Anti-Rhodotorula activity of mycophenolic acid enhanced in the presence of polyene antibiotic nystatin.

Rhodotorula species are opportunistic pathogens, which cause not only systemic fungaemia but also other localized infections. Despite serious side effects such as nephrotoxicity and hypokalemia, amphotericin B (a polyene antifungal) has been commonly prescribed for Rhodotorula infection because Rhodotorula species are resistant against a candin family of antifungal agents. In this study, novel active compounds against Rhodotorula species were screened from the extracts of entomopathogenic fungi based on the synergistic effect of polyene nystatin (NYS), which causes efficient targeting of compounds due to increased permeability through the fungal cell membrane. Around 37% of culture extracts from 31 entomopathogenic fungal strains showed anti-Rhodotorula activity in the synergistic bioassay system, suggesting that the coexistence assay with NYS enhanced the discovery of anti-Rhodotorula compounds. Judging from various physicochemical data, the active component from strain HF763 was identified as an immunosuppressant drug, mycophenolic acid (MPA). The minimum inhibitory concentration of MPA against three pathogenic Rhodotorula strains was determined, focusing on the synergistic effect with NYS. The results revealed that the values decreased by at least 87% in the presence of NYS, indicating that MPA showed a synergistic effect with NYS. SIGNIFICANCE AND IMPACT OF THE STUDY: This study aimed to screen active compounds against Rhodotorula species that are resistant to a candin family of antifungal agents, from entomopathogenic fungi. Assuming that most of the latent antifungal compounds do not exert their activity due to their inability to penetrate the membrane, we took advantage of polyene nystatin in the screening to increase permeability through the fungal cell membrane. The result of the screening revealed hidden antifungal activity of mycophenolic acid, demonstrating that the method applied in this study unlocks the potentials of bioresources, and proposes a new remedy for mycosis.

Improvement of compactin (ML-236B) production by genetic engineering in compactin high-producing Penicillium citrinum.

An increase in compactin (ML-236B) production was achieved by introducing a whole compactin biosynthetic gene cluster or the regulatory gene mlcR into compactin high-producing Penicillium citrinum. In the previous report, we introduced mlcR encoding the positive regulator of compactin biosynthetic genes into compactin high-producing strain no. 41520, and most of the transformants produced higher amounts of compactin. Here, we characterize one of the resulting high producers (strain TIR-35, which produced 50% more compactin) and reveal that TIR-35 contained five copies of mlcR and that early, enhanced expression of mlcR caused compactin overproduction. Similarly, the introduction of mlcR into strain T48.19, which was created previously from strain no. 41520 by introducing a partial compactin biosynthetic gene cluster, enhanced compactin production further. Our results indicated that genetic engineering is an effective tool to improve compactin production, even in compactin high producers.

Identification of the specific sequence recognized by Penicillium citrinum MlcR, a GAL4-type transcriptional activator of ML-236B (compactin) biosynthetic genes.

MlcR is a pathway-specific transcriptional activator of the ML-236B biosynthetic genes in Penicillium citrinum. The MlcR-binding sequences were identified by an in vitro gel-shift assay and an in vivo reporter assay for the region between mlcA and mlcC as a model. The gel-shift assay showed that recombinant MlcR bound to the DNA sequence 5'-ACGGCGTTATTCGG-3' and most of the bases in this motif were required for the interaction between MlcR and DNA. In the reporter assay using beta-glucuronidase (GUS), substitution of the bases in this binding sequence resulted in the drastic reduction of GUS activities. These data clearly indicate that this MlcR-binding sequence is essential for the transcriptional activation of mlcA and mlcC in P. citrinum. Similar motifs were found in other loci of the ML-236B biosynthetic gene cluster and the consensus-binding motif for MlcR was predicted to be a direct repeat, 5'-WCGG-N(6)-TCGG-3'.

Disruption of sscR encoding a gamma-butyrolactone autoregulator receptor in Streptomyces scabies NBRC 12914 affects production of secondary metabolites.

