Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli

Abstract

Cellulolytic strains of Bacillus stearothermophilus were isolated from nature and screened for the presence of activities associated with the degradation of plant cell walls. One isolate (strain XL-65-6) which exhibited strong activities with 4-methylumbelliferyl-4-D-glucopyranoside (MUG) and 4-methylumbelliferyl-p-D-cellobiopyranoside (MUC) was used to construct a gene library in Escherichia coli. Clones degrading these model substrates were found to encode the cellobiose-specific genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Both MUG and MUC activities were present together, and both activities were lost concurrently during subcloning experiments. A functional E. coli ptsI gene was required for MUC and MUG activities (presumably a ptsH gene also). The DNA fragment from B. stearothermophilus contained four open reading frames which appear to form a cel operon. Intergenic stop codons for celA, celB, and celC overlapped the ribosomal binding sites of the respective downstream genes. Frameshift mutations or deletions in celA, celB, and celD were individually shown to result in a loss of MUC and MUG activities. On the basis of amino acid sequence homology and hydropathy plots of translated sequences, celA and celB were identified as encoding PTS enzyme II and celD was identified as encoding PTS enzyme III. These translated sequences were remarkably similar to their respective E. coli homologs for cellobiose transport. No reported sequences exhibited a high level of homology with the celC gene product. The predicted carboxy-terminal region for celC was similar to the corresponding region of E. coli celF, a phospho-,-glucosidase. An incomplete regulatory gene (celR) and proposed promoter sequence were located 5' to the proposed cel operon. A stem-loop resembling a rho-independent terminator was present immediately downstream from celD. These results indicate that B. stearothermophilus XL-65-6 contains a cellobiose-specific PTS for cellobiose uptake. Similar systems may be present in other gram-positive bacteria.

Molecular Cloning and Expression of Cellulase and Polygalacturonase Genes in E. coli as a Promising Application for Biofuel Production

“…The suggestion of exo-acting activity of the celB enzyme is consistent with its original role inside the cell as a transmembrane channel for cellobiose [91,92]. CelC encodes the enzyme that is reported to be responsible for the hydrolysis of cellobiose-phosphate into hydrophilic glucose products [35].…”

“…celA, celB and celC encoded cellulases have been previously isolated and characterized from P. carotovorum LY43 [34]. The role of these cellulases in sugar uptake systems inside the cell was described in the cloned cellobiose phosphotransferase system operon from Bacillus stearothermophilus [35]. Sugar uptake is generally carried out through a phosphoenolpyruvate:phosphotransferase system (PEP:PTS) in which an enzyme I complex (EI), a histidine containing protein (HPr) as well as an enzyme II complex (EII) play important roles [36,37].…”

The Geobacillus paradox: why is a thermophilic bacterial genus so prevalent on a mesophilic planet?

“…Orthologues of a well-studied neopullulanase (Hondoh et al, 2003) are encoded within an apparent pullulan or starch utilization cluster found in eight of the ten genomes (Table 1 and Table S3). A previously studied system (Lai & Ingram, 1993) for the uptake and utilization of cellobiose, a disaccharide produced from partial cellulose hydrolysis, is found in four of the genomes, while a five-gene cluster highly similar to a well-characterized B. subtilis glucomannan utilization operon (Sadaie et al, 2008) is found in nine of the genomes.…”

Role of Periplasmic Trehalase in Uptake of Trehalose by the Thermophilic Bacterium Rhodothermus marinus

“…The weak inhibition caused by NaF suggests that the PTS is not involved. In fact, the occurrence of PTS in thermophiles and hyperthermophiles is extremely rare; the only exception is the thermophilic bacterium Bacillus stearothermophilus, which possesses PTSs for mannitol and cellobiose (8,12). On the other hand, all thermophilic and hyperthermophilic organisms examined thus far transport trehalose via ABC transport systems (5,9,11,18,24,25).…”