Decrypting the programming of ß-methylation in virginiamycin M biosynthesis.

During biosynthesis by multi-modular trans-AT polyketide synthases, polyketide structural space can be expanded by conversion of initially-formed electrophilic ß-ketones into ß-alkyl groups. These multi-step transformations are catalysed by 3-hydroxy-3-methylgluratryl synthase cassettes of enzymes. While mechanistic aspects of these reactions have been delineated, little information is available concerning how the cassettes select the specific polyketide intermediate(s) to target. Here we use integrative structural biology to identify the basis for substrate choice in module 5 of the virginiamycin M trans-AT polyketide synthase. Additionally, we show in vitro that module 7, at minimum, is a potential additional site for ß-methylation. Indeed, analysis by HPLC-MS coupled with isotopic labelling and pathway inactivation identifies a metabolite bearing a second ß-methyl at the expected position. Collectively, our results demonstrate that several control mechanisms acting in concert underpin ß-branching programming. Furthermore, variations in this control - whether natural or by design - open up avenues for diversifying polyketide structures towards high-value derivatives.

Genome shuffling and ribosome engineering of Streptomyces virginiae for improved virginiamycin production.

The production of virginiamycin (VGM) from Streptomyces virginiae was improved by genome shuffling and ribosome engineering companied with a high-throughput screening method integrating deep-well cultivation and the cylinder-plate detecting. First, a novel high-throughput method was developed to rapidly screen large numbers of VGM-producing mutants. Then, the starting population of genome shuffling was obtained through ultraviolet (UV) and microwave mutagenesis, and four mutants with higher productivity of VGM were selected for genome shuffling. Next, the parent protoplasts were inactivated by UV and heat when a fusant probability was about 98%. Streptomycin resistance was used as an evolutionary pressure to extend positive effects on VGM synthesis. Finally, after five rounds of genome shuffling, a genetically stable strain G5-103 was obtained and characterized to be able to yield 251 mg/L VGM, which was 3.1- and 11.6-fold higher than that of the mutant strain UV 1150 and the wild-type strain, respectively.

Streptogramins - two are better than one!

Streptogramins are potent drugs against numerous highly resistant pathogens and therefore are used as antibiotics of last-resort human therapy. They consist of a mixture of two different types of chemical substances - the group A streptogramins, which are polyunsaturated macrolactones, and the group B streptogramins, representing cyclic hexadepsipeptides. Streptogramins are unique in their mode of action: each component alone exhibits a moderate bacteriostatic activity by binding to the bacterial 50S ribosomal subunit and thereby blocking translation, whereas the synergic combination of both substances is up to hundred fold more effective than the single compounds, resulting in a bactericidal activity. The streptogramin biosynthetic genes are organized as large antibiotic superclusters. These clusters harbour numerous regulatory genes, which encode different types of regulators that together form a complex hierarchical signalling system, which governs the regulation of streptogramin biosynthesis. Resistance is also regulated by this cascade. However, whereas resistance against streptogramins is quite well understood in diverse pathogenic organisms, only little is known about how the natural producer strains protect themselves against these toxic compounds. Here, we give an overview about the recent advances in streptogramin investigations with a main focus on the best-studied representatives, pristinamycin and virginiamycin. We concentrate on the biosynthesis of these compounds, their regulation and resistance determinants as well as their application in medicine and food industry.

VisG is essential for biosynthesis of virginiamycin S, a streptogramin type B antibiotic, as a provider of the nonproteinogenic amino acid phenylglycine.

A streptogramin type B antibiotic, virginiamycin S (VS), is produced by Streptomyces virginiae, together with a streptogramin type A antibiotic, virginiamycin M1 (VM), as its synergistic counterpart. VS is a cyclic hexadepsipeptide containing a nonproteinogenic amino acid, Lphenylglycine (L-pheGly), in its core structure. We have identified, in the left-hand extremity of the virginiamycin supercluster, two genes that direct VS biosynthesis with L-pheGly incorporation. Transcriptional analysis revealed that visF, encoding a nonribosomal peptide synthetase, and visG, encoding a protein with homology to a hydroxyphenylacetyl-CoA dioxygenase, are under the transcriptional regulation of virginiae butanolide (VB), a small diffusing signalling molecule that governs virginiamycin production. Gene deletion of visG resulted in complete loss of VS production without any changes in VM production, suggesting that visG is required for VS biosynthesis. The abolished VS production in the visG disruptant was fully recovered either by the external addition of pheGly or by gene complementation, which indicates that VisG is involved in VS biosynthesis as the provider of an L-pheGly molecule. A feeding experiment with L-pheGly analogues suggested that VisF, which is responsible for the last condensation step, has high substrate specificity toward L-pheGly.

Null mutation analysis of an afsA-family gene, barX, that is involved in biosynthesis of the {gamma}-butyrolactone autoregulator in Streptomyces virginiae.