We report the cloning and sequence analysis of a gamma-butyrolactone autoregulator regulatory island that includes an sscR gene encoding the gamma-butyrolactone autoregulator receptor from Streptomyces scabies NBRC 12914, a plant pathogenic strain. gamma-Butyrolactone autoregulators trigger secondary metabolism, and sometimes morphological differentiation in the Gram-positive genus Streptomyces through binding to a specific autoregulator receptor. This gene cluster showed close similarity to other regulatory islands of Streptomyces origin that are responsible for the control of secondary metabolism. The recombinant SscR protein expressed in Escherichia coli prefers a gamma-butyrolactone autoregulator containing a long C-2 side chain and beta-hydroxyl group at the C-6 position. An inactivation experiment confirmed that this gamma-butyrolactone autoregulator receptor was involved in secondary metabolism but had no effects on the morphological differentiation. In the sscR-deleted mutant, the binding activity of the gamma-butyrolactone autoregulator was completely abolished, suggesting that its primary role is to detect the presence of an autoregulator in the environment. HPLC analysis of the culture broth showed that some peaks disappeared and new peaks that were not present in the broth of the wild-type strain appeared.

Fabrication of size-controlled polyimide nanoparticles.

Polyimide particles were fabricated through the two-steps imidization of poly(amic acid) particles prepared by using reprecipitation method. PAA and PI nanoparticles were all spherical, and the changes of particle size, its distribution, and morphology were not observed before and after the imidization. The preparation of PI nanoparticles size-controlled between ca. 20-500 nm was also achieved by changing the experimental conditions, temperature of the poor solvent, the composition of two kind of poor solvent, and PAA-NMP solution concentration.

Neuronal specificity of alpha-synuclein toxicity and effect of Parkin co-expression in primates.

Recombinant adeno-associated viral (rAAV) vector-mediated overexpression of alpha-synuclein (alphaSyn) protein has been shown to cause neurodegeneration of the nigrostriatal dopaminergic pathway in rodents and primates. Using serotype-2 rAAV vectors, we recently reported the protective effect of Parkin on alphaSyn-induced nigral dopaminergic neurodegeneration in a rat model. Here we investigated the neuronal specificity of alphaSyn toxicity and the effect of Parkin co-expression in a primate model. We used another serotype (type-1) of AAV vector that was confirmed to deliver genes of interest anterogradely and retrogradely to neurons in rats. The serotype-1 rAAV (rAAV1) carrying alphaSyn cDNA (rAAV1-alphaSyn), and a cocktail of rAAV1-alphaSyn and rAAV1 carrying parkin cDNA (rAAV1-parkin) were unilaterally injected into the striatum of macaque monkeys, resulting in protein expression in striatonigral GABAergic and nigrostriatal dopaminergic neurons. Injection of rAAV1-alphaSyn alone decreased tyrosine hydroxylase immunoreactivity in the striatum compared with the contralateral side injected with a cocktail of rAAV1-alphaSyn and rAAV1-parkin. Immunostaining of striatonigral GABAergic neurons was similar on both sides. Overexpression of Parkin in GABAergic neurons was associated with less accumulation of alphaSyn protein and/or phosphorylation at Ser129 residue. Our results suggest that the toxicity of accumulated alphaSyn is not induced in non-dopaminergic neurons and that the alphaSyn-ablating effect of Parkin is exerted in virtually all neurons in primates.

A complex role for the gamma-butyrolactone SCB1 in regulating antibiotic production in Streptomyces coelicolor A3(2).

Many streptomycetes produce extracellular gamma-butyrolactones. In several cases, these have been shown to act as signals for the onset of antibiotic production. Synthesis of these molecules appears to require a member of the AfsA family of proteins (AfsA is required for A-factor synthesis of the gamma-butyrolactone A-factor and consequently for streptomycin production in Streptomyces griseus). An afsA homologue, scbA, was identified in Streptomyces coelicolor A3(2) and was found to lie adjacent to a divergently transcribed gene, scbR, which encodes a gamma-butyrolactone binding protein. Gel retardation assays and DNase I footprinting studies revealed DNA binding sites for ScbR at - 4 to - 33 nt with respect to the scbA transcriptional start site, and at - 42 to - 68 nt with respect to the scbR transcriptional start site. Addition of the gamma-butyrolactone SCB1 of S. coelicolor resulted in loss of the DNA-binding ability of ScbR. A scbA mutant produced no gamma-butyrolactones, yet overproduced two antibiotics, actinorhodin (Act) and undecylprodigiosin (Red), whereas a deletion mutant of scbR also failed to make gamma-butyrolactones and showed delayed Red production. These phenotypes differ markedly from those expected by analogy with the S. griseus A-factor system. Furthermore, transcription of scbR increased, and that of scbA was abolished, in an scbR mutant, indicating that ScbR represses its own expression while activating that of scbA. In the scbA mutant, expression of both genes was greatly reduced. Addition of SCB1 to the scbA mutant induced transcription of scbR, but did not restore scbA expression, indicating that the deficiency in scbA transcription in the scbA mutant is not solely due to the inability to produce SCB1, and that ScbA is a positive autoregulator in addition to being required for gamma-butyrolactone production. Overall, these results indicate a complex mechanism for gamma-butyrolactone-mediated regulation of antibiotic biosynthesis in S. coelicolor.

