ABSTRACT
The first two genes pcbAB and pcbC of the penicillin biosynthesis pathway are expressed from a 1.01-kilobase bidirectional promoter region. A series of sequential deletions were made in the pcbAB promoter region, and the constructions with the modified promoters coupled to the lacZ reporter gene were introduced as single copies at the pyrG locus in Penicillium chrysogenum npe10. Three regions, boxes A, B, and C, produced a significant decrease in expression of the reporter gene when deleted. Protein-DNA complexes were observed by using the electrophoretic mobility shift assay with boxes A and B (complexes AG1, BG1, BG2, and BL1) but not with box C. Uracil interference assay showed that a protein in P. chrysogenum cell extracts interacts with the thymines in a palindromic heptanucleotide TTAGTAA. Point mutations and deletion of the entire TTAGTAA sequence supported the involvement of this sequence in the binding of a transcriptional activator named penicillin transcriptional activator 1 (PTA1). In vivo studies using constructions carrying point mutations in the TTAGTAA sequence (or a deletion of the complete heptanucleotide) confirmed that this intact sequence is required for high level expression of the pcbAB gene. The TTAGTAA sequence resembles the target sequence of BAS2 (PHO2), a factor required for expression of several genes in yeasts.
Subject(s)
Penicillins/biosynthesis , Penicillium chrysogenum/genetics , Peptide Synthases/genetics , Artificial Gene Fusion , Base Sequence , Binding Sites , DNA, Fungal , DNA-Binding Proteins/metabolism , Mutagenesis , Penicillium chrysogenum/metabolism , Peptide Synthases/metabolism , Point Mutation , Regulatory Sequences, Nucleic Acid , Sequence Deletion , Sequence Homology, Nucleic Acid , Transcription, GeneticABSTRACT
The interaction between the acidic exopolysaccharides produced by two Bradyrhizobium strains and several metal cations has been studied. Aqueous solutions in the millimolar range of Fe but not of Fe precipitated the exopolysaccharides from Bradyrhizobium (Chamaecytisus) strain BGA-1 and, to a lesser extent, Bradyrhizobium japonicum USDA 110. The precipitation was pH dependent, with a maximum around pH 3. The precipitate was redissolved by changing the pH and by Fe reduction or chelation. Deacetylation of B. japonicum polysaccharide increased its precipitation by Fe. At pH near neutrality, the polysaccharide from Bradyrhizobium (Chamaecytisus) strain BGA-1 stabilized Fe solutions, despite the insolubility of Fe(OH)(3). Aluminum precipitated Bradyrhizobium (Chamaecytisus) polysaccharide but not the polysaccharide produced by B. japonicum. The precipitation showed a maximum at about pH 4.8, and the precipitate was redissolved after Al chelation with EDTA. Precipitation was inhibited by increases in the ionic strength over 10 mM. Bradyrhizobium (Chamaecytisus) polysaccharide was also precipitated by Th, Sn, Mn, and Co. The presence of Fe increased the exopolysaccharide precipitation by aluminum. No precipitation, gelation, or increase in turbidity of polysaccharide solutions occurred when K, Na, Ca, Mg, Cu, Cd, Pb, Zn, Hg, or U was added at several pH values. The results suggest that the precipitation is based on the interaction between carboxylate groups from different polysaccharide chains and the partially hydrolyzed aquoions of Fe, Al, Th, and Sn.
