Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose.
Strains of Thermus thermophilus accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (O. C. Nunes, C. M. Manaia, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 61:2351-2357, 1995). A 2.4-kbp DNA fragment from T. thermophilus strain RQ-1 carrying otsA (encoding trehalose-phosphate synthase [TPS]), otsB (encoding trehalose-phosphate phosphatase [TPP]), and a short sequence of the 5' end of treS (trehalose synthase [TreS]) was cloned from a gene library. The sequences of the three genes (including treS) were amplified by PCR and sequenced, revealing that the genes were structurally linked. To understand the role of trehalose during salt stress in T. thermophilus RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (otsA) and TPP (otsB) genes were disrupted by gene replacement. Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCl. However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCl led to the accumulation of mannosylglycerate but not trehalose. The deletion of otsA and otsB reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCl for RQ-1 and 3% NaCl for RQ-1M6. The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media. In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities. The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in T. thermophilus RQ-1.
Silva
Zélia
Z
Departamento de Bioquímica and Centro de Neurociências de Coimbra, Universidade de Coimbra, 3004-517 Coimbra, Portugal.
Alarico
Susana
S
Nobre
Ana
A
Horlacher
Reinhold
R
Marugg
Joey
J
Boos
Winfried
W
Mingote
Ana I
AI
da Costa
Milton S
MS
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D003470:Culture Media; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D018869:Heat-Shock Response; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development* / Q000502:physiology; D014199:Trehalose / Q000378:metabolism*
D003470:Culture Media; D014199:Trehalose; D005964:Glucosyltransferases; C099692:trehalose synthase; C023180:trehalose-6-phosphate synthase; C023178:trehalose-phosphatase; D010744:Phosphoric Monoester Hydrolases