Complete genome sequence of "Bacillaceae sp. strain IKA-2": a cold-active, amylase-producing bacterium from ikaite columns in SW Greenland.

Bacillaceae sp. strain IKA-2 is a bacterium isolated from the permanently cold and alkaline ikaite columns in the Ikka Fjord in SW Greenland (61°12'05″N; 48°00'50″W). The bacterium grows well at 10°C in a substrate buffered to pH 10. It has a genome size of 4,424,890 bp and a guanine-cytosine (GC) content of 36.2%. The genome harbors genes involved in hydrolysis of long carbohydrates and in protection against cold shock.

Cloning, purification and characterization of a thermostable ß-galactosidase from Bacillus licheniformis strain KG9.

A thermo— and alkalitolerant Bacillus licheniformis KG9 isolated from Taşlıdere hot water spring in Batman/Turkey was found to produce a thermostable β—galactosidase. Phylogenetic analysis showed that the 16S rRNA gene from B. licheniformis strain KG9 was 99.9% identical to that of the genome sequenced B. licheniformis strain DSM 13. Analysis of the B. licheniformis DSM 13 genomic sequence revealed four putative β—galactosidase genes. PCR primers based on the genome sequence of strain DSM 13 were used to isolate the corresponding β—galactosidase genes from B. licheniformis strain KG9. The calculated molecular weights of the β—galactosidases I, II, III, and IV using sequencing data were 30, 79, 74, and 79 kDa, respectively. The genes were inserted into an expression vector and recombinant β—galactosidase was produced in Escherichia coli. Of the four β—galactosidase genes identified in strain KG9, three of them were expressed as active, intracellular enzymes in E. coli. One of the recombinant enzymes, β—galactosidase III, was purified and characterized. Optimal temperature and pH was determined to be at 60 ºC and pH 6.0, respectively. Km was determined to be 1.3 mM and 13.3 mM with oNPG (ortho—nitrophenyl—β—D—galactopyranoside) and lactose as substrates, respectively, and Vmax was measured to 1.96 μmol/min and 1.55 μmol/min with oNPG and lactose, respectively.

Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii.

The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

Optimization of the production of Chondrus crispus hexose oxidase in Pichia pastoris.

Hexose oxidase (D-hexose:O(2)-oxidoreductase, EC 1.1.3.5, HOX) normally found in the red alga Chondrus crispus was produced heterologously in different host systems. Full-length HOX polypeptide was produced in Escherichia coli, but no HOX activity could be detected. In contrast, active HOX could be produced in the methylotrophic yeast Pichia pastoris. Several growth physiological and genetic approaches for optimization of hexose oxidase production in P. pastoris were investigated. Our results indicate that specific growth conditions are essential in order to produce active HOX with the correct conformation. Furthermore, HOX seems to be activated by proteolytic cleavage of the full-length polypeptide chain into two fragments, which remain physically associated. Attempts to direct HOX to the extracellular compartment using the widely used secretion signals from Saccharomyces cerevisiae invertase or alpha-mating factor failed. However, we show in this study that HOX is transported out of P. pastoris via a hitherto unknown mechanism and that it is possible to enhance this secretion by mutagenesis from below the detection limit to at least 250 mg extracellular enzyme per liter.

Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization.

Three beta-galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 beta-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four beta-galactosidases were highly specific for hydrolysis of beta-D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.

High-efficiency synthesis of oligosaccharides with a truncated beta-galactosidase from Bifidobacterium bifidum.

An exceptionally large beta-galactosidase, BIF3, with a subunit molecular mass of 188 kDa (1,752 amino acid residues) was recently isolated from Bifidobacterium bifidum DSM20215 [Møller et al. (2001) Appl Environ Microbiol 67:2276-2283]. The BIF3 polypeptide comprises a signal peptide followed by an N-terminal beta-galactosidase region and a C-terminal galactose-binding motif. We have investigated the functional importance of the C-terminal part of the BIF3 sequence by deletion mutagenesis and expression of truncated enzyme variants in Escherichia coli. Deletion of approximately 580 amino acid residues from the C-terminal end converted the enzyme from a normal, hydrolytic beta-galactosidase into a highly efficient, transgalactosylating enzyme. Quantitative analysis showed that the truncated beta-galactosidase utilised approximately 90% of the reacted lactose for the production of galacto-oligosaccharides, while hydrolysis constituted a 10% side reaction. This 9:1 ratio of transgalactosylation to hydrolysis was maintained at lactose concentrations ranging from 10% to 40%, implying that the truncated beta-galactosidase behaved as a "true" transgalactosylase even at low lactose concentrations.

