Cloning and expression of an endocellulase gene from a novel streptomycete isolated from an East African soda lake.

Alkaline cellulase-producing actinomycete strains were isolated from mud samples collected from East African soda lakes. The strains were identified as novel Streptomyces spp. by 16S rDNA sequence analysis. A cellulase gene (cel12A) from Streptomyces sp. strain 11AG8 was cloned by expression screening of a genomic DNA library in Escherichia coli. From the nucleotide sequence of a 1.5-kb DNA fragment, an open reading frame of 1,113 nucleotides was identified encoding a protein of 371 amino acids. From computer analysis of the sequence, it was deduced that the Cel12A mature enzyme is a protein of 340 amino acids. The protein contained a catalytic domain, a glycine-rich linker region, and a cellulose-binding domain of 221, 12, and 107 amino acids, respectively. FASTA analysis of the catalytic domain of Cel12A classified the enzyme as a family 12 endoglucanase and the cellulose-binding domain as a family IIa CBD. Streptomyces rochei EglS was determined as nearest neighbor with a similarity of 75.2% and 61.0% to the catalytic domain and the cellulose-binding domain, respectively. The cell2A gene was subcloned in a Bacillus high-expression vector carrying the Bacillus amyloliquefaciens amylase regulatory sequences, and the construct was transformed to a Bacillus subtilis host strain. Crude enzyme preparations were obtained by ultrafiltration of cultures of the Bacillus subtilis recombinant strain containing the 11AG8 cell2A gene. The enzyme showed carboxymethylcellulase (CMCase) activities over a broad pH range (5-10) with an optimum activity at pH 8 and 50 degrees C. The enzyme retained more than 95% of its activity after incubation for 30 min under these conditions.

Characterization of two cold-sensitive mutants of the beta-galactosidase from Lactobacillus delbruckii subsp. bulgaricus.

Methoxylamine mutagenesis of the beta-galactosidase gene from Lactobacillus delbrückii subsp. bulgaricus was used to generate cold-sensitive variants. Two variants, P429S and L317F, were characterized kinetically in order to determine the enzymatic consequences of these mutations. The kinetic parameters Km and Vmax on the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside have been determined over a temperature range of 11-45 degrees C. Only the Vmax of the two variants was significantly different than the wild-type enzyme over the temperature range studied. The Vmax of the L317F variant is reduced proportionately at all temperatures compared to the wild-type enzyme while the value of Vmax for the P429S mutant deviates from wild-type only at lower temperatures (in 2 mM Mg2+). This temperature-dependent effect on the Vmax of P429S can be suppressed by increasing the Mg2+ concentration. The results suggest that the binding of this essential metal ion is altered in the P429S variant such that its dissociation is increased by lowering the temperature.

The catR gene encoding a catalase from Aspergillus niger: primary structure and elevated expression through increased gene copy number and use of a strong promoter.

Synthetic oligonucleotide probes based on amino acid sequence data were used to identify and clone cDNA sequences encoding a catalase (catalase-R) of Aspergillus niger. One cDNA clone was subsequently used to isolate the corresponding genomic DNA sequences (designated catR). Nucleotide sequence analysis of both genomic and cDNA clones suggested that the catR coding region consists of five exons interrupted by four small introns. The deduced amino acid sequence of catalase-R spans 730 residues which show significant homology to both prokaryotic and eukaryotic catalases, particularly in regions involved in catalytic activity and binding of the haem prosthetic group. Increased expression of the catR gene was obtained by transformation of an A. niger host strain with an integrative vector carrying the cloned genomic DNA segment. Several of these transformants produced three- to fivefold higher levels of catalase than the untransformed parent strain. Hybridization analyses indicated that these strains contained multiple copies of catR integrated into the genome. A second expression vector was constructed in which the catR coding region was functionally joined to the promoter and terminator elements of the A. niger glucoamylase (glaA) gene. A. niger transformants containing this vector produced from three- to 10-fold higher levels of catalase-R than the untransformed parent strain.

The nuclear-coded subunits of yeast cytochrome c oxidase. The amino acid sequences of subunits VII and VIIa, structural similarities between the three smallest polypeptides of the holoenzyme, and implications for biogenesis.

The complete amino acid sequences of subunits VII and VIIa from yeast cytochrome c oxidase are reported. Subunits VII and VIIa are 57 residues (Mr = 6603) and 54 residues (Mr = 6303) in length, respectively. Both polypeptides are amphiphilic, have an internal hydrophobic section and hydrophilic NH2 and COOH termini, and terminate at their COOH termini with a basic amino acid. This structural motif is similar to that possessed by subunit VIII of yeast cytochrome c oxidase. All three polypeptides have hydrophobic sections which are long enough to span the inner membrane; all three polypeptides lack methionine at their NH2 termini; and all three polypeptides have COOH termini which could result from proteolysis by a protease with trypsin or cathepsin B-like activity. These observations raise the interesting possibility that subunits VII, VIIa, and VIII are transmembranous polypeptides which are processed at both their NH2 and COOH termini during their biogenesis.

