Novel Xanthomonas campestris Long-Chain-Specific 3-Oxoacyl-Acyl Carrier Protein Reductase Involved in Diffusible Signal Factor Synthesis.

The precursors of the diffusible signal factor (DSF) family signals of Xanthomonas campestris pv. campestris are 3-hydroxyacyl-acyl carrier protein (3-hydroxyacyl-ACP) thioesters having acyl chains of 12 to 13 carbon atoms produced by the fatty acid biosynthetic pathway. We report a novel 3-oxoacyl-ACP reductase encoded by the X. campestris pv. campestris XCC0416 gene (fabG2), which is unable to participate in the initial steps of fatty acyl synthesis. This was shown by the failure of FabG2 expression to allow growth at the nonpermissive temperature of an Escherichia colifabG temperature-sensitive strain. However, when transformed into the E. coli strain together with a plasmid bearing the Vibrio harveyi acyl-ACP synthetase gene (aasS), growth proceeded, but only when the medium contained octanoic acid. In vitro assays showed that FabG2 catalyzes the reduction of long-chain (≥C8) 3-oxoacyl-ACPs to 3-hydroxyacyl-ACPs but is only weakly active with shorter-chain (C4, C6) substrates. FabG1, the housekeeping 3-oxoacyl-ACP reductase encoded within the fatty acid synthesis gene cluster, could be deleted in a strain that overexpressed fabG2 but only in octanoic acid-supplemented media. Growth of the X. campestris pv. campestris ΔfabG1 strain overexpressing fabG2 required fabH for growth with octanoic acid, indicating that octanoyl coenzyme A is elongated by X. campestris pv. campestrisfabH Deletion of fabG2 reduced DSF family signal production, whereas overproduction of either FabG1 or FabG2 in the ΔfabG2 strain restored DSF family signal levels.IMPORTANCE Quorum sensing mediated by DSF signaling molecules regulates pathogenesis in several different phytopathogenic bacteria, including Xanthomonas campestris pv. campestris DSF signaling also plays a key role in infection by the human pathogen Burkholderia cepacia The acyl chains of the DSF molecules are diverted and remodeled from a key intermediate of the fatty acid synthesis pathway. We report a Xanthomonas campestris pv. campestris fatty acid synthesis enzyme, FabG2, of novel specificity that seems tailored to provide DSF signaling molecule precursors.

Production of tyrosine through phenylalanine hydroxylation bypasses the intrinsic feedback inhibition in Escherichia coli.

Tyrosine is a proteinogenic aromatic amino acid that is often used as a supplement of food and animal feed, as well as a (bio-)synthetic precursor to various pharmaceutically or industrially important molecules. Extensive metabolic engineering efforts have been made towards the efficient and cost-effective microbial production of tyrosine. Conventional strategies usually focus on eliminating intrinsic feedback inhibition and redirecting carbon flux into the shikimate pathway. In this study, we found that continuous conversion of phenylalanine into tyrosine by the action of tetrahydromonapterin (MH4)-utilizing phenylalanine 4-hydroxylase (P4H) can bypass the feedback inhibition in Escherichia coli, leading to tyrosine accumulation in the cultures. First, expression of the P4H from Xanthomonas campestris in combination with an MH4 recycling system in wild-type E. coli allowed the strain to accumulate tyrosine at 262 mg/L. On this basis, enhanced expression of the key enzymes associated with the shikimate pathway and the MH4 biosynthetic pathway resulted in the elevation of tyrosine production up to 401 mg/L in shake flasks. This work demonstrated a novel approach to tyrosine production and verified the possibility to alleviate feedback inhibition by creating a phenylalanine sink.

Tomato TFT1 is required for PAMP-triggered immunity and mutations that prevent T3S effector XopN from binding to TFT1 attenuate Xanthomonas virulence.

XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344-733) is sufficient to bind TFT1. Removal of amino acids 605-733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis.

Low molecular weight plant extract induces metabolic changes and the secretion of extracellular enzymes, but has a negative effect on the expression of the type-III secretion system in Xanthomonas campestris pv. campestris.