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.

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

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.

Identification of the bkdAB gene cluster, a plausible source of the starter-unit for virginiamycin M production in Streptomyces virginiae.

The bkdAB gene cluster, which encodes plausible E1 and E2 components of the branched-chain alpha-keto acid dehydrogenase (BCDH) complex, was isolated from Streptomyces virginiae in the vicinity of a regulatory island for virginiamycin production. Gene disruption of bkdA completely abolished the production of virginiamycin M (a polyketide-peptide antibiotic), while the production of virginiamycin S (a cyclodepsipeptide antibiotic) was unaffected. Complementation of the bkdA disruptant by genome-integration of intact bkdA completely restored the virginiamycin M production, indicating that the bkdAB cluster is essential for virginiamycin M biosynthesis, plausibly via the provision of isobutyryl-CoA as a primer unit. In contrast to a feature usually seen in the Streptomyces E1 component, namely, the separate encoding of the alpha and beta subunits, S. virginiae bkdA seemed to encode the fused form of the alpha and beta subunits, which was verified by the actual catalytic activity of the fused protein in vitro using recombinant BkdA overexpressed in Escherichia coli. Supply of an additional bkdA gene under the strong and constitutive promoter ermE* in the wild-type strain of S. virginiae resulted in enhanced production of virginiamycin M, suggesting that the supply of isobutyryl-CoA is one of the rate-limiting factors in the biosynthesis of virginiamycin M.

Characterization of biosynthetic gene cluster for the production of virginiamycin M, a streptogramin type A antibiotic, in Streptomyces virginiae.

Virginiamycin M (VM) of Streptomyces virginiae is a hybrid polyketide-peptide antibiotic with peptide antibiotic virginiamycin S (VS) as its synergistic counterpart. VM and VS belong to the Streptogramin family, which is characterized by strong synergistic antibacterial activity, and their water-soluble derivatives are a new therapeutic option for combating vancomycin-resistant Gram-positive bacteria. Here, the VM biosynthetic gene cluster was isolated from S. virginiae in the 62-kb region located in the vicinity of the regulatory island for virginiamycin production. Sequence analysis revealed that the region consists of 19 complete open reading frames (ORFs) and one C-terminally truncated ORF, encoding hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS), typical PKS, enzymes synthesizing precursors for VM, transporters for resistance, regulatory proteins, and auxiliary enzymes. The involvement of the cloned gene cluster in VM biosynthesis was confirmed by gene disruption of virA encoding a hybrid PKS-NRPS megasynthetase, which resulted in complete loss of VM production without any effect on VS production. To assemble the VM core structure, VirA, VirF, VirG, and VirH consisting, as a whole, of 24 domains in 8 PKS modules and 7 domains in 2 NRPS modules were predicted to act as an acyltransferase (AT)-less hybrid PKS-NRPS, whereas VirB, VirC, VirD, and VirE are likely to be essential for the incorporation of the methyl group into the VM framework by a HMG-CoA synthase-based reaction. Among several uncommon features of gene organization in the VM gene cluster, the lack of AT domain in every PKS module and the presence of a discrete AT encoded by virI are notable. AT-overexpression by an additional copy of virI driven by ermEp() resulted in 1.5-fold increase of VM production, suggesting that the amount of VirI is partly limiting VM biosynthesis.

Phenotypic and genotypic characterization of mutants of the virginiamycin producing strain 899 and its relatedness to the type strain of Streptomyces virginiae.

A representative set of 19 mutants, with a known genealogy, of the virginiamycin producing strain Streptomyces virginiae 899 was investigated phenotypically and genotypically. Colour of the aerial and substrate mycelium were very variable both among spontaneous variants and those obtained after induced mutagenesis. At genotypic level, all mutants showed nearly identical BOX patterns, not reflecting the phenotypic heterogeneity observed. More than 40 years of forced mutational pressure did not cause huge chromosomal distortions but was most likely limited to base substitutions. The species S. virginiae, including besides producers of virginiamycin the type strain and non-type strains producing other bioactive compounds, is genomically heterogeneous on the basis of BOX-PCR fingerprinting and DNA-DNA hybridizations. The virginiamycin producing strain 899 does not belong to the species S. virginiae despite its phenotypic similarity to the latter.

Identification by gene deletion analysis of barB as a negative regulator controlling an early process of virginiamycin biosynthesis in Streptomyces virginiae.

