Article Figures & Data
Figures
- FIG. 1.
Methionine biosynthesis in S. meliloti. The probable methionine biosynthetic pathway as annotated previously (http://bioinfo.genopole-toulouse.prd.fr/annotation/iANT/bacteria/rhime/ ) is shown. MetA, homoserine O-succinyltransferase; MetB, cystathionine gamma-synthase; MetC, cystathionine beta-lyase; MetH, 5-methyltetrahydrofolate-homocysteine methyltransferase; BHMT, betaine-homocysteine methyl transferase, as proposed in this study. M-THF represents methyltetrahydrofolate.
- FIG. 2.
Phenotype of the metH mutant. metH (A and B) and wild-type (C and D) strains were grown in LAS medium (A and C) or in 0.5 M NaCl-LAS medium (B and D). Growth was analyzed in regular medium (open squares) or in medium supplemented with 1 mM methionine (open triangles), 1 mM glycine betaine (closed circles), or 1 mM DMG (closed diamonds). OD570, optical density at 570 nm.
- FIG. 3.
Alignment of bacterial BHMTs with the human enzyme. BLAST scanning of the SMc04325 sequence against microbial genomes (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi ) allowed the identification of various putative bacterial BHMT-encoding sequences. The corresponding proteins were aligned with human BHMT using CLUSTALW (http://www.ebi.ac.uk/clustalw/ ). The amino acids interacting with homocysteine and glycine betaine and zinc binding domains in human BHMT are boxed and annotated as HC, GB, and Zn, respectively. Each organism is identified by a three-letter code: Sme, Sinorhizobium meliloti; Mel, Mesorhizobium loti MAFF303099; Sil, Silicibacter pomeroyi DSS-3 (Silicibacter sp. strain TM1040); Jan, Jannaschia sp. strain CCS1; Rds, Rhodobacter sphaeroides 2.4.1; Prd, Paracoccus denitrificans PD1222; Unc, uncultured alphaproteobacterium EBAC2C11; Hsa, Homo sapiens. Asterisks indicate amino acids identical in all sequences, colons indicate conserved substitutions, and periods indicate semiconserved substitutions.
- FIG. 4.
Growth of wild-type and bmt strains on glycine betaine as the carbon and energy source. S. meliloti 102F34 (closed symbols) and the bmt mutant (open symbols) were grown in minimal medium AS supplemented with 10 mM of lactate (squares), glycine betaine (circles), or DMG (triangles) as the carbon and energy source. OD570, optical density at 570 nm.
- FIG. 5.
BHMT assay. BHMT assays were performed on bmt (A) and wild-type (B) strains grown in Vincent medium to mid-exponential growth phase. Reaction products were separated by two-dimensional chromatography. The positions of spots corresponding to glycine betaine (GB), DMG, methionine (met), and sarcosine are indicated.
- FIG. 6.
Effects of betaines on the growth of the bmt mutant. Wild-type (A) and bmt mutant (B) strains were grown in LAS medium lacking NaCl (open circles) or containing 0.5 M NaCl (closed circles) or 0.5 M NaCl medium supplemented with 1 mM glycine betaine (open triangles), 1 mM trigonelline (closed triangles), 1 mM HB (open squares), or 1 mM DMSP (open diamonds). OD570, optical density at 570 nm.
- FIG. 7.
Influences of glycine betaine and methionine on the growth of the bmt mutant. The bmt mutant was grown in LAS medium lacking NaCl (A) or containing 0.5 M of NaCl (B) without any supplementation (open triangles) or in the presence of 1 mM glycine betaine (open circles), 1 mM methionine (open squares), 1 mM glycine betaine plus 1 mM methionine (closed diamonds), or 1 mM glycine betaine plus 1 mM homocysteine (closed circles). The growth rates of the bmt mutant in LAS medium were identical in the absence and in the presence of 1 mM homocysteine. OD570, optical density at 570 nm.
- FIG. 8.
Fate of glycine betaine in the wild-type strain and the bmt mutant. The wild type (A, B, C, and D) and the bmt mutant (E, F, G, and H) were grown in LAS medium containing 1 mM [C14]glycine betaine in the absence (diamonds) or in the presence (circles) of 0.5 M NaCl. Growth was in the absence (closed symbols) or in the presence of 1 mM methionine (open symbols). Growth (A and E) was monitored, and aliquots were periodically harvested and extracted in 80% ethanol. The radioactivities remaining in the growth medium (B and F) and in the ethanol-soluble fraction (C and G) and incorporated in insoluble material (D and H) were quantified by scintillation counting. OD570, optical density at 570 nm.