Gene replacement analysis of the butyrolactone autoregulator receptor (FarA) reveals that FarA acts as a Novel regulator in secondary metabolism of Streptomyces lavendulae FRI-5.

IM-2 [(2R,3R,1'R)-2-1'-hydroxybutyl-3-hydroxymethyl gamma-butanolide] is a gamma-butyrolactone autoregulator which, in Streptomyces lavendulae FRI-5, switches off the production of D-cycloserine but switches on the production of a blue pigment and several nucleoside antibiotics. To clarify the in vivo function of an IM-2-specific receptor (FarA) in the IM-2 signaling cascade of S. lavendulae FRI-5, a farA deletion mutant was constructed by means of homologous recombination. On several solid media, no significant difference in morphology was observed between the wild-type strain and the farA mutant (strain K104), which demonstrated that the IM-2-FarA system does not participate in the morphological control of S. lavendulae FRI-5. In liquid media, the farA mutant overproduced nucleoside antibiotics and produced blue pigment earlier than did the wild-type strain, suggesting that the FarA protein acts primarily as a negative regulator on the biosynthesis of these compounds in the absence of IM-2. However, contrary to the IM-2-dependent suppression of D-cycloserine production in the wild-type strain, overproduction of D-cycloserine was observed in the farA mutant, indicating for the first time that the presence of both IM-2 and intact FarA are necessary for the suppression of D-cycloserine biosynthesis.

Identification of the varR gene as a transcriptional regulator of virginiamycin S resistance in Streptomyces virginiae.

A gene designated varR (for virginiae antibiotic resistance regulator) was identified in Streptomyces virginiae 89 bp downstream of a varS gene encoding a virginiamycin S (VS)-specific transporter. The deduced varR product showed high homology to repressors of the TetR family with a conserved helix-turn-helix DNA binding motif. Purified recombinant VarR protein was present as a dimer in vitro and showed clear DNA binding activity toward the varS promoter region. This binding was abolished by the presence of VS, suggesting that VarR regulates transcription of varS in a VS-dependent manner. Northern blot analysis revealed that varR was cotranscribed with upstream varS as a 2.4-kb transcript and that VS acted as an inducer of bicistronic transcription. Deletion analysis of the varS promoter region clarified two adjacent VarR binding sites in the varS promoter.

Identification by gene deletion analysis of a regulator, VmsR, that controls virginiamycin biosynthesis in Streptomyces virginiae.

Virginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newly discovered pairs of a butyrolactone autoregulator and a corresponding receptor protein of Streptomyces species and regulates the production of the antibiotic virginiamycin (VM) in S. virginiae. The gene vmsR was found to be situated 4.7 kbp upstream of the barA gene, which encodes the VB-specific receptor. The vmsR product was predicted to be a regulator of VM biosynthesis based on its high homology to some Streptomyces pathway-specific transcriptional regulators for the biosynthetic gene clusters of polyketide antibiotics, such as Streptomyces peucetius DnrI (47.5% identity, 84. 3% similarity), which controls daunorubicin biosynthesis. A vmsR deletion mutant was created by homologous recombination. Neither virginiamycin M(1) nor virginiamycin S was produced in the vmsR mutant, while amounts of VB and BarA similar to those produced in the wild-type strain were detected. Reverse transcription-PCR analyses confirmed that the vmsR deletion had no deleterious effects on the transcription of the vmsR-surrounding genes, indicating that VmsR is a positive regulator of VM biosynthesis in S. virginiae.

Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus.

