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Biosynthesis of gamma-butyrolactone autoregulators that switch on secondary metabolism and morphological development in Streptomyces.

ABSTRACT: A factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is a representative of the gamma-butyrolactone autoregulators that trigger secondary metabolism and morphogenesis in the Gram-positive, filamentous bacterial genus Streptomyces. Here, we report the A factor biosynthesis pathway in Streptomyces griseus. The monomeric AfsA, containing a tandem repeat domain of approximately 80 aa, catalyzed beta-ketoacyl transfer from 8-methyl-3-oxononanoyl-acyl carrier protein to the hydroxyl group of dihydroxyacetone phosphate (DHAP), thus producing an 8-methyl-3-oxononanoyl-DHAP ester. The fatty acid ester was nonenzymatically converted to a butenolide phosphate by intramolecular aldol condensation. The butenolide phosphate was then reduced by BprA that was encoded just downstream of afsA. The phosphate group on the resultant butanolide was finally removed by a phosphatase, resulting in formation of A factor. The 8-methyl-3-oxononanoyl-DHAP ester produced by the action of AfsA was also converted to A factor in an alternative way; the phosphate group on the ester was first removed by a phosphatase and the dephosphorylated ester was converted nonenzymatically to a butenolide, which was then reduced by a reductase different from BprA, resulting in A factor. Because introduction of afsA alone into Escherichia coli caused the host to produce a substance having A factor activity, the reductase(s) and phosphatase(s) were not specific to the A factor biosynthesis but commonly present in bacteria. AfsA is thus the key enzyme for the biosynthesis of gamma-butyrolactones.

SUBMITTER: Kato JY

PROVIDER: S-EPMC1892969 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Streptomyces γ-butyrolactones (GBLs) are quorum sensing communication signals triggering antibiotic production. The GBL system of Streptomyces filipinensis, the producer of the antifungal agent filipin, has been investigated. Inactivation of sfbR (for S. filipinensis γ-butyrolactone receptor), a GBL receptor, resulted in a strong decrease in production of filipin, and deletion of sfbR2, a pseudo-receptor, boosted it, in agreement with lower and higher levels of transcription of filipin biosynthetic genes, respectively. It is noteworthy that none of the mutations affected growth or morphological development. While no ARE (autoregulatory element)-like sequences were found in the promoters of filipin genes, suggesting indirect control of production, five ARE sequences were found in five genes of the GBL cluster, whose transcription has been shown to be controlled by both S. filipinensis SfbR and SfbR2. In vitro binding of recombinant SfbR and SfbR2 to such sequences indicated that such control is direct. Transcription start points were identified by 5' rapid amplification of cDNA ends, and precise binding regions were investigated by the use of DNase I protection studies. Binding of both regulators took place in the promoter of target genes and at the same sites. Information content analysis of protected sequences in target promoters yielded an 18-nucleotide consensus ARE sequence. Quantitative transcriptional analyses revealed that both regulators are self-regulated and that each represses the transcription of the other as well as that of the remaining target genes. Unlike other GBL receptor homologues, SfbR activates its own transcription whereas SfbR2 has a canonical autorepression profile. Additionally, SfbR2 was found here to bind the antifungal antimycin A as a way to modulate its DNA-binding activity.IMPORTANCEStreptomyces GBLs are important signaling molecules that trigger antibiotic production in a quorum sensing-dependent manner. We have characterized the GBL system from S. filipinensis, finding that two key players of this system, the GBL receptor and the pseudo-receptor, each counteracts the transcription of the other for the modulation of filipin production and that such control over antifungal production involves an indirect effect on the transcription of filipin biosynthetic genes. Additionally, the two regulators bind the same sites, are self-regulated, and repress the transcription of three other genes of the GBL cluster, including that encoding the GBL synthase. In contrast to all the GBL receptors known, SfbR activates its own synthesis. Moreover, the pseudo-receptor was identified as the receptor of antimycin A, thus extending the range of examples supporting the idea of signaling effects of antibiotics in Streptomyces The intricate regulatory network depicted here should provide important clues for understanding the regulatory mechanism governing secondary metabolism.

Project description:Many microorganisms, including bacteria of the class Streptomycetes, produce various secondary metabolites including antibiotics to gain a competitive advantage in their natural habitat. The production of these compounds is highly coordinated in a population to expedite accumulation to an effective concentration. Furthermore, as antibiotics are often toxic even to their producers, a coordinated production allows microbes to first arm themselves with a defense mechanism to resist their own antibiotics before production commences. One possible mechanism of coordination among individuals is through the production of signaling molecules. The gamma-butyrolactone system in Streptomyces coelicolor is a model of such a signaling system for secondary metabolite production. The accumulation of these signaling molecules triggers antibiotic production in the population. A pair of repressor-amplifier proteins encoded by scbA and scbR mediates the production and action of one particular gamma-butyrolactone, SCB1. Based on the proposed interactions of scbA and scbR, a mathematical model was constructed and used to explore the ability of this system to act as a robust genetic switch. Stability analysis shows that the butyrolactone system exhibits bistability and, in response to a threshold SCB1 concentration, can switch from an OFF state to an ON state corresponding to the activation of genes in the cryptic type I polyketide synthase gene cluster, which are responsible for production of the hypothetical polyketide. The switching time is inversely related to the inducer concentration above the threshold, such that short pulses of low inducer concentration cannot switch on the system, suggesting its possible role in noise filtering. In contrast, secondary metabolite production can be triggered rapidly in a population of cells producing the butyrolactone signal due to the presence of an amplification loop in the system. S. coelicolor was perturbed experimentally by varying concentrations of SCB1, and the model simulations match the experimental data well. Deciphering the complexity of this butyrolactone switch will provide valuable insights into how robust and efficient systems can be designed using "simple" two-protein networks.

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Biosynthesis of gamma-butyrolactone autoregulators that switch on secondary metabolism and morphological development in Streptomyces.

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