Cellulomonas fimi secretes several cellulase activities as well as protease activity into the culture medium. In contrast, few activities are bound to the cellulose in the culture. To characterize the cellulase system and to Identify cloned gene products, it was necessary to purify native, intact cellulases. We hypothesized that the cellulose-bound cellulases would be protected from proteolysis, and therefore represent the intact enzymes. Two cellulases were purified from Cellulomonas fimi. Avicel was recovered from cultures and the proteins were eluted from it with guanidine-HCl (Gdn-HCl). The Gdn-HCl extract was fractionated by Concanavalin A-Sepharose affinity column chromatography and by Mono Q anion exchange column chromatography. The cellulases purified by this procedure were an endoglucanase, EngA, and an exoglucanase, Exg. The purified enzymes were characterized. EngA has Mr 57,000, pi 8.2, and is 10 % mannose by weight. Exg has Mv 56,000, pi 5.8, and is 8 % mannose by weight. Two recombinant DNA plasmids were identified as encoding EngA and Exg. The recombinant gene products were not glycosylated. The role of glycosylation was studied by comparing some properties of the recombinant EngA and Exg with the native EngA and Exg. Both glycosylated and unglycosylated forms bound to Avicel. Sensitivity to the C. fimi protease was also compared. The glycosylated enzymes were protected from proteolysis when bound to cellulose. In contrast, the unglycosylated forms were processed to yield active, truncated products with greatly reduced affinity for cellulose. The cleavage site was predicted based on size of the products and reactivity with anti-PT serum. The N-terminal region of EngA and the C-terminal of Exg show 50 % conservation of sequence (Warren et al., 1986). This region appears to be the cellulose-binding domain and is not required for the hydrolysis of soluble substrates. The C. fimi protease can partially degrade glycosylated EngA when it is not bound to cellulose. Some of the multiple CMCase activities in culture supernatants are derived from EngA by partial proteolysis. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In Cellulomonas fimi the cenB gene encodes a secreted endoglucanase (EngB) involved in the degradation of cellulose. The cenB gene carried on a 5.6 kb C fimi DNA fragment encodes a polypeptide of Mr 110,000 in Escherichia coli. The level of expression of the gene was significantly increased by replacing its normal transcriptional and translational regulatory signals with those of the E. coli lac operon. The intact EngB polypeptide is not required for enzymatic activity: active polypeptides of Mr 95,000 and 82,000 also appear in E. coli and a deletion mutant of cenB encodes an active polypeptide of Mr 72,000. The intact and truncated EngB both bind to microcrystalline cellulose. A simple, rapid affinity chromatography procedure on Avicel was developed for the purification of intact EngB and of the 72,000 deletion derivative. Alignment of the amino-terminal amino acid sequence of the purified intact EngB from E. coli with the partial nucleotide sequence of the cloned C. fimi DNA showed that the mature EngB is preceded by a sequence encoding a putative signal polypeptide of 32 amino acids, a translational initiation codon and a sequence resembling an E. coli ribosome binding site 4 nucleotides before the initiation codon. The signal peptide functions and is correctly processed in E. coli, even when its first 15 amino acids are replaced by the first 7 amino acids of β-galactosidase. The truncation of EngB does not affect its export to the periplasm of E.coli. In the intact EngB, 25% of the residues are hydroxyamino acids. It displays features common to endo-β-1 ,4-glucanases, since it has a high activity on carboxymethylcellulose. The kinetic parameters for carboxymethylcellulose hydrolysis of both intact and truncated EngB are not significantly different. C. fimi protease cleaves intact EngB, in a specific manner, to generate two polypeptides of Mr 65,000 and 43,000; the former has the capacity to bind Avicel. A polyclonal antibody raised against the purified intact EngB recognizes a C. fimi extracellular protein of M 110,000 as well as 5 polypeptides of lower molecular weight. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Transcriptional analysis was used to investigate the molecular mechanisms which effect cellulase gene expression in the gram-positive bacterium Cellulomonas fimi strain ATCC 484 and the gram-negative bacterium Agrobacterium sp. strain ATCC 21400. The cenA, cex and cenB genes of C. fimi encoding the extracellular β-1,4-endoglucanase, EngA (EC 3.2.1.4; Mr 48,700), the extracellular β-1, 4-exoglucanase, Exg (EC 3.2.1.91; Mr 47,300) and the extracellular β-1,4-endoglucanase EngB (EC 3.2.1.4; Mr 110,000) respectively, were characterised. By northern blot analysis, cenA mRNA was detected in C. fimi RNA prepared from glycerol- and carboxymethylcellulose (CMC)-grown cells but not in RNA from glucose-grown cells. The cex mRNA was found only in RNA from CMC-grown cells. The cenB mRNA was found in all three preparations of RNA. Therefore, the expression of these genes is subject to regulation by the carbon source provided to C. fimi. High resolution nuclease SI protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, were used to map the 5' termini of cenA and cex mRNAs. Two cenA mRNA 5' ends, 11 bases apart, mapped 51 and 62 bases upstream of the cenA start codon, suggesting that in vivo, cenA transcription was directed from two promoters in tandem. The cex mRNA 5' end was found to map 28 bases upstream of the cex start codon. Using SI mapping with unlabeled DNA probes and C. fimi RNA which had been isolatedin vivo but which had been 5'-labeled in vitro with vaccinia virus capping enzyme confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. The SI mapping revealed mRNA 3' termini 1,438, 1,449, and 1, 464 bases from the major cenA start site, and one 3' terminus 1,564 bases from the major cex mRNA start site, in good agreement with the northern blot data. High resolution SI studies were also used to show that abundant mRNA 5' ends mapped upstream of the cenB start codon in RNA prepared from CMC-grown cells, while less-abundant species mapped 52 bases closer to the ATG codon in RNA prepared from C. fimi grown on any one of the three substrates. These results seem to indicate a tandem promoter arrangement with an ATG-proximal promoter directing low-level constitutive cenB transcription and a more distal promoter directing higher levels of cenB transcription as a result of C. fimi growth on cellulosic substrate. Steady- state levels were determined for cenA, cex and cenB mRNAs with RNA prepared from glycerol-, glucose-, and CMC-grown cultures of C. fimi in slot-blot hybridisations with radiolabeled oligodeoxyribonucleotide probes. A cex-linked gene (clg) was identified by sequence inspection and SI mapping. Transcripts of the abg gene encoding the β-glucosidase (Abg, EC 3.2.2.21/ Mr 50,000) of Agrobacterium sp. strain ATCC 21400 were also characterised. Northern blot analysis of Agrobacterium RNA revealed the size of the in vivo abgmRNA was approximately 1,500 bases in length. High resolution SI mapping determined abg mRNA 5' ends 22 bases upstream of the abg ATG codon and 3' ends 71 bases downstream of the abg stop codon. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate