Dennis J. Henner

Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis

A single-copy integration vector was used for the in vitro construction of translational fusions to the lacZ gene of Escherichia coli. Insertion of a single copy of the lacZ fusion into the B. subtilis chromosome leads to an easily detected Amy- phenotype. A trpE-lacZ fusion was constructed in which the trp promoter directs hybrid beta-galactosidase (beta Gal) synthesis. The level of beta Gal in a wild-type strain carrying the trpE-lacZ fusion in the chromosome is regulated by exogenous tryptophan, while a 5-methyltryptophan-resistant mutant constitutively synthesizes betaGal. A trpF-lacZ fusion was constructed and used to determine the effect of a frameshift mutation in the trpE gene on expression of the trpF-lacZ fusion. The frameshift mutation in trpE led to a three-fold reduction in the levels of the trpF-lacZ fusion. The levels of the betaGal activity of these integrated lacZ fusions appear to provide a quantitative measure of the expression of B. subtilis genes under single-copy conditions.

Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph

Aspergillus nidulans strain G191 was transformed to hygromycin resistance using plasmid pDH25, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the A. nidulans trpC gene. Southern hybridizations of transformants revealed multiple, integrated copies of the vector. A pleiotropic effect conferring increased hygromycin B sensitivity was found to be associated with the A. nidulans pyrG89 allele. Plasmid pDH25 features a ClaI site immediately preceding the hph start codon thus permitting convenient replacement of the trpC sequences with other eukaryotic promoters.

Nucleotide sequence of the Bacillus subtilis tryptophan operon.

In Bacillus subtilis, tryptophan biosynthesis is one of the most thoroughly characterized biosynthetic pathways. Recombinant DNA methodology has permitted a rapid characterization of the tryptophan (trp) gene cluster at the molecular level. In this report the nucleotide sequence of the six structural genes together with the intercistronic regions and flanking regulatory regions are presented.

Selection of an anti-IGF-1 Fab from a Fab phage library created by mutagenesis of multiple CDR loops

Diverse Fab libraries containing 2-3 x 10(8) members were generated by randomizing amino acid residues within four of the six complementarity determining regions of a humanized version of an anti-HER-2 Ab (hu4D5). These libraries were subsequently displayed on the surface of the filamentous bacteriophage M13 and selected for binding to three proteins: CD4, insulin-like growth factor 1 (IGF-1), and tissue plasminogen activator. An Fab-bacteriophage was isolated that showed specific binding to IGF-1. The affinity of this Fab was determined to be 3.5 microM.

High-level expression in Escherichia coli and rapid purification of phosphatidylinositol-specific phospholipase C from Bacillus cereus and Bacillus thuringiensis

The construction of four vectors for high-level expression in Escherichia coli of the phosphatidylinositol-specific phospholipase C from Bacillus cereus or Bacillus thuringiensis is described. In all constructs the coding sequence for the mature phospholipase is precisely fused to the E. coli heat-stable enterotoxin II signal sequence for targeting of the protein to the periplasm. In one set of plasmids expression of the B. cereus or B. thuringiensis enzyme is under control of the E. coli alkaline phosphatase promoter, while in a second set of plasmids expression is under control of a lac-tac-tac triple tandem promoter. A simple and rapid procedure for complete purification of the phospholipase C overproduced in E. coli, involving isolation of the periplasmic proteins by osmotic shock followed by a single column chromatography step, is described. The largest quantity of purified enzyme, 40-60 mg per liter culture, is obtained with the plasmid expressing the B. cereus enzyme under control of the lac-tac-tac promoter. Lower quantities are obtained with the plasmids containing the alkaline phosphatase promoter (15-20 and 4-6 mg/liter for the B. cereus and B. thuringiensis enzymes, respectively) and with the plasmid expressing the B. thuringiensis phospholipase under control of the lac-tac-tac promoter (15-20 mg/liter). A comparison of the functional properties of the recombinant phospholipases with the native enzymes isolated from B. cereus or B. thuringiensis culture supernatant shows that they are identical with respect to their catalytic functions, viz., cleavage of phosphatidylinositol and cleavage of the glycosyl-phosphatidylinositol membrane anchor of bovine erythrocyte acetylcholinesterase.

The organization and nucleotide sequence of the Bacillus subtilis hisH, tyrA and aroE genes

The nucleotide sequence of approximately 3 kb of Bacillus subtilis DNA distal to the trp operon was determined. Three open reading frames were found and these were shown to encode the hisH, tyrA and aroE genes. Integrative plasmids were constructed to interrupt transcription through this region. These data suggest that these three genes can be transcribed from both the trp promoter preceding the trp operon and from a promoter within the trpA structural gene.

Construction of a vector for cloning promoters in Bacillus subtilis

A versatile vector for cloning DNA fragments containing promoter activity in Bacillus subtilis was derived from plasmids pBR322, pUB110 and pC194. Selection is based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The plasmid contains a second selectable marker, neomycin resistance, and contains functional origins of replication for both B. subtilis and Escherichia coli.

The promoter of the glucoamylase-encoding gene of Aspergillus niger functions in Ustilago maydis.

Promoter sequences from the Aspergillus niger glucoamylase-encoding gene (glaA) were linked to the bacterial hygromycin (Hy) phosphotransferase-encoding gene (hph) and this chimeric marker was used to select Hy-resistant (HyR) Ustilago maydis transformants. This is an example of an Ascomycete promoter functioning in a Basidiomycete. HyR transformants varied with respect to copy number of integrated vector, mitotic stability, and tolerance to Hy. Only 216 bp of glaA promoter sequence is required for expression in U. maydis but this promoter is not induced by starch as it is in Aspergillus spp. The transcriptional start points are the same in U. maydis and A. niger.

Nucleotide sequence of the Bacillus subtilis trpE and trpD genes

Several overlapping portions of the tryptophan (trp) operon of Bacillus subtilis have been cloned into plasmid pBR322. The nucleotide sequence of the region comprising the trpE and trpD genes and a portion of the trpC gene has been determined. When the deduced amino acid sequences of these genes are compared with their counterparts in Escherichia coli, several regions of striking homology are seen. The probable initiation codons for the trpE, D and C genes are each preceded by a recognizable Shine-Dalgarno sequence. The coding sequences for the trpE and trpD genes and for the trpD and trpC genes overlap slightly, leaving no intercistronic regions between the genes.

An SfiI restriction map of the Bacillus subtilis 168 genome.

A restriction map of 24 SfiI (GGCCN4/NGGCC) restriction fragments has been constructed for the Bacillus subtilis genome. The combined sizes of the fragments indicate a genome size of approx. 4.2 Mb. The SfiI fragments range in size from 7-730 kb. Genetic markers have been located on 19 of the SfiI fragments, and 69 genetic markers have been assigned to the SfiI restriction map.