Rhizobium meliloti NodP and NodQ form a multifunctional sulfate-activating complex requiring GTP for activity.

The nodulation genes nodP and nodQ are required for production of Rhizobium meliloti nodulation (Nod) factors. These sulfated oligosaccharides act as morphogenic signals to alfalfa, the symbiotic host of R. meliloti. In previous work, we have shown that nodP and nodQ encode ATP sulfurylase, which catalyzes the formation of APS (adenosine 5'-phosphosulfate) and PPi. In the subsequent metabolic reaction, APS is converted to PAPS (3'-phosphoadenosine 5'-phosphosulfate) by APS kinase. In Escherichia coli, cysD and cysN encode ATP sulfurylase; cysC encodes APS kinase. Here, we present genetic, enzymatic, and sequence similarity data demonstrating that nodP and nodQ encode both ATP sulfurylase and APS kinase activities and that these enzymes associate into a multifunctional protein complex which we designate the sulfate activation complex. We have previously described the presence of a putative GTP-binding site in the nodQ sequence. The present report also demonstrates that GTP enhances the rate of PAPS synthesis from ATP and sulfate (SO4(2-)) by NodP and NodQ expressed in E. coli. Thus, GTP is implicated as a metabolic requirement for synthesis of the R. meliloti Nod factors.     

Reduction of adenosine-5'-phosphosulfate instead of 3'-phosphoadenosine-5'-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and other members of the family Rhizobiaceae.

We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizobium meliloti) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the Escherichia coli cysDN genes, which code for an ATP sulfurylase (EC 2.7.7.4). The third gene has homology to the E. coli cysH gene, a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase (EC 1.8.99.4), but has greater homology to a set of genes found in Arabidopsis thaliana that encode an adenosine-5'-phosphosulfate (APS) reductase. In order to determine the specificity of the R. meliloti reductase, the R. meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the A. thaliana reductases, the histidine-tagged R. meliloti cysH gene product appears to favor APS over PAPS as a substrate, with a Km for APS of 3 to 4 microM but a Km for PAPS of >100 microM. In order to determine whether this preference for APS is unique to R. meliloti among members of the family Rhizobiaceae or is more widespread, cell extracts from R. leguminosarum, Rhizobium sp. strain NGR234, Rhizobium fredii (Sinorhizobium fredii), and Agrobacterium tumefaciens were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS.     

Characterization of two Azospirillum brasilense Sp7 plasmid genes homologous to Rhizobium meliloti nodPQ

Bacteria belonging to the Azospirillum genus are nitrogen fixers that colonize the roots of grasses, but do not cause the formation of differentiated structures. Sequences from total DNA of several Azospirillum strains are homologous to restriction fragments containing Rhizobium meliloti nodulation genes. A 10-kilobase (kb) EcoRI fragment from A. brasilense Sp7, sharing homology with a 6.8-kb EcoRI fragment carrying nodGEFH and part of nodP of R. meliloti 41, was cloned in pUC18 to yield pAB502. The nucleotide sequence of a 3.5-kb EcoRI-SmaI fragment of the pAB502 insert revealed 60% homology with R. meliloti nodP and nodQ genes. The nodP gene product shares no homology to any known protein sequence. The Azospirillum nodQ gene product shares homology with a family of initiation and elongation factors as does the R. meliloti nodQ gene product. Since the nodQ gene overlaps the nodP gene, the two genes might be cotranscribed. Azospirillum contains large plasmids, and the nodPQ genes were found on the 90-MDa plasmid (p90). A translational nodP-lacZ fusion was constructed in the broad host range plasmid pGD926. No beta-galactosidase activity was detected in Escherichia coli, but the fusion was functional in Azospirillum and constitutively expressed. Deletions and mutations of nodPQ did not modify growth, nitrogen fixation, or interaction with wheat seedlings.     

Identification of a third sulfate activation system in Sinorhizobium sp. strain BR816: the CysDN sulfate activation complex

Sinorhizobium sp. strain BR816 possesses two nodPQ copies, providing activated sulfate (3'-phosphoadenosine-5'-phosphosulfate [PAPS]) needed for the biosynthesis of sulfated Nod factors. It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain. In this study, we describe the characterization of a third sulfate activation locus. Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes. The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined. FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent. On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors. Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources. In this way, it could be demonstrated that the "household" CysDN sulfate activation complex of Sinorhizobium sp. strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids. Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution.     

The DNA sequence of the sulfate activation locus from Escherichia coli K-12.