Secretion and autoproteolytic maturation of subtilisin.

The sequence of the cloned Bacillus amyloliquefaciens subtilisin gene suggested that this secreted serine protease is produced as a larger precursor, designated preprosubtilisin [Wells, J. A., Ferrari, E., Henner, D. J., Estell, D. A. & Chen, E. Y. (1983) Nucleic Acids Res. 11, 7911-7925]. Biochemical evidence presented here shows that a subtilisin precursor is produced in Bacillus subtilis hosts. The precursor is first localized in the cell membrane, reaching a steady-state level of approximately equal to 1000 sites per cell. Mutations in the subtilisin gene that alter a catalytically critical residue (i.e., aspartate +32----asparagine), or delete the carboxyl-terminal portion of the enzyme that contains catalytically critical residues, block the maturation of this precursor. This block occurs when these mutant genes are expressed in B. subtilis hosts where the chromosomal subtilisin gene has been deleted. When the mutant B. amyloliquefaciens subtilisins are expressed in B. subtilis hosts that contain an intact chromosomal subtilisin gene, the mutant precursors are processed to a mature form and released to the medium. Such processing, in trans, of the precursor is also demonstrated in vitro by addition of active subtilisin. Thus, the release of subtilisin from the cell membrane is dependent on an autoproteolytic process that appears to be novel among secreted proteins.

Bacteriophage T4 regA protein. Purification of a translational repressor.

The bacteriophage T4 regA protein translationally regulates its own synthesis and the synthesis of several other T4 early proteins. In order to study the mechanism of translational regulation, we have purified the regA protein. Initially a mutant protein, incapable of autogenous repression, was placed under lambda PL transcriptional control and amplified to approximately 10% of total cell protein. The membrane-associated mutant protein was extracted with organic solvent mixtures and purified by reverse phase-high performance liquid chromatography. Polyclonal antibodies prepared against the mutant protein were used in Western blot assays to monitor purification of the wild-type protein from T4-infected cells. Phosphocellulose and poly(U)-agarose chromatography were important steps in its purification. The binding properties of regA protein to polyribonucleotides are discussed in relation to the mechanism by which the protein recognizes its mRNA targets.

The nuclear-coded subunits of yeast cytochrome c oxidase. I. Fractionation of the holoenzyme into chemically pure polypeptides and the identification of two new subunits using solvent extraction and reversed phase high performance liquid chromatography.

Previously, cytochrome c oxidase from the yeast Saccharomyces cerevisiae has been thought to be composed of seven different polypeptide subunits. Four of these are small polypeptides (4,000-15,000 daltons), subunits IV-VII, which are encoded by nuclear DNA. Studies described here reveal the presence of two new polypeptides in this size range. These polypeptides, designated as subunits VIIa and VIII, co-migrate with subunit VII (R.O. Poyton and G. Schatz (1975) J. Biol. Chem. 250, 752-761) on low resolution sodium dodecyl sulfate (SDS) polyacrylamide gels, can be partially resolved on high resolution SDS polyacrylamide gels, and can be completely separated from one another by reversed phase high performance liquid chromatography. In order to determine the sequences of each of these six nuclear-coded polypeptides (subunits IV, V, VI, VII, VIIa, and VIII), we have developed new methods for the large scale purification of the holoenzyme and have employed a new strategy for the isolation of each polypeptide. By using octyl-Sepharose chromatography to isolate holocytochrome c oxidase and by extracting the holoenzyme with aprotic organic solvents and fractionating these extracts by reversed phase high performance liquid chromatography, it is possible to isolate several milligrams of each of these subunits. Each subunit preparation gives a single peak during reversed phase high performance liquid chromatography, a single band during SDS-polyacrylamide gel electrophoresis, a single NH2-terminal sequence, and a unique amino acid composition and tryptic peptide map. Since each purified subunit preparation gives close to a 100% yield of its NH2-terminal amino acid during quantitative Edman degradation, we conclude that no subunit has a blocked NH2 terminus and that no subunit preparation contains either blocked or unblocked contaminating polypeptides. Thus, each consists of a single unique polypeptide species. Together, these results demonstrate that yeast cytochrome c oxidase contains six, rather than four, small subunit polypeptides. Thus, it appears that these polypeptides, in combination with the three polypeptides encoded by mitochondrial DNA, constitute a holoenzyme which contains nine subunits, instead of seven as proposed earlier.

