Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli

Abstract

It has been shown previously that Escherichia coli accumulates endogenously synthesized trehalose under osmotic stress. We report here that E. coli contained an osmotically regulated trehalose-phosphate synthase which utilized UDP-glucose and glucose 6-phosphate as substrates. In the wild type, the synthase was induced by growth in glucose-mineral medium of elevated osmotic strength and the synthase itself was strongly stimulated by K+ and other monovalent cations. A laboratory strain which expressed the synthase at a high constitutive level was found. GalU mutants, defective in synthesis of UDP-glucose, did not accumulate trehalose. Two genes governing the synthase were identified and named otsA and otsB (osmoregulatory trehalose synthesis). They mapped near 42 min in theflbB-uvrC region. Mutants with an otsA-lacZ or otsB-lacZ operon fusion displayed osmotically inducible I8-galactosidase activity; i.e., the activity was increased fivefold by growth in medium of elevated osmotic strength. Mutants unable to synthesize trehalose (galU, otsA, and otsB) were osmotically sensitive in glucose-mineral medium. But an osmotically tolerant phenotype was restored in the presence of glycine betaine, which also partially repressed the synthesis of synthase in the wild type and of 0-galactosidase in ots-lacZ fusion mutants.

Biochemical and genetic characterization of the pathways for trehalose metabolism in Propionibacterium freudenreichii, and their role in stress response

“…In cell extracts, the OtsA of P. freudenreichii was absolutely specific for ADP-glucose, a finding that appears to be unique as far as OtsA proteins are concerned. In fact, known OtsA proteins either can use the four glucosyl donors (Lapp et al, 1971;Killick, 1979;Pan et al, 2002;Silva et al, 2005), or are specific for UDP-or GDPglucose (Elbein, 1968;Giaever et al, 1988). Most surprising was the observation that the pure (native or recombinant) OtsA of P. freudenreichii lacked the absolute specificity shown in cell extracts, and recognized any of the four sugar-nucleotides as a substrate.…”

Differential interaction of Salmonella enterica serovars with lettuce cultivars and plant-microbe factors influencing the colonization efficiency

“…The genes related to a sugar-like carbon source were OtsA (trehalose-6-phosphate synthase), which utilizes glucose-6-phosphate as substrate (Gizver et al, 1988), UhpC (hexose phosphate utilization protein), which is a sensor for external glucose-6-phosphate) (Schwjppe et al, 2003), MetE (methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase), a vitamin B 12 -independent enzyme (Urbanowski and Stauffer, 1989), DsrA (putative anti-silencer RNA), a regulator of transcription to express RcsA promoter, which in its turn is responsible for capsular polysaccharide synthesis (Sledjeski and Gottesman, 1995), RseA (sigma-E factor regulatory protein), which is involved in the storage of sigma which is released during stress (Ades et al, 1999), SsaH and SsaM (putative effector proteins), both regulators of secretion of the type III secretion system (Lee et al, 2000) and SpaO, involved in surface presentation of antigens, secretory proteins. Other genes that were differentially expressed were related to specific limiting factors like iron (gene SitD) or anaerobic respiration (TtrA).…”

Ubiquinone accumulation improves osmotic-stress tolerance in Escherichia coli

“…Third, in case of prolonged osmotic stress, E. coli can take up the osmoprotectants glycine betaine and proline from the environment via the proVWX-encoded ABC transporter or synthesize glycine betaine from the extracellular precursor choline 2,9,10 . Fourth, if no extracellular compatible solutes are available, E. coli induces expression of trehalose-6-phosphate synthase (OtsA) and phosphatase (OtsB) to produce high intracellular concentrations of the nonreducing disaccharide trehalose from the precursors UDP-glucose and glucose 6-phosphate, conveying long-term resistance to sustained osmotic stress [11][12][13] . Quantitative studies indicated, however, that the amounts of compatible solutes produced by E. coli may not be sufficient to maintain cell turgor exclusively based on increasing intracellular osmolality, implying accompanying effects such as molecular crowding 14 .…”