Presence of a gene encoding choline sulfatase in Sinorhizobium meliloti bet operon: choline-O-sulfate is metabolized into glycine betaine

Abstract

Glycine betaine is a potent osmoprotectant accumulated by Sinorhizobium meliloti to cope with osmotic stress. The biosynthesis of glycine betaine from choline is encoded by an operon of four genes, betICBA, as determined by sequence and mutant analysis. The betI and betC genes are separated by an intergenic region containing a 130-bp mosaic element that also is present between the betB and betA genes. In addition to the genes encoding a presumed regulatory protein (betI), the betaine aldehyde dehydrogenase (betB), and the choline dehydrogenase (betA) enzymes also found in Escherichia coli, a new gene (betC) was identified as encoding a choline sulfatase catalyzing the conversion of choline-O-sulfate and, at a lower rate, phosphorylcholine, into choline. Choline sulfatase activity was absent from betC but not from betB mutants and was shown to be induced indifferently by choline or choline-O-sulfate as were the other enzymes of the pathway. Unlike what has been shown in other bacteria and plants, choline-O-sulfate is not used as an osmoprotectant per se in S. meliloti, but is metabolized into glycine betaine. S. meliloti also can use this compound as the sole carbon, nitrogen, and sulfur source for growth and that depends on a functional bet locus. In conclusion, choline-O-sulfate and phosphorylcholine, which are found in higher plants and fungi, appear to be substrates for glycine betaine biosynthesis in S. meliloti.

Figure 1

(A) Physical and genetic map of the pROX341 plasmid containing the bet genes of S. meliloti. The insertions with the allele number are shown by arrowheads below the map. The arrowhead content indicates a minus (solid), leaky (gray), or wild-type (open) choline growth phenotype. The position of the two mosaic elements SmbetIC and SmbetBA is indicated by shaded boxes. Restriction sites shown are ApaI (A), BglII (B), EcoRI (E), HindIII (H), PstI (P), and SmaI (S). (B) Sequence alignment of the four mosaic elements and the four palindromes that were identified in Rhizobiaceae. The numbers on the left show the position of the elements within the published sequences. The conserved left and right domains are boxed, and the three palindromes (A, B, and C) are indicated by solid arrows. The flanking left (L) and right (R) heptamers are shown by open arrows. Only the 5-bp flanking sequence of the central region is given with the number of remaining nucleotides (N). Sm, S. meliloti; Rlt, R. leguminosarum bv. trifolii; Sf, S. fredii; Rt, R. tropicii; Bj, B. japonicum.

Figure 2

clustalv alignment of the deduced amino acid sequence for S. meliloti BetI (A) and BetC (B). The BetI regulator (SmBetI) is compared with its homologue in E. coli (EcBetI). The choline sulfatase (SmBetC) is compared with the phosphonate monoester hydrolase of Burkholderia caryophilli (BcPehA), the hypothetical YidJ protein of E. coli (EcYidJ), and the arylsulfatases of Pseudomonas aeruginosa (PaAtsa), E. coli (EcAslA), and Klebsiella pneumoniae (KpAtsA). Boxes numbered 1–6 indicate conserved domains in all six sequences. Strictly conserved residues are shown in black, and residues conserved in at least four sequences are shown in gray.

Figure 3

Osmoprotection growth phenotype of S. meliloti strains 102F34R (A), UNA156 (B), and UNA208 (C) grown in LAS + 0.5 M NaCl (•) supplemented with 1 mM of glycine betaine (○), choline (X), choline-O-sulfate (■), or phosphorylcholine (□). Growth phenotype (D) of S. meliloti strain 102F34R in sulfur-free M9 medium (NaCl, 0) supplemented with 10 mM of glycine betaine + MgSO₄ (•), glycine betaine (○), choline-O-sulfate + MgSO₄ (■), or choline-O-sulfate (□), and of strain UNA208 in the same M9 medium supplemented with choline-O-sulfate + MgSO₄ (X).

Figure 4

(A) Autoradiography after electrophoresis of cell extracts incubated 30 min with ¹⁴C-choline-O-sulfate. Cell extracts were obtained from wild-type strain 102F34R grown in MCAA (lane 1), MCAA + choline (lane 2), MCAA + choline-O-sulfate (lane 4), and sulfur-free M9 with 0.1% (wt/vol) methionine (lane 3). Cell extracts from mutants UNA208 (lane 5) and UNA156 (lane 6) were obtained after growth in MCAA + choline-O-sulfate. (B) Choline sulfatase activity determined from wild-type 102F34R cell extracts grown under different conditions. Each value was obtained from duplicate assays. (C) Competition assay with 10- (open) and 100-fold (solid) excess competitor compared with choline-O-sulfate using wild-type cell extract grown in MCAA + choline. Shaded column, no competitor. Cho, choline; Cho-O-S, choline-O-sulfate; GB, glycine betaine; Pcho, phosphorylcholine; AcCho, acetylcholine; PdylCho, phosphatidylcholine; GlyCho, glycerophosphatidylcholine.