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1 International Center for Biotechnology, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan
2 MU-OU Collaborative Research Center for Bioscience and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand
Correspondence
Takuya Nihira
nihira{at}icb.osaka-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary table of primers and two supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), although the details of the regulatory mechanism for this synchronized production remain unclear. In general, the genes involved in regulating the production of antibiotics exert their effects in a hierarchical manner at various levels to turn on antibiotic production, and these types of regulator appear to fall into two categories. The first type consists of higher-level regulators regarded as global regulators that transmit signals from the environment. Genes that encode the higher-level regulators are most commonly situated outside of biosynthetic gene clusters. The second type of regulator consists of lower-level regulators regarded as pathway-specific regulators that control the transcription of biosynthetic structural genes associated with the synthesis of individual antibiotics, and these genes are usually found within the respective biosynthetic gene cluster. In the case of Streptomyces coelicolor A3(2), the factors AbsB (Adamidis & Champness, 1992) and BldA (Lawlor et al., 1987) are typical higher-level regulators that sequentially control ActII-ORF4, RedD and CdaR, i.e. pathway-specific regulators that activate biosynthetic gene clusters for actinorhodin, undecylprodigiosin and calcium-dependent antibiotics, respectively (Bibb, 1996).
The biosynthesis of VM and VS in S. virginiae is initiated by the production of virginiae butanolide (VB), a small diffusible signalling molecule that binds to VB-specific receptor protein (BarA), a DNA-binding transcriptional repressor (Kinoshita et al., 1997). The flanking regions of barA contain other important genes that are involved in VB biosynthesis (barX, barS1 and barS2) and mechanisms of resistance to VM and VS (varM, varR and varS) (Kawachi et al., 2000a; Namwat et al., 2001) (Fig. 1). These genes are clustered in a 10 kb region, and thus are considered as the virginiamycin regulatory island. This 10 kb regulatory island includes one pathway-specific regulatory gene, vmsR, which is essential for both VM and VS production and is transcriptionally controlled by higher-level regulators such as barA, thus indicating that VmsR is a lower-level regulator for virginiamycin production in the VB-BarA regulatory system (Kawachi et al., 2000b). Recently, we characterized a 62 kb virginiamycin biosynthesis gene cluster in the flanking region of the virginiamycin regulatory island (Pulsawat et al., 2007). This gene cluster encodes six multi-functional enzymes that catalyse the formation of a hybrid peptide-polyketide and a cyclohexadepsipeptide skeleton, as well as 14 additional proteins that are responsible for the introduction of carboxylic acid and amino acid precursors into the skeleton, transporters for self-resistance, and two plausible regulators of gene expression. Expression of these genes is also under the control of higher-level factors such as BarA and BarX, both of which are involved in a number of cell functions, including virginiamycin production, VM biosynthesis and VM resistance.
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) has been demonstrated. The SARP-family proteins are known to be pathway-specific regulators that positively control the production of secondary metabolites. Meanwhile, VmsT was found to resemble the response-regulator proteins of a bacterial two-component signal transduction system able to detect and respond to changes in the environment (Hutchings et al., 2004). The phosphorylated response regulators possess the ability to positively or negatively regulate gene expression. These findings led us to investigate the potential roles played by vmsS and vmsT in the regulation of virginiamycin biosynthesis. In this study, we characterized the functions of vmsS and vmsT in the regulatory cascade of virginiamycin biosynthesis, and demonstrated that VmsS and VmsT are pathway-specific transcriptional activators of virginiamycin production. The present report also specifies the hierarchical correlation among three pathway-specific regulatory genes (vmsS, vmsT and vmsR) responsible for controlling the biosynthetic expression of virginiamycin. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. For routine cultivation on solid media, S. virginiae strains were grown at 28 °C on ISP medium 2 (Becton Dickinson). AS-1 medium, minimal medium with mannitol (Kieser et al., 2000) and ISP medium 2 were used for observing the morphological development of gene disruptants. For virginiamycin production, liquid f-medium was used as described previously (Nihira et al., 1988). For routine cloning and conjugation, Escherichia coli strains were grown on LB medium with the appropriate antibiotics, when necessary. Plasmid DNA was transferred from E. coli into Streptomyces by intergenic conjugation as previously described (Kitani et al., 2000). PCR was performed using high-fidelity PrimeSTAR HS DNA polymerase (Takara Bio) with genomic DNA of S. virginiae as a template, according to the manufacturer's recommendations. Southern blot hybridization was carried out using the AlkPhos Direct Labelling and Detection System with CDP-Star (GE Healthcare Bio-Sciences). For DNA manipulation in E. coli and Streptomyces, standard procedures were performed according to the methods of Sambrook & Russell (2001) and Kieser et al. (2000), respectively.
