Biomedical subjects
Publications and source records attributed to D G Kilburn.
A cartridge was constructed which contained the divergent tet promoters of transposon Tn10 between an exoglucanase gene (cex) and an endoglucanase gene (cenA) of Cellulomonas fimi. When carried in a broad-host-range vector, the cartridge gave expression of cex and cenA in Escherichia coli, Rhodobacter capsulatus and Klebsiella pneumoniae.
CenA is an endo-beta 1,4-glucanase from the cellulolytic bacterium Cellulomonas fimi. It is a bifunctional enzyme comprising an amino-terminal cellulose-binding domain and a carboxyl-terminal catalytic domain joined by a short sequence of prolyl and threonyl residues (the Pro-Thr box). Additional structural and functional information was revealed by a detailed analysis of the products generated by proteolytic cleavage of a nonglycosylated form of CenA. An extracellular C. fimi protease attacked nonglycosylated CenA at the junctions between the Pro-Thr box and the two functional domains. A stable "core" peptide (p30), corresponding to the catalytic domain, remained after extensive proteolysis. p30 was resistant to further attack even in the presence of 2-mercaptoethanol plus urea or dithiothreitol, but treatment in the presence of sodium dodecyl sulfate allowed complete fragmentation to small peptides. Stable peptides, identical, or closely related to p30, were generated by alpha-chymotrypsin or papain. These results indicated that the catalytic domain adopts a tightly folded conformation affording protection from proteolytic attack. In contrast, the cellulose-binding domain showed a relatively loose conformation. Progressive proteolytic truncation from the amino terminus was apparent during incubation with alpha-chymotrypsin or papain, or with C. fimi protease under reducing conditions. Affinity for cellulose was retained by products missing up to 64 amino-terminal amino acids. The remaining carboxyl-proximal region of the cellulose-binding domain with affinity (47 amino acids) contained sequences highly conserved in analogous domains from other bacterial endo-beta 1,4-glucanases. By analogy with other systems, the properties of the Pro-Thr box are consistent with an elongated conformation. The results of this investigation suggest that CenA has a tertiary structure which resembles that of certain fungal cellulases.
Endoglucanase CenA of Cellulomonas fimi comprises an N-terminal cellulose-binding domain and a C-terminal catalytic domain joined together by a sequence of 23 proline and threonine residues (the Pro-Thr box). The domains function independently when separated by proteolysis. TnphoA has been used to generate cenA'-'phoA fusions. CenA'-'PhoA fusion polypeptides which contain the entire cellulose-binding domain of CenA bind to cellulose, allowing their purification from periplasmic extracts in a single, facile step. This result has implications for purification or immobilisation of chimeric proteins on a cheap cellulose matrix.
Two nonglycosylated endoglucanases which bind to Sephadex were purified from culture supernatants of Cellulomonas fimi grown on microcrystalline cellulose. Their Mrs were 120,000 and 130,000. The N-terminal amino acid sequences of the enzymes were identical, suggesting that the enzymes were related. A DNA fragment encoding this N-terminal sequence was cloned in Escherichia coli. The nucleotide sequence corresponding to the N-terminal amino acid sequence was preceded by a sequence encoding a typical leader peptide. Transcripts hybridizing to the cloned fragment were detected in total RNA isolated from C. fimi cells grown on carboxymethyl cellulose but not from cells grown on glycerol or glucose. Transcription started at a cluster of sites 53 to 59 nucleotides upstream of a GUG translation initiation codon and terminated at either of two closely spaced C residues immediately downstream of a region of potential secondary structure. The size of the transcript was approximately 3.5 kilobases, sufficient to encode a polypeptide of 130 kilodaltons. The 130-kilodalton polypeptide is designated endoglucanase C (CenC), and the gene encoding it is designated cenC.
An endo-beta-1,4-glucanase (CenA) and an exo-beta-1,4-glucanase (Cex) were prepared from Escherichia coli expressing recombinant DNA of the cellulolytic bacterium Cellulomonas fimi. Purification was facilitated by the high affinities of these enzymes for cellulose. An extracellular C. fimi protease cleaved both enzymes in vivo in a highly specific manner. The affinity of the parent enzyme for cellulose was contained independently in an amino-terminal fragment (p20) of CenA and a carboxyl-terminal fragment (p8) of Cex. These fragments contained homologous amino acid sequences which were proposed to comprise cellulose binding domains. Corresponding fragments, p30 from CenA and p35 from Cex, which were unable to bind to cellulose, contained catalytic domains. In both enzymes, the two functional domains were joined by a hinge region consisting solely of prolyl and threonyl residues. The binding domain was excised from CenA by proteolytic cleavage immediately adjacent to the carboxyl terminus of this hinge. Cex was cleaved at an exactly analogous site. p30 and p35 retained several of the catalytic functions of their parent enzymes. However, p30 was less active than intact CenA against microcrystalline cellulose implying a critical role for the binding domain of CenA in the hydrolysis of crystalline substrate.
