Cloning and analysis of the simocyclinone biosynthetic gene cluster of <Emphasis Type="Italic">Streptomyces antibioticus</Emphasis> Tü 6040

The biosynthetic gene cluster of the aminocoumarin antibiotic simocyclinone D8 was cloned by screening a cosmid library of Streptomyces antibioticusTü 6040 with a heterologous probe from a gene encoding a cytochrome P450 enzyme involved in the biosynthesis of the aminocoumarin antibiotic novobiocin. Sequence analysis of a 39.4-kb region revealed the presence of 38 ORFs. Six of the identified ORFs showed striking similarity to genes from the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin and coumermycin A(1). Simocyclinone also contains an angucyclinone moiety, and 12 of the ORFs showed high sequence similarity to biosynthetic genes of other angucyclinone antibiotics. Possible functions within the biosynthesis of simocyclinone D8 could be assigned to 23 ORFs by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by a gene inactivation experiment, which resulted in the abolishment of the formation of the aminocoumarin moiety of simocyclinone. Feeding of the mutant with the aminocoumarin moiety of novobiocin led to a new, artificial simocyclinone derivative.     

Characterization of the aminocoumarin ligase SimL from the simocyclinone pathway and tandem incubation with NovM,P,N from the novobiocin pathway

Simocyclinone D(8) consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics novobiocin, clorobiocin, and coumermycin A(1), the phenolic hydroxyl group of the aminocoumarin in simocyclinone is not glycosylated with a decorated noviosyl moiety that is the pharmacophore for targeting bacterial DNA gyrase. We have expressed the Streptomyces antibioticus simocyclinone ligase SimL, purified it from Escherichia coli, and established its ATP-dependent amide bond forming activity with a variety of polyenoic acids including retinoic acid and fumagillin. We have then used the last three enzymes from the novobiocin pathway, NovM, NovP, and NovN, to convert a SimL product to a novel novobiocin analogue, in which the 3-prenyl-4-hydroxybenzoate of novobiocin is replaced with a tetraenoate moiety, to evaluate antibacterial activity.     

Mass spectrometry reveals that the antibiotic simocyclinone D8 binds to DNA gyrase in a "bent-over" conformation: evidence of positive cooperativity in binding

DNA topoisomerases are enzymes that control DNA topology and are vital targets for antimicrobial and anticancer drugs. Here we present a mass spectrometry study of complexes formed between the A subunit of the topoisomerase DNA gyrase and the bifunctional inhibitor simocyclinone D8 (SD8), an antibiotic isolated from Streptomyces. These studies show that, in an alternative mode of interaction to that found by X-ray crystallography, each subunit binds a single bifunctional inhibitor with separate binding pockets for the two ends of SD8. The gyrase subunits form constitutive dimers, and fractional occupancies of inhibitor-bound states show that there is strong allosteric cooperativity in the binding of two bifunctional ligands to the dimer. We show that the mass spectrometry data can be fitted to a general model of cooperative binding via an extension of the "tight-binding" approach, providing a rigorous determination of the dissociation constants and degree of cooperativity. This general approach will be applicable to other systems with multiple binding sites and highlights mass spectrometry's role as a powerful emerging tool for unraveling the complexities of biomolecular interactions.     

A New Crystal Structure of the Bifunctional Antibiotic Simocyclinone D8 Bound to DNA Gyrase Gives Fresh Insight into the Mechanism of Inhibition

Simocyclinone D8 (SD8) is an antibiotic produced by Streptomyces antibioticus that targets DNA gyrase. A previous structure of SD8 complexed with the N-terminal domain of the DNA gyrase A protein (GyrA) suggested that four SD8 molecules stabilized a tetramer of the protein; subsequent mass spectrometry experiments suggested that a protein dimer with two symmetry-related SD8s was more likely. This work describes the structures of a further truncated form of the GyrA N-terminal domain fragment with and without SD8 bound. The structure with SD8 has the two SD8 molecules bound within the same GyrA dimer. This new structure is entirely consistent with the mutations in GyrA that confer SD8 resistance and, by comparison with a new apo structure of the GyrA N-terminal domain, reveals the likely conformation changes that occur upon SD8 binding and the detailed mechanism of SD8 inhibition of gyrase. Isothermal titration calorimetry experiments are consistent with the crystallography results and further suggest that a previously observed complex between SD8 and GyrB is ~ 1000-fold weaker than the interaction with GyrA.     