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(i) vmsS deletion.
A 1.8 kb vmsS-upstream DNA fragment was amplified by the primer pair vmsSA-E/vmsSA-S, and digested by EcoRI and SpeI. Similarly, a 1.8 kb vmsS-downstream DNA fragment was amplified by the primer pair vmsSB-S/vmsSB-H, and digested by SpeI and HindIII. The two resultant fragments were cloned together into the EcoRI and HindIII sites of pUC19 and were recovered as a 3.7 kb EcoRI/HindIII fragment, which was then transferred into pKC1132 at the EcoRI and HindIII sites, thereby yielding the vmsS-disruption plasmid pLT202.
(ii) vmsT deletion.
A 2.1 kb vmsT-upstream DNA fragment was amplified by the primer pair vmsTA-X/vmsTA-K and was digested by XbaI and KpnI. Similarly, a 2.2 kb vmsT-downstream DNA fragment was amplified by the primer pair vmsTB-K/vmsTB-H and was digested by KpnI and HindIII. The two resultant fragments were cloned together into the XbaI and HindII sites of pUC19 and were recovered as a 4.3 kb XbaI/HindIII fragment, which was then transferred into pKC1132 at the XbaI and HindIII sites, thereby yielding the vmsT-disruption plasmid pLT203.
(iii) vmsR disruption.
A 2.0 kb vmsR-upstream DNA fragment was amplified by the primer pair vmsRA-H/vmsRA-S and was digested by HindIII and SpeI, while a 2.1 kb vmsR-downstream DNA fragment was amplified by the primer pair vmsRB-S/vmsRB-X and was digested by SpeI and XbaI. The two resultant fragments were cloned together into the HindIII and XbaI sites of pUC19 and were recovered as a 4.1 kb HindIII/XbaI fragment, which was then transferred into pKC1132 at the HindIII and XbaI sites, thereby yielding the vmsR-disruption plasmid pLT204.
Each of the disruption plasmids was introduced into S. virginiae via conjugation, and the wild-type gene was replaced with the disrupted allele by homologous recombination. The genotype of the corresponding mutants was confirmed by PCR and Southern blot hybridization (Supplementary Fig. S1), and representatives of the vmsS, vmsT and vmsR mutants were designated S. virginiae strains IC102, IC103 and IC104, respectively.
Complementation of the S. virginiae mutant strains.
A 1.0 kb vmsS gene was PCR-amplified by the primer pair vmsS-N2/vmsS-C2, and inserted into the EcoRV site of pBluescript II SK. The resulting plasmid was digested by BamHI, and was then cloned into the BamHI site of pLT101, yielding the vmsS-complementing plasmid pLT205 to place vmsS under the control of ermEp*. A 1.5 kb DNA fragment containing the entire vmsT gene and the vmsT-upstream region was PCR-amplified by the primer pair vmsT-CF/vmsT-CR, digested by EcoRV and XbaI, and cloned into the EcoRV and XbaI sites of pSET152 to obtain pLT206. A 1.3 kb DNA fragment containing the entire vmsR gene and the vmsR-upstream region was PCR-amplified by the primer pair vmsR-CF/vmsR-CF, digested by EcoRV and BamHI, and inserted into the EcoRV and BamHI sites of pSET152 to obtain pLT207. The fidelity of the amplified fragments was confirmed by DNA sequencing. By intergenic conjugation, pLT205, pLT206 and pLT207 were introduced into S. virginiae strains IC102 (
vmsS), IC103 (vmsT) and IC104 (vmsR), respectively. The correct integration in the exconjugants was confirmed by PCR analysis and Southern blot hybridization to yield the vmsS-, vmsT- and vmsR-complemented strains IC105, IC106 and IC107, respectively.
Constitutive expression of vmsS in the mutant IC104.
By intergenic conjugation, pLT205 was introduced into S. virginiae strain IC104 (vmsR). The correct integration in the exconjugants was confirmed by PCR analysis to yield a strain in which vmsS is constitutively expressed under the genetic background of vmsR; this strain was designated S. virginiae strain IC108.
Transcriptional analysis by RT-PCR.
