrp genes (for HR and pathogenicity) were enhanced in the Xoo hpaP deletion mutant compared to the wild type. By contrast, in the same growth conditions, hrp gene expression was decreased in the Xoc hpaP deletion mutant compared to the wild type. However, an opposite expression pattern was observed when the pathogens grew in planta, where the expression of hrp genes was reduced in the Xoo hpaP mutant but increased in the Xoc hpaP mutant. These findings indicate that HpaP plays a divergent role in Xoo and Xoc, which may lead to the different infection strategies employed by these two pathogens.

Preoperative inhalation of milrinone attenuates inflammation in patients undergoing cardiac surgery with cardiopulmonary bypass.

OBJECTIVES: The purpose of this study was to evaluate the effect of preoperative inhalation of milrinone on cardiopulmonary bypass (CPB)-related inflammation. SUBJECTS AND METHODS: A total of 30 patients undergoing cardiac surgery were recruited and randomized for preoperative inhalation of milrinone (Mil group) or normal saline (NS group), respectively. Each group had 15 patients. Their hemodynamic parameters were measured and blood samples were obtained longitudinally. The levels of serum interleukin (IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinase (MMP)-9 were determined by ELISA. RESULTS: The levels of serum IL-6, TNF-α, and MMP-9 significantly increased at the end of cardiac surgery and remained high for 24 h in both groups of patients. However, the levels of proinflammatory cytokines at the end of cardiac surgery in the Mil group of patients were significantly lower than those of the NS group of patients. CONCLUSIONS: Our data indicated that preoperative inhalation of milrinone significantly mitigated CPB-related inflammation.

McvR, a single domain response regulator regulates motility and virulence in the plant pathogen Xanthomonas campestris.

Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions in most bacteria. Comparatively little is known about the mechanism(s) by which single-domain response regulators (SD-RRs), which lack a dedicated output domain but harbour a phosphoryl receiver domain, exert their various regulatory effects in bacteria. Here we have examined the role of the SD-RR proteins encoded by the phytopathogen Xanthomonas campestris pv. campestris (Xcc). We describe the identification and characterization of a SD-RR protein named McvR (motility, chemotaxis, and virulence-related response regulator) that is required for virulence and motility regulation in Xcc. Deletion of the mcvR open reading frame caused reduced motility, chemotactic movement, and virulence in Xcc. Global transcriptome analyses revealed the McvR had a broad regulatory role and that most motility and pathogenicity genes were down-regulated in the mcvR mutant. Bacterial two-hybrid and protein pull-down assays revealed that McvR did not physically interact with components of the bacterial flagellum but interacts with other SD-RR proteins (like CheY) and the subset of DNA-binding proteins involved in gene regulation. Site-directed mutagenesis and phosphor-transfer experiments revealed that the aspartyl residue at position 55 of the receiver domain is important for phosphorylation and the regulatory activity of McvR protein. Taken together, the findings describe a previously unrecognized class of SD-RR protein that contributes to the regulation of motility and virulence in 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.

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.

A HU-like protein is required for full virulence in Xanthomonas campestris pv. campestris.

Bacteria harbour several abundant small DNA-binding proteins known as nucleoid-associated proteins (NAPs) that contribute to the structure of the bacterial nucleoid as well as to gene regulation. Although the function of NAPs as global transcriptional regulators has been comprehensively studied in the model organism Escherichia coli, their regulatory functions in other bacteria remain relatively poorly understood. Xanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium that causes black rot disease in almost all members of the crucifer family. In previous work, we demonstrated that a Fis homologue protein, which we named Fis-like protein (Flp), contributes to the regulation of virulence, type III secretion, and a series of other phenotypes in Xcc. Here we have examined the role of XC_1355, which is predicted to encode a DNA-binding protein belonging to the HU family herein named HU-like protein (Hlp). We show that mutation of XC_1355 in Xcc reduces the virulence, extracellular polysaccharide production, and cell motility, but has no effect on the production of extracellular enzymes and induction of the hypersensitive response. These data together with transcriptome analysis indicate that hlp is a previously uncharacterized gene involved in virulence that has partially overlapping and complementary functions with flp in Xcc, although the two regulators have opposite effects on the expression of genes involved in type III secretion. The findings add to our understanding of the complex regulatory pathways that act to regulate virulence in Xcc.