Xanthomonas campestris pathovar campestris (Xcc) is a plant pathogenic bacterium and as such has to adapt to a variety of environments. During the course of disease, Xcc colonizes the surface of its host, infects the xylem in the early stages, and develops a fully saprophytic life-style, aided by secreted degradative enzymes, in the late stages. To get some insight into this complex regulation, Xcc was cultivated in the presence of low molecular weight host plant extract (<10 kDa). From this experiments it could be observed, that malate and sucrose are taken up preferably in such an environment. Furthermore, it was demonstrated, that the plant extract has a negative effect on the gene expression of the hrp-gene cluster, although the activator hrpG was induced. Also, the secretion of degradative enzymes was shown to be upregulated. These observations indicate, that a low molecular weight plant extract (<10 kDa) is a sufficient signal to regulate metabolic pathways and the secretion of enzymes relevant for the development of virulence in Xanthomonas, but has a negative effect on the expression of genes involved in type-III secretion.

Regulation of the pehA gene encoding the major polygalacturonase of Xanthomonas campestris by Clp and RpfF.

Exopolysaccharide and several extracellular enzymes of Xanthomonas campestris pv. campestris (Xcc), the causative agent of black rot in crucifers, are virulence factors. In this study, sequence and mutational analysis has demonstrated that Xcc pehA encodes the major polygalacturonase, a member of family 28 of the glycosyl hydrolases. Using the 5' RACE (rapid amplification of cDNA ends) method, the pehA transcription initiation site was mapped at 102 nt downstream of a Clp (cAMP receptor protein-like protein)-binding site. Transcriptional fusion assays showed that pehA transcription is greatly induced by polygalacturonic acid, positively regulated by Clp and RpfF (an enoyl-CoA hydratase homologue which is required for the synthesis of cis-11-methyl-2-dodecenoic acid, a low-molecular-mass diffusible signal factor), subjected to catabolite repression, which is independent of Clp or RpfF, and repressed under conditions of oxygen limitation or nitrogen starvation. Our findings extend previous work on Clp and RpfF regulation to show that they both influence the expression of pehA in Xcc.

Production of inulinase by Xanthomonas campestris pv phaseoli using onion (Allium cepa) and garlic (Allium sativum) peels in solid state cultivation.

AIMS: To access inulinase production by Xanthomonas campestris pv phaseoli using the submerged and solid state cultivation (SSC) methods. METHODS AND RESULTS: Various carbon sources, inulin-rich solid substrates and pure synthetic inulin were tested for their efficiency in inulinase induction. The highest inulinase production (17.42 IU ml(-1)) in submerged cultures of X. campestris was observed with inulin as a carbon source with an initial pH, temperature and agitation of 7.0, 37 degrees C and 150 rev min(-1) respectively. Among the various substrates, garlic peels (117 IU gds(-1)) and onion peels (101 IU gds(-1)) were found to be the best for inulinase production. CONCLUSION: The inulinase production level of X. campestris was 6.7-fold higher in garlic and 5.8-fold in onion, under optimized SSC conditions compared with the submerged culture. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on inulinase production from garlic and onion peels by X. campestris using SSC. SSC is an efficient method for inulinase production by X. campestris for commercial applications.

Phosphomannose isomerase of Xanthomonas campestris: a zinc activated enzyme.

Phosphomannose isomerase (pmi, EC 5.3.1.8) was purified to homogeneity from a wild strain of Xanthomonas campestris. The apparent molecular weight as determined by SDS-PAGE and Sephadex G-100 Superfine was found to be 58 kDa. The purified enzyme showed a single band on acrylamide gel electrophocusing with pI = 5.25. The optimum pH was 7.0 and the Km for D-mannose-6-phosphate was 2 mM. Pmi can be activated by bivalent cations with the order of Co2+>Zn2+>Mn2+>Ni2+>Ca2+. Addition of low concentration of ZnCl2 (2 x 10[-7] M) in the growth medium resulted in the enhancement of pmi activity to around 2.5 x fold. The half life of pmi, as it was measured by the addition of chloramphenicol, was 110 min, whereas in the medium supplemented with ZnCl2 was 270 min. Chemical modification experiments implied the existence of one histidyl residue located at or near the active site.