RT-PCR was conducted as described by Pulsawat et al. (2007). Total RNAs were extracted from fresh mycelia harvested after 8, 10, 12 and 14 h cultivation for the wild-type strain, after 8 and 14 h cultivation for strains IC102, IC103 and IC104, and after 14 h cultivation for strain IC108. The cDNAs were amplified from 34 gene transcripts in the virginiamycin biosynthetic gene cluster using the previously described primers. The primers used for the co-transcription analysis of virN-virM-vmsS genes were virN-F, virM-R and vmsS-R as previously described (Pulsawat et al., 2007), and vmsS-F1, vmsS-F2 and vmsS-F3 as listed in Table S1. For PCR, the amplification conditions were a single round of denaturation at 94 °C for 5 min and 25 cycles of denaturation (98 °C for 10 s), annealing (58 °C for 5 s) and extension (72 °C for 1 min), followed by a single extension at 72 °C for 7 min.
Detection of virginiamycin production.
VM and VS in the culture broth were detected by a bioassay against Bacillus subtilis PCI219 (Yanagimoto, 1983) and reverse-phase C18-HPLC analysis as described by Pulsawat et al. (2007).
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RESULTS |
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) clearly demonstrated the presence of vmsR mRNA after 12 h and 14 h of cultivation, whereas vmsS mRNA was detected only after 14 h of cultivation, when all of the virginiamycin biosynthetic genes were actively transcribed along with the maximal virginiamycin production. This result suggested that the transcription of vmsS is preceded by that of vmsR. As regards gene organization, vmsS runs in the same direction as the upstream virM, which encodes a homologue of sarcosine oxidase, and virN encodes flavin-dependent oxidoreductase, both of which seem to participate in the post-modification steps of virginiamycin biosynthesis (Pulsawat et al., 2007). Because virN and virM overlap in a 47 bp region, and there is only a narrow (50 bp) intergenic region between virM and vmsS, vmsS may form a tricistronic operon with virN and virM. To examine this possibility, primers were designed to detect the tricistronic (virN-virM-vmsS) as well as bicistronic transcripts (virN-virM and virM-vmsS) (Fig. 2b). RT-PCR analysis using RNA from the 14 h mycelia gave the expected band for each primer set, implying that vmsS forms a tricistronic operon, virN-virM-vmsS.
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) (O'Connor & Nodwell, 2005) although the DNA-binding domain is significantly conserved. Furthermore, the N-terminus of VmsT is approximately 70 amino acids longer than that of other response regulators. These results, taken together, suggest that VmsT might exert different regulatory functions other than those typically carried out by response regulators. Similar to the case of the vmsS transcript, the vmsT transcript was barely detectable until 14 h cultivation (Fig. 2a
), indicating that the active expression of vmsT occurred only at the stage of virginiamycin production.
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). The culture filtrate of the wild-type strain showed an inhibition zone of virginiamycin, whereas no clear zone was observed in the case of the culture filtrate of mutant IC102. On the other hand, a culture filtrate of the mutant IC103 exhibited an inhibition zone, albeit smaller than that of the wild-type strain. Because this phenomenon might have been due to the composition or change in the quantity of virginiamycin, HPLC analysis was performed in order to investigate in more detail virginiamycin production in mutants IC102 and IC103. As shown in Fig. 4(b), both VM and VS peaks were missing in the culture filtrate of the mutant IC102, whereas only the VS peak was present in the culture filtrate of the mutant IC103, indicating that VmsS is necessary for both VM and VS biosynthesis, but VmsT is only necessary for VM biosynthesis.
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), thus confirming that VmsS is indeed involved in both VM and VS biosynthesis. Meanwhile, the clear inhibition zone formation by mutant IC103 harbouring pLT206, which contains the entire vmsT gene and the upstream region, was greater than that of the parent strain (mutant IC103), and was identical to that of the wild-type strain (Fig. 4a). HPLC analysis revealed that the reintroduction of vmsT into the vmsT mutant restored VM production (Fig. 4b), whereas VS production was retained in the mutant IC106 (Table 1); these results clearly indicated that vmsT is essential only for VM biosynthesis.