The Streptomyces virginiae gamma-butyrolactone autoregulator virginiae butanolide is a low-molecular-weight Streptomyces hormone eliciting virginiamycin biosynthesis through its binding to the specific receptor protein, BarA. Immediately downstream of barA lies barB, the transcription of which is tightly repressed by BarA in the absence of virginiae butanolide and derepressed in its presence. Thus, BarB is next to BarA on the virginiae butanolide-BarA signaling cascade. An in-frame 279-bp deletion was introduced into the barB allele, which rendered it inactive by eliminating the majority of the coding region, including the helix-turn-helix DNA-binding motif. No significant change was observed with the Delta barB mutant with respect to the timing or amount of virginiae butanolide production, or the morphological differentiation on solid media, indicating that barB neither participates in virginiae butanolide biosynthesis nor in cytodifferentiation. In contrast, analysis of virginiamycin production in the Delta barB mutant revealed that production of both virginiamycin M(1) and virginiamycin S occurred immediately after virginiae butanolide production, 2-3 h earlier than in the wild-type strain, indicating that BarB participates in the temporal retardation of virginiamycin production after virginiae butanolide inactivates the repressor function of BarA. RT-PCR analysis of the transcription of several genes surrounding barA-barB by the Delta barB mutant indicated that BarB plays a negative regulatory role, directly or indirectly, in the transcription of barZ, vmsR, and orf5 located upstream of barB.

barS1, a gene for biosynthesis of a gamma-butyrolactone autoregulator, a microbial signaling molecule eliciting antibiotic production in Streptomyces species.

From Streptomyces virginiae, in which production of streptogramin antibiotic virginiamycin M(1) and S is tightly regulated by a low-molecular-weight Streptomyces hormone called virginiae butanolide (VB), which is a member of the gamma-butyrolactone autoregulators, the hormone biosynthetic gene (barS1) was cloned and characterized by heterologous expression in Escherichia coli and by gene disruption in S. virginiae. The barS1 gene (a 774-bp open reading frame encoding a 257-amino-acid protein [M(r), 27,095]) is situated in the 10-kb regulator island surrounding the VB-specific receptor gene, barA. The deduced BarS1 protein is weakly homologous to beta-ketoacyl-acyl carrier protein/coenzyme A reductase and belongs to the superfamily of short-chain alcohol dehydrogenase. The function of the BarS1 protein in VB biosynthesis was confirmed by BarS1-dependent in vitro conversion of 6-dehydro-VB-A to VB-A, the last catalytic step in VB biosynthesis. Of the four possible enantiomeric products from racemic 6-dehydro-VB-A as a substrate, only the natural enantiomer of (2R,3R,6S)-VB-A was produced by the purified recombinant BarS1 (rBarS1), indicating that rBarS1 is the stereospecific reductase recognizing (3R)-isomer as a substrate and reducing it stereospecifically to the (6S) product. In the DeltabarS1 mutant created by homologous recombination, the production of VB as well as the production of virginiamycin was lost. The production of virginiamycin by the DeltabarS1 mutant was fully recovered by the external addition of VB to the culture, which indicates that the barS1 gene is essential in the biosynthesis of the autoregulator VBs in S. virginiae and that the failure of virginiamycin production was a result of the loss of VB production.

Identification by heterologous expression and gene disruption of VisA as L-lysine 2-aminotransferase essential for virginiamycin S biosynthesis in Streptomyces virginiae.

The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35 degrees C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into Delta(1)-piperidine 2-carboxylic acid. A visA deletion mutant of S. virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation. However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M(1) at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine.

Characterization of virginiamycin S biosynthetic genes from Streptomyces virginiae.

Streptomyces virginiae produces -butyrolactone autoregulators (virginiae butanolide, VB), which control the biosynthesis of virginiamycin M1 and S. A 6.3-kb region downstream of the virginiamycin S (VS)-resistance operon in S. virginiae was sequenced, and four plausible open reading frames (ORFs) (visA, 1,260 bp; visB, 1,656 bp; visC, 888 bp; visD, 1209 bp) were identified. Homology analysis revealed significant similarities with enzymes involved in the biosynthesis of cyclopeptolide antibiotics: VisA (53% identity, 65% similarity) to -lysine 2-aminotransferase (NikC) of nikkomycin D biosynthesis, VisB (66% identity, 72% similarity) to 3-hydroxypicolinic acid:AMP ligase of pristinamycin I biosynthesis, VisC (48% identity, 59% similarity) to lysine cyclodeaminase of ascomycin biosynthesis, and VisD (43% identity, 56% similarity) to erythromycin C-22 hydroxylase of erythromycin biosynthesis. Northern blotting as well as high-resolution S1 analysis of the ORFs revealed that they were transcribed as two bicistronic transcripts, namely 3.0-kb visB-visA and another 2.7-kb visC-visD transcript, with promoters locating upstream of visB and visC, respectively. Transcription of the two operons was observed only 1 h after the VB production, which was 2 h before the virginiamycin production. Furthermore, prompt induction of the transcription was observed as a result of external VB addition, suggesting that the expression of the two operons was under the control of VB.

Intracellular pH determination of pristinamycin-producing Streptomyces pristinaespiralis by image analysis.

