Shigeru Kitani

Avenolide, a Streptomyces hormone controlling antibiotic production in Streptomyces avermitilis

Gram-positive bacteria of the genus Streptomyces are industrially important microorganisms, producing >70% of commercially important antibiotics. The production of these compounds is often regulated by low-molecular-weight bacterial hormones called autoregulators. Although 60% of Streptomyces strains may use γ-butyrolactone–type molecules as autoregulators and some use furan-type molecules, little is known about the signaling molecules used to regulate antibiotic production in many other members of this genus. Here, we purified a signaling molecule (avenolide) from Streptomyces avermitilis—the producer of the important anthelmintic agent avermectin with annual world sales of

Characterization of a regulatory gene, aveR, for the biosynthesis of avermectin in Streptomyces avermitilis.

850 million—and determined its structure, including stereochemistry, by spectroscopic analysis and chemical synthesis as (4S,10R)-10-hydroxy-10-methyl-9-oxo-dodec-2-en-1,4-olide, a class of Streptomyces autoregulator. Avenolide is essential for eliciting avermectin production and is effective at nanomolar concentrations with a minimum effective concentration of 4 nM. The aco gene of S. avermitilis, which encodes an acyl-CoA oxidase, is required for avenolide biosynthesis, and homologs are also present in Streptomyces fradiae, Streptomyces ghanaensis, and Streptomyces griseoauranticus, suggesting that butenolide-type autoregulators may represent a widespread and another class of Streptomyces autoregulator involved in regulating antibiotic production.

Genome Sequence of Kitasatospora setae NBRC 14216T: An Evolutionary Snapshot of the Family Streptomycetaceae

Avermectin is an important macrocyclic polyketide produced by Streptomyces avermitilis and widely used as an anthelmintic agent in the medical, veterinary, and agricultural fields. The avermectin biosynthetic gene cluster contains aveR, which belongs to the LAL-family of regulatory genes. In this study, aveR was inactivated by gene replacement in the chromosome of S. avermitilis, resulting in the complete loss of avermectin production. The aveR mutant was unable to convert an avermectin intermediate to any avermectin derivatives, and complementation by intact aveR and its proper upstream region restored avermectin production in the mutant, suggesting that AveR is a positive regulator controlling the expression of both polyketide biosynthetic genes and postpolyketide modification genes in avermectin biosynthesis. Despite the general concept that an increased amount of a positive pathway-specific regulator leads to higher production, a higher amount of aveR resulted in complete loss of avermectin, indicating that there is a maximum threshold concentration of aveR for the production of avermectin.

The autoregulator receptor homologue AvaR3 plays a regulatory role in antibiotic production, mycelial aggregation and colony development of Streptomyces avermitilis

Kitasatospora setae NBRC 14216T (=KM-6054T) is known to produce setamycin (bafilomycin B1) possessing antitrichomonal activity. The genus Kitasatospora is morphologically similar to the genus Streptomyces, although they are distinguishable from each other on the basis of cell wall composition and the 16S rDNA sequence. We have determined the complete genome sequence of K. setae NBRC 14216T as the first Streptomycetaceae genome other than Streptomyces. The genome is a single linear chromosome of 8 783 278 bp with terminal inverted repeats of 127 148 bp, predicted to encode 7569 protein-coding genes, 9 rRNA operons, 1 tmRNA and 74 tRNA genes. Although these features resemble those of Streptomyces, genome-wide comparison of orthologous genes between K. setae and Streptomyces revealed smaller extent of synteny. Multilocus phylogenetic analysis based on amino acid sequences unequivocally placed K. setae outside the Streptomyces genus. Although many of the genes related to morphological differentiation identified in Streptomyces were highly conserved in K. setae, there were some differences such as the apparent absence of the AmfS (SapB) class of surfactant protein and differences in the copy number and variation of paralogous components involved in cell wall synthesis.

Rakicidin D, an inhibitor of tumor cell invasion from marine-derived Streptomyces sp.

The γ-butyrolactone autoregulator receptor has been shown to control secondary metabolism and/or morphological differentiation across many Streptomyces species. Streptomyces avermitilis produces an important anthelmintic agent (avermectin) and two further polyketide antibiotics, filipin and oligomycin. Genomic analysis of S. avermitilis revealed that this micro-organism has the clustered putative autoregulator receptor genes distant from the antibiotic biosynthetic gene clusters. Here, we describe the characterization of avaR3, one of the clustered receptor genes, which encodes a protein containing an extra stretch of amino acid residues that has not been found in the family of autoregulator receptors. Disruption of avaR3 resulted in markedly decreased production of avermectins, with delayed expression of avermectin biosynthetic genes, suggesting that AvaR3 positively controls the avermectin biosynthetic genes. Moreover, the disruption caused increased production of filipin without any changes in the transcriptional profile of the filipin biosynthetic genes, suggesting that filipin production is indirectly controlled by AvaR3. The avaR3 disruptant displayed fragmented growth in liquid culture and conditional morphological defects on solid medium. These findings demonstrated that AvaR3 acts as a global regulator that controls antibiotic production and cell morphology.

Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR

Tumor metastasis is the leading cause of death in cancer patients. It is the process by which a tumor cell leaves the primary tumor, disseminates to a distant site through the circulatory system and establishes a secondary tumor.1 During the metastatic cascade, tumor cells must pass through the extracellular matrix barriers to accomplish the metastasis. Although the genetic basis of tumorigenesis can vary greatly, the steps required for metastasis are similar for all tumor cells. Therefore, interruption of metastasis is a promising approach to the treatment of cancers of various genetic origins. In our continuing search for anti-invasive compounds from microbial secondary metabolites,2–5 rakicidin D (1) was isolated from the culture broth of an actinomycete strain of the genus Streptomyces. Rakicidins are the 15-membered depsipeptides consisting of three amino acids and a 3-hydroxyfatty acid (Figure 1). To date, three congeners, rakicidins A (2) and B (3) from Micromonospora and rakicidin C (4) from Streptomyces, have been reported.6,7 Rakicidins contain a rare unusual amino acid, 4-amino-2,4-pentadienoate, which has been found only in the secondary metabolites from actinomycetes. Except for rakicidins, only two classes of cyclic peptides, BE435478 and vinylamycin,9 are reported to date to contain this unusual amino acid. Herein, we describe the isolation, structure elucidation and biological properties of rakicidin D (1). The producing strain Streptomyces sp. MWW064 was isolated from a marine sediment sample collected in Samut Sakhon province, Thailand. The strain was cultured in our standard medium for actinomycetes and the whole culture broth was extracted with 1-butanol. The extract showed inhibitory activity toward tumor cell invasion into Matrigel, the reconstituted extracellular matrix proteins.10 Bioassay-guided fractionation of the extract led to the isolation of a new compound, rakicidin D (1). Compound 1 was obtained as a colorless amorphous powder. The high-resolution ESITOFMS indicated a molecular formula of C24H38N4O7, which was consistent with the 1H and 13C NMR data. The IR spectrum of 1 indicated the presence of OH or NH (3356 cm 1) and carbonyl (1688 and 1648 cm 1) functionalities. The UV spectrum and the 1H and 13C NMR spectra of 1 showed high similarity to those for rakicidins A and B.7 The 13C NMR and HMQC analysis confirmed the presence of 24 carbons attributable to six deshielded signals including carbonyl carbons, two sp2 methines, one sp2 methylene, five sp3 methines, six sp3 methylenes, four methyl carbons and five exchangeable protons. 2-Amino-2,4-pentadienoate moiety was elucidated by a COSY correlation for H-9/H-10 and a series of HMBC correlations from H-12 to C-10 and C-11, from H-10 to C-8, C-11 and C-12, and from H-9 to C-8 and C-10 (Figure 2). An NOE between H-10 and an exomethylene proton at dH 5.41 indicated that the latter proton and C-10 were located on the same side of the C-11/C-12 double bond. E configuration for the double bond between C-9 and C-10 was confirmed by a large coupling constant for H-9 and H-10 (JHH1⁄415.0 Hz). HMBC correlations from H-7 to C-6 and C-8, and from H-6 to C-5 and C-8 established the connectivity of N-methylglycine and the pentadienoate. COSY correlations for 2-NH/H-2/H-3 and HMBC correlations from H-2 to C-1, H-3 to C-4 and 4-NH2 to C-3 confirmed the presence of a b-hydroxyasparagine moiety. Connection of this amino acid to the glycine moiety was established by an HMBC correlation from 2-NH to C-5. Three additional fragments, H-23/H-14/H-15, H-24/H-16 and H-21/H-22, were recognized from the COSY spectrum. HMBC correlations from H-23 to C-13, C-14 and C-15, and from H-24 to C-15, C-16 and C-17, established the 2,4-dimethyl-3-hydroxyalkanoate substructure. Connectivity of this fragment to the peptide unit was elucidated by HMBC correlations from 11-NH to C-13 and from H-15 to C-1. Finally, the fragment C-22/C-21 containing the triplet methyl group, the methylene fragment C-20 that had an HMBC correlation from H-22 and

Pleiotropic control of secondary metabolism and morphological development by KsbC, a butyrolactone-autoregulator receptor homologue in Kitasatospora setae

