The Gene Cluster for the Biosynthesis of the Glycopeptide Antibiotic A40926 by Nonomuraea Species

Sosio, Margherita; Stinchi, Sofia; Beltrametti, Fabrizio; Lazzarini, Ameriga; Donadio, Stefano

doi:10.1016/s1074-5521(03)00120-0

Cited by 157 publications

(157 citation statements)

References 43 publications

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“…The thin arrows represent the experimentally determined transcriptional units; the thick arrows indicate the Dbv4-controlled dbv14-dbv8 and dbv30-dbv35 operons; triangles denote DNA fragments used in gel retardation experiments; asterisks and the symbol "Q" indicate genes targeted by RT-PCR and quantitative RT-PCR, respectively. dbv genes are grouped by category as indicated (33,35). (B) RT-PCR analysis of intergenic regions.…”

Section: Methodsmentioning

confidence: 99%

“…Nonomuraea sp. ATCC 39727 was grown for 70 h at 30°C in 200 ml of Rare3 medium (33). The cells were then harvested by centrifugation, washed twice with crack buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 0.3 mM DTT), resuspended in 5 ml of crack buffer, and disrupted by sonication.…”

Section: Vol 189 2007 Phosphate Regulation Of A40926 Biosynthesis 8121mentioning

confidence: 99%

“…The dbv gene cluster for the biosynthesis of A40926 (33,35) includes 37 open reading frames participating in antibiotic biosynthesis, regulation, resistance, and export (Fig. 1A).…”

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Phosphate-Controlled Regulator for the Biosynthesis of the Dalbavancin Precursor A40926

et al. 2007

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The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of the novel antibiotic dalbavancin. Previous studies have shown that phosphate limitation results in enhanced A40926 production. The A40926 biosynthetic gene (dbv) cluster, which consists of 37 genes, encodes two putative regulators, Dbv3 and Dbv4, as well as the response regulator (Dbv6) and the sensor-kinase (Dbv22) of a putative two-component system. Reverse transcription-PCR (RT-PCR) and real-time RT-PCR analysis revealed that the dbv14-dbv8 and the dbv30-dbv35 operons, as well as dbv4, were negatively influenced by phosphate. Dbv4 shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, the transcriptional activator of streptomycin biosynthesis in Streptomyces griseus. Dbv4 was expressed in Escherichia coli as an N-terminal His 6 -tagged protein. The purified protein bound the dbv14 and dbv30 upstream regions but not the region preceding dbv4. Bbr, a Dbv4 ortholog from the gene cluster for the synthesis of the glycopeptide balhimycin, also bound to the dbv14 and dbv30 upstream regions, while Dbv4 bound appropriate regions from the balhimycin cluster. Our results provide new insights into the regulation of glycopeptide antibiotics, indicating that the phosphate-controlled regulator Dbv4 governs two key steps in A40926 biosynthesis: the biosynthesis of the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine and critical tailoring reactions on the heptapeptide backbone.

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Section: Methodsmentioning

confidence: 99%

Section: Vol 189 2007 Phosphate Regulation Of A40926 Biosynthesis 8121mentioning

confidence: 99%

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Phosphate-Controlled Regulator for the Biosynthesis of the Dalbavancin Precursor A40926

et al. 2007

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“…The heptapeptide is assembled by a nonribosomal peptide synthetase (NRPS) and then modified by oxidative cross-linking of the aromatic side chains to yield a rigid peptide scaffold. Further tailoring steps include halogenation, glycosylation, oxidation, methylation, acetylation, and acylation (5). The A40926 components differ by the type of N-acyl chain attached to the glucuronic acid moiety and by the presence or absence of an O-linked acetyl residue at position 6 of the mannose (6)(7)(8).…”

