Antibiotic Actinorhodin Research Articles

Two overlapping antiparallel genes encoding the iron regulator DmdR1 and the Adm proteins control sidephore and antibiotic biosynthesis in Streptomyces coelicolor A3(2)

The dmdR1 gene of Streptomyces coelicolor encodes an important regulator of iron metabolism. An antiparallel gene (adm) homologous to a development‐regulated gene of Streptomyces aureofaciens has been found to overlap with dmdR1. Both proteins DmdR1 and Adm are formed in solid and liquid cultures of S. coelicolor A3(2). The purpose of this study was to assess possible interaction between the products of these two antiparallel genes. Two mutants with stop codons resulting in arrested translation of either DmdR1 or Adm were obtained by gene replacement and compared with a deletion mutant (ΔdmdR1/adm) that was defective in both genes. The deletion mutant was unable to form either protein, did not sporulate and lacked desferrioxamine, actinorhodin and undecylprodigiosin biosynthesis; biosynthesis of these compounds was recovered by complementation with dmdR1/adm genes. The mutant in which formation of Adm protein was arrested showed normal levels of DmdR1, lacked Adm and over‐produced the antibiotics undecylprodigiosin and actinorhodin (in MS medium), suggesting that Adm plays an important role in secondary metabolism. The mutant in which DmdR1 formation was arrested synthesized desferrioxamines in a constitutive (deregulated) manner, and produced relatively normal levels of antibiotics. In conclusion, our results suggest that there is a fine interplay of expression of these antiparallel genes, as observed for other genes that encode lethal proteins such as the toxin/antitoxin systems. The Adm protein seems to have a major effect on the control of secondary metabolism, and its formation is probably tightly controlled, as expected for a key regulator.

Structures and Mechanisms of Polyketide Synthases

Nearly a quarter-century ago, the advent of molecular genetic tools in the field of natural product biosynthesis led to the remarkable revelation that the genes responsible for the biosynthesis, regulation, and self-resistance of complex polyketide antibiotics were clustered in the genomes of the bacteria that produced these compounds. This in turn facilitated rapid cloning and sequencing of genes encoding a number of polyketide synthases (PKSs). By now, it is abundantly clear that, notwithstanding extraordinary architectural and biocatalytic diversity, all PKSs are evolutionarily related enzyme assemblies. As such, understanding the molecular logic for the biosynthesis of literally thousands of amazing polyketide natural products made by nature can benefit enormously from detailed investigations into a few "model systems". For nearly the past two decades, our laboratory has focused its efforts on two such PKSs. One of them synthesizes two polyketides in approximately equal ratios, SEK4 and SEK4b, and both shunt products from the pathway that leads to the biosynthesis of the pigmented antibiotic actinorhodin. The other synthesizes 6-deoxyerythronolide B, the first isolable intermediate in the biosynthetic pathway for the widely used antibacterial agent erythromycin. Our present-day knowledge of the structures and mechanisms of these two PKSs is summarized here.

Cross-talk between an orphan response regulator and a noncognate histidine kinase inStreptomyces coelicolor

Two-component systems (TCSs), typically consisting of a histidine kinase (HK) and a cognate response regulator (RR), are the most common signaling systems in bacteria. Besides paired genes encoding TCSs, there also exists unpaired HKs and orphan RRs. In Streptomyces coelicolor, 13 orphan RRs have been annotated. Because of lack of cognate HKs, little is known as yet about the regulation of orphan RRs. Bioinformatic analysis revealed that several orphan RRs had high amino acid sequence identities with RRs from typical TCSs in S. coelicolor. Among them, the orphan RR SCO3818 and RR SCO0204, which paired with HK SCO0203, showed the highest identity (65%), suggesting that the two RRs might both be under the regulation of SCO0203. Following studies showed that SCO0203 could phosphorylate not only SCO0204 but also SCO3818. Deletion of either sco0203 or sco3818 led to enhanced production of blue-pigmented antibiotic actinorhodin, which indicated a functional correlation between SCO0203 and SCO3818. These results suggested that SCO3818 might be regulated by SCO0203. This is the first report describing the regulation of an orphan RR by an HK. Moreover, this is also the first identification of cross-talk between different TCS components in S. coelicolor.