Virginiae butanolides (VBs) and IM-2 are members of Streptomyces hormones called 'butyrolactone autoregulators' which regulate the antibiotic production in Streptomyces species at nanomolar concentrations. Cell-free extract of a VB-A overproducer, Streptomyces antibioticus NF-18, is capable of catalyzing the final step of the autoregulator biosynthesis, namely, the NADPH-dependent reduction of 6-dehydroVB-A. However, physico-chemical analyses of the purified enzymatic products revealed that, in addition to the VB-type isomer [(2R,3R,6S)-enantiomer], IM-2-type isomers [(2R,3R, 6R)- and (2S,3S,6S)-enantiomers] were also produced from (+/-)-6-dehydroVB-A, suggesting the existence of several 6-dehydroVB-A reductases with respective stereoselectivities. The reductase activity of the crude extracts was separated into two activity peaks, peak I (major) and peak II (minor), by DEAE-5PW HPLC. Chiral HPLC analyses demonstrated that peak I enzyme and peak II enzyme catalyzed the production of (2R,3R,6S), (2R,3R,6R) and (2S,3S, 6S) isomers at ratios of 46:1:3.2 and 4.9:1:1.5, respectively, indicating clearly that S. antibioticus NF-18 possesses at least two 6-dehydroVB-A reductases: one much favored toward VB-A biosynthesis, the other with relaxed stereoselectivity capable of synthesizing both VB-type and IM-2-type autoregulators.

Identification of an AfsA homologue (BarX) from Streptomyces virginiae as a pleiotropic regulator controlling autoregulator biosynthesis, virginiamycin biosynthesis and virginiamycin M1 resistance.

Virginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newly discovered pairs of a gamma-butyrolactone autoregulator and the corresponding receptor protein of the Streptomyces species, and has been shown to regulate the production of antibiotic virginiamycin (VM) in S. virginiae. A divergently transcribed barX gene is situated 259 bp upstream of the barA gene, and the BarX protein has been shown to be highly homologous (39.8% identity, 74. 6% similarity) to S. griseus AfsA. Although AfsA is thought to be a biosynthetic enzyme for A-factor, another member of the family of gamma-butyrolactone autoregulators, the in vivo function of S. virginiae BarX was investigated in this study by phenotypic and transcriptional comparison between wild-type S. virginiae and a barX deletion mutant. With the same growth rate as wild-type S. virginiae on both solid and liquid media, the barX mutant showed no apparent changes in its morphological behaviour, indicating that barX does not participate in morphological control in S. virginiae. However, the barX mutant became more sensitive to virginiamycin M1 than did the wild-type strain (minimum inhibitory concentration, 50 microgram ml-1 compared with > 200 microgram ml-1) and exhibited reduced VB and VM production. The VM production was not restored by exogenous addition of VB, suggesting that BarX per se is not a biosynthetic enzyme of VBs but a pleiotropic regulatory protein controlling VB biosynthesis. DNA sequencing of a 5.6 kbp downstream region of barX revealed the presence of five open reading frames (ORFs): barZ, encoding a BarB-like regulatory protein; orf2, encoding a Streptomyces coelicolor RedD-like pathway specific regulator; varM, encoding a homologue of ATP-dependent transporters for macrolide antibiotics; orf4, encoding a homologue of beta-ketoacyl ACP/CoA reductase; and orf5, encoding a homologue of dNDP-glucose dehydratase. Reverse transcription polymerase chain reaction (RT-PCR) analyses of the downstream five genes together with those of the three upstream genes (barA, barB, encoding a regulatory protein; and varS, encoding a virginiamycin S specific transporter) revealed that, in the barX mutant, the transcriptions of barZ, orf2, varM and orf5 were completely repressed and those of barB and varS were derepressed. Because free BarA (BarA in the absence of VB) in wild-type S. virginiae represses the transcription of bicistronic barB-varS operon through binding to a specific DNA sequence (BarA-responsive element, BARE) overlapping the barB transcriptional start site, the derepression of barB-varS transcription in the barX mutant suggested that the in vivo function of BarA was impaired by the lack of BarX protein. Gel-shift assays revealed that BarA easily lost its DNA-binding activity in the absence of BarX but that the defect was restored by the presence of recombinant BarX as a fusion with maltose-binding protein (MBP-BarX), whereas MBP-BarX itself showed no DNA-binding activity, indicating that BarX is likely to be a co-repressor of BarA, enforcing the DNA-binding activity of BarA through protein-protein interactions.