Overexpression,purification and characterization of SimL,an amide synthetase involved in simocyclinone biosynthesis

Simocyclinone D8 is a potent inhibitor of bacterial gyrase, produced by Streptomyces antibioticus Tü 6040. It contains an aminocoumarin moiety, similar to that of novobiocin, which is linked by an amide bond to a structurally complex acyl moiety, consisting of an aromatic angucycline polyketide nucleus, the deoxysugar olivose and a tetraene dicarboxylic acid. We have now investigated the enzyme SimL, responsible for the formation of the amide bond of simocyclinone. The gene was cloned, expressed in S. lividans T7, and the protein was purified to near homogeneity, and characterized. The 60 kDa protein catalyzed both the ATP-dependent activation of the acyl component as well as its transfer to the amino group of the aminocoumarin ring, with no requirement for a 4-phosphopantetheinyl cofactor. Besides its natural substrate, simocyclinone C4, SimL also accepted a range of cinnamic and benzoic acid derivatives and several other, structurally very diverse acids. These findings make SimL a possible tool for the creation of new aminocoumarin antibiotics.     

High-level fluoroquinolone resistance in Pseudomonas aeruginosa due to interplay of the MexAB-OprM efflux pump and the DNA gyrase mutation

Fluoroquinolone resistance in Pseudomonas aeruginosa is mainly attributable to the constitutive expression of the xenobiotic efflux pump and mutation in DNA gyrase or topoisomerase IV. We constructed cells with a double-mutation in gyrA and mexR encoding DNA gyrase and repressor for the mexAB-oprM operon, respectively. The mutant showed 1,024 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM. Cells with a single mutation in gyrA and producing a wild-type level of the MexAB-OprM efflux pump showed 128 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM. In contrast, a single mutation in gyrA or mexR caused only 4 and 64 times higher resistance, respectively. These findings manifested the interplay between the MexAB-OprM efflux pump and the target mutation in fluoroquinolone resistance.     

Flavone-based analogues inspired by the natural product simocyclinone D8 as DNA gyrase inhibitors

The increasing occurrence of drug-resistant bacterial infections in the clinic has created a need for new antibacterial agents. Natural products have historically been a rich source of both antibiotics and lead compounds for new antibacterial agents. The natural product simocyclinone D8 (SD8) has been reported to inhibit DNA gyrase, a validated antibacterial drug target, by a unique catalytic inhibition mechanism of action. In this work, we have prepared simplified flavone-based analogues inspired by the complex natural product and evaluated their inhibitory activity and mechanism of action. While two of these compounds do inhibit DNA gyrase, they do so by a different mechanism of action than SD8, namely DNA intercalation.     

Initiation of actinorhodin export in Streptomyces coelicolor

Many microorganisms produce molecules having antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbours. Given that these molecules are often toxic to the producer, mechanisms must exist to ensure that the assembly of the export apparatus accompanies or precedes biosynthesis. Streptomyces coelicolor produces the polyketide antibiotic actinorhodin in a multistep pathway involving enzymes encoded by genes that are clustered together. Embedded within the cluster are genes for actinorhodin export, two of which, actR and actA resemble the classic tetR and tetA repressor/efflux pump-encoding gene pairs that confer resistance to tetracycline. Like TetR, which represses tetA, ActR is a repressor of actA. We have identified several molecules that can relieve repression by ActR. Importantly (S)-DNPA (an intermediate in the actinorhodin biosynthetic pathway) and kalafungin (a molecule related to the intermediate dihydrokalafungin), are especially potent ActR ligands. This suggests that along with the mature antibiotic(s), intermediates in the biosynthetic pathway might activate expression of the export genes thereby coupling export to biosynthesis. We suggest that this could be a common feature in the production of many bioactive natural products.     

Role of MbtH-like proteins in the adenylation of tyrosine during aminocoumarin and vancomycin biosynthesis