Identification of four novel small non-coding RNAs from Xanthomonas campestris pathovar campestris.

BACKGROUND: In bacteria, small non-coding RNAs (sRNAs) have been recognized as important regulators of various cellular processes. Approximately 200 bacterial sRNAs in total have been reported. However, very few sRNAs have been identified from phytopathogenic bacteria. RESULTS: Xanthomons campestris pathovar campestris (Xcc) is the causal agent of black rot disease of cruciferous crops. In this study, a cDNA library was constructed from the low-molecular weight RNA isolated from the Xcc strain 8004 grown to exponential phase in the minimal medium XVM2. Seven sRNA candidates were obtained by sequencing screen of 2,500 clones from the library and four of them were confirmed to be sRNAs by Northern hybridization, which were named sRNA-Xcc1, sRNA-Xcc2, sRNA-Xcc3, and sRNA-Xcc4. The transcription start and stop sites of these sRNAs were further determined. BLAST analysis revealed that the four sRNAs are novel. Bioinformatics prediction showed that a large number of genes with various known or unknown functions in Xcc 8004 are potential targets of sRNA-Xcc1, sRNA-Xcc3 and sRNA-Xcc4. In contrast, only a few genes were predicted to be potential targets of sRNA-Xcc2. CONCLUSION: We have identified four novel sRNAs from Xcc by a large-scale screen. Bioinformatics analysis suggests that they may perform various functions. This work provides the first step toward understanding the role of sRNAs in the molecular mechanisms of Xanthomonas campestris pathogenesis.

The Zur of Xanthomonas campestris functions as a repressor and an activator of putative zinc homeostasis genes via recognizing two distinct sequences within its target promoters.

It has been long considered that zinc homeostasis in bacteria is maintained by export systems and uptake systems, which are separately controlled by their own regulators and the uptake systems are negatively regulated by Zur which binds to an about 30-bp AT-rich sequence known as Zur-box present in its target promoters to block the entry of RNA polymerase. Here, we demonstrated in vivo and in vitro that in addition to act as a repressor of putative Zn(2+)-uptake systems, the Zur of the bacterial phytopathogen Xanthomonas campestris pathovar campestris (Xcc) acts as an activator of a Zn(2+) efflux pump. The Xcc Zur binds to a similar Zur-box with approximately 30-bp AT-rich sequence in the promoters of the genes encoding putative Zn(2+)-uptake systems but a 59-bp GC-rich sequence with a 20-bp inverted repeat overlapping the promoter's -35 to -10 sequence of the gene encoding a Zn(2+)-export system. Mutagenesis of the inverted repeat sequence resulted in abolishment of the in vitro binding and the in vivo and in vitro activation of the export gene's promoter by Zur. These results reveal that the Xcc Zur functions as a repressor and an activator of putative zinc homeostasis genes via recognizing two distinct sequences within its target promoters.

[Effect of cardiopulmonary bypass on the expression of survivin in neutrophils].

OBJECTIVE: To investigate the effects of cardiopulmonary bypass on neutrophils apoptosis and the expression of survivin. METHODS: Ten patients who scheduled for cardiac surgery under cardiopulmonary bypass were recruited as study group and 10 healthy volunteers as control. Blood samples were obtained before operation, at the end of surgery, and at 24 hours postoperatively. Neutrophils were isolated by density gradient centrifugation and its apoptosis were evaluated by fluorescent microscope and flow cytometry. The expression of survivin protein was examined by Western blotting analysis. Expression level of survivin mRNA was detected by RT-PCR. RESULTS: The apoptotic rate of neutrophils decreased significantly at the end of surgery (P < 0.01), and was still lower at 24 hours postoperatively than before operation (P < 0.05). The expression ratios of survivin protein and mRNA were increased at the end of surgery (P < 0.01), and decreased gently at 24 hours postoperatively but was still higher than before operation (P < 0.05). CONCLUSION: Cardiopulmonary bypass could inhibit neutrophils apoptosis and increase the expression of survivin. The decrease of neutrophils apoptosis was correlated with high expression of survivin.

[Identification of a gene involved in the expression of the pathogenicity-related gene XC3814 in Xanthomonas campestris].

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of the black rot disease of cruciferous plants. Our previous work had demonstrated that XC3814 is required for full virulence and extracellular polysaccharide production. In this work, the reporter plasmid pL3814sac was constructed by fusing the promoter region of XC3814 to the coding region of the gene sacB, and introduced into Xcc wild-type strain 8004. The resulted strain 8004/pL3814sac was mutagenized randomly by the transposon EZ::Tn5, and 3 mutant strains insensitive to sucrose were isolated. One of the mutants was due to the disruption of the open reading frame XC3882, which was assigned to code a hypothetical protein. To verify whether XC3882 has an impact on the expression level of XC3814, the reporter plasmid pGUS3814 was constructed by fusing the promoter region of XC3814 to the coding region of the gusA gene. This construct was introduced into the wild-type strain 8004 and the XC3882 mutant strain 190A10, which was derived from the transposon Tn5gusA5 insertion. The GUS activity, produced by pGUS3814 in the XC3882 mutant background, was reduced by 81.3% compared to that in the wild type background. These results indicate that the expression of XC3814 is influenced by XC3882.

Xanthomonas campestris sensor kinase HpaS co-opts the orphan response regulator VemR to form a branched two-component system that regulates motility.

Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacts with VemR. Phos-tag SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection.

An adenosine kinase exists in Xanthomonas campestris pathovar campestris and is involved in extracellular polysaccharide production, cell motility, and virulence.

Adenosine kinase (ADK) is a purine salvage enzyme and a typical housekeeping enzyme in eukaryotes which catalyzes the phosphorylation of adenosine to form AMP. Since prokaryotes synthesize purines de novo and no endogenous ADK activity is detectable in Escherichia coli, ADK has long been considered to be rare in bacteria. To date, only two prokaryotes, both of which are gram-positive bacteria, have been reported to contain ADK. Here we report that the gram-negative bacterium Xanthomonas campestris pathovar campestris, the causal agent of black rot of crucifers, possesses a gene (designated adk(Xcc)) encoding an ADK (named ADK(Xcc)), and we demonstrate genetically that the ADK(Xcc) is involved in extracellular polysaccharide (EPS) production, cell motility, and pathogenicity of X. campestris pv. campestris. adk(Xcc) was overexpressed as a His(6)-tagged protein in E. coli, and the purified His(6)-tagged protein exhibited ADK activity. Mutation of adk(Xcc) did not affect bacterial growth in rich and minimal media but led to an accumulation of intracellular adenosine and diminutions of intracellular ADK activity and ATP level, as well as EPS. The adk(Xcc) mutant displayed significant reductions in bacterial growth and virulence in the host plant.

The Zur of Xanthomonas campestris is involved in hypersensitive response and positively regulates the expression of the hrp cluster via hrpX but not hrpG.

In bacteria, Zur is a key regulator for zinc homeostasis. Our previous work has shown that, in the phytopathogen Xanthomonas campestris pv. campestris, in addition to regulating zinc homeostasis, Zur is essential for full virulence. Here, we demonstrate that the X. campestris pv. campestris Zur is involved in hypersensitive response (HR) and positively regulates the transcription of hrpA to hrpF operons and hrpX but not hrpG. Constitutively expressing hrpX but not hrpG in the zur mutant could bypass the requirement of Zur for the expression of hrpA to hrpF operons and the induction of wild-type HR, indicating that Zur controls the expression of hrp cluster via hrpX. Promoter-gusA reporter and semiquantitative reverse-transcription polymerase chain reaction analyses revealed that HrpG controls the expression of hrpX and HrpX regulates the expression of all the six hrp operons (hrpA to hrpF) in X. campestris pv. campestris.