A Novel Insertion Mutation in Streptomyces coelicolor Ribosomal S12 Protein Results in Paromomycin Resistance and Antibiotic Overproduction

We identified a novel paromomycin resistance-associated mutation in rpsL, caused by the insertion of a glycine residue at position 92, in Streptomyces coelicolor ribosomal protein S12. This insertion mutation (GI92) resulted in a 20-fold increase in the paromomycin resistance level. In combination with another S12 mutation, K88E, the GI92 mutation markedly enhanced the production of the blue-colored polyketide antibiotic actinorhodin and the red-colored antibiotic undecylprodigiosin. The gene replacement experiments demonstrated that the K88E-GI92 double mutation in the rpsL gene was responsible for the marked enhancement of antibiotic production observed. Ribosomes with the K88E-GI92 double mutation were characterized by error restrictiveness (i.e., hyperaccuracy). Using a cell-free translation system, we found that mutant ribosomes harboring the K88E-GI92 double mutation but not ribosomes harboring the GI92 mutation alone displayed sixfold greater translation activity relative to that of the wild-type ribosomes at late growth phase. This resulted in the overproduction of actinorhodin, caused by the transcriptional activation of the pathway-specific regulatory gene actII-orf4, possibly due to the increased translation of transcripts encoding activators of actII-orf4. The mutant with the K88E-GI92 double mutation accumulated a high level of ribosome recycling factor at late stationary phase, underlying the high level of protein synthesis activity observed.

Ligand Recognition by ActR, a TetR-Like Regulator of Actinorhodin Export

TetR-like transcriptional repressors interact with small-molecule ligands to control many facets of prokaryotic biology, including clinical antibiotic resistance. ActR is a TetR-like protein encoded in the biosynthetic gene cluster for the antibiotic actinorhodin and controls the expression of two actinorhodin exporters. We showed previously that actinorhodin and its precursor 4-dihydro-9-hydroxy-1-methyl-10-oxo-3- H-naphtho-[2,3-c]-pyran-3-( S)-acetic acid can bind ActR and prevent its interaction with DNA. Here, we compare ActR's interaction with naturally occurring and synthetic molecules to show that pathway intermediates bind to ActR 5- to 10-fold more tightly than actinorhodin itself, consistent with our suggestion that they are the biologically relevant triggers for actinorhodin export. We also find that the ligand-binding cavity of this protein can accommodate a surprisingly large diversity of ligands, many of which can release ActR from DNA in vitro and in vivo. These data suggest that the actR locus could be activated by, and perhaps adapted to confer resistance to other antibiotics.

Antibiotic Overproduction in Streptomyces coelicolor A3(2) Mediated by Phosphofructokinase Deletion

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by (13)C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

Spontaneous Amplification of the Actinorhodin Gene Cluster in Streptomyces coelicolor Involving Native Insertion Sequence IS 466

We observed a spontaneous amplification of the Streptomyces coelicolor chromosome, including genes encoding biosynthetic enzymes of the antibiotic actinorhodin. A new junction of two tandem segments has, inserted within it, a third copy of a transposable element existing in two places elsewhere in the chromosome, suggesting its involvement in the amplification mechanism.

Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor

The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. First, a C(4a)-FMN-hydroperoxide species is formed after binding of reduced FMN to the monooxygenase and reaction with O(2). This intermediate hydroxylates the substrate and is transformed to a second reaction intermediate, a C(4a)-FMN-hydroxy species. In addition, we demonstrate that reduced FMN can be transferred efficiently from the reductase to the monooxygenase without involving any protein.protein complexes. The rate of transfer of reduced FMN from ActVB to ActVA was found to be controlled by the release of NAD(+) from ActVB and was strongly affected by NAD(+) concentration, with an IC(50) of 40 microm. This control of reduced FMN transfer by NAD(+) was associated with the formation of a strong charge.transfer complex between NAD(+) and reduced FMN in the active site of ActVB. These results suggest that, in Streptomyces coelicolor, the reductase component ActVB can act as a regulatory component of the monooxygenase activity by controlling the transfer of reduced FMN to the monooxygenase.