Intracellular pH (pH(i)) is an essential parameter in the regulation of intracellular processes. Thus, its measurement might provide clues regarding the physiological state of cells cultivated in vitro. pH(i) of the filamentous, pristinamycin-producing Streptomyces pristinaespiralis was determined by epifluorescence microscopy and image analysis using the pH-sensitive fluorescent probe BCECF-AM [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester]. Staining cell culture samples (OD(660)=1) of S. pristinaespiralis with 20 microM BCECF at 28 degrees C for 30 min yielded a green/red fluorescence ratio (R:(527/600)) that correlated with the pH(i) of the cells for values ranging from 6.5 to 8.5. When S. pristinaespiralis was cultivated in pristinamycin-producing conditions (in batch mode, with a constant external pH of 6.8), the measured pH(i) varied between 6.3 and 8.7. In fact, pH(i) correlated with the excretion of pristinamycins and glucose consumption during the production process.

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 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.

Ecological approach of macrolide-lincosamides-streptogramin producing actinomyces from Cuban soils.

We report in this study the frequency of Streptomyces strains to produce macrolide-lincosamide-streptogramin (MLS) antibiotics isolated from Cuban soils. The screening assay is based on the induction of MLS-resistance phenotype in a clinical isolated strain of Staphylococcus aureus S-18. Our results suggest that of 800 Streptomyces strains isolated from different soil samples, 6% were positives in the screening test used. The ferralitic red soil from Pinar del Río (north) provided the major percentage (3.6%) of MLS producing strains. The other soil samples tested belonging to Guira de Melena and Bauta in Havana, Matanzas City, Topes De Collantes (Villa Clara), and Soroa Mountains (Pinar del Rio) hill reached very low percentages.

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.

Optimization of agitation and aeration conditions for maximum virginiamycin production.

To maximize the productivity of virginiamycin, which is a commercially important antibiotic as an animal feed additive, an empirical approach was employed in the batch culture of Streptomyces virginiae. Here, the effects of dissolved oxygen (DO) concentration and agitation speed on the maximum cell concentration at the production phase, as well as on the productivity of virginiamycin, were investigated. To maintain the DO concentration in the fermentor at a certain level, either the agitation speed or the inlet oxygen concentration of the supply gas was manipulated. It was found that increasing the agitation speed had a positive effect on the antibiotic productivity independent of the DO concentration. The optimum DO concentration, agitation speed and addition of an autoregulator, virginiae butanolide C (VB-C), were determined to maximize virginiamycin productivity. The optimal strategy was to start the cultivation at 450 rpm and to continue until the DO concentration reached 80%. After reaching 80%, the DO concentration was maintained at this level by changing the agitation speed, up to a maximum of 800 rpm. The addition of an optimal amount of the autoregulator VB-C in an experiment resulted in the maximal production of virginiamycin M (399 mg/l), which was about 1.8-fold those obtained previously.

Gene replacement analysis of the Streptomyces virginiae barA gene encoding the butyrolactone autoregulator receptor reveals that BarA acts as a repressor in virginiamycin biosynthesis.

Virginiae butanolides (VBs), which are among the butyrolactone autoregulators of Streptomyces species, act as a primary signal in Streptomyces virginiae to trigger virginiamycin biosynthesis and possess a specific binding protein, BarA. To clarify the in vivo function of BarA in the VB-mediated signal pathway that leads to virginiamycin biosynthesis, two barA mutant strains (strains NH1 and NH2) were created by homologous recombination. In strain NH1, an internal 99-bp EcoT14I fragment of barA was deleted, resulting in an in-frame deletion of 33 amino acid residues, including the second helix of the probable helix-turn-helix DNA-binding motif. With the same growth rate as wild-type S. virginiae on both solid and liquid media, strain NH1 showed no apparent changes in its morphological behavior, indicating that the VB-BarA pathway does not participate in morphological control in S. virginiae. In contrast, virginiamycin production started 6 h earlier in strain NH1 than in the wild-type strain, demonstrating for the first time that BarA is actively engaged in the control of virginiamycin production and implying that BarA acts as a repressor in virginiamycin biosynthesis. In strain NH2, an internal EcoNI-SmaI fragment of barA was replaced with a divergently oriented neomycin resistance gene cassette, resulting in the C-terminally truncated BarA retaining the intact helix-turn-helix motif. In strain NH2 and in a plasmid-integrated strain containing both intact and mutated barA genes, virginiamycin production was abolished irrespective of the presence of VB, suggesting that the mutated BarA retaining the intact DNA-binding motif was dominant over the wild-type BarA. These results further support the hypothesis that BarA works as a repressor in virginiamycin production and suggests that the helix-turn-helix motif is essential to its function. In strain NH1, VB production was also abolished, thus indicating that BarA is a pleiotropic regulatory protein controlling not only virginiamycin production but also autoregulator biosynthesis.