MbtH-like proteins consist of ～70 amino acids and are encoded in the biosynthetic gene clusters of non-ribosomally formed peptides and other secondary metabolites derived from amino acids. Recently, several MbtH-like proteins have been shown to be required for the adenylation of amino acid in non-ribosomal peptide synthesis. We now investigated the role of MbtH-like proteins in the biosynthesis of the aminocoumarin antibiotics novobiocin, clorobiocin, and simocyclinone D8 and of the glycopeptide antibiotic vancomycin. The tyrosine-adenylating enzymes CloH, SimH, and Pcza361.18, involved in the biosynthesis of clorobiocin, simocyclinone D8, and vancomycin, respectively, required the presence of MbtH-like proteins in a 1:1 molar ratio, forming heterotetrameric complexes. In contrast, NovH, involved in novobiocin biosynthesis, showed activity in the absence of MbtH-like proteins. Comparison of the active centers of CloH and NovH showed only one amino acid to be different, i.e. Leu-383 versus Met-383. Mutation of this amino acid in CloH (L383M) indeed led to MbtH-independent adenylating activity. All investigated tyrosine-adenylating enzymes exhibited remarkable promiscuity for MbtH-like proteins from different pathways and organisms. YbdZ, the MbtH-like protein from the expression host Escherichia coli, was found to bind to adenylating enzymes during expression and to influence their biochemical properties markedly. Therefore, the use of ybdZ-deficient expression hosts is important in biochemical studies of adenylating enzymes.     

The DrrAB Efflux System of Streptomyces peucetius Is a Multidrug Transporter of Broad Substrate Specificity

The soil bacterium Streptomyces peucetius produces two widely used anticancer antibiotics, doxorubicin and daunorubicin. Present within the biosynthesis gene cluster in S. peucetius is the drrAB operon, which codes for a dedicated ABC (ATP binding cassette)-type transporter for the export of these two closely related antibiotics. Because of its dedicated nature, the DrrAB system is believed to belong to the category of single-drug transporters. However, whether it also contains specificity for other known substrates of multidrug transporters has never been tested. In this study we demonstrate under both in vivo and in vitro conditions that the DrrAB system can transport not only doxorubicin but is also able to export two most commonly studied MDR substrates, Hoechst 33342 and ethidium bromide. Moreover, we demonstrate that many other substrates (including verapamil, vinblastine, and rifampicin) of the well studied multidrug transporters inhibit DrrAB-mediated Dox transport with high efficiency, indicating that they are also substrates of the DrrAB pump. Kinetic studies show that inhibition of doxorubicin transport by Hoechst 33342 and rifampicin occurs by a competitive mechanism, whereas verapamil inhibits transport by a non-competitive mechanism, thus suggesting the possibility of more than one drug binding site in the DrrAB system. This is the first in-depth study of a drug resistance system from a producer organism, and it shows that a dedicated efflux system like DrrAB contains specificity for multiple drugs. The significance of these findings in evolution of poly-specificity in drug resistance systems is discussed.     

Metabolic engineering of the heterologous production of clorobiocin derivatives and elloramycin in Streptomyces coelicolor M512

The aminocoumarin antibiotic clorobiocin is a potent inhibitor of bacterial gyrase. Two new analogs of clorobiocin could be obtained by deletion of a methyltransferase gene, involved in deoxysugar biosynthesis, from the biosynthetic gene cluster of clorobiocin, followed by expression of the modified cluster in the heterologous host Streptomyces coelicolor M512. However, only low amounts of the desired glycosides were formed, and aminocoumarins accumulated predominantly in form of aglyca. In the present study, we clarified the limiting steps for aminocoumarin glycoside formation, and devised strategies to improve glycosylation efficiency. Heterologous expression of a partial elloramycin biosynthetic gene cluster indicated that the rate of dTDP-l-rhamnose synthesis, rather than the rate of glycosyl transfer, was limiting for glycoside formation in this strain. Introduction of plasmid pRHAM which contains four genes from the oleandomycin biosynthetic gene cluster, directing the synthesis of dTDP-rhamnose, led to a 26-fold increase of the production of glycosylated aminocoumarins. Expression of the 4-ketoreductase gene oleU alone resulted in an 8-fold increase. Structural investigation of the resulting deoxysugars confirmed that both the endogeneous and the heterologous pathway involve a 3,5-epimerization of the deoxysugar, a hypothesis which had recently been questioned.     

Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens

For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.     

Biosynthesis of spectinomycin: heterologous production of spectinomycin and spectinamine in an aminoglycoside-deficient host, Streptomyces venezuelae YJ003

Aims:  To obtain spectinomycin and spectinamine by heterologous expression into the biosynthetic deoxysugar (desosamine) gene-deleted host Streptomyces venezuelae YJ003.
Methods and Results:  The 17-kb spectinomycin biosynthetic gene cluster from Streptomyces spectabilis ATCC 27741 was heterologously expressed into Streptomyces venezuelae YJ003. Furthermore, the speA , speB and spcS2 encoded in the spectinomycin biosynthetic gene cluster of cosmid pSPC8 were also heterologously characterized to be responsible for the production of spectinamine.
Conclusions:  The results of this study indicated that pSPC8 contains all the genes necessary for the biosynthesis of spectinomycin. We also concluded that SpeA, SpeB and SpcS2 are sufficient for the biosynthesis of spectinamine. We also verified that SpeB and SpcS2 show dual character in the biosynthetic pathway of spectinomycin in Streptomyces spectabilis .
Significance and Impact of the Study:  This is the report regarding the expression of a biosynthetic gene cluster that gives rise to the production of aminoglycoside antibiotics in Streptomyces venezuelae YJ003. Therefore, this work may serve as a foundation for further research on spectinomycin biosynthesis and other aminoglycosides.     

