A-factor and phosphate depletion signals are transmitted to the grixazone biosynthesis genes via the pathway-specific transcriptional activator GriR

Abstract

Grixazone (GX), which is a diffusible yellow pigment containing a phenoxazinone chromophore, is one of the secondary metabolites under the control of A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) in Streptomyces griseus. GX production is also induced by phosphate starvation. The whole biosynthesis gene cluster for GX was cloned and characterized. The gene cluster consisting of 13 genes contained six transcriptional units, griT, griSR, griR, griAB, griCDEFG, and griJIH. During cultivation in a phosphate-depleted medium, the six promoters were activated in the order (i) griR, (ii) griC and griJ, and (iii) griT, griS, and griA. Disruption of griR, which encodes a SARP family transcriptional regulator, abolished the transcriptional activation of all other genes in the cluster. In addition, ectopic expression of griR from a constitutively active promoter resulted in GX overproduction even in the absence of AdpA, a key transcriptional activator in the A-factor regulatory cascade, and in the presence of phosphate at a high concentration. GriR monomers bound direct repeat sequences in the griC and griJ promoters in a cooperative manner. Therefore, the early active genes (griCDEFG and griJIH), all of which, except for griG (which encodes a transporter-like protein), encode the GX biosynthesis enzymes, were directly activated by GriR. The transcription of griR was greatly reduced in the presence of phosphate at a high concentration and was hardly detected in the absence of AdpA. These findings showed that both A-factor and phosphate depletion signals were required for griR transcription and both signals were transmitted to the GX biosynthesis genes solely via the griR promoter.

FIG. 1.

Organization of the GX biosynthesis gene cluster (A) and a proposed GX biosynthesis pathway (B). (A) Bold arrows indicate the positions and directions of the GX biosynthesis genes (griT, -S, -R, and -A to -J) and two neighboring genes (orfA and orf1). The extents of the DNA fragments cloned on vectors are shown below the arrows. Six transcriptional units are also shown above the arrows. Restriction enzyme abbreviations: B, BamHI; Bg, BglII; E, EcoRI; N, NcoI; P, PstI; S, Sau3AI; Sp, SphI. (B) The catalytic functions of GriI, GriH, GriE, and GriF have been elucidated by in vitro analysis with purified enzymes, and their steps in GX biosynthesis are indicated by solid arrows. The functions of GriC and GriD have been elucidated by in vivo analysis, and their steps are indicated by dotted-line arrows. A key intermediate in GX biosynthesis, 3,4-AHBA, is synthesized from two primary metabolites, aspartate-4-semialdehyde (ASA) and dihydroxyacetone phosphate (DHAP), by the action of GriI and GriH. 3,4-AHBA is reduced to 3,4-AHBAL by the action of GriC and GriD, probably in an ATP-dependent manner. GX-A is synthesized from two molecules of 3,4-AHBAL and N-acetylcysteine (AcCys) by the action of GriE and GriF. Conversion of GX-A to GX-B is presumably catalyzed by an oxidase that is not specific to GX-A and is encoded out of the GX biosynthesis gene cluster.

FIG. 2.

Time courses of griR, griT, griS, griA, griC, and griJ transcription as determined by low-resolution S1 nuclease mapping. RNA was prepared from the wild-type (wt) strain grown in SMM containing 0.25 mM (A and E) or 2.5 mM (B) KH₂PO₄. Transcription of the gri genes in SMM containing 0.25 mM phosphate in mutants ΔadpA (C) and ΔgriR (D) was also determined. Cultivation times (hours) are shown above the panels.

FIG. 3.

RT-PCR analysis of the polycistronic mRNA in the GX biosynthesis gene cluster. (A) Schematic representation of the positions of primers for RT reactions and DNA fragments amplified by PCR. The positions of primers used for synthesis of cDNA in RT reactions are shown by open triangles. The lengths and positions of the DNA fragments amplified by PCR in panel B are indicated by bars below the ORFs. P represents a promoter. (B) RNA was prepared from the wild-type strain grown in SMM containing 0.25 mM KH₂PO₄. Cultivation times in hours are shown above the panel. Control experiments with no RT (lanes −) confirmed that the RNA samples contained no chromosomal DNA. The amplified fragments were analyzed by agarose gel electrophoresis and stained with ethidium bromide.

