Hormonal control by A-factor of morphological development and secondary metabolism in Streptomyces

Abstract

Streptomyces griseus, a well-known industrial producer of streptomycin, is a member of the genus Streptomyces, which shows a complex life cycle resembling that of fungi. A-factor, a C13 γ-butyrolactone compound, was discovered as a self-regulatory factor or a bacterial hormone to induce morphological differentiation and production of secondary metabolites, including streptomycin, in this organism. Accumulating evidence has revealed an A-factor-triggered signal cascade, which is composed of several key steps or components. These include: (i) AfsA catalyzing a crucial step of A-factor biosynthesis, (ii) the A-factor-specific receptor (ArpA), which acts as a transcriptional repressor for adpA, (iii) adpA, a sole target of ArpA, which encodes a global transcriptional activator AdpA, and (iv) a variety of members of the AdpA regulon, a set of the genes regulated by AdpA. A-factor is biosynthesized via five reaction steps, in which AfsA catalyzes acyl transfer between a β-ketoacyl-acyl carrier protein and the hydroxyl group of dihydroxyacetone phosphate. The receptor ArpA, belonging to the TetR family, is a homodimer, each subunit of which contains a helix-turn-helix DNA-binding motif and an A-factor-binding pocket. The three-dimensional structure and conformational change upon binding A-factor are elucidated, on the basis of X-ray crystallography of CprB, an ArpA homologue. AdpA, belonging to the AraC/XylS transcriptional activator family, binds operators upstream from the promoters of a variety of the target genes and activates their transcription, thus forming the AdpA regulon. Members of the AdpA regulon includes the pathway-specific transcriptional activator gene strR that activates the whole streptomycin biosynthesis gene cluster, in addition to a number of genes that direct the multiple cellular functions required for cellular differentiation in a concerted manner. A variety of A-factor homologues as well as homologues of afsA/arpA are distributed widely among Streptomyces, indicating the significant role of this type of molecular signaling in the ecosystem and evolutional processes.

Keywords: A-factor; A-factor receptor; Streptomyces; morphological differentiation; secondary metabolism; streptomycin biosynthesis.

Fig. 1

Life cycle of S. griseus. A transmission electron microgram of a matured spore and scanning electron micrograms of substrate and aerial hyphae are also shown. Sm, streptomycin; GX, grixazone. Reproduced from Ohnishi and Horinouchi.

Fig. 2

The A-factor regulatory cascade leading to morphological development and secondary metabolite production. Sm, streptomycin; GX, grixazone; PK, a polyketide compound. See text for details.

Fig. 3

The whole A-factor biosynthesis pathway. The major pathway in S. griseus is hatched.

Fig. 4

A-factor and its homologues in Streptomyces. These include A-factor from S. griseus, controlling both secondary metabolism and morphological differentiation; IM-2 from S. lavendulae, controlling pigment and antibiotic production; SCBs from S. coelicolor A3(2), controlling antibiotic production; and virginiae butanolides (VBs) from S. virginiae, controlling virginiamycin production. Two differences in chemical structure among the γ-butyrolactones are the length and branching of the acyl chains and the reduction state, either a keto or a hydroxyl group, at position 6.

Fig. 5

Overall structure of CprB, an ArpA homologue. Each subunit of the CprB dimer contains a C-terminal ligand-binding pocket and an N-terminal helix-turn-helix DNA-binding motif. A model structure of the CprB-DNA complex, provided by R. Natsume, is shown. One subunit of the CprB dimer is drawn by a ribbon model and the other subunit is drawn by a tube model. Helices α1, α2, α3, and α4 are indicated in red, orange, yellow, and green. The ligand-binding pocket is 5 Å diameter and 20 Å long, which is enough to accommodate the entire molecule of a γ-butyrolactone in an extended form, shown by a ball model. When the ligand is embedded in the pocket, a long helix-4 is relocated to move the DNA-binding domain (DBD) outside, as shown, resulting in dissociation of the receptor from the DNA, shown by a stick model. Reproduced in a modified form from Horinouchi.

Fig. 6

The A-factor signal relay to the streptomycin biosynthesis genes. Construction and transcriptional organization of the gene cluster for streptomycin biosynthesis, including the pathway-specific transcriptional activator strR, are shown. The A-factor signal relay starts with A-factor and then transmitted to ArpA to AdpA to StrR, and finally to the nine transcriptional units covering the whole gene cluster. StrR is required for the transcriptional activation of all the nine transcriptional units by directly binding to the promoters (indicated by solid lines) and by a still unknown mechanism (indicated by dotted lines).

Fig. 7

A model for strict control of adpA transcription by the A-factor receptor ArpA in the early stage of growth and by AdpA itself after the middle of exponential growth phase. (A) Architecture of the adpA promoter and operators. The number on the sequence indicate the nucleotide positions with respect to the transcriptional start point +1 of adpA. The −35 and −10 promoter elements of adpA overlap with the ArpA-binding site and one (site 2) of the AdpA-binding sites (sites 1 to 3). (B) Control of the adpA transcription by ArpA and AdpA. Stage 1. In the early growth stage when the concentration of A-factor is still low, ArpA sits exactly on the promoter elements and represses its transcription to avoid production of AdpA. Stage 2. When the concentration of A-factor (shown by a triangle) reaches a critical level, it binds the DNA-bound ArpA and dissociates ArpA from the DNA, thus allowing RNA polymerase to bind the adpA promoter and initiate transcription. Stage 3. When the intracellular concentration of AdpA is low, it probably binds site 1 having the highest affinity to AdpA among the three AdpA-binding sites. However, the binding of AdpA only to site 1 exerts no effect on the adpA transcription. Stage 4. When the intracellular concentration of AdpA exceeds an appropriate level required for ordered gene expression in a concerted manner, the AdpA dimer bound to site 1 recruits another AdpA dimer to a rather weak binding site 2, thus forming a DNA loop in a way that RNA polymerase cannot gain access to the promoter. A third AdpA dimer also bind to a weak binding site 3, located downstream of +1, inhibits elongation of the adpA transcript that has escaped from the repression by the AdpA dimers that bind sites 1 and 2.

Fig. 8

Comparison of the phylogenetic positions of several ArpA homologues (γ-butyrolactone receptors) and AfsA homologues (γ-butyrolactone synthases) from the same Streptomyces species. Phylogenetic trees were constructed by the maximum likelihood method. Reproduced in a modified form from Nishida et al.