SummaryThe 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 endproduct 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.

Seven xanthomonadin transcriptional units (pigA through pigG) were identified by transposon saturation mutagenesis within an 18.6-kbp portion of the previously identified 25.4-kbp pig region from Xanthomonas campestris pv. campestris (strain B-24). Since marker exchange mutant strains with insertions in one 3.7-kbp portion of pig could not be obtained, mutations in this region may be lethal to the bacterium. Complementation analyses with different insertion mutations further defined and confirmed the seven transcriptional units. Insertional inactivation of one of the transcriptional units, pigB, resulted in greatly reduced levels of both xanthomonadins and extracellular polysaccharide slime, and a pigB-encoding plasmid restored both traits to these strains. pigB mutant strains could also be restored extracellularly by growth adjacent to strains with insertion mutations in any of the other six xanthomonadin transcriptional units, the parent strain (B-24), or strains of five different species of Xanthomonas. Strain B-24 produced a nontransforming diffusible factor (DF), which could be restored to pigB mutants by the pigB-encoding plasmid. Several lines of evidence indicate that DF is a novel bacterial pheromone, different from the known signal molecules of Vibrio, Agrobacterium, Erwinia, Pseudomonas, and Burkholderia spp.Members of the genus Xanthomonas are distributed worldwide and are the causal agents for disease for at least 124 monocot and 268 dicot plant hosts (20). Many of the phytopathogenic xanthomonads are disseminated on seed (22), and at least some of these pathogens survive and multiply as epiphytes prior to plant infection (13,35). Two common traits of Xanthomonas spp. are the production of yellow pigments (xanthomonadins) and extracellular polysaccharide slime (EPS) (32). The xanthomonadins are yellow, membrane-bound, brominated aryl-polyene pigments unique to the genus Xanthomonas (35). Thus, these pigments are commonly used as chemotaxonomic (2, 36) and diagnostic (32) markers. Although the biological role of xanthomonadins is not well understood, a correlation between these pigments and protection against photobiological damage has been observed (14). A correlation between EPS production and virulence has been widely reported for Xanthomonas campestris (7,9,33,38,39).In a previous study with X. campestris pv. campestris (causal agent of black rot of crucifers), six functional domains for xanthomonadin production were identified within a 25-kbp genomic clone (pIG102) (25). Sequences from this region (pig) restored pigment production to all 19 induced and naturally occurring xanthomonadin mutants tested (25). Clone pIG102 also conferred xanthomonadin production on a strain of Pseudomonas. Thus, it was concluded that at least some of these functional domains must be structural genes. The original purpose of this work was to perform a transcriptional analysis of pig by using transcriptional lacZ fusions created with the transposon Tn3HoHo1. In the course of this study, we made two unexpected observat...

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenicXanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.Xanthomonas bacteria are the causal agents of disease on at least 124 monocot and 268 dicot plant hosts (12), and many of them can survive and multiply as epiphytes (8,24). Most Xanthomonas bacteria produce yellow, membrane-bound, brominated aryl-polyene pigments referred to as xanthomonadins (24). Xanthomonadins are unique to Xanthomonas bacteria and serve as useful chemotaxonomic (2, 25) and diagnostic (22) markers. With methods of artificial infection, xanthomonadin-deficient strains were not affected in pathogenicity, symptomatology, or in planta growth (15). Thus, the xanthomonadins apparently are not important to the pathogen after infection of the host plant.Xanthomonas campestris pv. campestris, the causal agent of black rot of crucifers and one of the most serious disease problems in crucifer production, naturally infects its host via hydathodes or wounds in the leaves (28). A cluster of seven transcriptional units required for xanthomonadin production (pigA to pigG) was previously identified in X. campestris pv. campestris (13,15). In addition to a loss of xanthomonadin production, pigB mutant strains were also greatly impaired in the production of extracellular polysaccharide (EPS) and a pheromone (DF). Mutations in the other pig transcriptional units did not appear to have pleiotropic affects. When tested on the host plant, pigB mutants were significantly reduced in epiphytic survival and natural host infection via hydathodes (16). DF extracellularly restored all of these traits to a pigB mutant strain (4, 15, 16), indicating that DF is needed for xanthomonadin and EPS production, as well as for epiphytic survival and host infection. These results suggest that DF acts as a signal for the initiation of xanthomonadin and EPS production and that xanthomonadins and EPS may play a role in host infection and/or epiphytic survival.The results of other studies suggest an association of xanthomonadins with protec...

When cauliflower plants (Brassica oleraceae) were misted with bacterial suspensions of Xanthomonas campestris pv. campestris (causal agent of black rot of cruciferous plants), two separate populations of the pathogen were associated with the leaves. Initially, bacteria removable by sonication and sensitive to sodium hypochlorite treatment predominated (easily removable epiphytic bacteria, EREB). However, after 2 weeks, bacteria not removable by sonication and insensitive to sodium hypochlorite treatment were dominant. Although the exact location of this second population of the pathogen was not determined, evidence is presented to support its location in protected sites on the leaf surface, pigB of this pathogen is required for production of extracellular polysaccharide (EPS), xanthomonadin pigments, and the diffusible signal molecule, DF (diffusible factor). DF can extracellularly restore EPS and xanthomonadin production to pigB mutant strains. Parent strain B-24 and pigB mutant strain B24-B2 were identical for in planta growth and symptomatology after artificial infection by injection in leaf mid-veins. Subsequently, X. campestris pv. campestris parent strain B-24, Tn3HoHo1 pigB insertion mutation strain B24-B2, chromosomally restored pigB mutation strain B24-B2R, and strain B24-79 with a Tn3HoHo1 insertion in an unrelated part of the genome were compared for epiphytic survival on, and natural infection of, cauliflower. After application, strains B-24, B24-B2R, and B24-79 all maintained leaf EREB populations of between approximately 3 and 6 (log [1 + CFU per g of fresh weight]) over a 3-week period, whereas B24-B2 populations fell to nearly undetectable levels. Plants sprayed with strains B-24, B24-B2R, and B24-79 averaged between 1.0 and 1.2 lesions, whereas those sprayed with B24-B2 averaged only 0.03 lesions per plant after 3 weeks. Differences in EREB population levels did not explain the observed differences in host infection frequencies, and the results indicated that strain B24-B2 was reduced in its ability to infect the host via the hydathodes, but unaffected in infection via wounds. When strains B-24 and B24-B2 were mixed in equal numbers and sprayed on plants together, B24-B2 epiphytic populations were intermediate between those of B-24 applied alone and B24-B2 applied alone. These results indicate that a functional pigB is required for epiphytic survival and natural host infection under the experimental conditions tested, and suggest that DF, xanthomonadins, and EPS could all be important for survival of this pathogen on the leaf surface, and/or for host infection.

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.