RESUMEN
Bacillithiol (BSH), an α-anomeric glycoside of l-cysteinyl-d-glucosaminyl-l-malate, is a major low molecular weight thiol found in low GC Gram-positive bacteria, such as Staphylococcus aureus. Like other low molecular weight thiols, BSH is likely involved in protection against a number of stresses. We examined S. aureus transposon mutants disrupted in each of the three genes associated with BSH biosynthesis. These mutants are sensitive to alkylating stress, oxidative stress, and metal stress indicating that BSH and BSH-dependent enzymes are involved in protection of S. aureus. We further demonstrate that BshB, a deacetylase involved in the second step of BSH biosynthesis, also acts as a BSH conjugate amidase and identify S. aureus USA 300 LAC 2626 as a BSH-S-transferase, which is able to conjugate chlorodinitrobenzene, cerulenin, and rifamycin to BSH.
Asunto(s)
Cisteína/análogos & derivados , Glucosamina/análogos & derivados , Mutación , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas/genética , Cromatografía Líquida de Alta Presión , Cisteína/metabolismo , Glucosamina/metabolismo , Yodoacetamida/farmacología , Metales/farmacología , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/genética , Oxidantes/farmacología , Piruvaldehído/farmacología , Staphylococcus aureus/enzimología , Compuestos de Sulfhidrilo/metabolismo , Factores de TiempoRESUMEN
The validamycin biosynthetic gene cluster in Streptomyces hygroscopicus var. limoneus contains vldI, the gene encoding a glucoamylase (1,4-alpha-D-glucan glucohydrolase). The knock-out of vldI (vldI::neo) reduced the yield of validamycin-A, thus indicating that VldI contributes to validamycin-A productivity by supplying glucose with the hydrolysis of 1,4-alpha-D-glucan(s). Promoter-probe assays employing xylE fusions indicated that the transcription of vldI correlates to those of other biosynthetic genes, which are organized with two divergently arranged vldABC and vldDEFGH sets. These results reveal that the contiguous region covering nine genes of vldABCDEFGHI represents the core of the validamycin biosynthetic cluster. After confirming that genes vldABCDEFGH confer validamycin production ability to Streptomyces lividans, genes vldBCHI were eliminated from the expression construct and the remaining genes, vldADEFG, were tested for the ability to confer validamycin-A production to S. lividans. Ion-exchange chromatographic purification and a subsequent HPLC analysis confirmed that S. lividans/vldADEFG yielded a 75 microg/l of validamycin-A, showing that the validamycin pathway involves a single NDP-sugar glycosyltransferase reaction. It was also demonstrated that VldE is capable of coupling validoxylamine-A and UDP-glucose to generate validamycin-A. The proposal that VldADEFG catalyze the biosynthesis of validamycin-A from sedo-heptulose 7-phosphate and UDP-glucose and include a N-bridge-forming catalyst will serve as a guideline for future biochemical studies and a platform to explore this m-C7N cyclitol pathway for biotechnological applications.
Asunto(s)
Genes Bacterianos , Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/metabolismo , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Secuencia de Bases , Mapeo Cromosómico , Cartilla de ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Glucosa/metabolismo , Inositol/análogos & derivados , Inositol/biosíntesis , Inositol/química , Inositol/genética , Cinética , Modelos Biológicos , Familia de Multigenes , Plásmidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
AfsKav is a eukaryotic-type serine/threonine protein kinase, required for sporulation and avermectin production in Streptomyces avermitilis. In terms of their ability to complement SJW4001 (DeltaafsK-av), afsK-av mutants T165A and T168A were not functional, whereas mutants T165D and T168D retained their ability, indicating that Thr-165 and Thr-168 are the phosphorylation sites required for the role of AfsKav. Expression of the S-adenosylmethione synthetase gene promoted avermectin production in the wild-type S. avermitilis, yet not in the mutant harboring T168D or T165D, demonstrating that tandem phosphorylation on Thr-165 and Thr-168 in AfsKav is the mechanism modulating avermectin production in response to S-adenosylmethione accumulation in S. avermitilis.
Asunto(s)
Ivermectina/análogos & derivados , Morfogénesis/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Streptomyces/genética , Treonina/fisiología , Regulación Bacteriana de la Expresión Génica , Ivermectina/metabolismo , Mutación/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , S-Adenosilmetionina/metabolismo , Streptomyces/citología , Streptomyces/enzimología , Streptomyces/fisiologíaRESUMEN
The validamycin biosynthetic gene cluster was isolated from Streptomyces hygroscopicus var. limoneus KTCC 1715 (IFO 12704) using a pair of degenerated PCR primers designed from the sequence of AcbC, 2-epi-5-epi-valiolone synthase in the acarbose biosynthesis. The nucleotide sequence analysis of the 37-kb DNA region revealed 22 complete ORFs including vldA, the acbC ortholog. Located around vldA, vldB to K were predicted to encode adenyltransferase, kinase, ketoreductase (or epimerase/dehydratase), glycosyltransferase, aminotransferase, dehydrogenase, phosphatase/phosphomutase, glycosyl hydrolase, transport protein, and glycosyltransferase, respectively. Apparently absent were any regulatory components within the sequenced region. The disruption of vldA abolished the validamycin biosynthesis and the plasmid-based complementation with vldABC restored production to the vldA-mutant; this substantiated that vldABC are essential to validamycin biosynthesis. This finding enabled us to discover the complete validamycin biosynthetic cluster. The cosmid clone of pJWS3001 harboring the 37-kb DNA region conferred validamycin-accumulation to Streptomyces lividans, indicating that the entire gene cluster of validamycin biosynthesis had been isolated. Additionally, Streptomyces albus, transformed with pJWS3001, produced a high level of alpha-glucosidase inhibitory activity in a R2YE liquid culture, which highlights the portability of the cluster within Streptomyces. The product of vldI was characterized as a glucoamylase (kcat, 32 s(-1); K(m), 5 mg/ml of starch) that does not play any apparent role in the validamycin biosynthesis. In order to characterize the upstream region, a vldW knockout was achieved via gene-replacement. A phenotypic study of the resulting mutant revealed that vldW is not essential for the host's ability to control Pellicularia filamentosa growth. The current information suggests that vldA to vldH is the genetic region essential to validamycin biosynthesis. This promises excellent opportunities to elucidate biosynthetic route(s) to the validamycin complex and to engineer the pathway for industrial application.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , Familia de Multigenes/genética , Sistemas de Lectura Abierta/genética , Streptomyces lividans/genética , Secuencia de Bases , Clonación Molecular , Cósmidos/genética , Técnicas de Transferencia de Gen , Inositol/análogos & derivados , Inositol/biosíntesis , Inositol/genética , Eliminación de Secuencia/genética , Streptomyces lividans/enzimologíaRESUMEN
Adenosine kinase (ADK) catalyses phosphorylation of adenosine (Ado) and generates adenosine monophosphate (AMP). ADK gene (adk(Sli), an ortholog of SCO2158) was disrupted in Streptomyces lividans by single crossover-mediated vector integration. The adk(Sli) disruption mutant (Deltaadk(Sli)) was devoid of sporulation and a plasmid copy of adk(Sli) restored sporulation ability in Deltaadk(Sli), thus indicating that loss of adk(Sli) abolishes sporulation in S. lividans. Ado supplementation strongly suppressed sporulation ability in S. lividans wild-type (wt), supporting that disruption of adk(Sli) resulted in Ado accumulation, which in turn suppressed sporulation. Cell-free experiments demonstrated that Deltaadk(Sli) lacked ADK activity and in vitro characterization confirms that adk(Sli) encodes ADK. The intracellular level of Ado was highly elevated while the AMP level was significantly reduced after loss of adk(Sli) while Deltaadk(Sli) displayed no significant derivation from wt in the levels of S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Notably, Ado supplementation to wt lowered AMP content, albeit not to the level of Deltaadk(Sli), implying that the reduction of AMP level is partially forced by Ado accumulation in Deltaadk(Sli). In Deltaadk(Sli), actinorhodin (ACT) production was suppressed and undecylprodigiosin (RED) production was dramatically enhanced; however, Ado supplementation failed to exert this differential control. A promoter-probe assay verified repression of actII-orf4 and induction of redD in Deltaadk(Sli), substantiating that unknown metabolic shift(s) of ADK-deficiency evokes differential genetic control on secondary metabolism in S. lividans. The present study is the first report revealing the suppressive role of Ado in Streptomyces development and the differential regulatory function of ADK activity in Streptomyces secondary metabolism, although the underlying mechanism has yet to be elucidated.