Distribution of genes for synthesis of trehalose and Mannosylglycerate in Thermus spp. and direct correlation of these genes with halotolerance

Abstract

In this study we correlate the presence of genes leading to the synthesis of trehalose and mannosylglycerate (MG) in 17 strains of the genus Thermus with the ability of the strains to grow and accumulate these compatible solutes in a defined medium containing NaCl. The two sets of genes, namely, otsA/otsB for the synthesis of trehalose and mpgS/mpgP for the synthesis of MG, were necessary for the growth of Thermus thermophilus in a defined medium containing up to 6% NaCl. Strains lacking a complete otsA gene did not grow in defined medium containing >2% NaCl. One strain of T. thermophilus lacking the genes for the synthesis of MG did not grow in a medium with >1% NaCl. We did not identify any of these genes in the type strains of the other seven species of Thermus, and none of those strains grew in defined medium with 1% NaCl. The results strongly indicate that the combined accumulation of trehalose and MG is required for optimal osmotic adjustment.

FIG. 1.

Schematic representation of primers used for PCR amplifications (A) and comparison of the organizations of genes for the synthesis of trehalose and mannosylglycerate (B) in T. thermophilus strains RQ-1, HB27, Fiji3 A1, PRQ-14, AT-62, B, T-2, CC-16, HB8, and GK24. The mpgS and mpgP sequences of strain HB27; the otsA, otsB, and treS sequences of strain RQ-1; the treS sequence of strain AT-62; and the otsB and treS sequences of strain GK24 were previously described (12, 16, 26, 31). The identities (%) of the deduced amino acid sequences to the corresponding proteins from RQ-1 and HB27 are shown inside the arrows. Unshaded arrows represent the α-glucosidase gene (EC 3.2.1.20).

FIG. 2.

Southern blot analysis of BamHI-digested genomic DNA (5 μg) probed with the otsA gene of strain RQ-1 (A) and the mpgS gene of strain HB27 (B) at 60°C. Lane 1, strain RQ-1; lane 2, strain Fiji3 A1; lane 3, strain HB27; lane 4, strain HB8; lane 5, strain AT-62; lane 6, strain CC-16; lane 7, Thermus aquaticus (DSM 625); lane 8, Thermus scotoductus (DSM 8553); lane 9, Thermus filiformis (DSM 4687); lane 10, strain B.

FIG. 3.

Effect of NaCl concentration in TD medium on the growth rate of T. thermophilus. (A) Strains B (•), Fiji3 A1 (○), T-2 (▪), PRQ-14 (□), and RQ-1 (▴); (B) strains HB8 (▵), HB27 (♦), AT-62 (⋄), and GK24 (▾); (C) strain CC-16 (▿).

FIG. 4.

Accumulation of compatible solutes in TD medium by Thermus thermophilus strains B, Fiji3 A1, T-2, PRQ-14, and RQ-1. The concentrations of compatible solutes were determined for cultures grown in medium containing 4% NaCl. Bars represent the intracellular concentrations of mannosylglycerate (black), trehalose (white), and glycine betaine (gray).

FIG. 5.

Accumulation of compatible solutes by Thermus thermophilus strains AT-62, HB27, GK24, and HB8. The concentrations of compatible solutes were determined for cultures grown in TD medium containing 2% NaCl (A) and TD medium containing 2% NaCl and exogenous trehalose (B). Bars represent the intracellular concentrations of mannosylglycerate (black) and trehalose (white).

FIG. 6.

Representation of maximum NaCl concentrations for strains of Thermus thermophilus during growth in TD medium at 70°C. The strains clustered into three groups according to their halotolerance and the nature of accumulated compatible solutes. MG, mannosylglycerate; Tre, trehalose.