Endoglucanase B (EB) of Cellulomonas fimi has an M(r) of 110,000 when it is produced in Escherichia coli. The level of expression of the cenB gene (encoding EB) was significantly increased by replacing its normal transcriptional and translational regulatory signals with those of the E. coli lac operon. EB was purified to homogeneity from the periplasmic fraction of E. coli in one step by affinity chromatography on microcrystalline cellulose (Avicel). Alignment of the NH(2)-terminal amino acid sequence with the partial nucleotide sequence of a fragment of C. fimi DNA showed that EB is preceded by a putative signal polypeptide of 33 amino acids. The signal peptide functions and is processed correctly in E. coli, even when its first 15 amino acids are replaced by the first 7 amino acids of beta-galactosidase. The intact EB polypeptide is not required for enzymatic activity. Active polypeptides with M(r)s of 95,000 and 82,000 also appear in E. coli, and a deletion mutant of cenB encodes an active polypeptide with an M(r) of 72,000.
The DNA sequence was determined for the cloned Agrobacterium sp. strain ATCC 21400 beta-glucosidase gene, abg. High-resolution nuclease S1 protection studies were used to map the abg mRNA 5' and 3' termini. A putative abg promoter was identified whose sequence shows similarities to the consensus promoter of Escherichia coli and with the nif promoter regions of Klebsiella. The abg coding sequence was 1,374 nucleotides long. The molecular weight of the enzyme, based on the predicted amino acid sequence, was 51,000. The observed Mr was 50,000 to 52,000. A region of deduced protein sequence was homologous to a region from two other beta-glucosidase sequences. This region of homology contained a putative active site by analogy with the active site of hen egg white lysozyme.
Glycosylated cellulases from Cellulomonas fimi were compared with their non-glycosylated counterparts synthesized in Escherichia coli from recombinant DNA. Glycosylation of the enzymes does not significantly affect their kinetic properties, or their stabilities towards heat and pH. However, the glycosylated enzymes are protected from attack by a C. fimi protease when bound to cellulose, while the non-glycosylated enzymes yield active, truncated products with greatly reduced affinity for cellulose.
The exoglucanase gene (cex) and the endoglucanase A gene (cenA) from Cellulomonas fimi were subcloned into the Escherichia coli/Brevibacterium lactofermentum shuttle vector pBK10. Both genes were expressed to five to ten times higher levels in B. lactofermentum than in E. coli, probably because these genes were expressed from C. fimi promoters. In B. lactofermentum virtually all of the enzyme activities were in the culture supernatant. This system will facilitate analysis of the expression of the C. fimi genes in and secretion of their products from a Gram-positive bacterium.
A fusion was constructed between the cex gene of Cellulomonas fimi, which encodes an exoglucanase, and the cenA gene of the same organism, which encodes an endoglucanase. The cex-cenA fusion was expressed in Escherichia coli to give a fusion protein with both exoglucanase and endoglucanase activities. The fusion protein, unlike the cex and the cenA gene products from E. coli, did not bind to microcrystalline cellulose, presumably because it lacked an intact substrate-binding region. The fusion protein was exported to the periplasm in E. coli.
We characterized the in vivo transcription of the Cellulomonas fimi cenB gene, which encodes an extracellular endo-beta-1,4-glucanase (EC 3.2.1.4). By Northern blot (RNA blot) analysis, cenB mRNA was detected in C. fimi RNA preparations from glycerol-, glucose-, and carboxymethyl cellulose (CMC)-grown cells. The relative abundance of the specific mRNAs in these preparations appeared to depend on the carbon source provided, with the preparations from CMC-grown cells having the greatest amount of cenB transcripts, followed by glycerol- and glucose-grown cells. Therefore, the transcription of this gene could be regulated by the carbon source provided to C. fimi. High-resolution nuclease S1 protection studies were used to map cenB mRNA 5' termini with a unique 5'-labeled DNA probe and C. fimi RNA isolated in vivo. With this procedure, three 5' termini were found in abundance upstream of the translational initiation ATG codon in RNA preparations from C. fimi grown on CMC, while less-abundant 5' termini were found 52 bases closer to the ATG codon in RNA prepared from C. fimi grown on any one of the three substrates. These results are indicative of a tandem promoter arrangement, with the ATG-proximal promoter directing constitutive low-level cenB transcription and the more distal promoter directing higher levels of transcription under the inducing effects of the cellulosic substrate. The corresponding transcripts were not detected in S1 mapping experiments with RNA isolated in vivo from Escherichia coli clones harboring recombinant plasmids carrying C. fimi genomic inserts. Comparative analysis of the 5' -flanking DNA sequences of the cenB gene and the cenA and cex genes of C. fimi (N. M. Greenberg, R. A. J. Warren, D. G. Kilburn, and R. C. Miller, Jr., J. Bacteriol. 169:646-653, 1987) revealed a region of 50 bases in which these sequences displayed at least 64% homology.
