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Corncob is a kind of abundant lignocellulosic biomass. One of the bottlenecks in utilization of corncob in fermentation industry is many microorganisms cannot ferment xylose, which is the main component of corncob hydrolysate. Here we identified a novel oily yeast strain AM2352 of Rhodotorula taiwanensis, which could effectively convert corncob hydrolysate into microbial lipid. Notably, the fatty acid synthase alpha-subunit of R. taiwanensis has two acyl carrier protein (ACP) domains, which may be involved in high-level oil contents in its cells. Based on a 5-L fermentation analysis, the coefficient of lipid production was 55.8 g oil per kilogram of corncob. Long-chain fatty acids (C16–C18) were the main components of the intracellular lipid accumulated by the yeast strain AM2352. Over 81.5% of the extracted oil could be converted into biodiesel. This work not only provides an inspiring information into the utilization of corncob hydrolysate by the yeast strain AM2352 for microbial lipid production, but also probably promotes biofuel production.

Phenazine-1-carboxamide (PCN) produced by multifarious Pseudomonas strains represents a promising candidate as a new metabolite pesticide due to its broad-spectrum antifungal activity and capacity to induce systemic resistance in plants. The rice rhizosphere Pseudomonas strain PA1201 contains two reiterated gene clusters, phz1 and phz2, for phenazine-1-carboxylic acid (PCA) biosynthesis; PCA is further converted into PCN by this strain using a functional phzH-encoding glutamine aminotransferase. However, PCN levels in PA1201 constitute approximately one-fifth of PCA levels and the optimal temperature for PCN synthesis is 28 °C. In this study, the phzH open reading frame (ORF) and promoter region were investigated and reannotated. phzH promoter PphzH was found to be a weak promoter, and PhzH levels were not sufficient to convert all of the native PCA into PCN. Following RNA Seq and promoter-lacZ fusion analyses, a strong quorum sensing (QS)- and thermo-regulated promoter PrhlI was identified and characterized. The activity of PphzH is approximately 1% of PrhlI in PA1201. After three rounds of promoter editing and swapping by PrhlI, a new PCN-overproducing strain UP46 was generated. The optimal fermentation temperature for PCN biosynthesis in UP46 was increased from 28 to 37 °C and the PCN fermentation titer increased 179.5-fold, reaching 14.1 g/L, the highest ever reported.

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.

Coenzyme Q (CoQ) is a lipid-soluble membrane component found in organisms ranging from bacteria to mammals. The biosynthesis of CoQ has been intensively studied in Escherichia coli, where 12 genes (ubiA, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, and -X) are involved. In this study, we first investigated the putative genes for CoQ8 biosynthesis in the phytopathogen Xanthomonas campestris pv. campestris using a combination of bioinformatic, genetic, and biochemical methods. We showed that Xc_0489 (coq7Xc) encodes a di-iron carboxylate monooxygenase filling the E. coli UbiF role for hydroxylation at C-6 of the aromatic ring. Xc_0233 (ubiJXc) encodes a novel protein with an E. coli UbiJ-like domain organization and is required for CoQ8 biosynthesis. The X. campestris pv. campestris decarboxylase gene remains unidentified. Further functional analysis showed that ubiB and ubiK homologs ubiBXc and ubiKXc are required for CoQ8 biosynthesis in X. campestris pv. campestris. Deletion of ubiJXc, ubiBXc, and ubiKXc led to the accumulation of an intermediate 3-octaprenyl-4-hydroxybenzoic acid. UbiKXc interacts with UbiJXc and UbiBXc to form a regulatory complex. Deletion analyses of these CoQ8 biosynthetic genes indicated that they are important for virulence in Chinese radish. These results suggest that the X. campestris pv. campestris CoQ8 biosynthetic reactions and regulatory mechanisms are divergent from those of E. coli. The variations provide an opportunity for the design of highly specific inhibitors for the prevention of infection by the phytopathogen X. campestris pv. campestris.

Actinobacteria constitute prolific sources of novel and vital bioactive metabolites for pharmaceutical utilization. In recent years, research has focused on exploring actinobacteria that thrive in extreme conditions to unearth their beneficial bioactive compounds for natural product drug discovery. Natural products have a significant role in resolving public health issues such as antibiotic resistance and cancer. The breakthrough of new technologies has overcome the difficulties in sampling and culturing extremophiles, leading to the outpouring of more studies on actinobacteria from extreme environments. This review focuses on the diversity and bioactive potentials/medically relevant biomolecules of extremophilic actinobacteria found from various unique and extreme niches. Actinobacteria possess an excellent capability to produce various enzymes and secondary metabolites to combat harsh conditions. In particular, a few strains have displayed substantial antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), shedding light on the development of MRSA-sensitive antibiotics. Several strains exhibited other prominent bioactivities such as antifungal, anti-HIV, anticancer, and anti-inflammation. By providing an overview of the recently found extremophilic actinobacteria and their important metabolites, we hope to enhance the understanding of their potential for the medical world.

An actinomycete strain CSR-4 was isolated from the rhizosphere soil of Zingiber montanum. Taxonomic characterization revealed strain CSR-4 was a member of the genus Microbispora. Whole-genome sequence analysis exhibited the highest average nucleotide identity (ANI) value (95.34%) and digital DNA–DNA hybridization (DDH) value (74.7%) between strain CSR-4 and the closest relative M. hainanensis DSM 45428T, which was in line with the assignment to same species. In addition, a new diterpene compound, 2α-hydroxy-8(14), 15-pimaradien-17, 18-dioic acid, and nine known compounds were isolated from the ethyl acetate crude extract of fermentation broth. Interestingly, a new diterpene displayed the suppressive effect on the recombinant human acetylcholinesterase (rhAChE) enzymes (IC50 96.87 ± 2.31 μg/ml). In silico studies based on molecular docking and molecular dynamics (MD) simulations were performed to predict a binding mode of the new compound into the binding pocket of the rhAChE enzyme and revealed that some amino acids in the peripheral anions site (PAS), anionic subsite, oxyanion site and catalytic active site (CAS) of the rhAChE have interacted with the compound. Therefore, our new compound could be proposed as a potential active human AChE inhibitor. Moreover, the new compound can protect significantly the neuron cells (% neuron viability = 88.56 ± 5.19%) from oxidative stress induced by serum deprivation method at 1 ng/ml without both neurotoxicities on murine P19-derived neuron cells and cytotoxicity against Vero cells.

Xanthomonadins are membrane-bound yellow pigments that are typically produced by phytopathogenic bacterial Xanthomonas spp., Xylella fastidiosa, and Pseudoxanthomonas spp. They are also produced by a diversity of environmental bacterial species. Considerable research has revealed that they are a unique group of halogenated, aryl-polyene, water-insoluble pigments. Xanthomonadins have been shown to play important roles in epiphytic survival and host-pathogen interactions in the phytopathogen Xanthomonas campestris pv. campestris, which is the causal agent of black rot in crucifers. Here, we review recent advances in the understanding of xanthomonadin chemical structures, physiological roles, biosynthetic pathways, regulatory mechanisms, and crosstalk with other signaling pathways. The aim of the present review is to provide clues for further in-depth research on xanthomonadins from Xanthomonas and other related bacterial species.

Xanthomonadins are yellow pigments that are produced by the phytopathogen Xanthomonas campestris pv. campestris (Xcc). A pig cluster is responsible for xanthomonadin biosynthesis. Previously, Xcc4014 of the cluster was characterized as a bifunctional chorismatase that produces 3-hydroxybenzoic acid (3-HBA) and 4-HBA. In this study, genetic analysis identified 11 genes within the pig cluster to be essential for xanthomonadin biosynthesis. Biochemical and bioinformatics analysis suggest that xanthomonadins are synthesized via an unusual type II polyketide synthase pathway. Heterologous expression of the pig cluster in non-xanthomonadin-producing Pseudomonas aeruginosa strain resulted in the synthesis of chlorinated xanthomonadin-like pigments. Further analysis showed that xanC encodes an acyl carrier protein (ACP) while xanA2 encodes a ATP-dependent 3-HBA:ACP ligase. Both of them act together to catalyse the formation of 3-HBA-S-ACP from 3-HBA to initiate xanthomonadin biosynthesis. Finally, we showed that xanH encodes a FabG-like enzyme and xanK encodes a novel glycosyltransferase. Both xanH and xanK are not only required for xanthomonadin biosynthesis, but also required for the balanced biosynthesis of extracellular polysaccharides and DSF-family quorum sensing signals. These findings provide us with a better understanding of xanthomonadin biosynthetic mechanisms and directly demonstrate the presence of extensive cross-talk among xanthomonadin biosynthetic pathways and other metabolic pathways.

Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc hereafter) is the causal agent of black rot of crucifers. Whole genome sequencing has revealed an abundance of GGDEF-, EAL-, and HD-GYP-domain-containing proteins in Xcc. Most GGDEF, EAL, and HD-GYP domains are linked to a wide range of signal-input domains, suggesting that numerous environmental and internal signals can be potentially integrated into the cyclic di-GMP metabolism network. This chapter summarizes these interesting findings with a focus on diffusible signaling factor (DSF)-dependent quorum sensing, RavS/RavR-dependent hypoxia sensing and the identified cyclic di-GMP effectors in Xcc.

Coenzyme Q (ubiquinone) is a redox-active isoprenylated benzoquinone commonly found in living organisms. The biosynthetic pathway for this lipid has been extensively studied in Escherichia coli and Saccharomyces cerevisiae; however, little is known in Pseudomonas aeruginosa. In this study, we observed that CoQ9 is the predominant coenzyme Q synthesized by the Shenqinmycin-producing strain M18. BLASTP and domain organization analyses identified 15 putative genes for CoQ biosynthesis in M18. The roles of 5 of these genes were genetically and biochemically investigated. PAM18_4662 encodes a nonaprenyl diphosphate synthase (Nds) and determines the number of isoprenoid units of CoQ9 in M18. PAM18_0636 (coq7PA) and PAM18_5179 (ubiJPA) are essential for aerobic growth and CoQ9 biosynthesis. Deletion of ubiJPA, ubiBPA and ubiKPA led to reduced CoQ biosynthesis and an accumulation of the CoQ9 biosynthetic intermediate 3-nonaprenylphenol (NPP). Moreover, we also provide evidence that the truncated UbiJPA interacts with UbiBPA and UbiKPA to affect CoQ9 biosynthesis by forming a regulatory complex. The genetic diversity of coenzyme Q biosynthesis may provide targets for the future design of specific drugs to prevent P. aeruginosa-related infections.

Xanthomonas campestris pv. campestris uses the diffusible signal factor (DSF) family of quorum-sensing (QS) signals to coordinate virulence and adaptation. DSF family signals have been well-characterized using laboratory-based cell cultures. The in-planta QS signal used during X. campestris pv. campestris infection remains unclear. To achieve this goal, we first mimic in-planta X. campestris pv. campestris growth conditions by supplementing the previously developed XYS medium with cabbage hydrolysate and found that the dominant signal produced in these conditions was BDSF. Secondly, by using XYS medium supplemented with diverse plant-derived compounds, we examined the effects of diverse plant-derived compounds on the biosynthesis of DSF family signals. Several compounds were found to promote biosynthesis of BDSF. Finally, using an X. campestris pv. campestris ΔrpfB–Chinese cabbage infection model and an ultra-performance liquid chromatographic-time of flight-mass spectrometry–based assay, BDSF was found to comprise >70% of the DSF family signals present in infected cabbage tissue. BDSF at a concentration of 2.0 μM induced both protease activity and engXCA expression. This is the first report to directly show that BDSF is the predominant in-planta QS signal used during X. campestris pv. campestris infection. It provides a better understanding of the molecular interactions between X. campestris pv. campestris and its cruciferous hosts and also provides the logical target for designing strategies to counteract BDSF signaling and, thus, infection. Further studies are needed to get an exact idea about the DSF production dynamics of the wild-type strain inside the plant.

Two almost identical gene clusters (phz1 and phz2) are responsible for phenazine-1-carboxylic acid (PCA) production in Pseudomonas aeruginosa (P. aeruginosa) strain MSH (derived from strain PA1201). Here, we showed that the anti-activator QslA negatively regulated PCA biosynthesis and phz1 expression in strain PA1201 but had little effect on phz2 expression. This downregulation was mediated by a 56-bp region within the 5′-untranslated region (5′-UTR) of the phz1 promoter and was independent of LasR and RsaL signaling. QslA also negatively regulated Pseudomonas quinolone signal (PQS) production. Indeed, QslA controlled the PQS threshold concentration needed for PQS-dependent PCA biosynthesis. The quorum sensing regulator MvfR was required for the QslA-dependent inhibition of PCA production. We identified a direct protein–protein interaction between QslA and MvfR. The ligand-binding domain of MvfR (residues 123–306) was involved in this interaction. Our results suggested that MvfR bound directly to the promoter of the phz1 cluster. QslA interaction with MvfR prevented the binding of MvfR to the phz1 promoter regions. Thus, this study depicted a new mechanism by which QslA controls PCA and PQS biosynthesis in P. aeruginosa.

