Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11394–11399, September 1998 Microbiology Presence of a gene encoding choline sulfatase in Sinorhizobium meliloti bet operon: Choline-O-sulfate is metabolized into glycine betaine MAGNE ØSTERÅS*, ERIC BONCOMPAGNI†,NADINE VINCENT,MARIE-CHRISTINE POGGI, AND DANIEL LE RUDULIER‡ Laboratoire de Biologie Ve´ge´tale et Microbiologie, Centre National de la Recherche Scientifique Equipe en Restructuration 590, Universite´de Nice–Sophia Antipolis, 06108 Nice Cedex, France Edited by Roland Douce, University of Grenoble, Grenoble, France, and approved July 17, 1998 (received for review April 24, 1998) ABSTRACT Glycine betaine is a potent osmoprotectant been shown that S. meliloti has the ability to use different accumulated by Sinorhizobium meliloti to cope with osmotic compatible solutes in presence of an osmotic stress, it appears that stress. The biosynthesis of glycine betaine from choline is en- glycine betaine is the most potent osmoprotectant and strongly coded by an operon of four genes, betICBA, as determined by stimulates the growth rate of the bacteria in high-salt medium (8). sequence and mutant analysis. The betI and betC genes are Unlike enteric bacteria such as Escherichia coli, S. meliloti also separated by an intergenic region containing a 130-bp mosaic catabolizes glycine betaine and uses it both as a carbon and a element that also is present between the betB and betA genes. In nitrogen source for growth (9, 10). The importance of this addition to the genes encoding a presumed regulatory protein compound in response to osmotic stress is confirmed by the (betI), the betaine aldehyde dehydrogenase (betB), and the cho- observation that the catabolic pathway is repressed in presence of line dehydrogenase (betA) enzymes also found in Escherichia coli, high-salt concentration, favoring the accumulation of the com- pound over its metabolization (10). Glycine betaine either can be a new gene (betC) was identified as encoding a choline sulfatase taken up directly from the environment by specific transport O catalyzing the conversion of choline- -sulfate and, at a lower systems or synthesized from choline by a two-step pathway with rate, phosphorylcholine, into choline. Choline sulfatase activity betaine aldehyde as intermediate (10). This pathway appears to was absent from betC but not from betB mutants and was shown be conserved in bacteria and plants, but shows divergence in the to be induced indifferently by choline or choline-O-sulfate as were enzymes involved. Gram-positive bacteria such as Arthrobacter the other enzymes of the pathway. Unlike what has been shown pascens and A. globiformis and the fungus Cylindrocarpon didy- in other bacteria and plants, choline-O-sulfate is not used as an mun use a soluble choline oxidase to catalyze both steps (11, 12). osmoprotectant per se in S. meliloti, but is metabolized into Higher plants and Gram-negative bacteria both are using a glycine betaine. S. meliloti also can use this compound as the sole conserved betaine aldehyde dehydrogenase to catalyze the be- carbon, nitrogen, and sulfur source for growth and that depends taine aldehyde to glycine betaine reaction. The choline-to-betaine on a functional bet locus. In conclusion, choline-O-sulfate and aldehyde reaction, however, is catalyzed by a choline monooxy- phosphorylcholine, which are found in higher plants and fungi, genase in plants and by a choline dehydrogenase in bacteria such appear to be substrates for glycine betaine biosynthesis in S. as E. coli, Pseudomonas aeruginosa, and S. meliloti (9, 13–15). The meliloti. glycine betaine biosynthesis pathway has been characterized at the molecular level in E. coli and Bacillus subtilis (16, 17), and the betBA genes of S. meliloti have been characterized previously (18). When subjected to an environmental osmotic stress, the most Recently, choline-O-sulfate, synthesized from choline and 39- common response mechanism developed by living organisms is to phosphoadenosine 59-phosphosulfate (PAPS), has been identi- accumulate compatible solutes called osmolytes to maintain a fied as a potent osmoprotectant in some plants (4, 19, 20) and positive cellular turgor essential for metabolic functions and fungi (21, 22) in which it seems to be accumulated essentially for structural integrity. Osmolytes identified until now are either ions sulfate storage. Choline-O-sulfate also is accumulated as an (potassium) or small organic molecules such as amino acids osmolyte in E. coli and B. subtilis that is subjected to an osmotic (glutamate, proline), sugars (trehalose, glucosylglycerol), ectoine, stress but, like glycine betaine in these organisms, cannot be dipeptides, and quartenary amines (stachydrine, glycine betaine) metabolized further (ref. 19; E. Bremer, personal communica- (1–5). Among these compatible solutes, glycine betaine (N,N,N- tion). We report here a genetic and molecular analysis of the S. trimethylglycine) has been shown to be a very