Isolation of a developmental gene of Bacillus subtilis and its expression in Escherichia coli.

Glucose dehydrogenase of Bacillus subtilis is a developmental enzyme that is not found in growing (vegetative) cells but is synthesized after the differentiation process that leads to the production of endospores has started. We have isolated the gene coding for this enzyme from a lambda Charon 4A phage library of B. subtilis DNA. It is transcribed and translated in vegetative cells of the nondifferentiating organism Escherichia coli into enzymatically active glucose dehydrogenase that has the same physicochemical properties as the enzyme produced in B. subtilis during sporulation. Subcloning of the lambda DNA insert into pBR322 plasmid derivatives showed that the glucose dehydrogenase gene was transcribed in E. coli from a promoter within the B. subtilis genome.     

DNA polymerase III gene of Bacillus subtilis.

The Bacillus subtilis dnaF (polC) gene that codes for the alpha subunit of the DNA polymerase III holoenzyme has been sequenced. It consists of 4005 base pairs coding for 1335 amino acids (from the start to the stop codon), giving a molecular weight of 151,273. A mutation (azp-12) that confers resistance to the antimicrobial drug 6-(p-hydroxyphenylazo)-uracil is due to a single base change at nucleotide 3523, from TCA to GCA, resulting in a change of the 1175th amino acid, serine, to alanine. It is in the active site and located at the C-terminal part of the enzyme. The amino acid composition in an N-terminal domain has 26% homology to the epsilon subunit coded by the dnaQ gene of Escherichia coli, which is a 3'----5' proofreading exonuclease, supporting an earlier observation that this function is an integral part of the polymerase molecule in B. subtilis.     

An ordered collection of Bacillus subtilis DNA segments cloned in yeast artificial chromosomes.

A collection of 772 Bacillus subtilis DNA segments was obtained by cloning in yeast artificial chromosomes. The B. subtilis inserts of 288 clones were mapped by hybridization using as probes 65 cloned genes and 188 isolated insert ends. In this way, 59 inserts were ordered in four contigs that cover > 98% of the B. subtilis chromosome. This ordered collection is now available for further genetic and physical analysis of the B. subtilis genome.     

Translational block to expression of the Escherichia coli Tn9-derived chloramphenicol-resistance gene in Bacillus subtilis.

The Gram-negative product-encoding Tn9-derived chloramphenicol-resistance (Cmr) gene can be cloned but not phenotypically expressed in Bacillus subtilis. We show that, even when transcribed from B. subtilis promoters, the ribosomal binding site for the Cmr gene does not function well in B. subtilis. The Cmr gene product, chloramphenicol acetyltransferase (CmAcTase; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28), is detected in B. subtilis when the promoters, ribosomal binding sites, and initiation codons of B. subtilis genes are fused to the Cmr gene. These gene fusions lead to the in vivo production of mRNAs containing B. subtilis translation start signals followed in an open reading frame by the translation start site normally used by Escherichia coli to initiate translation of Cmr mRNA. Both fusion and native CmAcTase proteins are produced in E. coli, but only fusion CmAcTase is produced in B. subtilis. We conclude that the absence of native CmAcTase in B. subtilis is due to inability of the E. coli ribosomal binding site to function well in B. subtilis. Since fusion CmAcTase polypeptides are produced in E. coli, we conclude that these particular B. subtilis regulatory elements function heterologously in E. coli. The absence of a suitable binding site on the Cmr gene for B. subtilis ribosomes is consistent with reports that many E. coli genes are not expressed in B. subtilis and that E. coli mRNA functions poorly in B. subtilis in vitro translation systems. The functioning of B. subtilis regulatory sequences in E. coli is consistent with in vivo and in vitro data showing the expression of B. subtilis genes in E. coli. To confirm the hypothesis that the large CmAcTase proteins are NH2-terminal fusions of native CmAcTase we partially determined the sequence of one CmAcTase fusion protein.     

Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis.

The Escherichia coli lac operator has been placed on the 3' side of the promoter for the penicillinase gene of Bacillus licheniformis, creating a hybrid promoter controllable by the E. coli lac repressor. The E. coli lac repressor gene has been placed under the control of a promoter and ribosome-binding site that allows expression in Bacillus subtilis. When the penicillinase gene that contains the lac operator is expressed in B. subtilis on a plasmid that also produces the lac repressor, the expression of the penicillinase gene can be modulated by isopropyl beta-D-thiogalactoside (IPTG), an inducer of the lac operon in E. coli. A similar system was constructed from a promoter of the B. subtilis phage SPO-1 and the leukocyte interferon A gene, which allowed the controlled expression of interferon in B. subtilis. These two examples show that a functional control system can be introduced into B. subtilis from E. coli.     

A general method for maximizing the expression of a cloned gene.

We present a method, utilizing a combination of restriction endonuclease cleavage and digestion with Escherichia coli exonuclease III and Aspergillus orizae nuclease S1, that allows us to position a restriction fragment bearing the promoter of the lacZ gene of E. coli at virtually any distance in front of any cloned gene. In particular, we have used this method to examine the effect on protein production of gene-promoter separation for the cro gene of phage lambda and to produce plasmids that, upon transformation into appropriate E. coli hosts, direct the synthesis of up to 190,000 cro protein monomers per cell.     

Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis.

One S. aureus plasmid coding for tetracycline resistance, pT127, and four plasmids (pC194, pC221, pC223, and pUB112) coding for chloramphenicol resistance have been introduced by transformation into B, subtilis. The plasmids replicate in--and confer antibiotic resistance upon--their new host. These experiments show that the potential for genetic exchange between diverse bacterial species is greater than has been commonly assumed.