Glutathione as an essential factor for chaperon-mediated activation of lactonizing lipase (LipL) from Pseudomonas sp. 109.

Pseudomonas sp. 109 produces a unique lipase (LipL) which efficiently catalyzes intramolecular transesterification of omega-hydroxyesters to form macrocyclic lactones. The production of the enzymatically active LipL requires a specific molecular chaperon (LimL protein) together with a low-M(r) lipase-activation-factor (LAF) of unknown structure. From 50 g of Pseudomonas cells, 2.15 mg of LAF was purified as a sulfobenzofurazanyl derivative after methanol extraction, derivatization, and C(18) reverse-phase HPLC. One-dimensional and two-dimensional 600 MHz (1)H-NMR and fast atom bombardment mass spectrometry (FAB-MS) revealed that LAF is glutathione. Because several SH compounds (L-cysteine and mercaptoethanol) were similarly effective to native LAF in the activation of LipL, and because only LipL contains two cysteinyl residues forming an intramolecular disulfide bond, it is concluded that the reduction of and reformation of the intramolecular disulfide bond of LipL is essential to liberate free and fully active LipL.

A null mutant of the Streptomyces virginiae barA gene encoding a butyrolactone autoregulator receptor and its phenotypic and transcriptional analysis.

The Streptomyces virginiae barA gene encodes a specific receptor protein for virginiae butanolide (VB), one of the gamma-butyrolactone autoregulators of Streptomyces species. By homologous recombination, a barA null strain was constructed to clarify the in vivo function of BarA protein in S. virginiae. The deltabarA mutant showed no difference in terms of growth, but lost VB production and produced virginiamycin 7 h earlier than the wild-type strain. These results indicated that, phenotypically, BarA protein acts negatively in virginiamycin biosynthesis and positively in VB biosynthesis. Furthermore, Northern (RNA) blot analysis of the DeltabarA mutant revealed that transcription of the BarA target gene (barB) was derepressed, confirming that BarA acts as a transcriptional repressor in S. virginiae.

Purification and structural determination of SCB1, a gamma-butyrolactone that elicits antibiotic production in Streptomyces coelicolor A3(2).

Early stationary phase culture supernatants of Streptomyces coelicolor A3(2) contained at least four small diffusible signaling molecules that could elicit precocious antibiotic synthesis in the producing strain. The compounds were not detected in exponentially growing cultures. One of these compounds, SCB1, was purified to homogeneity and shown to be a gamma-butyrolactone of structure (2R, 3R,1'R)-2-(1'-hydroxy-6-methylheptyl)-3-hydroxymethylbutanolide . Bioassays of chemically synthesized SCB1, and of its purified stereoisomers, suggest that SCB1 acts in a highly specific manner to elicit the production of both actinorhodin and undecylprodigiosin, the two pigmented antibiotics made by S. coelicolor.

A low-Mr lipase activation factor cooperating with lipase modulator protein LimL in Pseudomonas sp. strain 109.

Pseudomonas sp. strain 109 produces a unique lipase (LipL) which efficiently catalyses intramolecular transesterification of omega-hydroxyesters to form macrocyclic lactones. In vivo production of enzymically active LipL requires lipase modulator protein (LimL), which functions as a molecular chaperone for the correct folding of LipL. However, previous work has shown that LipL forms a tight complex with LimL in vitro and the resulting LipL-LimL complex is only partially active, suggesting an additional mechanism that facilitates the dissociation of the complex to form enzymically active LipL. In the present work, a low-Mr compound (lipase activation factor, LAF) was found in Pseudomonas sp. strain 109 that when added to the LipL-LimL complex resulted in the activation of LipL. Ca2+ ions also enhanced lipase activity, but the instantaneous activation by Ca2+ was different from the gradual and time-dependent activation by LAF, indicating the novel nature of this compound. LAF passed through an ultrafiltration membrane with an Mr cut-off of 3000 and showed an apparent Mr of 330+/-30 on Superdex Peptide gel-filtration chromatography. Treatment of the LipL-LimL complex with LAF liberated free active LipL, indicating that LAF was necessary to dissociate the LipL-LimL complex.