We characterized the in vivo transcripts of two Cellulomonas fimi genes, the cenA gene, which encodes an extracellular endo-beta-1,4-glucanase (EC 3.2.1.4) and the cex gene, which encodes an extracellular exo-beta-1,4-glucanase (EC 3.2.1.91). By Northern blot analysis, cenA mRNA was detected in C. fimi RNA preparations from glycerol- and carboxymethyl cellulose-grown cells but not from glucose-grown cells. In contrast, cex mRNA was detected only in the preparations from carboxymethyl cellulose-grown cells. Therefore, the transcription of these genes is subject to regulation by the carbon source provided to C. fimi. By nuclease S1 protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, 5' termini were found 51 and 62 bases before the cenA translational initiation codon and 28 bases before the cex translational initiation codon. S1 mapping with unlabeled DNA probes and C. fimi RNA which had been isolated in vivo but which had been 5' labeled in vitro with guanylyltransferase and [alpha-32P]GTP confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. Comparative analysis of the DNA sequences immediately upstream of the initiation sites of the cenA and cex mRNAs revealed a 30-base-pair region where these two sequences display at least 66% homology. S1 mapping was also used to locate the 3' termini of the cenA and cex transcripts. Three 3' termini were found for cenA messages, whereas only one 3' terminus was identified for cex mRNA. The transcripts of both genes terminate in regions where their corresponding DNA sequences contain inverted repeats.
The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.
Cellulomonas fimi produces an endoglucanase and an exoglucanase which bind strongly to cellulose. Each enzyme contains three distinct regions: a short sequence of about 20 amino acids containing only proline and threonine (the Pro-Thr box); an irregular region, rich in hydroxyamino acids, of low charge density, and which is predicted to have little secondary structure; and an ordered region of higher charge density which contains a potential active site, and which is predicted to have secondary structure. The Pro-Thr box is conserved almost perfectly in the two enzymes. The irregular regions are 50% conserved, and the conserved sequences include four Asn-Xaa-Ser/Thr sites. The ordered regions appear not to be conserved, but the potential active sites both have the sequence Glu-Xaa7-Asn-Xaa6-Thr; they occur at widely separated sites in the two regions. The order of the regions is reversed in the two enzymes: irregular-Pro-Thr box-ordered in the endoglucanase; ordered-Pro-Thr box-irregular in the exoglucanase. The genes for the two enzymes appear to have arisen by shuffling of two conserved sequences and either one or two other sequences.
Two BamHI fragments (0.8 and 5.2 kb) of Cellulomonas fimi containing an endoglucanase (Eng) gene (cenA) were individually cloned into the BamHI site of pBR322; they expressed carboxymethylcellulase activity in Escherichia coli. The nucleotide (nt) sequence of the cenA gene was determined by sequencing overlapping deletions. The cenA gene is 1350 bp long encoding a polypeptide of 449 amino acids (aa) and stop codon. The 0.8-kb BamHI component encodes the first 76 aa, whereas the 5.2-kb BamHI component encodes the rest of the Eng. The Eng lacking the N-terminal 76 aa retains its activity and antigenicity, and it forms an active fusion protein with the N-terminal portion of the TcR determinant. The C-terminal region of the Eng is crucial for activity and a deletion of as little as 12 aa from that end results in the loss of all Eng activity. The N-terminal 31 aa of the Eng constitute a leader peptide which appears to be functional in exporting the enzyme to the periplasm in E. coli.
In Cellulomonas fimi the cex gene encodes an exoglucanase (Exg) involved in the degradation of cellulose. The gene now has been sequenced as part of a 2.58-kb fragment of C. fimi DNA. The cex coding region of 1452 bp (484 codons) was identified by comparison of the DNA sequence to the N-terminal amino acid (aa) sequence of the Exg purified from C. fimi. The Exg sequence is preceded by a putative signal peptide of 41 aa, a translational initiation codon, and a sequence resembling a ribosome-binding site five nucleotides (nt) before the initiation codon. The nt sequence immediately following the translational stop codon contains four inverted repeats, two of which overlap, and which can be arranged in stable secondary structures. The codon usage in C. fimi appears to be quite different from that of Escherichia coli. A dramatic (98.5%) bias occurs for G or C in the third position for the 35 codons utilized in the cex gene.
A leader sequence of 41 amino acids (aa) has been proposed as the signal sequence for the exoglucanase (Exg) from Cellulomonas fimi. The ability of this 41-aa peptide to function as a leader sequence has been shown here by gene fusion experiments in Escherichia coli. A hybrid leader sequence containing C-terminal 37 aa of the leader peptide and N-terminal 6 aa of beta-galactosidase (beta Gal) directed export of the Exg into the periplasm of E. coli. In contrast, hybrid beta Gal-Exg proteins in which the leader sequence is not present are retained in the cytoplasm.
A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism.