Some features of DNA-binding proteins involved in the regulation of the Streptomyces aureofaciens gap gene, encoding glyceraldehyde-3-phosphate dehydrogenase.

A gapR gene, encoding a protein similar to the AraC/XylS family of bacterial transcriptional regulators, was previously identified upstream of the gap gene, coding for glyceraldehyde-3-phosphate dehydrogenase in Streptomyces aureofaciens. The GapR protein overproduced in Escherichia coli was shown to bind to the gap-P promoter region. Using the gel mobility shift assay with cell-free protein extracts from different developmental stages of S. aureofaciens, we identified several other proteins, in addition to GapR, that specifically bound to the S. aureofaciens gap-P promoter region. When cell-free extracts from S. aureofaciens cultivated in liquid medium with glucose were analyzed, only one complex corresponding to GapR was detected. A new protein interacting with the gap-P promoter was detected in stationary culture of S. aureofaciens grown in the presence of mannitol as carbon sources. The GapR protein was partially purified from S. aureofaciens cultivated in liquid medium containing glucose and used for binding studies. DNA footprinting analysis revealed an identical protected region as previously identified for the GapR protein overproduced from Escherichia coli. The direct role of the GapR protein in the regulation of gap expression in S. aureofaciens in vivo was confirmed but regulation of gap expression seems to be more complex, possibly involving other regulatory protein(s), depending on the developmental stage of S. aureofaciens.

Expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase of Streptomyces aureofaciens requires GapR, a member of the AraC/XylS family of transcriptional activators.

Expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is developmentally regulated, and induced by glucose in Streptomyces aureofaciens. A gene, gapR, encoding a protein similar to the AraC/XylS family of bacterial transcriptional regulators was identified upstream of gap. The gapR gene was constitutively expressed from a single promoter during the course of differentiation. By integrative transformation, via double crossover, a stable null mutant of the gapR gene was obtained. The mutation only slightly affected growth, and had no effect on differentiation of S. aureofaciens. However, transcription of the GAPDH-encoding gap gene was substantially reduced in the S. aureofaciens DeltagapR null mutant, irrespective of carbon source used. Though GAPDH activity was about 1.5-fold lower in the mutant, the substantial enzyme activity remained, suggesting the presence of a second GAPDH which is sufficient to ensure growth. The GapR protein, overproduced in Escherichia coli, was shown to bind upstream of the gap-P promoter region. The results indicate a direct role of GapR in regulation of gap expression in S. aureofaciens.

Identification of DNA-binding proteins involved in regulation of expression of the Streptomyces aureofaciens sigF gene, which encodes sporulation sigma factor sigma(F).

Expression of the sigF gene encoding a sporulation-specific sigma factor, sigma(F), in Streptomyces aureofaciens is restricted only to sporulation. Gel mobility-shift assays using protein fractions from different developmental stages of S. aureofaciens revealed two different putative proteins specifically bound to the sigF promoter region: a protein (designated RsfA) present in young substrate mycelium, and a protein (designated RsfB) present in the course of sporulation. Based on the characteristic profiles of their appearance during differentiation, RsfA might be a repressor and RsfB an activator of sigF expression. The location of a specific binding site of the repressor-like protein (RsfA) was determined by gel mobility-shift assays of promoter deletion fragments and by DNase I footprinting analysis. The binding site mapped from nucleotides -87 to -25 relative to the transcription start point of the sigF promoter, and overlapped the -35 promoter region. Given the dependence of sigF expression upon whiH, the putative sporulation transcription factor WhiH was overproduced in Escherichia coli and used in the mobility-shift assays with the sigF promoter. However, no specific binding was detected, indicating an indirect dependence of sigF upon whiH.

Cloning of the putative aldehyde dehydrogenase, aldA, gene from Streptomyces aureofaciens.

A Streptomyces aureofaciens gene, gap, encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was previously identified. Hybridization studies suggested the presence of a second gap gene in S. aureofaciens. To clone the gene, S. aureofaciens subgenomic library was screened with an oligonucleotide probe encoding a peptide motif conserved in all GAPDH. 3352 bp positive BamHI fragment was identified, the length of which correlated with the hybridization signal. The nucleotide sequence of the fragment was determined, and analysis of the sequence revealed the presence of three open reading frames (ORF). However, none of the genes coded for GAPDH. All three genes formed an operon, consisting of gene orf251, with a high homology to a conserved gene present only in archaeabacteria, and the aldA and adhA genes homologous to various eukaryotic and prokaryotic aldehyde- and alcohol-dehydrogenases, with maximum homology to the phenylacetaldehyde dehydrogenases and arylalcohol dehydrogenases, respectively.

The gene downstream of Streptomyces aureofaciens whiB encodes a large protein with proposed transmembrane localization, and is induced by glucose.

The sequence analysis of the region downstream of the Streptomyces aureofaciens whiB sporulation gene revealed a long open reading frame (1219 amino acids; Mr 128 209) encoding protein with potential transmembrane structure. By integrative transformation, via double cross-over, a stable null mutant of the gene, orf1219, was prepared. This mutation appeared to have no obvious effect on vegetative growth and differentiation. In vitro and in vivo transcriptional analysis of the downstream gene revealed a single apparent promoter induced by glucose.

The Streptomyces aureofaciens homologue of the whiB gene is essential for sporulation; its expression correlates with the developmental stage.

In previous experiments, a Streptomyces aureofaciens gene highly similar to the sporulation-specific whiB gene of Streptomyces coelicolor was identified. By integrative transformation, via double cross-over, a stable null mutant of the whiB-homologous gene of S. aureofaciens was obtained. The disruption blocked differentiation at a stage between the formation of aerial mycelium and the development of mature spores, producing white aerial hyphae without septation. Expression of the whiB gene was investigated during differentiation by S1 nuclease mapping, using RNA prepared from S. aureofaciens in various developmental stages. Two putative promoters were identified upstream of the whiB coding region. The stronger promoter, whiB-P2, was induced at the beginning of aerial mycelium formation, and the weaker promoter, whiB-P1, was expressed fairly constantly during differentiation. No differences in the expression of the whiB promoters were detected in an rpoZ-disrupted S. aureofaciens strain. The promoter bearing DNA fragment was inserted into the promoter-probe vector pARC1 to produce an expression pattern consistent with the results of direct RNA analysis.