Characterization of binding sequences for butyrolactone autoregulator receptors in streptomycetes.

BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autoregulators which binds to an upstream region of target genes to control transcription, leading to the production of the antibiotic virginiamycin M(1) and S. BarA-binding DNA sequences (BarA-responsive elements [BAREs]), to which BarA binds for transcriptional control, were restricted to 26 to 29-nucleotide (nt) sequences on barA and barB upstream regions by the surface plasmon resonance technique, gel shift assay, and DNase I footprint analysis. Two BAREs (BARE-1 and BARE-2) on the barB upstream region were located 57 to 29 bp (BARE-1) and 268 to 241 bp (BARE-2) upstream from the barB translational start codon. The BARE located on the barA upstream region (BARE-3) was found 101 to 76 bp upstream of the barA start codon. High-resolution S1 nuclease mapping analysis revealed that BARE-1 covered the barB transcription start site and BARE-3 covered an autoregulator-dependent transcription start site of the barA gene. Deletion and mutation analysis of BARE-2 demonstrated that at least a 19-nt sequence was required for sufficient BarA binding, and A or T residues at the edge as well as internal conserved nucleotides were indispensable. The identified binding sequences for autoregulator receptor proteins were found to be highly conserved among Streptomyces species.

In vitro analysis of the butyrolactone autoregulator receptor protein (FarA) of Streptomyces lavendulae FRI-5 reveals that FarA acts as a DNA-binding transcriptional regulator that controls its own synthesis.

FarA of Streptomyces lavendulae FRI-5 is a specific receptor protein for IM-2, a butyrolactone autoregulator that controls the production of a blue pigment and the nucleoside antibiotics showdomycin and minimycin. Gel shift assays demonstrated that FarA binds to the farA upstream region and that this binding is abolished in the presence of IM-2. The FarA binding sequence was localized by DNase I footprinting to a 28-bp sequence located approximately 70 bp upstream of the farA translational start site. High-resolution S1 nuclease mapping of farA transcripts revealed a putative transcription start site, located at an A residue positioned 64 bp upstream from the farA translation start codon and 4 bp downstream from an Escherichia coli sigma(70)-like -10 recognition region. The FarA-binding sequence overlaps this -10 region and contains the farA transcription initiation site, suggesting that FarA acts as a repressor that, in the absence of IM-2, represses transcription of farA.

Increase production of lactonizing lipase (LipL) from Pseudomonas sp. strain 109 by lipids and detergents.

LipL of Pseudomonas sp. strain 109 is a unique lipase capable of catalyzing macrocyclic lactone synthesis using omega-hydroxyfatty acid esters as substrates. Several fatty acid esters were tested as inducers of LipL production. The addition of either soybean oil or a non-ionic detergent (Noigen HC) resulted in a 44 to 45-fold increase in extracellular LipL, and the presence of both resulted in a further 56-fold increase. Among the triglycerides tested, triolein was the most effective, with a 50-fold increase in LipL production. A Northern blot hybridization analysis found that the lipL transcript increased in the presence of soybean oil or Noigen HC, indicating that the production of LipL is regulated at the transcriptional level.

Identification and in vivo functional analysis of a virginiamycin S resistance gene (varS) from Streptomyces virginiae.

BarA of Streptomyces virginiae is a specific receptor protein for virginiae butanolide (VB), one of the gamma-butyrolactone autoregulators of the Streptomyces species, and acts as a transcriptional regulator controlling both virginiamycin production and VB biosynthesis. The downstream gene barB, the transcription of which is under the tight control of the VB-BarA system, was found to be transcribed as a polycistronic mRNA with its downstream region, and DNA sequencing revealed a 1,554-bp open reading frame (ORF) beginning at 161 bp downstream of the barB termination codon. The ORF product showed high homology (68 to 73%) to drug efflux proteins having 14 transmembrane segments and was named varS (for S. virginiae antibiotic resistance). Heterologous expression of varS with S. lividans as a host resulted in virginiamycin S-specific resistance, suggesting that varS encoded a virginiamycin S-specific transport protein. Northern blot analysis indicated that the bicistronic transcript of barB-varS appeared 1 to 2 h before the onset of virginiamycin M1 and S production, at which time VB was produced, while exogenously added virginiamycin S apparently induced the monocistronic varS transcript.