Hexose oxidase from the red alga Chondrus crispus. Purification, molecular cloning, and expression in Pichia pastoris.

Hexose oxidase from Chondrus crispus catalyzes the oxidation of a variety of mono- and disaccharides including D-glucose, D-galactose, maltose, and lactose. The enzyme has previously been partially purified and was reported to be a highly glycosylated, copper-containing protein with a relative molecular mass of approximately 130,000 (Sullivan, J. D., and Ikawa, M. (1973) Biochim. Biophys. Acta 309, 11-22). We report here the purification to homogeneity of hexose oxidase from C. crispus. The purified enzyme was cleaved with cyanogen bromide and endoproteinase Lys-C and the peptide fragments were subjected to amino acid sequence analysis. Oligonucleotides were designed on the basis of the peptide sequences and a cDNA clone encoding C. crispus hexose oxidase was obtained using polymerase chain reaction on reverse transcribed cDNA. The nucleotide sequence of the hexose oxidase cDNA contained an open reading frame of 546 amino acid residues with a predicted relative molecular mass of 61,898. No significant sequence similarity was found between hexose oxidase and other protein sequences available in data bases. Expression of the hexose oxidase cDNA in Pichia pastoris as an active enzyme confirmed the identity of the DNA sequence. Native hexose oxidase from C. crispus was characterized and compared with purified, recombinant enzyme.

A new glucose oxidase from Aspergillus niger: characterization and regulation studies of enzyme and gene.

A new glucose oxidase from Aspergillus niger was isolated and characterized. The enzyme showed different kinetic and stability characteristics when compared to a commercially available batch of A. niger glucose oxidase. The gene encoding the new glucose oxidase was isolated and DNA sequence analysis of the coding region showed 80% identity to the sequence of a glucose oxidase gene previously published. However, the similarity of the non-coding sequences up- and downstream of the open reading frame was much less, showing only 66% and 50% identity respectively. Despite the low degree of similarity between the promotor region of the new gene and the previously published one, the new glucose oxidase was likewise induced by calcium carbonate. In addition, we showed that this induction occurred on the transcriptional level. Observations concerning the effect of gluconolactone and the levels of glucose-6 phosphate isomerase upon calcium carbonate induction suggested that the enhancement of glucose oxidase biosynthesis by calcium carbonate was accompanied by a metabolic shift from glycolysis to the pentose phosphate pathway.

Purification and properties of Saccharomyces cerevisiae acetolactate synthase from recombinant Escherichia coli.

The yeast ilv2 gene, encoding acetolactate synthase, was subcloned in an Escherichia coli expression vector. Although a major part of the acetolactate synthase synthesized by E. coli cells harbouring this vector was packaged into protein inclusion bodies, we used these recombinant E. coli cells to produce large quantities of the yeast enzyme. The yeast acetolactate synthase was purified to homogeneity using first streptomycin and ammonium sulfate precipitations, followed by T-gel thiophilic interaction, Sephacryl S-300 gel filtration, Mono Q anion exchange, and Superose 12 gel filtration chromatography. SDS/PAGE and gel filtration of the purified enzyme showed that it is a dimer composed of two subunits, each with the molecular mass of 75 kDa. The purified yeast acetolactate synthase was further characterized with respect to pH optimum, dependence of the substrate, pyruvate, and requirements of the cofactors, thiamin diphosphate, Mg2+, and FAD.

Identification and characterization of mutations responsible for a runaway replication phenotype of plasmid R1.

Initiation of replication of the resistance plasmid R1 is carefully regulated by the two negatively acting factors, CopA and CopB. It is shown here that the temperature-dependent runaway-replication phenotype of an R1 plasmid mutant is caused by two point mutations in each of the promoters for the genes of these control factors. Expression of the two genes is affected in the following way: (1) one C-to-T transition in the putative -35 box of the copB-repA operon creates a two- to three-fold stronger promoter from which expression is temperature-dependent; (2) another C-to-T transition in a G + C-rich area immediately downstream from the -10 box of the copA promoter reduces expression of the copA gene three-fold. The phenotypic consequences of the two mutations are discussed in the light of the current model for R1 replication control.

Molecular cloning and functional characterization of a copy number control gene (copB) of plasmid R1.

