Cloning, overexpression, and purification of Escherichia coli quinolinate synthetase.

Quinolinate synthetase catalyzes the second step of the de novo biosynthetic pathway of pyridine nucleotide formation. In particular, quinolinate synthetase is involved in the condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinic acid. To study the mechanism of action, the specificity of the enzyme and the interaction with l-aspartate oxidase, the other component of the so-called "quinolinate synthetase complex," the cloning, the overexpression, and the purification to homogeneity of Escherichia coli quinolinate synthetase were undertaken. The results are presented in this paper. Since the overexpression of the enzyme resulted in the formation of inclusion bodies, a procedure of renaturation and refolding had to be set up. The overexpression and purification procedure reported in this paper allowed the isolation of 12 mg of electrophoretically homogeneous quinolinate synthetase from 1 liter of E. coli culture. A new, continuous, method for the evaluation of quinolinate synthetase activity was also devised and is presented. Finally, our data definitely exclude the possibility that other enzymes are involved in the biosynthesis of quinolinic acid in E. coli, since it is possible to synthesize quinolinic acid from l-aspartate, dihydroxyacetone phosphate, and O(2) by using only nadA and nadB gene overexpressed products.

The UP element of the promoter for the flagellin gene, hag, stimulates transcription from both SigD- and SigA-dependent promoters in Bacillus subtilis.

DNA sequences upstream (UP element) of the core promoter (-10, -35 region) of the Bacillus subtilis flagellin gene hag stimulate transcription in vivo and in vitro. We constructed a number of hybrids, placing the UP element of hagp upstream of the core of one SigD-dependent (fliDp) and two SigA-dependent (tmsp, vegp) B. subtilis promoters. The hybrid promoters were fused to a lacZ reporter gene and their activity tested in vivo. The presence of the UP module enhanced transcription at both types of promoters. We conclude that the hagp UP sequence can act as a promoter module independently of the core sequence.

Role of FlgM in sigma D-dependent gene expression in Bacillus subtilis.

The alternative sigma factor sigma D directs transcription of a number of genes involved in chemotaxis, motility, and autolysis in Bacillus subtilis (sigmaD regulon). The activity of SigD is probably in contrast to that of FlgM, which acts as an antisigma factor and is responsible for the coupling of late flagellar gene expression to the assembly of the hook-basal body complex. We have characterized the effects of an in-frame deletion mutation of flgM. By transcriptional fusions to lacZ, we have shown that in FlgM-depleted strains there is a 10-fold increase in transcription from three different sigmaD-dependent promoters, i.e., Phag, PmotAB, and PfliDST. The number of flagellar filaments was only slightly increased by the flgM mutation. Overexpression of FlgM from a multicopy plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible spac promoter drastically reduced the level of transcription from the hag promoter. On the basis of these results, we conclude that, as in Salmonella typhimurium, FlgM inhibits the activity of SigD, but an additional element is involved in determining the number of flagellar filaments.

Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element.

Flagellin is one of the most abundant proteins in motile bacteria, yet its expression requires a low abundance sigma factor (sigma 28). We show that transcription from the Bacillus subtilis flagellin promoter is stimulated 20-fold by an upstream A+T-rich region [upstream promoter (UP) element] both in vivo and in vitro. This UP element is contacted by sigma 28 holoenzyme bound at the flagellin promoter and binds the isolated alpha 2 subassembly of RNA polymerase. The UP element increases the affinity of RNA polymerase for the flagellin promoter and stimulates transcription when initiation is limited by the rate of RNA polymerase binding. Comparison with other promoters in the flagellar regulon reveals a bipartite architecture: the -35 and -10 elements confer specificity for sigma 28, while promoter strength is determined largely by upstream DNA sequences.

Sequence around the 159 degree region of the Bacillus subtilis genome: the pksX locus spans 33.6 kb.

The nucleotide sequence of 20 kb contiguous to the pksX locus of Bacillus subtilis was determined. Six ORFs were recognized, one of which extended for 13,341 nucleotides. Their predicted products have significant similarities to proteins with known functions involved in the synthesis of polypeptides and polyketides or in fatty acid metabolism. At the nucleotide level, three regions with a high level of sequence identity (49-54%) to the Aspergillus nidulans wA gene, responsible for the synthesis of a polyketide pigment, were recognized. The observed similarities suggest that the 20 kb region and the previously reported 13.6 kb region containing pksX are part of the same locus, possibly involved in secondary metabolism.

Coupling of flagellin gene transcription to flagellar assembly in Bacillus subtilis.

The regulation of flagellin gene expression in Bacillus subtilis was examined in vivo by means of a lacZ translational fusion to the flagellin structural gene (hag). We have tested the effects of two known mutations (flaA4 and flaA15) in the major flagellar operon and of three deletions. One deletion was in frame in the fliI cistron, one was out of frame in the fliK cistron, and the last spanned about 21 kb of the flaA operon. In all instances, the expression of the flagellin gene was defective. Flagellin gene expression was restored in the strain with the 21-kb deletion by overexpression of the sigD gene under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible spac promoter. These results indicate that transcription of the flagellin gene is dependent on the formation of the flagellar basal body but that such a requirement can be bypassed by overexpression of sigD. Lack of expression of hag was observed in the presence of flaD1, flaD2, and delta sin mutations as well.

Functional analysis of the outB gene of Bacillus subtilis.

Three additional alleles of the outB gene of Bacillus subtilis, whose activity is required for spore outgrowth, were identified. The nucleotide sequence of three mutant genes was determined. Analyses of dominance-recessivity showed that the wild-type allele is dominant over the mutant ones. When the outB gene was placed under the control of the inducible spac-1 promoter, the presence of IPTG was necessary to obtain normal growth. The results suggested that the outB gene is required for growth of B. subtilis. Expression of outB from the sporulation promoter spoIID negatively affected subsequent spore outgrowth, without altering vegetative growth and sporulation.

The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide.

We cloned and sequenced 8.3 kb of Bacillus subtilis DNA corresponding to the flaA locus involved in flagellar biosynthesis, motility, and chemotaxis. The DNA sequence revealed the presence of 10 complete and 2 incomplete open reading frames. Comparison of the deduced amino acid sequences to data banks showed similarities of nine of the deduced products to a number of proteins of Escherichia coli and Salmonella typhimurium for which a role in flagellar functioning has been directly demonstrated. In particular, the sequence data suggest that the flaA operon codes for the M-ring protein, components of the motor switch, and the distal part of the basal-body rod. The gene order is remarkably similar to that described for region III of the enterobacterial flagellar regulon. One of the open reading frames was translated into a protein with 48% amino acid identity to S. typhimurium FliI and 29% identity to the beta subunit of E. coli ATP synthase.

In vivo generation of hybrids between two Bacillus thuringiensis insect-toxin-encoding genes.

The parasporal crystal of Bacillus thuringiensis is composed of polypeptides highly toxic to a number of insect larvae. The structural genes (cryIA) encoding the Lepidoptera-specific toxin from different bacterial strains diverge primarily in a single hypervariable region, whereas the N-terminal and C-terminal parts of the proteins are highly conserved. In this report, we describe the generation of hybrid genes between two cryIA genes. Two truncated cryIA genes were cloned in a plasmid vector in such way as to have only the hypervariable region in common. The two truncated cryIA genes were separated by the tetracycline-resistance determinant (or part of it). In vivo recombination between the hypervariable regions of the cryIA genes reconstituted an entire hybrid cryIA gene. Direct sequence analysis of 17 recombinant plasmids identified eleven different crossover regions which did not alter the reading frame and allowed the production of eight different hybrid proteins. The recombination events were independent from the RecA function of Escherichia coli. Some of the hybrid gene products were more specific in their insecticidal action and one had acquired a new biological activity.

The outB gene of Bacillus subtilis regulates its own transcription.

The outB gene of Bacillus subtilis is under the control of two promoters (P1 and P2). To study the regulation of expression from the P1 promoter we have constructed a set of multicopy plasmids carrying different portions of the outB region and analyzed the transcripts present in vivo by RNase protection experiments. The data indicate that the product of gene outB regulates its own transcription from the P1 promoter. We also constructed an outB-lacZ fusion in an insertional plasmid. The plasmid was inserted into the chromosome adjacent to or distal from the outB gene. Assays of beta-galactosidase activity and RNase protection experiments are in accordance with a model implying that the product of gene outB regulates the initiation of transcription from the P1 promoter acting in the cis configuration.

Nucleotide sequence of the outB locus of Bacillus subtilis and regulation of its expression.

The outB gene is one of the genes involved in the process of spore outgrowth in Bacillus subtilis. The gene has been cloned in bacteriophage lambda and subcloned in plasmids. We have determined the sequence of 2,553 base pairs around the outB locus. The locus was found to code for a protein of about 30,000 daltons. Analysis of the in vivo transcripts from this region by RNase protection experiments revealed the presence of two start sites for transcription. Two potential promoters for these transcripts can be tentatively assigned from the sequence data. The amount of one transcript is highest during outgrowth and vegetative growth and absent during the stationary phase. The second transcript is present at a low level throughout the cell cycle.