Dramatic Activation of Antibiotic Production in Streptomyces coelicolor by Cumulative Drug Resistance Mutations

We recently described a new method to activate antibiotic production in bacteria by introducing a mutation conferring resistance to a drug such as streptomycin, rifampin, paromomycin, or gentamicin. This method, however, enhanced antibiotic production by only up to an order of magnitude. Working with Streptomyces coelicolor A3(2), we established a method for the dramatic activation of antibiotic production by the sequential introduction of multiple drug resistance mutations. Septuple and octuple mutants, C7 and C8, thus obtained by screening for resistance to seven or eight drugs, produced huge amounts (1.63 g/liter) of the polyketide antibiotic actinorhodin, 180-fold higher than the level produced by the wild type. This dramatic overproduction was due to the acquisition of mutant ribosomes, with aberrant protein and ppGpp synthesis activity, as demonstrated by in vitro protein synthesis assays and by the abolition of antibiotic overproduction with relA disruption. This new approach, called "ribosome engineering," requires less time, cost, and labor than other methods and may be widely utilized for bacterial strain improvement.

Manipulating and understanding antibiotic production in Streptomyces coelicolor A3(2) with decoy oligonucleotides

We have adapted and extended the decoy oligonucleotide technique for use in prokaryotes. To identify cis-acting regulatory elements within a promoter, we developed a DNase I/T7 exonuclease footprinting technique and applied it to actII-orf4 from Streptomyces coelicolor A3(2), which encodes the pathway-specific activator for production of the antibiotic actinorhodin. Our in vivo mapping data allowed us to create decoy oligonucleotides incorporating the identified regulatory elements and to test whether their introduction into S. coelicolor affected antibiotic production. We mapped the promoter region when in a transcriptionally inactive state before the onset of actinorhodin production with the aim of designing decoy oligonucleotides capable of interfering with potential repressor binding and so stimulate actinorhodin production. Mapping identified five candidates for decoy oligonucleotides, and these were tested in a plate-based assay to rapidly validate their activity. A transfection protocol was developed for liquid cultures that enabled efficient uptake of decoys, and quantitative real-time PCR demonstrated decoy persistence for >70 h. Measurement of the effects on growth, expression of actII-orf4, and antibiotic production demonstrated that one of the decoys, in concordance with the plate assay, was more efficacious than the others in increasing actinorhodin production. Two of the identified regulatory elements occurred upstream of gene SCO5812, deletion of which reduced actinorhodin production, confirming that experimental analysis of regulatory motifs can provide new insights into factors influencing antibiotic production in streptomycetes.

Actinoperylone, a novel perylenequinone-type shunt product, from a deletion mutant of the actVA-ORF5 and ORF6 genes for actinorhodin biosynthesis in Streptomyces coelicolor A3(2)

A novel shunt product, actinoperylone, has been isolated from a deletion mutant of the actVA-ORF5 and ORF6 genes involved in the biosynthesis of a benzoisochromanequinone (BIQ) antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2). Spectroscopic analysis revealed its perylenequinone-type skeleton with the four chiral centers, obviously derived from the dimerization of an ACT intermediate. The structure of actinoperylone indicates the essential role of ActVA-ORF5 in the oxygen introduction at C-6, which is common to the formation of BIQ chromophore. The present results also agree with the distribution of the actVA-ORF5 homologues in all known BIQ biosynthetic clusters in streptomycetes.

A New TetR Family Transcriptional Regulator Required for Morphogenesis inStreptomyces coelicolor

Both morphogenesis and antibiotic production in the streptomycetes are initiated in response to starvation, and these events are coupled. We previously described a transposon-generated mutant in Streptomyces coelicolor, SE293, that resulted in a bld strain that overproduced the antibiotic actinorhodin. The SCO1135 open reading frame identified by the insertion encodes a member of the TetR family of transcriptional regulators. Here we show that a constructed deletion of the SCO1135 open reading frame resulted in the same morphological and antibiotic production phenotype as the insertion mutant. The constructed deletion also resulted in constitutive expression of SCO1135 transcript, as well as that of the gene cluster immediately adjacent to it, SCO1134-1132, which encodes a putative molybdopterin binding complex. A His6-tagged version of the SCO1135 protein product was shown to bind the intergenic region between SCO1135 and SCO1134, which contains the apparent transcription start sites for each gene mapped by primer extension analysis. Increased expression of the SCO1134-1132 transcript in the SCO1135 deletion mutant also resulted in increased expression of xanthine dehydrogenase activity, confirming the predictions about these open reading framed based on protein similarity. We have designated the SCO1134-1142 gene cluster xdhABC and the regulator encoded by SCO1135 xdhR. We speculate that the inappropriate expression of xanthine dehydrogenase affects purine salvaging pathways at the onset of development, creating artificially high concentrations of both GTP and ppGpp and perturbing the pathways these molecules participate in for the initiation of morphogenesis and antibiotic production.

FtsY Affects Sporulation and Antibiotic Production by whiH in Streptomyces coelicolor

FtsY, the Signal Recognition Particle (SRP) receptor in bacteria, is known to facilitate the cotranslational protein targeting by recruiting SRP-protein complex to secYEG. We show in this work that deletion of the ftsY gene in Streptomyces coelicolor would also lead to complete blockage of sporulation process and reduced production of antibiotic actinorhodin. These defects cannot be complemented by only the NG domain of FtsY, while full-length ftsY was able to restore spore generation and increase production of actinorhodin in ftsY-disrupted strains. Further transcriptional analysis on sporulation controlling genes, i.e., whiG, whiB, whiH, and prox, indicated that the regulation of sporulation by ftsY is likely to take effect through whiH.

Mutations in rsmG , Encoding a 16S rRNA Methyltransferase, Result in Low-Level Streptomycin Resistance and Antibiotic Overproduction in Streptomyces coelicolor A3(2)

Certain str mutations that confer high- or low-level streptomycin resistance result in the overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif. Deletion of rsmG from wild-type Streptomyces coelicolor resulted in the acquisition of streptomycin resistance and the overproduction of the antibiotic actinorhodin. Introduction of wild-type rsmG into the deletion mutant completely abrogated the effects of the rsmG deletion, confirming that rsmG mutation underlies the observed phenotype. Consistent with earlier work using a spontaneous rsmG mutant, the strain carrying DeltarsmG exhibited increased SAM synthetase activity, which mediated the overproduction of antibiotic. Moreover, high-performance liquid chromatography analysis showed that the DeltarsmG mutant lacked a 7-methylguanosine modification in the 16S rRNA (possibly at position G518, which corresponds to G527 of Escherichia coli). Like certain rpsL mutants, the DeltarsmG mutant exhibited enhanced protein synthetic activity during the late growth phase. Unlike rpsL mutants, however, the DeltarsmG mutant showed neither greater stability of the 70S ribosomal complex nor increased expression of ribosome recycling factor, suggesting that the mechanism underlying increased protein synthesis differs in the rsmG and the rpsL mutants. Finally, spontaneous rsmG mutations arose at a 1,000-fold-higher frequency than rpsL mutations. These findings provide new insight into the role of rRNA modification in activating secondary metabolism in Streptomyces.

The global role of ppGpp synthesis in morphological differentiation and antibiotic production in Streptomyces coelicolor A3(2).

The induction of ppGpp synthesis in Streptomyces coelicolor influenced the expression of several genomic elements characteristic of streptomycete biology, including antibiotic gene clusters, conservons, and morphogenetic proteins.

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.