Identification of 8-amino-2,5,7-trihydroxynaphthalene-1,4-dione, a novel intermediate in the biosynthesis of Streptomyces meroterpenoids

Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) produced by Streptomyces sp. strain KO-3988. All of the fur genes required for furaquinocin biosynthesis have been cloned, and the heterologous production of furaquinocin has been demonstrated in Streptomyces albus. Here, we report the identification of 8-amino-2,5,7-trihydroxynaphthalene-1,4-dione (8-amino-flaviolin) produced by the S. albus heterologous expression of the three contiguous genes encoding type III polyketide synthase (Fur1), monooxygenase (Fur2), and aminotransferase (Fur3) in the furaquinocin biosynthetic gene cluster. An S. albus transformant (S. albus/pWHM-Fur2_del3) harboring the fur gene cluster and lacking the fur3 gene did not produce furaquinocin, whereas furaquinocin production was restored when 8-amino-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del3. These results demonstrate that Fur3 aminotransferase is essential for furaquinocin biosynthesis and that 8-amino-flaviolin is an early-stage intermediate in furaquinocin biosynthesis. We propose that the biosynthetic pathway generating 8-amino-flaviolin is the common route for the biosynthesis of Streptomyces meroterpenoids.     

Genetic organization of the biosynthetic gene cluster for the indolocarbazole K-252a in <Emphasis Type="Italic">Nonomuraea longicatena</Emphasis> JCM 11136

Indolocarbazole metabolite K-252a is a natural product that was previously reported as a potent protein kinase C inhibitor with in vitro and in vivo potency. From a biosynthetic viewpoint, this compound possesses structurally interesting features such as an unusual furanosyl sugar moiety, which are absent in the well-studied staurosporine and rebeccamycin. A cosmid library from genomic DNA of Nonomuraea longicatena JCM 11136 was constructed and screened for the presence of genes to be involved in the biosynthesis of indolocarbazole K-252a. Using as a probe an internal fragment of vioB, a Chromobacterium violaceum gene encoding a multifunctional enzyme that catalyzes tryptophan decarboxylation and condensation reaction in violacein biosynthesis, we isolated a DNA region that directed the biosynthesis of K-252a when introduced into the heterologous expression host Streptomyces albus. Sequence analysis of 45 kb revealed genes for indolocarbazole core formation, glycosylation, and sugar methylation, as well as a regulatory gene and two resistance/secretion genes. The cloned genes should help to elucidate the molecular basis for indolocarbazole biosynthesis and generate new indolocarbazole analogues by genetic engineering.     

Mechanism of action of and resistance to quinolones

Fluoroquinolones are an important class of wide‐spectrum antibacterial agents. The first quinolone described was nalidixic acid, which showed a narrow spectrum of activity. The evolution of quinolones to more potent molecules was based on changes at positions 1, 6, 7 and 8 of the chemical structure of nalidixic acid. Quinolones inhibit DNA gyrase and topoisomerase IV activities, two enzymes essential for bacteria viability. The acquisition of quinolone resistance is frequently related to (i) chromosomal mutations such as those in the genes encoding the A and B subunits of the protein targets (gyrA, gyrB, parC and parE), or mutations causing reduced drug accumulation, either by a decreased uptake or by an increased efflux, and (ii) quinolone resistance genes associated with plasmids have been also described, i.e. the qnr gene that encodes a pentapeptide, which blocks the action of quinolones on the DNA gyrase and topoisomerase IV; the aac(6′)‐Ib‐cr gene that encodes an acetylase that modifies the amino group of the piperazin ring of the fluoroquinolones and efflux pump encoded by the qepA gene that decreases intracellular drug levels. These plasmid‐mediated mechanisms of resistance confer low levels of resistance but provide a favourable background in which selection of additional chromosomally encoded quinolone resistance mechanisms can occur.