The nuclear-coded subunits of yeast cytochrome c oxidase. II. The amino acid sequence of subunit VIII and a model for its disposition in the inner mitochondrial membrane.

The amino acid sequence of subunit VIII from yeast cytochrome c oxidase is reported. This 47-residue (Mr = 5364) amphiphilic polypeptide has a polar NH2 terminus, a hydrophobic central section, and a dilysine COOH terminus. An analysis of local hydrophobicity and predicted secondary structure along the peptide chain predicts that the hydrophobic central region is likely to be transmembranous. Subunit VIII from yeast cytochrome c oxidase exhibits 40.4% homology to bovine heart cytochrome c oxidase subunit VIIc , at the level of primary structure. Secondary structures and hydrophobic domains predicted from the sequences of both polypeptides are also highly conserved. From the location of hydrophobic domains and the positions of charged amino acid residues we have formulated a topological model for subunit VIII in the inner mitochondrial membrane.

The nuclear-coded subunits of yeast cytochrome c oxidase. III. Identification of homologous subunits in yeast, bovine heart, and Neurospora crassa cytochrome c oxidases.

Sequences for the NH2-terminal halves of subunits IV, V, VI, VII, and VIIa from yeast cytochrome c oxidase have been determined and used to identify homologous subunits in bovine heart and Neurospora crassa cytochrome c oxidases. In conjunction with the complete sequence of subunit VIII (S. D. Power, M. A. Lochrie , T. E. Patterson, and R. O. Poyton (1984) J. Biol. Chem. 259, 6571-6574), we have been able to identify counterparts to yeast subunits IV, V, VI, and VIII in bovine heart cytochrome c oxidase and counterparts to yeast subunits IV and V in Neurospora crassa cytochrome c oxidase. The sequences of these nuclear-coded subunits are conserved between species at a level of 30-50%. Thus, they are conserved to the same extent as the three mitochondrially coded subunits (I, II, and III). The similar degree of homology between species for both the nuclear and mitochondrially coded subunits of cytochrome c oxidase suggests that both sets of polypeptides are conserved coordinately and are, therefore, important components of the functional holoenzyme.

Mitochondrial membrane biogenesis: characterization and use of pet mutants to clone the nuclear gene coding for subunit V of yeast cytochrome c oxidase.

A nuclear pet mutant of Saccharomyces cerevisiae that is defective in the structural gene for subunit V of cytochrome c oxidase has been identified and used to clone the subunit V gene (COX5) by complementation. This mutant, E4-238 [24], and its revertant, JM110, produce variant forms of subunit V. In comparison to the wild-type polypeptide (Mr = 12,500), the polypeptides from E4-238 and JM110 have apparent molecular weights of 9,500 and 13,500, respectively. These mutations directly alter the subunit V structural gene rather than a gene required for posttranslational processing or modification of subunit V because they are cis-acting in diploid cells; that is, both parental forms of subunit V are produced in heteroallelic diploids formed from crosses between the mutant, revertant, and wild type. Several plasmids containing the COX5 gene were isolated by transformation of JM28, a derivative of E4-238, with DNA from a yeast nuclear DNA library in the vector YEp13. One plasmid, YEp13-511, with a DNA insert of 4.8 kilobases, was characterized in detail. It restores respiratory competency and cytochrome oxidase activity in JM28, encodes a new form of subunit V that is functionally assembled into mitochondria, and is capable of selecting mRNA for subunit V. The availability of mutants altered in the structural gene for subunit V (COX5) and of the COX5 gene on a plasmid, together with the demonstration that plasmid-encoded subunit V is able to assemble into a functional holocytochrome c oxidase, enables molecular genetic studies of subunit V assembly into mitochondria and holocytochrome c oxidase.

Reversed-phase high-performance liquid chromatographic purification of subunits of oligomeric membrane proteins. The nuclear coded subunits of yeast cytochrome c oxidase.

Reversed-phase chromatography of the subunits of an oligomeric membrane protein such as yeast cytochrome c oxidase requires additional sample handling techniques which are not necessary for soluble proteins. This paper considers these and discusses (1) methods for the removal of ballast material by preliminary batchwise extraction with solvent mixtures similar to those used for reversed-phase elution; (2) the chromatographic heterogeneity induced by partial cysteine oxidation; (3) the removal of tightly bound proteins from the stationary phase; and (4) the generation of an elution system with continuously variable selectivity based on acetonitrile-1-propanol ratios (0.05% triethylamine, 0.05% trifluoroacetic acid). These methods are designed to simplify complex mixtures of hydrophobic proteins prior to chromatography and to purify them chromatographically in high yield.