The environmentally friendly antibiotic phenazine-1-carboxylic acid (PCA) protects plants, mammals and humans effectively against various fungal pathogens. However, the mechanism by which PCA inhibits or kills fungal pathogens is not fully understood. We analyzed the effects of PCA on the growth of two fungal model organisms, Saccharomyces cerevisiae and Candida albicans, and found that PCA inhibited yeast growth in a dose-dependent manner which was inversely dependent on pH. In contrast, the commonly used antibiotic hygromycin B acted in a dose-dependent manner as pH increased. We then screened a yeast mutant library to identify genes whose mutation or deletion conferred resistance or sensitivity to PCA. We isolated 193 PCA-resistant or PCA-sensitive mutants in clusters, including vesicle-trafficking- and autophagy-defective mutants. Further analysis showed that unlike hygromycin B, PCA significantly altered intracellular vesicular trafficking under growth conditions and blocked autophagy under starvation conditions. These results suggest that PCA inhibits or kills pathogenic fungi in a complex way, in part by disrupting vesicular trafficking and autophagy.

Pseudomonas aeruginosa strain PA1201 is a newly identified rhizobacterium that produces high levels of the secondary metabolite phenazine-1-carboxylic acid (PCA), the newly registered biopesticide Shenqinmycin. PCA production in liquid batch cultures utilizing a specialized PCA-promoting medium (PPM) typically occurs after the period of most rapid growth and production is regulated in a quorum sensing (QS)-dependent manner. PA1201 contains two PCA biosynthetic gene clusters phz1 and phz2; both clusters contribute to PCA production, with phz2 making a greater contribution. PA1201 also contains a complete set of genes for four QS systems (LasI/LasR, RhlI/RhlR, PQS/MvfR and IQS). By using several methods including gene deletion, the construction of promoter-lacZ fusion reporter strains and RNA-Seq analysis, this study investigated the effects of the four QS systems on bacterial growth, QS signal production, the expression of phz1 and phz2 and PCA production. The possible mechanisms for the strain- and condition-dependent expression of phz1 and phz2 were discussed and a schematic model was proposed. These findings provide a basis for further genetic engineering of the QS systems to improve PCA production.

The diffusible factor synthase XanB2, originally identified in Xanthomonas campestris pv. campestris (Xcc), is highly conserved across a wide range of bacterial species, but its substrate and catalytic mechanism have not yet been investigated. Here, we show that XanB2 is a unique bifunctional chorismatase that hydrolyses chorismate, the end-product of the shikimate pathway, to produce 3-hydroxybenzoic acid (3-HBA) and 4-HBA. 3-HBA and 4-HBA are respectively associated with the yellow pigment xanthomonadin biosynthesis and antioxidant activity in Xcc. We further demonstrate that XanB2 is a structurally novel enzyme with three putative domains. It catalyses 3-HBA and 4-HBA biosynthesis via a unique mechanism with the C-terminal YjgF-like domain conferring activity for 3-HBA biosynthesis and the N-terminal FGFG motif-containing domain responsible for 4-HBA biosynthesis. Furthermore, we show that Xcc produces coenzyme Q8 (CoQ8) via a new biosynthetic pathway independent of the key chorismate-pyruvate lyase UbiC. XanB2 is the alternative source of 4-HBA for CoQ8 biosynthesis. The similar CoQ8 biosynthetic pathway, xanthomonadin biosynthetic gene cluster and XanB2 homologues are well conserved in the bacterial species within Xanthomonas, Xylella, Xylophilus, Pseudoxanthomonas, Rhodanobacter, Frateuria, Herminiimonas and Variovorax, suggesting that XanB2 may be a conserved metabolic link between the shikimate pathway, ubiquinone and xanthomonadin biosynthetic pathways in diverse bacteria.

Summary The diffusible signal factor (DSF)-dependent quorum sensing (QS) system adopts a novel protein-protein interaction mechanism to autoregulate the production of signal DSF. Here, we present the crystal structures of DSF synthase RpfF and its complex with the REC domain of sensor protein RpfC. RpfF is structurally similarity to the members of the crotonase superfamily and contains an N-terminal α/β spiral core domain and a C-terminal α-helical region. Further structural and mutational analysis identified two catalytic glutamate residues, which is the conserved feature of the enoyl-CoA hydratases/dehydratases. A putative substrate-binding pocket was unveiled and the key roles of the residues implicated in substrate binding were verified by mutational analysis. The binding of the REC domain may lock RpfF in an inactive conformation by blocking the entrance of substrate binding pocket, thereby negatively regulating DSF production. These findings provide a structural model for the RpfC-RpfF interaction-mediated QS autoinduction mechanism.

Bacterial phenazine metabolites belong to a group of nitrogen-containing heterocyclic compounds with antimicrobial activities. In this study, a rhizosphere Pseudomonas aeruginosa strain PA1201 was isolated and identified through 16S rDNA sequence analysis and fatty acid profiling. PA1201 inhibited the growth of various pathogenic microorganisms, including Rhizotonia solani, Magnaporthe grisea, Fusarium graminearum, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Staphylococcus aureus. High Performance Liquid Chromatography showed that PA1201 produced high levels of phenazine-1-carboxylic acid (PCA), a registered green fungicide ‘Shenqinmycin’ with the fermentation titers of 81.7 mg/L in pigment producing medium (PPM) and 926.9 mg/L in SCG medium containing soybean meal, corn steep liquor and glucose. In addition, PA1201 produced another antifungal metabolite, phenazine-1-carboxaminde (PCN), a derivative of PCA, with the fermentation titers of 18.1 and 489.5 mg/L in PPM and SCG medium respectively. To the best of our knowledge, PA1201 is a rhizosphere originating P. aeruginosa strain that congenitally produces the highest levels of PCA and PCN among currently reported P. aeruginosa isolates, which endows it great biotechnological potential to be transformed to a biopesticide-producing engineering strain.

Background Small-molecule hormones are well known to play key roles in the plant immune signaling network that is activated upon pathogen perception. In contrast, little is known about whether phytohormones also directly influence microbial virulence, similar to what has been reported in animal systems. Results In this paper, we tested the hypothesis that hormones fulfill dual roles in plant-microbe interactions by orchestrating host immune responses, on the one hand, and modulating microbial virulence traits, on the other. Employing the rice-Xanthomonas oryzae pv. oryzae (Xoo) interaction as a model system, we show that Xoo uses the classic immune hormone salicylic acid (SA) as a trigger to activate its virulence-associated quorum sensing (QS) machinery. Despite repressing swimming motility, sodium salicylate (NaSA) induced production of the Diffusible Signal Factor (DSF) and Diffusible Factor (DF) QS signals, with resultant accumulation of xanthomonadin and extracellular polysaccharides. In contrast, abscisic acid (ABA), which favors infection by Xoo, had little impact on DF- and DSF-mediated QS, but promoted bacterial swimming via the LuxR solo protein OryR. Moreover, we found both DF and DSF to influence SA- and ABA-responsive gene expression in planta. Conclusions Together our findings indicate that the rice SA and ABA signaling pathways cross-communicate with the Xoo DF and DSF QS systems and underscore the importance of bidirectional interkingdom signaling in molding plant-microbe interactions. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0411-3) contains supplementary material, which is available to authorized users.

Lysobacter enzymogenes is a ubiquitous environmental bacterium that is emerging as a potentially novel biological control agent and a new source of bioactive secondary metabolites, such as the heat-stable antifungal factor (HSAF) and photoprotective polyene pigments. Thus far, the regulatory mechanism(s) for biosynthesis of these bioactive secondary metabolites remains largely unknown in L. enzymogenes . In the present study, the diffusible signal factor (DSF) and diffusible factor (DF)-mediated cell-cell signaling systems were identified for the first time from L. enzymogenes . The results show that both Rpf/DSF and DF signaling systems played critical roles in modulating HSAF biosynthesis in L. enzymogenes . Rpf/DSF signaling and DF signaling played negative and positive effects in polyene pigment production, respectively, with DF playing a more important role in regulating this phenotype. Interestingly, only Rpf/DSF, but not the DF signaling system, regulated colony morphology of L. enzymgenes . Both Rpf/DSF and DF signaling systems were involved in the modulation of expression of genes with diverse functions in L. enzymogenes , and their own regulons exhibited only a few loci that were regulated by both systems. These findings unveil for the first time new roles of the Rpf/DSF and DF signaling systems in secondary metabolite biosynthesis of L. enzymogenes .

Xanthomonas oryzae pv. oryzae, the causal agent of rice bacterial blight, produces membrane-bound yellow pigments, referred to as xanthomonadins. Xanthomonadins protect the pathogen from photodamage and host-induced perioxidation damage. They are also required for epiphytic survival and successful host plant infection. Here, we show that XanB2 encoded by PXO_3739 plays a key role in xanthomonadin and coenzyme Q8 biosynthesis in X. oryzae pv. oryzae PXO99A. A xanB2 deletion mutant exhibits a pleiotropic phenotype, including xanthomonadin deficiency, producing less exopolysaccharide (EPS), lower viability and H2O2 resistance, and lower virulence. We further demonstrate that X. oryzae pv. oryzae produces 3-hydroxybenzoic acid (3-HBA) and 4-hydroxybenzoic acid (4-HBA) via XanB2. 3-HBA is associated with xanthomonadin biosynthesis while 4-HBA is mainly used as a precursor for coenzyme Q (CoQ)8 biosynthesis. XanB2 is the alternative source of 4-HBA for CoQ8 biosynthesis in PXO99A. These findings suggest that the roles of XanB2 in PXO99A are generally consistent with those in X. campestris pv. campestris. The present study also demonstrated that X. oryzae pv. oryzae PXO99A has evolved several specific features in 3-HBA and 4-HBA signaling. First, our results showed that PXO99A produces less 3-HBA and 4-HBA than X. campestris pv. campestris and this is partially due to a degenerated 4-HBA efflux pump. Second, PXO99A has evolved unique xanthomonadin induction patterns via 3-HBA and 4-HBA. Third, our results showed that 3-HBA or 4-HBA positively regulates the expression of gum cluster to promote EPS production in PXO99A. Taken together, the results of this study indicate that XanB2 is a key metabolic enzyme linking xanthomonadin, CoQ, and EPS biosynthesis, which are collectively essential for X. oryzae pv. oryzae pathogenesis.

Xanthomonas campestris pv. campestris produces a membrane-bound yellow pigment called xanthomonadin. A diffusible factor (DF) has been reported to regulate xanthomonadin biosynthesis. In this study, DF was purified from bacterial culture supernatants using a combination of solvent extraction, flash chromatography, and high-performance liquid chromatography. Mass spectrometry and nuclear magnetic resonance analyses resolved the DF chemical structure as 3-hydroxybenzoic acid (3-HBA), which was further confirmed by synthetic 3-HBA. Significantly, bioassay and in silico analysis suggest that DF production is widely conserved in a range of bacterial species. Analysis of DF derivatives established the hydroxyl group and its position as the key structural features for the role of DF in xanthomonadin biosynthesis. In addition, we showed that DF is also associated with bacterial survival, H2O2 resistance, and systemic invasion. Furthermore, evidence was also presented that DF and diffusible signaling factor have overlapping functions in modulation of bacterial survival, H2O2 resistance, and virulence. Utilization of different mechanisms to modulate similar virulence traits may provide X. campestris pv. campestris with plasticity in response to various environmental cues.

The hybrid sensor kinase RpfC positively regulates the expression of a range of virulent genes and negatively modulates the synthesis of the quorum sensing signal diffusible signal factor (DSF) in Xanthomonas campestris. Three conserved amino acid residues of RpfC implicated in phosphorelay (His(198) in the histidine kinase domain, Asp(512) in the receiver domain, and His(657) in the histidine phosphotransfer domain) were essential for activation of the production of extracellular enzymes and extracellular polysaccharide (EPS) virulence factors but were not essential for repression of DSF biosynthesis. Domain deletion and subsequent in trans expression analysis revealed that the receiver domain of RpfC alone was sufficient to repress DSF overproduction in an rpfC deletion mutant. Further deletion and alanine scanning mutagenesis analyses identified a peptide of 107 amino acids and three amino acid residues (Gln(496), Glu(504), and Ile(552)) involved in modulating DSF production. Co-immunoprecipitation and far Western blot analyses suggested an interaction between the receiver domain and RpfF, the enzyme involved in DSF biosynthesis. These data support a model in which RpfC modulates two different functions (virulence factor synthesis and DSF synthesis) by utilization of a conserved phosphorelay system and a novel domain-specific protein-protein interaction mechanism, respectively. This latter mechanism represents an added dimension to conventional two-component signaling paradigms.

HD-GYP is a protein domain of unknown biochemical function implicated in bacterial signaling and regulation. In the plant pathogen Xanthomonas campestris pv. campestris , the synthesis of virulence factors and dispersal of biofilms are positively controlled by a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC and by cell–cell signaling mediated by the diffusible signal molecule DSF (diffusible signal factor). The RpfG/RpfC two-component system has been implicated in DSF perception and signal transduction. Here we show that the role of RpfG is to degrade the unusual nucleotide cyclic di-GMP, an activity associated with the HD-GYP domain. Mutation of the conserved H and D residues of the isolated HD-GYP domain resulted in loss of both the enzymatic activity against cyclic di-GMP and the regulatory activity in virulence factor synthesis. Two other protein domains, GGDEF and EAL, are already implicated in the synthesis and degradation respectively of cyclic di-GMP. As with GGDEF and EAL domains, the HD-GYP domain is widely distributed in free-living bacteria and occurs in plant and animal pathogens, as well as beneficial symbionts and organisms associated with a range of environmental niches. Identification of the role of the HD-GYP domain thus increases our understanding of a signaling network whose importance to the lifestyle of diverse bacteria is now emerging.

The effects of triacontanol (TRIA), applied singly or in combination with cerium nitrate and lanthanum nitrate, on bolting of Arabidopsis thaliana were studied. Triacontanol (0.1 to 0.6 microM) added to the culture medium induced early bolting. TRIA (0.3 microM) applied with low concentrations of cerium and lanthanum caused a synergistic stimulation of bolting. In medium containing 0.3 microM TRIA, 0.1 microM cerium nitrate and 0.1 mM lanthanum nitrate, 82% of the plants bolted 20 d after seed sowing compared to only 8.6% in basal medium and 47.8% in medium with TRIA only. The changes in the endogenous concentrations of total cytokinins of the isopentenyl adenine (IP) subfamily in the leaf and root tissues were correlated with TRIA-induced early bolting. The combined treatment of TRIA (0.3 microM), cerium nitrate (0.1 microM) and lanthanum nitrate (0.1 mM) resulted in a significant increase in the endogenous concentrations of total cytokinins of the IP subfamily in the root and leaf tissues compared to plants growing in the basal medium and medium with TRIA. The exogenous application of six natural cytokinins to the plants revealed that only isopentenyl adenosine (iPAdos) was as effective as TRIA on floral bud formation. iPAdos was also found to have similar effects as TRIA on root growth and reproductive growth. These results suggest a correlation between the early bolting induced by TRIA, cerium and lanthanum and the production of higher concentrations of endogenous iPAdos.

The effects of cerium and lanthanum on the vegetative growth, floral initiation and reproductive growth of Arabidopsis thaliana were studied. Addition of cerium nitrate (0.5-10 µM) or lanthanum nitrate (0.5-50 µM) to the culture medium significantly increased the lengths of primary roots, but had no significant effects on the number of rosette leaves produced per plant, plant heights and dry weights during the vegetative growth stage (17 days after seed germination). The percentage of plants bolted was significantly increased with the addition of 0.5-10.0 µM cerium nitrate or lanthanum nitrate. The combination of 0.5 µM cerium nitrate and 0.5 µM lanthanum nitrate was found to be most effective on the induction of floral initiation. The height, dry weight and average number of flower numbers of 35-day-old plants growing in media containing cerium nitrate or/and lanthanum nitrate (0.5-10.0 µM) were found to be significantly higher than those in the control medium. The endogenous levels of cytokinins (zeatin riboside, dihydrozeatin riboside and isopentenyl adenosine) and carbohydrates (sucrose, glucose and fructose) in leaf and root tissues of plants growing in the medium supplemented with 0.5 µM cerium nitrate and 0.5 µM lanthanum nitrate were not significantly different from those of plants in the control medium. Application of 0.5 µM cerium nitrate and 0.5 µM lanthanum nitrate enhanced the effects of 10(-6) M IPA on root growth, plant height and flowering. The role of cerium and lanthanum in promoting floral initiation and reproductive growth and the possibility of developing non-hormonal flowering promoting agents are discussed.