efficient osmolyte meliloti bet region that revealed two additional genes encoding a found in a wide range of bacterial and plant species, where it is BetI-like putative repressor and a choline sulfatase. We present accumulated at high cytoplasmic concentrations in response to evidence that the betC gene codes for a choline sulfatase activity osmotic stress. The potential importance of this molecule for that allows S. meliloti to convert choline-O-sulfate and, to a lesser stress resistance among agronomically important organisms has extent, phosphorylcholine into choline. led to investigations of betaine biosynthesis and transport in Sinorhizobium meliloti. This soil bacterium has the ability to MATERIALS AND METHODS develop a symbiotic relationship with alfalfa within specific root Bacterial Strains, Plasmids, and Media. All Sinorhizobium nodules, where it can reduce atmospheric nitrogen into ammonia meliloti strains used in this study are derived from 102F34R, a rif taken up by the plant host (6). However, variations of the osmotic environment within the rhizosphere may affect all steps of the This paper was submitted directly (Track II) to the Proceedings office. plant–microbe interaction, from the root colonization to nodule Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. U39940). development and function, and some osmoadaptative responses *Present address: Biozentrum, University of Basel, 4056 Basel, Swit- of the two partners have been characterized (7). Although it has zerland. †Present address: Department of Biological Sciences, Dartmouth The publication costs of this article were defrayed in part by page charge College, Hanover, NH 03755. ‡To whom reprint requests should be addressed at: Laboratoire de payment. This article must therefore be hereby marked ‘‘advertisement’’ in Biologie Ve´ge´tale et Microbiologie, Centre National de la Recherche accordance with 18 U.S.C. §1734 solely to indicate this fact. Scientifique ERS 590, Faculte´des Sciences, Universite´de Nice– © 1998 by The National Academy of Sciences 0027-8424y98y9511394-6$2.00y0 Sophia Antipolis, Parc Valrose, 06108 Nice Ce´dex,France. e-mail: PNAS is available online at www.pnas.org. [email protected]. 11394 Downloaded by guest on September 26, 2021 Microbiology: Østeras et al. Proc. Natl. Acad. Sci. USA 95 (1998) 11395 derivative of wild-type 102F34 (18). The pCHO341 plasmid an IS50-specific primer (59-TCACATGGAAGTCAGATCCT- contains the S. meliloti bet genes cloned as a 8.6-kb BglII fragment 39). into the TcR vector pRK415 (18). For growth, Luria broth Enzymology. Bacterial cultures were grown in MCAA or (Luria-Bertani; LB) was used as complex medium for E. coli and MCAA 1 0.3 M NaCl, supplemented, when desired, with 7 mM supplemented with 2.5 mM MgSO4 and 2.5 mM CaCl2 (LBmc) choline or choline-O-sulfate, to late log phase. The cells were for S. meliloti. Defined minimal medium used for S. meliloti was washed once with the same volume of S medium and resuspended z either carbon-free M9 (23) or carbon- and nitrogen-free S in 0.1 M K2HPO4, pH 7.6 (for BADH and MDH) or 0.1 M Tris Cl, medium (10). LAS [S medium 1 0.1% (volyvol) lactic acid 1 pH 7.6 (for CHS) at 1y100th of the original volume. Choline 0.1% (wtyvol) aspartic acid] (9) was used for analysis of the dehydrogenase, CDH (EC 1.1.99.1) activity was determined on osmotolerance phenotype. Cells were grown in MCAA [S me- toluene-permeabilized cells by monitoring the production of dium 1 0.1% (wtyvol) malic acid 1 0.1% (wtyvol) casamino [1,2-14C]glycine betaine aldehyde as described in Landfald and acids] (10) for preparation of cell extracts for enzyme assays. Strøm (14). For betaine aldehyde dehydrogenase, BADH (EC Sulfur-free M9 medium was prepared by replacing 1 mM MgSO4 1.2.1.8), choline sulfatase, CHS (EC 3.1.6.6), and malate dehy- with 1 mM MgCl2. Choline, glycine betaine, and choline-O- drogenase, MDH (EC 1.1.1.37) assays, the cells were disrupted by sulfate were prepared as 0.5 M solutions and sterilized by two passages through a French press (8,500 lbyin2), followed by filtration before incorporation into defined media. Their final centrifugation at 12,000 3 g for 10 min at 4°C to remove cell concentration was 1 mM for osmoprotection assays and 10 mM debris. BADH and MDH activities were determined by measur- when used as the carbon, nitrogen, and sulfur source. When ing the NADH production on a spectrophotometer at 340 nm as required, antibiotics were added at concentrations described described previously (10, 37). CHS activity was determined by previously (18, 24). monitoring the [14C]choline formation from [1,2-14C]choline-O- Chemicals. Choline chloride and the choline esters were pur- sulfate or [methyl-14C]phosphorylcholine. No CDH activity chased from SigmayAldrich. Choline-O-sulfate was prepared (which would further metabolize choline) was present in the from choline chloride and sulfuric acid according to Stevens and supernatant of the extract as this enzyme is bound to the cell Vohra (25).