Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose

Abstract

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.

FIG. 1.

Alignment of TPS of T. thermophilus RQ-1. The deduced amino acid sequence of otsA of RQ-1 was aligned with those of homologous proteins: Styph, Salmonella enterica serovar Typhimurium (GenPept AF213176); Rhisp., Rhizobium sp. (GenPept AAB91813); Mtube, M. tuberculosis (GenPept CAB08713); Rsola, Ralstonia solanacearum (GenPept CAD17882); Ecoli, E. coli (GenPept AAC74966). Black shading represents identity, and gray shading represents conservative changes.

FIG. 2.

Verification of the internal deletion in otsA and otsB. Southern blots of genomic DNA from T. thermophilus RQ-1 and two kanamycin-resistant clones (RQ-1M6 and RQ-1M8) are shown. Bands were identified by hybridization with a 2.05-kbp fragment corresponding to otsA and otsB (A) or kat (B). Lanes 1, 4, and 7, RQ-1M6; lanes 2, 5, and 8, RQ-1M8; lanes 3, 6, and 9, RQ-1. (C) Genetic organization of trehalose-synthesizing genes in the chromosomes of the wild type and an RQ-1 deletion mutant.

FIG. 3.

Growth of wild-type and mutant T. thermophilus strains in TD medium with 0% (○), 3% (•), 4% (▵), 5% (▴), and 6% (▪) NaCl. RQ-1 (A and B) and RQ-1M6 (C and D) were grown in TD medium in the absence (A and C) or presence (B and D) of exogenous trehalose. OD₆₁₀, optical density at 610 nm.

FIG. 4.

Effect of the presence of exogenous organic solutes on the cell yield of RQ-1 and RQ-1M6. RQ-1 (A) and mutant RQ-1M6 (B) were grown in TD medium containing 4, 5, and 6% NaCl. Within each group, bars represent (from left to right) TD medium without exogenous solutes and TD medium supplemented with mannosylglycerate, glycine betaine, trehalose, maltose, and glucose. OD₆₁₀, optical density at 610 nm.

FIG. 5.

Accumulation of compatible solutes by RQ-1 and RQ-1M6. RQ-1 (A) and RQ-1M6 (B) were grown in TD medium containing 3% NaCl without the addition of exogenous organic solutes (no addition) or with trehalose (Tre), maltose (Mal), or glucose (Glu). Bars represent intracellular concentrations of mannosylglycerate (▪) and trehalose (▧).

FIG. 6.

Effect of salinity on the accumulation of solutes by RQ-1M6. RQ-1M6 was grown in TD medium without (A) or with (B) exogenous trehalose. Bars represent intracellular concentrations of mannosylglycerate (▪) and trehalose (▧).