The DNA sequence of the sulfate activation locus from Escherichia coli K-12 has been determined. The sequence includes the structural genes encoding the enzymes ATP sulfurylase (cysD and cysN) and APS kinase (cysC) which catalyze the synthesis of activated sulfate. These are the only genes known to reside in the sulfate activation operon. Consensus elements of the operon promoter were identified, and the start codons and open reading frames of the Cys polypeptides were determined. During this work, another gene, iap, was partially sequenced and mapped. The activity of ATP sulfurylase is stimulated by an intrinsic GTPase. Comparison of the primary sequences of CysN and Ef-Tu revealed that CysN has conserved many of the residues integral to the three-dimensional structure important for guanine nucleotide binding in Ef-Tu and RAS. nodP and nodQ, from Rhizobium meliloti, are essential for nodulation in leguminous plants. The Cys and Nod proteins are remarkably similar. NodP appears to be the smaller subunit of ATP sulfurylase. NodQ encodes homologues of both CysN and CysC; thus, these enzymes may be covalently associated in R. meliloti. The consensus GTP-binding sequences of NodQ and CysN are identical suggesting that NodQ encodes a regulatory GTPase.     

Characterization of the gene encoding the autotrophic ATP sulfurylase from the bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

ATP sulfurylase is a key enzyme in the energy-generating sulfur oxidation pathways of many chemoautotrophic bacteria. The utilization of reduced sulfur compounds to fuel CO2 fixation by the still-uncultured bacterial endosymbionts provides the basis of nutrition in invertebrates, such as the tubeworm Riftia pachyptila, found at deep-sea hydrothermal vents. The symbiont-containing trophosome tissue contains high levels of ATP sulfurylase activity, facilitating the recent purification of the enzyme. The gene encoding the ATP sulfurylase from the Riftia symbiont (sopT) has now been cloned and sequenced by using the partial amino acid sequence of the purified protein. Characterization of the sopT gene has unequivocally shown its bacterial origin. This is the first ATP sulfurylase gene to be cloned and sequenced from a sulfur-oxidizing bacterium. The deduced amino acid sequence was compared to those of ATP sulfurylases reported from organisms which assimilate sulfate, resulting in the discovery that there is substantial homology with the Saccharomyces cerevisiae MET3 gene product but none with the products of the cysDN genes from Escherichia coli nor with the nodP and nodQ genes from Rhizobium meliloti. This and emerging evidence from other sources suggests that E. coli may be atypical, even among prokaryotic sulfate assimilators, in the enzyme it employs for adenosine 5'-phosphosulfate formation. The sopT gene probe also was shown to specifically identify chemoautotrophic bacteria which utilize ATP sulfurylase to oxidize sulfur compounds.     

ATP sulfurylase from the hyperthermophilic chemolithotroph Aquifex aeolicus

ATP sulfurylase from the hyperthermophilic chemolithotroph Aquifex aeolicus is a bacterial ortholog of the enzyme from filamentous fungi. (The subunit contains an adenosine 5'-phosphosulfate (APS) kinase-like, C-terminal domain.) The enzyme is highly heat stable with a half-life >1h at 90 degrees C. Steady-state kinetics are consistent with a random A-B, ordered P-Q mechanism where A=MgATP, B=SO4(2-), P=PP(i), and Q=APS. The kinetic constants suggest that the enzyme is optimized to act in the direction of ATP+sulfate formation. Chlorate is competitive with sulfate and with APS. In sulfur chemolithotrophs, ATP sulfurylase provides an efficient route for recycling PP(i) produced by biosynthetic reactions. However, the protein possesses low APS kinase activity. Consequently, it may also function to produce PAPS for sulfate ester formation or sulfate assimilation when hydrogen serves as the energy source and a reduced inorganic sulfur source is unavailable.     

Structural and functional analysis of a truncated form of Saccharomyces cerevisiae ATP sulfurylase: C-terminal domain essential for oligomer formation but not for activity

ATP sulfurylase catalyzes the first step in the activation of sulfate by transferring the adenylyl-moiety (AMP approximately ) of ATP to sulfate to form adenosine 5'-phosphosulfate (APS) and pyrophosphate (PP(i)). Subsequently, APS kinase mediates transfer of the gamma-phosphoryl group of ATP to APS to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and ADP. The recently determined crystal structure of yeast ATP sulfurylase suggests that its C-terminal domain is structurally quite independent from the other domains, and not essential for catalytic activity. It seems, however, to dictate the oligomerization state of the protein. Here we show that truncation of this domain results in a monomeric enzyme with slightly enhanced catalytic efficiency. Structural alignment of the C-terminal domain indicated that it is extremely similar in its fold to APS kinase although not catalytically competent. While carrying out these structural and functional studies a surface groove was noted. Careful inspection and modeling revealed that the groove is sufficiently deep and wide, as well as properly positioned, to act as a substrate channel between the ATP sulfurylase and APS kinase-like domains of the enzyme.     

Enzymatic synthesis of PAPS with an ATP-regeneration system.

Sulfate activating enzymes, ATP sulfurylase and APS kinase, were newly isolated from a thermophile, Bacillus stearothermophilus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS) was synthesized by these enzymes. The reaction proceeded more efficiently when an ATP-regeneration system, using acetate kinase, was coupled to the reaction system.     

Crystal structure of the bifunctional ATP sulfurylase-APS kinase from the chemolithotrophic thermophile Aquifex aeolicus

The thermophilic chemolithotroph, Aquifex aeolicus, expresses a gene product that exhibits both ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase activities. These enzymes are usually segregated on two separate proteins in most bacteria, fungi, and plants. The domain arrangement in the Aquifex enzyme is reminiscent of the fungal ATP sulfurylase, which contains a C-terminal domain that is homologous to APS kinase yet displays no kinase activity. Rather, in the fungal enzyme, the motif serves as a sulfurylase regulatory domain that binds the allosteric effector 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the product of true APS kinase. Therefore, the Aquifex enzyme may represent an ancestral homolog of a primitive bifunctional enzyme, from which the fungal ATP sulfurylase may have evolved. In heterotrophic sulfur-assimilating organisms such as fungi, ATP sulfurylase catalyzes the first committed step in sulfate assimilation to produce APS, which is subsequently metabolized to generate all sulfur-containing biomolecules. In contrast, ATP sulfurylase in sulfur chemolithotrophs catalyzes the reverse reaction to produce ATP and sulfate from APS and pyrophosphate. Here, the 2.3 A resolution X-ray crystal structure of Aquifex ATP sulfurylase-APS kinase bifunctional enzyme is presented. The protein dimerizes through its APS kinase domain and contains ADP bound in all four active sites. Comparison of the Aquifex ATP sulfurylase active site with those from sulfate assimilators reveals similar dispositions of the bound nucleotide and nearby residues. This suggests that minor perturbations are responsible for optimizing the kinetic properties for the physiologically relevant direction. The APS kinase active-site lid adopts two distinct conformations, where one conformation is distorted by crystal contacts. Additionally, a disulfide bond is observed in one ATP-binding P-loop of the APS kinase active site. This linkage accounts for the low kinase activity of the enzyme under oxidizing conditions. The thermal stability of the Aquifex enzyme can be explained by the 43% decreased cavity volume found within the protein core.     

Induction of positive cooperativity by amino acid replacements within the C-terminal domain of Penicillium chrysogenum ATP sulfurylase

ATP sulfurylase from Penicillium chrysogenum is an allosteric enzyme in which Cys-509 is critical for maintaining the R state. Cys-509 is located in a C-terminal domain that is 42% identical to the conserved core of adenosine 5'-phosphosulfate (adenylylsulfate) (APS) kinase. This domain is believed to provide the binding site for the allosteric effector, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Replacement of Cys-509 with either Tyr or Ser destabilizes the R state, resulting in an enzyme that is intrinsically cooperative at pH 8 in the absence of PAPS. The kinetics of C509Y resemble those of the wild type enzyme in which Cys-509 has been covalently modified. The kinetics of C509S resemble those of the wild type enzyme in the presence of PAPS. It is likely that the negative charge on the Cys-509 side chain helps to stabilize the R state. Treatment of the enzyme with a low level of trypsin results in cleavage at Lys-527, a residue that lies in a region analogous to a PAPS motif-containing mobile loop of true APS kinase. Both mutant enzymes were cleaved more rapidly than the wild type enzyme, suggesting that movement of the mobile loop occurs during the R to T transition.     

The Rhizobium meliloti host range nodQ gene encodes a protein which shares homology with translation elongation and initiation factors.