Increased expression of ribosome recycling factor is responsible for the enhanced protein synthesis during the late growth phase in an antibiotic‐overproducing Streptomyces coelicolor ribosomal rpsL mutant

K88E mutation within rpsL, which encodes the S12 ribosomal protein, enhanced the protein synthetic activity of Streptomyces coelicolor during the late growth phase, resulting in overproduction of the deep blue-pigmented polyketide antibiotic actinorhodin. In vitro cross-mixing experiments using the ribosomal and S-150 fractions derived from wild-type and K88E mutant strains suggested that one or more translation factors are enriched in the mutant's S-150 fraction, while Western analysis using antibodies against various translation factors revealed ribosome recycling factor (RRF) to be one of the enriched mediators. RRF purified from overexpressing cells stimulated mRNA-directed green fluorescent protein (GFP) synthesis in an in vitro protein synthesis system. GFP synthesis rates were complemented by RRF addition into wild-type cell's S-150 fraction, eliminating the difference between wild-type and mutant S-150 fractions. The frr gene encoding RRF was found to be transcribed from two distinct start points (frrp1 and frrp2), and increased expression from frrp1 could account for the elevated level of RRF in the K88E mutant during the late growth phase. Moreover, introduction of a plasmid harbouring a high copy number of frr gene into wild-type S. coelicolor induced remarkable overproduction of antibiotic, demonstrating that the increased levels of RRF caused by the K88E mutation is responsible for an aberrant stationary-phase event: overproduction of antibiotic.

An Aromatic Hydroxylation Reaction Catalyzed by a Two-component FMN-dependent Monooxygenase: THE ActVA-ActVB SYSTEM FROM STREPTOMYCES COELICOLOR

The ActVA-ActVB system from Streptomyces coelicolor isatwo-component flavin-dependent monooxygenase that belongs to an emerging class of enzymes involved in various oxidation reactions in microorganisms. The ActVB component is a NADH:flavin oxidoreductase that provides a reduced FMN to the second component, ActVA the proper monooxygenase. In this work, we demonstrate that the ActVA-ActVB system catalyzes the aromatic monohydroxylation of dihydrokalafungin by molecular oxygen. In the presence of reduced FMN and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic flavin FMN-OOH hydroperoxide intermediate species as the oxidant. Surprisingly, we demonstrate that the quinone form of dihydrokalafungin is not oxidized by the ActVA-ActVB system, whereas the corresponding hydroquinone is an excellent substrate. The enantiomer of dihydrokalafungin, nanaomycin A, as well as the enantiomer of kalafungin, nanaomycin D, are also substrates in their hydroquinone forms. The previously postulated product of the ActVA-ActVB system, the antibiotic actinorhodin, was not found to be formed during the oxidation reaction.

A Tailoring Activity Is Responsible for Generating Polyene Amide Derivatives in Streptomyces diastaticus var. 108

We recently characterized rimocidin B (3b) and CE-108B (4b) as two polyene amides with improved pharmacological properties, produced by genetically modified Streptomyces diastaticus var. 108. In this work, genetic and biochemical analysis of the producer strain show that the two amides are derived from the parental polyenes rimocidin (3a) and CE-108 (4a) by a post-PKS modification of the free side chain carboxylic acid. This modification is mediated by an amidotransferase activity operating after the biosynthesis of rimocidin (3a) and CE-108 (4a) are completed. Two polyenes, intermediates of the biosynthetic pathway of rimocidin (3a) and CE-108 (4a), were also isolated and shown to have some improved pharmacological properties compared with the final products.

Enantioselective Reduction of β‐Keto Acids with Engineered Streptomyces coelicolor

Compelling evidence for the intermediacy of the free β-keto acid 1, rather than the corresponding enzyme-bound thiolate as previously proposed, in the biosynthesis of the antibiotic actinorhodin (2) was obtained from studies of the enantioselective reduction of a range of β-keto acids by the engineered strain of S. coelicolor CH999/pIJ5675. This excellent whole-cell biotransformation system gave the desired S β-hydroxy acids with >95 % ee.