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The DNA sequence of the BaciUus subtlis DLG endo-,B-1,4-glucanase gene was determined, and the in vivo site of transcription initiation was located. Immediately upstream from the transcription start site were sequences closely resembling those recognized by B. subtilis &43-RNA polymerase. Two possible ribosomebinding sites were observed downstream from the transcription start site. These were followed by a long open reading frame capable of encoding a protein of ca. 55,000 daltons. A signal sequence, typical of those present in gram-positive organisms, was observed at the amino terminus of the open reading frame. Purification of the mature exocellular 0-1,4-glucanase and subsequent amino-terminal protein sequencing defined the site of signal sequence processing to be between two alanine residues following the hydrophobic portion of the signal sequence. The probability of additional carboxy-terminal processing of the 0-1,4-glucanase precursor is discussed. Si nuclease protection studies showed that the amount of ,B-1,4-glucanase mRNA in cells increased significantly as the culture entered the stationary phase. In addition, glucose was found to dramatically stimulate the amount of ,B-1,4-glucanase mRNA in vivo. Finally, the specffic activities of purified B. subtilis DLG endo-0-1,4-glucanase and Trichoderma reesei QM9414 endo-f-1,4-glucanase (EC 3.2.1.4) were compared by using the noncrystalline cellulosic substrate trinitrophenyl-carboxymethyl cellulose.Many members of the family Bacillaceae produce extracellular ,B-glucanases. The linkage specificities of these enzymes are varied, the most common being ,-1,3-1,4-glucanases and 13-1,3-glucanases (6,20,32,41,57). There are relatively few reports of members of the family Bacillaceae producing ,B-1,4-glucanases (13,21,41,44). The two relatively well characterized examples, the alkalophilic Bacillus strain N-4 (21, 48) and Bacillus subtilis DLG (44, 45), produce cellulolytic activity in the form of a carboxymethyl cellulase (CMC) (i.e., P-1,4-glucanase). Although incapable of degrading crystalline forms of cellulose individually, bacterial P-1,4-glucanases (most likely endo acting) may be able to act synergistically with cellulases of other specificities, such as exo-acting P-1,4-glucanases or P-glucosidases, or both, to achieve the enzymatic bioconversion of cellulose to more commercially useful products. A Bacillus P-1,4-glucanase may also have a role to play in the brewing industry (5,11,20). For B. subtilis DLG, ,B-1,4-glucanase production begins at the onset of the stationary phase and is not repressed by either glucose or cellobiose (44), as are many other cellulolytic systems (12,30,53,55). In fact, glucose stimulates enzyme production in some fashion. Additionally, the enzyme is initially translated as a large (ca. 51,500-dalton), enzymatically active intracellular precursor in B. subtilis before undergoing efficient secretion to the exterior of the cell (45). The mature exocellular ,B-1,4-glucanase is 35,200 ± 400 daltons.Cloning the ,3-1,4-glucanase gene ha...

The gene encoding beta-1,4-glucanase in Bacillus subtilis DLG was cloned into both Escherichia coli C600SF8 and B. subtilis PSL1, which does not naturally produce beta-1,4-glucanase, with the shuttle vector pPL1202. This enzyme is capable of degrading both carboxymethyl cellulose and trinitrophenyl carboxymethyl cellulose, but not more crystalline cellulosic substrates (L. M. Robson and G. H. Chambliss, Appl. Environ. Microbiol. 47:1039-1046, 1984). The beta-1,4-glucanase gene was localized to a 2-kilobase (kb) EcoRI-HindIII fragment contained within a 3-kb EcoRI chromosomal DNA fragment of B. subtilis DLG. Recombinant plasmids pLG4000, pLG4001a, pLG4001b, and pLG4002, carrying this 2-kb DNA fragment, were stably maintained in both hosts, and the beta-1,4-glucanase gene was expressed in both. The 3-kb EcoRI fragment apparently contained the beta-1,4-glucanase gene promoter, since transformed strains of B. subtilis PSL1 produced the enzyme in the same temporal fashion as the natural host B. subtilis DLG. B. subtilis DLG produced a 35,200-dalton exocellular beta-1,4-glucanase; intracellular beta-1,4-glucanase was undetectable. E. coli C600SF8 transformants carrying any of the four recombinant plasmids produced two active forms of beta-1,4-glucanase, an intracellular form (51,000 +/- 900 daltons) and a cell-associated form (39,000 +/- 400 daltons). Free exocellular enzyme was negligible. In contrast, B. subtilis PSL1 transformed with recombinant plasmid pLG4001b produced three distinct sizes of active exocellular beta-1,4-glucanase: approximately 36,000, approximately 35,200, and approximately 33,500 daltons. Additionally, B. subtilis PSL1(pLG4001b) transformants contained a small amount (5% or less) of active intracellular beta-1,4-glucanase of three distinct sizes: approximately 50,500, approximately 38,500 and approximately 36,000 daltons. The largest form of beta-1,4-glucanase seen in both transformants may be the primary, unprocessed translation product of the gene.

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was <100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58°C, although maximum thermal stability of the activity was observed only up to 45 to 50°C. Neither glucose nor cellobiose inhibited enzymatic activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly onefifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.

A cDNA encoding the 37-kilodalton (kDa) capsid assembly protein of cytomegalovirus (CMV) strain Colburn was isolated from a Xgtll library constructed from CMV Colburn-infected human fibroblast RNA. RNA transcribed in vitro from this cDNA was translated in vitro to give a 40-kDa protein whose electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fragmentation pattern following partial proteolysis were indistinguishable from those of authentic assembly protein precursor. The position of the assembly protein gene was mapped to the EcoRI F, XbaI R, and Sall U restriction fragments, near the middle of the CMV Colburn genome, by Southern hybridizations using the cloned assembly protein cDNA as a probe. Similar sequences were identified by cross-hybridizations in colinear regions of the genomes of human CMV strains Towne and AD169: specifically, in the Hindlll H, BamHI V, and EcoRI A fragments of Towne and in the HindIII L and BglII S fragments of AD169. The predominant transcript of the assembly protein gene was determined to be approximately 1 kilobase in size; however, a larger transcript (1.8 kilobases) was also identified. The nucleotide sequence of the assembly protein cDNA was determined and found to contain a single long open reading frame predicted to encode a polypeptide of 36.6 to 37 kDa, close to the 40-kDa size determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the assembly protein precursor. The presence of a single cysteine residue at the carboxy-terminal end of this open reading frame is consistent with data from biochemical studies and indicates that processing of the assembly protein precursor includes a proteolytic cleavage that removes its carboxyl end.