Identification of a Third Sulfate Activation System inSinorhizobiumsp. Strain BR816: the CysDN Sulfate Activation Complex

Abstract

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.Sulfur is a macronutrient that is required by all organisms. It forms constituents of proteins, lipids, carbohydrates, electron carriers, and numerous cellular metabolites. Sulfate is the most abundant source of utilizable sulfur in the aerobic biosphere. The sulfate assimilation complex, required for the formation of the sulfur-containing amino acid cysteine, has been the subject of intensive study in Escherichia coli (21). Cysteine is the central precursor of all organic molecules containing reduced sulfur, ranging from the amino acid methionine to peptides, proteins, vitamins, cofactors such as S-adenosylmethionine, and hormones.Like all inorganic nutrients, sulfate is transported into cells by highly specific membrane transport systems (18). Sulfate assimilation requires its prior activation to adenylate compounds via a pathway that seems to be similar in all organisms. The activation is achieved by the ATP sulfurylase-catalyzed reaction of sulfate with ATP to give adenosine 5Ј-phosphosulfate (APS), coupled with GTP hydrolysis. Subsequently, APS is phosphorylated by an APS kinase to produce 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS). In E. coli, ATP sulfurylase is encoded by cysD and cysN, whereas the APS kinase is encoded by cysC (27,28). PAPS is then enzymatically reduced by the cysH-encoded PAPS reductase (also known as ...

Interrelations between Glycine Betaine Catabolism and Methionine Biosynthesis in Sinorhizobium meliloti Strain 102F34

“…The nodulation efficiencies of methionine auxotrophs of other rhizobia are also affected, as in the cases of a metZ mutant of Rhizobium etli (44) and a cysD derivative of Sinorhizobium sp. strain BR816 (40). In contrast, the cysG derivative of R. etli forms effective nodules (45).…”

Regulation of Sulfur Assimilation Pathways in Burkholderia cenocepacia : Identification of Transcription Factors CysB and SsuR and Their Role in Control of Target Genes

“…The translated product, CysD2, shares 44% identity and 66% similarity with CysD1, and it is also similar (ϳ70%) to the NodP-type proteins of Rhizobiaceae. The product of cysNC is likely to be a fusion protein with ATP sulfurylase and APS kinase activities, as judged by comparisons of the CysNC Bc and CysNC proteins of P. aeruginosa and Mycobacterium tuberculosis (42) and the NodQ-type proteins of Rhizobiaceae (49) and RaxQ of Xanthomonas oryzae (46). It seems, therefore, that Burkholderia species, like Rhizobiaceae and Mycobacteriaceae but unlike Enterobacteriaceae, can channel the intracellular sulfate either to the reductive pathway (functions encoded in the cluster cysI cysH orf cysD1 cysN cysG) or to sulfatation processes that require phosphoadenosine 5Ј phosphosulfate as a sulfate donor (the latter being produced by the sulfate-activating complex CysD2/CysNC).…”

Regulatory Role of Rhizobium etli CNPAF512 fnrN during Symbiosis

“…Seeds were germinated for 3 days on water agar plates (15 g/liter) in the dark at 30°C. The seedlings were transferred to 250-ml conical bottles filled with 150-ml agar slants (1.2 g of agar/150 ml) of Snoeck medium, which is optimized for in vitro growth of common bean (46).…”