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confidence: 99%

Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

Grasso

Maffioli²,

Sosio³

et al. 2015

J Bacteriol

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The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of dalbavancin. Biosynthesis of A40926 is encoded by the dbv gene cluster, which contains 37 protein-coding sequences that participate in antibiotic biosynthesis, regulation, immunity, and export. In addition to the positive regulatory protein Dbv4, the A40926-biosynthetic gene cluster encodes two additional putative regulators, Dbv3 and Dbv6. Independent mutations in these genes, combined with bioassays and liquid chromatography-mass spectrometry (LC-MS) analyses, demonstrated that Dbv3 and Dbv4 are both required for antibiotic production, while inactivation of dbv6 had no effect. In addition, overexpression of dbv3 led to higher levels of A40926 production. Transcriptional and quantitative reverse transcription (RT)-PCR analyses showed that Dbv4 is essential for the transcription of two operons, dbv14-dbv8 and dbv30-dbv35, while Dbv3 positively controls the expression of four monocistronic transcription units (dbv4, dbv29, dbv36, and dbv37) and of six operons (dbv2-dbv1, dbv14-dbv8, dbv17-dbv15, dbv21-dbv20, dbv24-dbv28, and dbv30-dbv35). We propose a complex and coordinated model of regulation in which Dbv3 directly or indirectly activates transcription of dbv4 and controls biosynthesis of 4-hydroxyphenylglycine and the heptapeptide backbone, A40926 export, and some tailoring reactions (mannosylation and hexose oxidation), while Dbv4 directly regulates biosynthesis of 3,5-dihydroxyphenylglycine and other tailoring reactions, including the four cross-links, halogenation, glycosylation, and acylation. IMPORTANCEThis report expands knowledge of the regulatory mechanisms used to control the biosynthesis of the glycopeptide antibiotic A40926 in the actinomycete Nonomuraea sp. strain ATCC 39727. A40926 is the precursor of dalbavancin, approved for treatment of skin infections by Gram-positive bacteria. Therefore, understanding the regulation of its biosynthesis is also of industrial importance. So far, the regulatory mechanisms used to control two other similar glycopeptides (balhimycin and teicoplanin) have been elucidated, and beyond a common step, different clusters seem to have devised different strategies to control glycopeptide production. Thus, our work provides one more example of the pitfalls of deducing regulatory roles from bioinformatic analyses only, even when analyzing gene clusters directing the synthesis of structurally related compounds. T he glycopeptide antibiotic A40926 (Fig. 1A) is the precursor for dalbavancin, a semisynthetic lipoglycopeptide approved for clinical use in 2014 by the U.S. Food and Drug Administration to treat acute bacterial skin and skin structure infections caused by bacteria, such as Staphylococcus aureus (methicillin-susceptible and methicillin-resistant strains) and Streptococcus pyogenes. Compared with the structurally related glycopeptide teicoplanin, dalbavancin possesses better potency and pharmacokinetic properties.A40926 is produced by the actinomycete Nonomurae...

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“…One of these families comprises the strR-like genes and includes, besides novG, cloG and couG, also dbv4 of the glycopeptide antibiotic A40926 (Sosio et al, 2003), bbr of the balhimycin (Pelzer et al, 1999) and tcp28 of the teicoplanin (Sosio et al, 2004) clusters. Another family comprises the SARPs, i.e.…”

Section: Discussionmentioning

confidence: 99%

NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis

Eustáquio

Heide

2005

Microbiology

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The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e. novE and novG. The predicted gene product of novG shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, a well-studied pathway-specific transcriptional activator of streptomycin biosynthesis. Here functional proof is provided, by genetic and biochemical approaches, for the role of NovG as a positive regulator of novobiocin biosynthesis. The entire novobiocin cluster of the producer organism Streptomyces spheroides was expressed in the heterologous host Streptomyces coelicolor M512, and additional strains were produced which lacked the novG gene within the heterologously expressed cluster. These DnovG strains produced only 2 % of the novobiocin formed by the S. coelicolor M512 strains carrying the intact novobiocin cluster. The production could be restored by introducing an intact copy of novG into the mutant. The presence of novG on a multicopy plasmid in the strain containing the intact cluster led to almost threefold overproduction of the antibiotic, suggesting that novobiocin biosynthesis is limited by the availability of NovG protein. Furthermore, purified N-terminal His 6 -tagged NovG showed specific DNA-binding activity for the novG-novH and the cloG-cloY intergenic regions of the novobiocin and clorobiocin biosynthetic gene clusters, respectively. By comparing the DNA sequences of the fragments binding NovG, conserved inverted repeats were identified in both fragments, similar to those identified as the binding sites for StrR. The consensus sequence for the StrR and the putative NovG binding sites was GTTCRACTG(N) 11 CRGTYGAAC. Therefore, NovG and StrR apparently belong to the same family of DNA-binding regulatory proteins.

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The Gene Cluster for the Biosynthesis of the Glycopeptide Antibiotic A40926 by Nonomuraea Species

Cited by 157 publications

References 43 publications

Phosphate-Controlled Regulator for the Biosynthesis of the Dalbavancin Precursor A40926

Phosphate-Controlled Regulator for the Biosynthesis of the Dalbavancin Precursor A40926

Two Master Switch Regulators Trigger A40926 Biosynthesis in Nonomuraea sp. Strain ATCC 39727

NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis

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