Two regulatory genes encoding a Streptomyces antibiotic regulatory protein (vmsS) and a response regulator (vmsT) of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic virginiamycin, which is composed of virginiamycin M (VM) and virginiamycin S (VS), in Streptomyces virginiae. Disruption of vmsS abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for virginiamycin biosynthetic genes, whereas disruption of vmsT resulted in only a loss of VM biosynthesis, suggesting that vmsS is a pathway-specific regulator for both VM and VS biosynthesis, and that vmsT is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the virginiamycin biosynthetic gene cluster demonstrated that vmsS controls the biosynthetic genes for VM and VS, and vmsT controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of vmsR located in the clustered regulatory region in the virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of vmsS and vmsT is under the control of vmsR, and vmsR also contributes to the expression of VM and VS biosynthetic genes, independent of vmsS and vmsT. Therefore, coordinated virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.

Control of secondary metabolism by farX, which is involved in the γ-butyrolactone biosynthesis of Streptomyces lavendulae FRI-5

ABSTRACT The γ-butyrolactone autoregulator signaling cascades have been shown to control secondary metabolism and/or morphological development among many Streptomyces species. However, the conservation and variation of the regulatory systems among actinomycetes remain to be clarified. The genome sequence of Kitasatospora setae, which also belongs to the family Streptomycetaceae containing the genus Streptomyces, has revealed the presence of three homologues of the autoregulator receptor: KsbA, which has previously been confirmed to be involved only in secondary metabolism; KsbB; and KsbC. We describe here the characterization of ksbC, whose regulatory cluster closely resembles the Streptomyces virginiae barA locus responsible for the autoregulator signaling cascade. Deletion of the gene ksbC resulted in lowered production of bafilomycin and a defect of aerial mycelium formation, together with the early and enhanced production of a novel β-carboline alkaloid named kitasetaline. A putative kitasetaline biosynthetic gene cluster was identified, and its expression in a heterologous host led to the production of kitasetaline together with JBIR-133, the production of which is also detected in the ksbC disruptant, and JBIR-134 as novel β-carboline alkaloids, indicating that these genes were biosynthetic genes for β-carboline alkaloid and thus are the first such genes to be discovered in bacteria.

Null mutation analysis of an afsA-family gene, barx, that is involved in biosynthesis of the γ-butyrolactone autoregulator in Streptomyces virginiae

The γ-butyrolactone signaling system is distributed widely among streptomycetes as an important regulatory mechanism of antibiotic production and/or morphological differentiation. IM-2 [(2R,3R,1′R)-2-(1′-hydroxybutyl)-3-hydroxymethyl-γ-butanolide] is a γ-butyrolactone that switches off the production of d-cycloserine but switches on the production of several nucleoside antibiotics as well as blue pigment in Streptomyces lavendulae FRI-5. farX is a member of the afsA-family genes, which are proposed to encode enzymes involved in γ-butyrolactone biosynthesis. Disruption of farX caused overproduction of d-cycloserine, and abolished production of nucleoside antibiotic and blue pigment with the loss of IM-2 production. The finding that all phenotypic changes observed in the farX disruptant were restored by the addition of exogenous IM-2 suggested that FarX plays a biosynthetic role in IM-2 production. Transcriptional comparison between the wild-type strain and the farX disruptant revealed that, in addition to already known genes farR1 and farR2, several other genes (farR4, farD, and farE) are under the transcriptional regulation of IM-2. Furthermore, the fact that farX transcription is under the control of IM-2 suggested that S. lavendulae FRI-5 has a fine-tuning system to control γ-butyrolactone production.

Differential contributions of two SARP family regulatory genes to indigoidine biosynthesis in Streptomyces lavendulae FRI-5

Virginiae butanolide (VB) is a gamma-butyrolactone autoregulator that triggers production of the streptogramin antibiotic virginiamycin in Streptomyces virginiae. Our previous studies suggested that the barX gene, an afsA-family gene, is likely to participate in the regulatory pathway for the production of VB, rather than in the biosynthetic pathway of VB itself, in contrast to the function of other afsA-family genes. Mutation analysis now shows that BarX at least plays an enzymic role in the VB biosynthetic pathway. Heterologous expression of the afsA gene from Streptomyces griseus into the barX mutant partially restored the deficiency of virginiamycin production, suggesting that afsA-family genes have a common ability to synthesize the gamma-butyrolactone autoregulators. Taken together with previous works relating to the function of an afsA-family gene, these results support the idea that streptomycetes have two biosynthetic pathways for the gamma-butyrolactone autoregulators.