Strains of) 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.Thermophilic organisms, like the vast majority of other microorganisms, accumulate compatible solutes in response to water stress imposed by salt. However, the compatible solutes of thermophilic and hyperthermophilic prokaryotes are generally different from those of their mesophilic counterparts (34), and some compatible solutes, namely, di-myo-inositolphosphate, di-mannosyl-di-myo-inositol-phosphate, diglycerol phosphate, and mannosylglyceramide, are confined to organisms that grow at extremely high temperatures. Mannosylglycerate is also a common compatible solute of thermophiles and hyperthermophiles (21,28,35). Despite this association with organisms that grow at extremely high temperatures, mannosylglycerate was initially identified in red algae of the order Ceramiales (4, 15). Trehalose, a canonical compatible solute of mesophiles, also accumulates in a few thermophilic and hyperthermophilic organisms, where it appears to serve as a compatible solute (18,21,35). This nonreducing disaccharide has also been implicated in several stress responses in prokaryotes and eukaryotic microorganisms (10,32,36,37) and also serves as an intermediate in the synthesis of glycolipids, sulfolipids, and lipooligosaccharides in mycobacteria (3).The most common pathway for the synthesis of trehalose in bacteria involves trehalose-phosphate synthase (TPS), encoded by the gene otsA, which converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. This intermediate is subsequently dephosphorylated to yield trehalose via a specific trehalose-6-phosphate phosphatase (TPP), encoded by otsB (12). Another pathway converts maltose to trehalose via a trehalose synthase encoded by treS. The species of the...

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The nutritionally versatile and naturally competent soil bacterium Acinetobacter baylyi copes with salt stress by the accumulation of compatible solutes. NMR analyses revealed that cells amassed glutamate and the rather unusual sugar alcohol mannitol upon an increase of the external NaCl concentration. To unravel the path of mannitol biosynthesis, the genome was inspected for genes potentially involved in its biosynthesis. A gene encoding a potential mannitol-1-phosphate dehydrogenase (mtlD) was identified in the genome of A. baylyi. Expression of mtlD was highly induced at high salinity. mtlD was overexpressed and the purified protein indeed produced mannitol-1-phosphate from fructose-6-phosphate. The enzyme preferred NADPH over NADH and the specific activity of fructose-6-phosphate reduction with NADPH was 130 U mg(-1) . Enzymatic activity was strictly salt-dependent. Deletion of mtlD resulted in a complete loss of salt-dependent mannitol biosynthesis. We provide clear evidence that osmo-induced synthesis of the compatible solute mannitol is by a two-step pathway and that the mannitol-1-phosphate dehydrogenase mediating the first step of this pathway is regulated by salinity on the transcriptional as well as on the activity level.

␣-Phosphoglucomutase (␣-PGM) plays an important role in carbohydrate metabolism by catalyzing the reversible conversion of ␣-glucose 1-phosphate to glucose 6-phosphate. Isolation of ␣-PGM activity from cell extracts of Lactococcus lactis strain MG1363 led to the conclusion that this activity is encoded by yfgH, herein renamed pgmH. Its gene product has no sequence homology to proteins in the ␣-D-phosphohexomutase superfamily and is instead related to the eukaryotic phosphomannomutases within the haloacid dehalogenase superfamily. In contrast to known bacterial ␣-PGMs, this 28-kDa enzyme is highly specific for ␣-glucose 1-phosphate and glucose 6-phosphate and showed no activity for mannose phosphate. To elucidate the function of pgmH, the metabolism of glucose and galactose was characterized in mutants overproducing or with a deficiency of ␣-PGM activity. Overproduction of ␣-PGM led to increased glycolytic flux and growth rate on galactose. Despite several attempts, we failed to obtain a deletion mutant of pgmH. The essentiality of this gene was proven by using a conditional knock-out strain in which a native copy of the gene was provided in trans under the control of the nisin promoter. Growth of this strain was severely impaired when ␣-PGM activity was below the control level. We show that the novel L. lactis ␣-PGM is the only enzyme that mediates the interconversion of ␣-glucose 1-phosphate to glucose 6-phosphate and is essential for growth.Phosphoglucomutase (PGM 3 ; EC 5.4.2.2) is widespread in living organisms from bacteria to humans (1). It plays various roles in carbohydrate metabolism by catalyzing the reversible conversion of ␣-Glc-1-P to Glc-6-P. In higher organisms, its major function is mediating the mobilization of sugar moieties from energy reserves. Also, ␣-PGM activity is essential for the synthesis of UDP-glucose, a sugar donor for the production of glucose-containing polysaccharides. Therefore, PGM is a crucial link between catabolic and anabolic processes.Lactococcus lactis is used worldwide in the industrial manufacture of fermented milk products. The organism converts sugars primarily into lactic acid, thus providing an efficient means of food conservation. In L. lactis, PGM is assumed to be essential for the utilization of galactose via the Leloir pathway (2) and for the synthesis of cell wall polysaccharides and exopolysaccharides (3, 4). In a number of Gram-positive bacteria, pgm mutants show altered cell wall morphology and altered polysaccharide production as well as growth defects on glucose (5-7). Despite the wealth of knowledge on sugar metabolism of L. lactis (8), genes coding for ␣-PGM have not been identified in this organism.More than 1 decade ago, the presence of two distinct PGM activities in L. lactis ssp. lactis with specificity for ␣-and ␤-anomers of phosphoglucose was reported (9). A 28-kDa protein (designated ␤-PGM) was shown to catalyze the reversible conversion of ␤-Glc-1-P to Glc-6-P. L. lactis ␤-PGM, which belongs to the haloacid dehalogenase (HAD) superfamily (10 -12)...