Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase.

A resident-plasmid cloning system developed for Bacillus subtilis has been used to isolate recombinant plasmids carrying DNA from Bacillus licheniformis which confer alpha-amylase activity on alpha-amylase-negative mutants of B. subtilis. These plasmids contain a 3550-bp insert at the EcoRI site of the plasmid pBD64. Subcloning various lengths of the B. licheniformis DNA has localised the gene to a 2550-bp BclI fragment. We present evidence that the cloned fragment codes for a B. licheniformis heat-stable alpha-amylase with a temperature optimum of 93 degrees C. The foreign gene is expressed efficiently in B. subtilis and is stably maintained.

Accurate direct determination of low-density lipoprotein cholesterol using an immunoseparation reagent and enzymatic cholesterol assay.

Clinical laboratories currently estimate low-density lipoprotein cholesterol using the Friedewald formula, which requires fasting specimens and is subject to error with increasing triglyceride levels. We describe a rapid method for isolating low-density lipoproteins using the Direct LDL Immunoseparation Reagent for subsequent measurement of cholesterol by conventional assay. This method meets current guidelines for precision with within-run and run-to-run coefficients of variation of less than 3%. Results are in good agreement with the beta quantification reference method (Direct LDL-C = 1.03 [beta quantification] -0.06 mmol/L, [2.4 mg/dL] r = 0.980), there is minimal bias associated with increasing triglycerides or high-density lipoprotein cholesterol, and patient fasting is not required for accurate analysis. The Direct LDL Immunoseparation Reagent overcomes drawbacks of the Friedewald formula and appears to be suitable for accurate quantitation of low-density lipoprotein cholesterol in the routine laboratory.

A cloned gene that is turned on at an intermediate stage of spore formation in Bacillus subtilis.

Cells of Bacillus subtilis synthesize a relatively long-lived ribonucleic acid (RNA) of about 300 bases during the course of spore formation. This transcript does not appear until an intermediate stage (III or IV) of development but is the predominant sporulation-specific transcript among RNAs of discrete size in late (stages IV to VI) developing cells. Appearance of the 300-base RNA is under sporulation control as this transcript could not be detected in cells of an early-blocked sporulation mutant (Spo0A). We have located the coding sequence for the 300-base RNA within a cloned chromosomal segment from the purA-cysA region that was previously shown to contain a cluster of genes that are actively transcribed during sporulation. The coding sequence for the 300-base RNA (designated as the 0.3 kb gene) mapped between a gene (veg) that was actively transcribed during growth and development and a gene (0.4 kb) that was turned on at the onset of sporulation. Although clustered within a small segment of the chromosome, the veg, 0.3 kb, and 0.4 kb transcription units exhibited, therefore, distinct patterns of temporally programmed gene expression. Models for the activation of the 0.3 kb gene at an intermediate stage of development are discussed.

Developmentally regulated transcription in a cloned segment of the Bacillus subtilis chromosome.

We describe a model system for studying developmentally regulated transcription during spore formation in Bacillus subtilis. This model system is a cloned cluster of genes known as 0.4 kb, ctc, and veg from the purA-cysA region of the B. subtilis chromosome. Each gene exhibited a distinct pattern of transcription in cells growing in glucose medium and in cells deprived of nutrients in sporulation medium. The 0.4 kb gene was transcribed at a low level in growing cells but was actively transcribed during nutrient deprivation in sporulation medium. This ribonucleic acid (RNA) synthesis was dependent upon the products of five B. subtilis genes that are involved in the initiation of spore formation:spo0A, spo0A, spo0E, spo0F, and spo0H. A mutation in any one of these regulatory genes severely restricted transcription of the 0.4 kb sequence. Transcription of the ctc gene was also turned on by nutrient deprivation, but this RNA synthesis was not impaired in spo0 mutants. Although not under spo0 control, the ctc gene probably corresponds to a locus, spoVC, whose product is required at a late stage of sporulation. Finally, the veg gene was actively transcribed both in growing cells and in nutrient-deprived cells. Like ctc RNA synthesis, transcription of the veg gene was not dependent upon the spo0 gene products. We propose that the spo0A, spo0B, spo0E, spo0F, and spo0H gene products are components of a pathway(s) that senses nutrient deprivation in B. subtilis and translates this environmental signal into the transcriptional activation of a subset of developmental genes.

Nucleotide sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene: comparison with the B. amyloliquefaciens gene.

The DNA sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene is reported. Comparison of the inferred amino acid sequence of the B. licheniformis alpha-amylase gene with that of the Bacillus amyloliquefaciens gene shows that whereas the amino acid sequences of the mature proteins have considerable homology, the sequences for the signal peptides are distinct.

Cloning in Bacillus subtilis of an extremely thermostable alpha amylase: comparison with other cloned heatstable alpha amylases.

A heatstable alpha amylase gene was shotgun cloned from Bacillus licheniformis RPO1 into Bacillus subtilis. Restriction endonuclease analysis of the recombinant plasmid revealed a map which was identical to a previously cloned alpha amylase from B. licheniformis FDO2 and very similar to the restriction map of a high temperature amylase from Bacillus coagulans. The thermostability and temperature optimum of the cloned alpha amylase was measureably different from those of the previously reported cloned alpha amylases.

Mapping a cloned gene under sporulation control by inserttion of a drug resistance marker into the Bacillus subtilis chromosome.

A segment of Bacillus subtilis deoxyribonucleic acid (DNA) previously cloned in Escherichia coli contains a gene (the 0.4-kilobase [kb] gene) whose transcription is activated at an early stage of spore development. To map the genetic location of the 0.4-kb gene, we constructed a hybrid plasmid that inserts a chloramphenicol resistance determinant into the B. subtilis chromosome by recombination at a site of homology between cloned B. subtilis DNA and the chromosome. This hybrid plasmid (p1949-2) was constructed from the E. coli plasmid pMB9, the B. sultilis chloramphenicol resistance plasmid pCM194 (whose replication function was inactivated), and B. subtilis DNA from the vicinity of the 0.4-kb gene. Transformation of B. subtilis cells to drug resistance by p1949-2 was dependent upon the B. subtilis RecE+ phenotype and resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the 0.4-kb gene region of the chromosome. Chloramphenicol resistance in cells transformed by p1949-2 was mapped to the purA-cysA region of the B. subtilis chromosome, a region. In addition, DNA adjacent to the 0.4-kb gene was shown to contain the wild-type allele of genetic marker (tms-26) from that region.

Cloning and expression of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F in Escherichia coli.

The peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) gene from Flavobacterium meningosepticum was cloned into a high copy number Escherichia coli plasmid. Levels of PNGase F activity produced in cultures of the recombinant strain were up to 100-fold higher than those obtained in cultures of F. meningosepticum. The complete PNGase F gene sequence was determined. Comparison of the predicted amino acid sequence of pre-PNGase F to the N-terminal sequence of the native mature enzyme indicates that the protein is synthesized with a 40-amino acid signal sequence that is removed during secretion in F. meningosepticum. The recombinant PNGase F produced in E. coli is a mixture of products comprised predominantly of two proteins with molecular masses of 36.3 and 36.6 kDa. These proteins have a higher apparent molecular mass than the 34.7-kDa native enzyme. N-terminal amino acid sequencing demonstrated that these higher molecular mass products result from cleavage of the pre-PNGase F in E. coli upstream of the native N terminus. The PNGase F gene was engineered to encode a preenzyme that was processed in E. coli to give an N terminus identical to that of the native enzyme. Purified preparations of this form of recombinant PNGase F were shown to be suitable for glycoprotein analyses since they possess no detectable endo-beta-N-acetylglucosaminidase F, exoglycosidase, or protease activity.