Deletions or insertions in the copB gene of plasmid R1 result in a copy mutant phenotype. The wild-type copB gene has been cloned on various plasmid vectors. The presence of such chimeric plasmids reduced the copy number of R1 copB mutant plasmids to normal or subnormal levels, indicating the expression of a trans-acting inhibitor activity from the copB chimeras. However, the cloned copB gene did not affect the copy number of wild-type R1, and no incompatibility was exerted by the cloned copB gene against wild-type R1 (or R100). Although the copB gene is not normally required for the incompatibility exerted by copA, it is shown that the CopB function is required for expression of incompatibility by the copA gene from some types of chimeric plasmids. Mutant plasmids that have lost both Cop functions replicate in an uncontrolled fashion.

Convergent transcription interferes with expression of the copy number control gene, copA, from plasmid R1.

The copy number control gene, copA, of plasmid R1 codes for an 80-nucleotide untranslatable RNA. In Escherichia coli minicells, some copA hybrid plasmids and R1 miniplasmids also express a transcript of 200 nucleotides. Only the small RNA mediates the CopA phenotype. The switch between 80- and 200-nucleotide RNA synthesis is shown to be caused by convergent transcription; if transcription proceeds in both directions the inactive larger RNA is synthesised; the active small RNA is formed when copA transcription is not opposed by transcription from the other direction. The data presented indicate that convergent transcription interferes with copA expression by abolishing or reducing normal copA transcription termination.

RNAs involved in copy-number control and incompatibility of plasmid R1.

Replication of plasmid R1 is controlled by the products of two genes, copA and copB, that act as inhibitors of replication. Here it is shown that one small RNA synthesized from the copA gene acts as replication inhibitor. This RNA molecule was identified from analyses of RNAs synthesized in EScherichia coli minicells carrying R1 miniplasmids or chimeric plasmids containing the copA gene. In minicells, This RNA was found to be unstable with a half-life of less than a few minutes. Two mutant hybrid plasmids lacking the inhibitor function did not express the RNA normally made from plasmids carrying the wild-type copA allele. Nucleotide sequence analysis of one of the copA mutants showed that a base substitution had occurred within the promoter sequence in front of the copA gene. DNA sequence analysis of the other mutant showed that a putative transcription-termination sequence was affected. The DNA sequence analysis also showed that the RNA molecule synthesized from the copA gene is untranslatable but has the potential for a high degree of secondary structure.

The nucleotide sequence of the replication control region of the resistance plasmid R1drd-19.

The region of plasmid R 1 containing the replication control genes has been sequenced using the Maxam-Gilbert method. The nucleotide sequence of two small PstI restriction fragments (a total of about 1,000 base pairs) was determined for the wild-type R 1 plasmid as well as for two different copy mutants. It was found that one copy mutant has a single base substitution in the fragment which was recently shown to harbor an important inc/cop gene (Molin and Nordström 1980). Furthermore, the sequence indicates the presence of a structural gene that codes for a polypeptide of size 10,500 daltons. Possible gene products predicted from the nucleotide sequences and their role in replication control are discussed.

Isolation and characterization of new copy mutants of plasmid R1, and identification of a polypeptide involved in copy number control.

Site-specific deletions and insertions in the replication region of plasmid R1 have generated a new class of copy mutants that are present in the cell with 10-15-fold increased copy number. All mutations described inactivate a copy number control gene which is distinct from another cop inc gene that was identified previously (Molin and Nordström 1980). Insertion of the lac operon lacking the normal lac promoter has been used to determine the direction of transcription of this cop gene. The mutants may all be complemented by wild-type plasmid derivatives and are thus recessive. In incompatibility tests with wild-type R1 plasmids, these mutants are indistinguishable from the wild-type plasmid. It therefore seems that this cop function does not play an important role for the incompatibility function. A polypeptide, molecular weight 11,000, has been identified as being the product of this cop gene.

Clustering of genes involved in replication, copy number control, incompatibility, and stable maintenance of the resistance plasmid R1drd-19.

Plasmid R1drd-19 is present in a small number of copies per cell of Escherichia coli. The plasmid was reduced in size by in vivo as well as in vitro (cloning) techniques, resulting in a series of plasmid derivatives of different molecular weight. All plasmids isolated contain a small region (about 2 x 10(6) daltons of deoxyribonucleic acid) of the resistance transfer factor part of the plasmid located close to one of the IS1 sequences that separates the resistance transfer factor part from the resistance determinant. All these derivatives were present at the same copy number, retained the incompatibility properties of plasmid R1drd-19, and were stably maintained during cell division. Genes mutated to yield copy mutations also were found to be located in the same region.