Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can serve as a carbon source at both high and low osmolarity. The elucidation of the pathway of trehalose metabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins and degradative enzymes. The analysis of the phenotypes of these mutants and of the reactions catalyzed by the enzymes in vitro allowed the formulation of the degradative pathway at low osmolarity. Thus, trehalose utilization begins with phosphotransferase (IITreHIlI c)-mediated uptake delivering trehalose-6-phosphate to the cytoplasm. It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor. The enzyme catalyzing this reaction was named amylotrehalase. Amylotrehalase and EfiTre were induced by trehalose in the medium but not at high osmolarity. treC and treB encoding these two enzymes mapped at 96.5 min on the E. coli linkage map but were not located in the same operon. Use of a mutation in trehalose-6-phosphate phosphatase allowed demonstration of the phosphoenolpyruvate-and HTre-dependent in vitro phosphorylation of trehalose. The phenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system.The synthesis of internal trehalose in Escherichia coli in response to high osmolarity has been studied in detail on a genetic and biochemical level (12, 30), yet little is known about trehalose transport and metabolism. Early reports have described E. coli mutants that were partially defective in the utilization of trehalose. The mutations mapped at 26 min on the linkage map (3; for Salmonella typhimurium, see reference 29). Marechal (20) later reported the existence of a specific enzyme II of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) by demonstrating the PEP-dependent phosphorylation of trehalose. He also claimed, on the basis of biochemical studies, the existence of an enzyme able to hydrolyze trehalose-6-phosphate to glucose-6-phosphate and glucose (20). Different results were obtained by Postma et al. (23), who reported that trehalose is transported in S. typhimurium via the mannose-PTS without phosphorylation. Both studies reported the existence of a trehalose-inducible trehalase in crude extracts.A periplasmic trehalase was subsequently discovered and purified from E. coli, and mutants, termed treA, were isolated that lacked this enzyme. treA was mapped at 26 min (5), and the treA gene was cloned, sequenced, and found to be the only gene in the operon (14). Periplasmic trehalase synthesis is not induced by trehalose but rather by growth in the presence of 250 mM NaCl (5). Apparently, the function of the periplasmic trehalase is to ensure the utilization of trehalose under conditions of high osmolarity...

The gene encoding catalase-peroxidase was cloned from chromosomal DNA of Rhodobacter capsulatus B10. The nucleotide sequence of a 3.7-kb Sad-Hind111 fragment, containing the catalase-peroxidase gene (cpeA) and its flanking regions were determined. A 1728-bp open reading frame, coding for 576 amino acid residues (molecular mass 61 516 Da) of the enzyme, was observed. A Shine-Dalgarno sequence was found 5 bp upstream from the translational start site. The deduced amino acid sequence coincides with that of the amino terminus and of four peptides derived from trypsin digestion of the purified catalase-peroxidase of R. capsulatus B 10.The amino acid sequence of R. capsulatus catalase-peroxidase shows interesting similarities to the amino acid sequences of the hydroperoxidases of Escherichia coli (42.7%) and Salmonella typhimurium (39.9%), the peroxidase of Bacillus stearothermophilus (32.1 %) and the catalase-peroxidase of Mycobacterium intracellulare (42.2%). As shown by a cpeA::lacZ fusion in trans in R. capsulatus, the expression of the catalase-peroxidase gene is regulated by oxygen. The promoter of the cpeA gene was localized within 320 bp upstream of the ATG start codon.

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The expression of the Rhodobacter capsulatus catalase-peroxidase (cpeA) was studied by in-frame fusions of the upstream region of the cpeA gene to a promoter-less lacZ gene. The transcription of the cpeA gene is about 20-50-fold higher under aerobic-dark than under anaerobic-light conditions. The promoter was localized within a 69-bp upstream DNA region. The transcription start site, determined by primer extension, is 28 bases upstream from the initiation codon, confirming the postulated promoter localized by deletion analysis. Deletion of the part of the upstream region specifically responsible for oxygen regulation resulted in constitutive expression of the cpeA gene.

The expression of the Rhodobacter capsulatus catalase-peroxidase (cpeA) was studied by in-frame fusions of the upstream region of the cpeA gene to a promoter-less lacZ gene. The transcription of the cpeA gene is about 20-50-fold higher under aerobic-dark than under anaerobic-light conditions. The promoter was localized within a 69-bp upstream DNA region. The transcription start site, determined by primer extension, is 28 bases upstream from the initiation codon, confirming the postulated promoter localized by deletion analysis. Deletion of the part of the upstream region specifically responsible for oxygen regulation resulted in constitutive expression of the cpeA gene.