FIG. 4.

GX production on SMM agar medium. SMM agar contained 0.25 mM (A) or 2.5 mM (B) KH₂PO₄. The wild-type (wt) strain, the strain with adpA disrupted (ΔadpA), ΔadpA harboring pHR20 (pHR20/ΔadpA), the strain with griR disrupted (ΔgriR), and ΔgriR harboring pHR20 (pHR20/ΔgriR) were cultured at 28°C for 4 days. The wild-type strain produced a detectable amount of the yellow pigment GX on medium containing 0.25 mM phosphate but not on medium containing 2.5 mM phosphate. Mutants ΔadpA and ΔgriR produced no yellow pigments on medium containing 0.25 or 2.5 mM phosphate. Plasmid pHR20 carrying griR under the control of the hrdB promoter conferred yellow pigment productivity on both mutants. The amounts of GX produced by both mutants harboring pHR20 on medium containing 2.5 mM phosphate are larger than those produced on medium containing 0.25 mM phosphate. The mycelium of the strains grown on medium containing 2.5 mM phosphate is more abundant than that of the strain grown on medium containing 0.25 mM phosphate.

FIG. 5.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified GriR-H (A) and estimation of its molecular weight by gel filtration (B). (A) GriR-H used in this study was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and soybean trypsin inhibitor (20 kDa) were used as molecular mass standards. (B) The column used was a Sephadex G-200 column (Sigma) in a fast protein liquid chromatography system. Aldolase (158 kDa) (a), bovine serum albumin (67 kDa) (b), ovalbumin (43 kDa) (c), chymotrypsinogen A (25 kDa) (d), and RNase A (14 kDa) (e) were used for generation of a calibration line.

FIG. 6.

Binding of GriR-H to the griC and griJ promoters. (A) Schematic representation of the positions of probes used in panel C. The open boxes in the griC and griJ promoters represent the direct repeats. The transcriptional start point for each gene (see Fig. S1 in the supplemental material) is shown as +1. (B) Nucleotide sequences of the griC and griJ promoters. The direct repeats composed of an 8-bp sequence are indicated by arrows above the sequences. The nucleotides matching the consensus sequence, 5′-CGATACGC-3′, are highlighted. The transcriptional start points are shown in the boldface letters and numbered +1. The −10 element is indicated by a gray box. The mutation of one of the repeated sequences in the griJ promoter is also shown. (C) Gel mobility shift assay to determine the binding of GriR-H to the regions upstream of griC and griJ. GriR-H bound only to the C1 and J1 probes. GriR-H did not bind the regions upstream from griA (probe A) or griR (probe R) or the intervening region between griT and griS (probe TS). The amounts of GriR-H used were 0 μg (lane 1), 0.2 μg (lane 2), 0.4 μg (lane3), 0.8 μg (lane 4), and 1.5 μg (lane 5). (D) Gel mobility shift assay to determine the binding of GriR-H to the mutated sequence J1-M. GriR-H did not bind probe J1-M, whereas it bound control probes C1 and J1. The amounts of GriR-H used were 0 μg (lane 1), 0.3 μg (lane 2), 1.0 μg (lane3), and 3.0 μg (lane 4).

FIG. 7.

Model of the regulation of GX biosynthesis by A-factor and phosphate starvation. Both A-factor and phosphate depletion signals are required for the transcription of griR. GriR binds and activates the griC and griJ promoters, causing the transcription of all of the GX biosynthesis enzymes and production of GX. Because AdpA does not bind the upstream region of griR, AdpA is assumed to activate the griR promoter indirectly. The phosphate depletion signal is also transferred to the griR promoter, although its signaling pathway remains to be elucidated.