Glycine betaine biosynthesized from glycine provides an osmolyte for cell growth and spore germination during osmotic stress in Myxococcus xanthus

Abstract

Glycine sarcosine methyltransferase (Gsm) and sarcosine dimethylglycine methyltransferase (Sdm) catalyze glycine betaine synthesis from glycine. Disruption of the M. xanthus gsmA (MXAN 7068) or sdmA (MXAN 3190) gene, encoding Gsm or Sdm homologue proteins, respectively, generated mutants that exhibited a longer lag period of growth and delayed spore germination under osmostress.

FIG. 1.

(A) Alignment of the deduced amino acid sequences of M. xanthus GsmA (Mxa) with H. halochloris Gsm (Hha) and A. halophila Gsdm (Aha). (B) Alignment of the deduced amino acid sequences of M. xanthus SdmA (Mxa) with H. halochloris Sdm (Hha) and A. halophila Gsdm (Aha). Identical amino acid residues are indicated by asterisks. Putative S-adenosylmethionine binding motifs are overlined.

FIG. 2.

RT-PCR analysis of gsmA (lanes 1 and 2) and sdmA (lanes 3 and 4) gene expression in M. xanthus. RT-PCR analysis was performed on RNA prepared from cultures grown in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 0.2 M NaCl for 4 h. The molecular sizes of DNA fragments are given in base pairs (lane M).

FIG. 3.

(A) Changes in intracellular levels of glycine betaine in wild-type vegetative cells and gsmA and sdmA mutant vegetative cells during osmotic stress. Wild-type cells (circles) and gsmA (squares) and sdmA (triangles) mutant cells were incubated in CYE medium with 0.2 M NaCl (open symbols) or 0.2 M sucrose (closed symbols). (B) Changes in intracellular levels of glycine betaine in wild-type spores and gsmA and sdmA mutant spores under osmotic stress. Wild-type spores (circles) and gsmA (squares) and sdmA (triangles) mutant spores harvested from 6- to 8-day-old fruiting bodies on CF plates were incubated in CYE medium with 0.2 M NaCl (open symbols) or 0.2 M sucrose (closed symbols) for 72 h or without either (dashed lines) for 48 h. The spores were harvested by centrifugation, washed twice with distilled water, and sonicated with 0.2 g of 0.4-mm glass beads for 5 min. (C) Changes in intracellular levels of glycine betaine in wild-type cells during development under nonosmotic stress conditions. Cells were harvested from CF agar at various times during development, and glycine betaine was measured in the cell extracts. Experiments were repeated three times. The standard deviations are shown by error bars.