The Rhizobium meliloti nod region IIb is involved in host-range determination: (i) the presence of region IIb is necessary for transfer of alfalfa root hair curling ability to Rhizobium leguminosarum biovar trifolii; (ii) a mutation in region IIb extends the R. meliloti infection host range to Vicia sativa nigra; (iii) dominance of R. meliloti nod genes over R. leguminosarum biovar viciae nod genes is abolished by mutations in region IIb. The nucleotide sequence of this region has been determined. Genes corresponding to the two open reading frames identified are designated nodP and nodQ. The predicted amino acid sequence of the NodQ protein shows homology with translation initiation and elongation factors. The consensus sequence involved in the GTP-binding domain is conserved.     

Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.

Sulfation is a major modification of many molecules in eukaryotes that is dependent on the enzymatic synthesis of an activated sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). While sulfate activation has long been assumed to occur in the cytosol, we show in this study that human PAPS synthetase 1 (PAPSS1), a bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase enzyme sufficient for PAPS synthesis, accumulates in the nucleus of mammalian cells. Nuclear targeting of the enzyme is mediated by its APS kinase domain and requires a catalytically dispensable 21 amino acid sequence at the amino terminus. Human PAPSS1 and Drosophila melanogaster PAPSS localize to the nucleus in yeast and relieve the methionine auxotrophy of ATP sulfurylase- or APS kinase-deficient strains, suggesting that PAPSS1 is fully functional in vivo when targeted to the nucleus. A second PAPS synthetase gene, designated PAPSS2, has recently been described, mutations of which are responsible for abnormal skeletal development in human spondyloepimetaphyseal dysplasia and murine brachymorphism. We found that PAPSS2, which localizes to the cytoplasm when ectopically expressed in mammalian cells, is relocated to the nucleus when coexpressed with PAPSS1. Taken together, these results indicate that a sulfation pathway might exist in the nucleus of eukaryotic cells. -Besset, S., Vincourt, J.-B., Amalric, F., Girard, J.-P. Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.     

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase (isoform 1, brain): kinetic properties of the adenosine triphosphate sulfurylase and adenosine 5'-phosphosulfate kinase domains

Recombinant human 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase, isoform 1 (brain), was purified to near-homogeneity from an Escherichia coli expression system and kinetically characterized. The native enzyme, a dimer with each 71 kDa subunit containing an adenosine triphosphate (ATP) sulfurylase and an adenosine 5'-phosphosulfate (APS) kinase domain, catalyzes the overall formation of PAPS from ATP and inorganic sulfate. The protein is active as isolated, but activity is enhanced by treatment with dithiothreitol. APS kinase activity displayed the characteristic substrate inhibition by APS (K(I) of 47.9 microM at saturating MgATP). The maximum attainable activity of 0.12 micromol min(-1) (mg of protein)(-1) was observed at an APS concentration ([APS](opt)) of 15 microM. The theoretical K(m) for APS (at saturating MgATP) and the K(m) for MgATP (at [APS](opt)) were 4.2 microM and 0.14 mM, respectively. At likely cellular levels of MgATP (2.5 mM) and sulfate (0.4 mM), the overall endogenous rate of PAPS formation under optimum assay conditions was 0.09 micromol min(-1) (mg of protein)(-1). Upon addition of pure Penicillium chrysogenum APS kinase in excess, the overall rate increased to 0.47 micromol min(-1) (mg of protein)(-1). The kinetic constants of the ATP sulfurylase domain were as follows: V(max,f) = 0.77 micromol min(-1) (mg of protein)(-1), K(mA(MgATP)) = 0.15 mM, K(ia(MgATP)) = 1 mM, K(mB(sulfate)) = 0.16 mM, V(max,r) = 18.7 micromol min(-1) (mg of protein)(-1), K(mQ(APS)) = 4.8 microM, K(iq(APS)) = 18 nM, and K(mP(PPi)) = 34.6 microM. The (a) imbalance between ATP sulfurylase and APS kinase activities, (b) accumulation of APS in solution during the overall reaction, (c) rate acceleration provided by exogenous APS kinase, and (d) availability of both active sites to exogenous APS all argue against APS channeling. Molybdate, selenate, chromate ("chromium VI"), arsenate, tungstate, chlorate, and perchlorate bind to the ATP sulfurylase domain, with the first five serving as alternative substrates that promote the decomposition of ATP to AMP and PP(i). Selenate, chromate, and arsenate produce transient APX intermediates that are sufficiently long-lived to be captured and 3'-phosphorylated by APS kinase. (The putative PAPX products decompose to adenosine 3',5'-diphosphate and the original oxyanion.) Chlorate and perchlorate form dead-end E.MgATP.oxyanion complexes. Phenylalanine, reported to be an inhibitor of brain ATP sulfurylase, was without effect on PAPS synthetase isoform 1.     

Sulfate-activating enzymes of Penicillium chrysogenum. The ATP sulfurylase.adenosine 5'-phosphosulfate complex does not serve as a substrate for adenosine 5'-phosphosulfate kinase

At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a substrate for APS kinase, i.e. there is no "substrate channeling" of APS between the two sulfate-activating enzymes. APS kinase had no effect on the [S]0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound [35S]APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivation of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a "3'-phosphoadenosine 5'-phosphosulfate synthetase" complex.     

Activity and stability of recombinant bifunctional rearranged and monofunctional domains of ATP-sulfurylase and adenosine 5'-phosphosulfate kinase

Murine adenosine 3'-phosphate 5'-phosphosulfate (PAPS) synthetase consists of a COOH-terminal ATP-sulfurylase domain covalently linked through a nonhomologous intervening sequence to an NH2-terminal adenosine 5'-phosphosulfate (APS) kinase domain forming a bifunctional fused protein. Possible advantages of bifunctionality were probed by separating the domains on the cDNA level and expressing them as monofunctional proteins. Expressed protein generated from the ATP-sulfurylase domain alone was fully active in both the forward and reverse sulfurylase assays. APS kinase-only recombinants exhibited no kinase activity. However, extension of the kinase domain at the COOH terminus by inclusion of the 36 residue linker region restored kinase activity. An equimolar mixture of the two monofunctional enzymes catalyzed the overall reaction (synthesis of PAPS from ATP + SO42-) comparably to the fused bifunctional enzyme. The importance of the domain order and organization was demonstrated by generation of a series of rearranged recombinants in which the order of the two active domains was reversed or altered relative to the linker region. The critical role of the linker region was established by generation of recombinants that had the linker deleted or rearranged relative to the two active domains. The intrinsic stability of the various recombinants was also investigated by measuring enzyme deactivation as a function of time of incubation at 25 or 37 degrees C. The expressed monofunctional ATP-sulfurylase, which was initially fully active, was unstable compared with the fused bifunctional wild type enzyme, decaying with a t1/2 of 10 min at 37 degrees C. Progressive extension by addition of kinase sequence at the NH2-terminal side of the sulfurylase recombinant eventually stabilized sulfurylase activity. Sulfurylase activity was significantly destabilized in a time-dependent manner in the rearranged proteins as well. In contrast, no significant deactivation of any truncated kinase-containing recombinants or misordered kinase recombinants was observed at either temperature. It would therefore appear that fusion of the two enzymes enhances the intrinsic stability of the sulfurylase only.     

The Xanthomonas oryzae pv. lozengeoryzae raxP and raxQ genes encode an ATP sulphurylase and adenosine-5'-phosphosulphate kinase that are required for AvrXa21 avirulence activity

Xanthomonas oryzae pv. oryzae (Xoo) Philippine race 6 (PR6) is unable to cause bacterial blight disease on rice lines containing the rice resistance gene Xa21 but is virulent on non-Xa21 rice lines, indicating that PR6 carries avirulence (avrXa21) determinants required for recognition by XA21. Here we show that two Xoo genes, raxP and raxQ, are required for AvrXa21 activity. raxP and raxQ, which reside in a genomic cluster of sulphur assimilation genes, encode an ATP sulphurylase and APS (adenosine-5'-phosphosulphate) kinase. These enzymes function together to produce activated forms of sulphate, APS and PAPS (3'-phosphoadenosine-5'-phosphosulphate). Xoo PR6 strains carrying disruptions in either gene, PR6DeltaraxP or PR6DeltaraxQ, are unable to produce APS and PAPS and are virulent on Xa21-containing rice lines. RaxP and RaxQ are similar to the bacterial symbiont Sinorhizobium meliloti host specificity proteins, NodP and NodQ and the Escherichia coli cysteine synthesis proteins CysD, CysN and CysC. The APS and PAPS produced by RaxP and RaxQ are used for both cysteine synthesis and sulphation of other molecules. Mutation in Xoo xcysI, a homologue of Escherichia coli cysI that is required for cysteine synthesis, blocked APS- or PAPS-dependent cysteine synthesis but did not affect AvrXa21 activity, suggesting that AvrXa21 activity is related to sulphation rather than cysteine synthesis. Taken together, these results demonstrate that APS and PAPS production plays a critical role in determining avirulence of a phytopathogen and reveal a commonality between symbiotic and phytopathogenic bacteria.