Gene expression analysis in vmsS- and vmsT-disrupted strains
To examine whether VmsS is involved in regulation at the transcriptional level, the total RNA was extracted from the mycelia of the mutant IC102 after 8 h and 14 h cultivation, i.e. the time points before and after the onset of virginiamycin production in the wild-type strain. Semiquantitative RT-PCR analysis of the transcripts of the virginiamycin biosynthetic gene cluster (Fig. 5a) indicated that, at 8 h of cultivation, all of the virginiamycin biosynthetic genes in the mutant IC102 were transcribed at a low level; these results were similar to those observed with the wild-type strain (Fig. 2a). Moreover, no differences were observed between the mutant IC102 and the wild-type strain in terms of the expression profiles of genes in the regulatory island (e.g. barA and varR). After 14 h cultivation in the case of the mutant IC102, eight VM structural genes (virD to virK) showed markedly reduced transcription compared to that of other VM structural genes such as virA and virB. In addition, no transcripts of the three VS structural genes (visB, visC and visD) (Namwat et al., 2002) were detected in the case of mutant IC102, which would account for the lack of VS production in mutant IC102. These results indicated that vmsS regulates, either directly or indirectly, the expression of VM and VS structural genes without affecting the transcription level of genes in the regulatory island.
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) were similar to those of the corresponding genes in the wild-type strain. Surprisingly, after 14 h cultivation, all 15 genes designated as VM biosynthetic genes (bkdA, bkdB and virA to virN) were transcribed in a manner similar that of the corresponding genes in the wild-type strain, albeit with deficient VM production in the case of mutant IC103. This result indicated that the inactivation of vmsT does not exert a major influence on the transcription of vir genes in the 77 kb region; moreover, these findings suggested the possibility that vmsT may regulate unidentified gene(s) outside of this 77 kb region. In addition, the amount of transcripts associated with VS biosynthesis (visA to visD) remained unaffected by vmsT mutation, which is a finding consistent with observed VS production in mutant IC103 after a 14 h cultivation period. To investigate whether an autoregulatory mechanism operates on vmsS or vmsT, transcriptional analysis was performed with primer sets designed to detect transcript of the region downstream of the mutation (Supplementary Fig. S2). Although some reduction occurred, especially for vmsT, transcription of vmsS or vmsT was observed in the corresponding mutant, indicating that neither of the genes required intact protein product for their transcription.
Transcription of vmsS and vmsT is under the control of vmsR, a SARP-family gene
Neither the vmsS nor the vmsT mutation affected the expression of vmsR, which is an activator for the control of virginiamycin production. This finding suggested the possibility that vmsR regulates virginiamycin production by controlling the expression of vmsS and/or vmsT genes. To examine this possibility, we investigated the transcription profile of a vmsR-disrupted strain. As the previously created vmsR-disrupted strain (DR1 strain) contained a deletion of a 145 bp 5'-untranslated region (UTR) in addition to the deletion of a 698 bp region of vmsR (Kawachi et al., 2000b), we reconstructed an in-frame deletion mutant for vmsR (mutant IC104), as described in Methods, to completely avoid any indirect effects due to the deletion of the 5'-UTR. The behaviour of mutant IC104 was similar to that of the DR1 strain: it did not produce any virginiamycin throughout the cultivation period. Transcriptional analysis to examine the possibility of autoregulation by vmsR suggested that vmsR, similar to the cases of vmsS and vmsT, does not require intact VmsR for its expression (Fig. S2). At 14 h cultivation, only scant amounts of vmsS and vmsT transcripts were detected in the case of mutant IC104 (Fig. 5b), in clear contrast to the results obtained with the wild-type strain (Fig. 2a). These findings suggested that vmsR upregulates the expression of both vmsS and vmsT. In addition, similar to the case of the vmsS mutant (IC102), a remarkable decrease in the amounts of the transcripts of three VS structural genes (visB to visD) was observed, implying that the expression of these three genes is controlled by vmsR via vmsS. Moreover, it was of note that the levels of transcripts of four other genes (visE and visF as VS structural genes, and virM and virN with putative involvement in VM biosynthesis) decreased dramatically, in clear contrast to the unaltered transcriptional profiles of these four genes among mutants IC102, IC103 and the wild-type strain. To further examine which genes are controlled by vmsS, we constructed mutant IC108, in which ermEp*-driven vmsS was integrated in the genome of the vmsR mutant (strain IC104), and analysed the transcriptional profile of the virginiamycin biosynthetic genes. As shown in Fig. 5(c), six VS biosynthetic genes (visA to visF) that showed reduced (visA) or missing (visB to visF) transcription in the vmsR mutant became clearly transcribed by the introduction of vmsS, which was considered to be transcribed constitutively in the mutant IC108, but the amount of the vmsT transcript was not affected. These results suggested that these VS biosynthetic genes are plausible direct targets of VmsS (see Discussion). Intriguingly, transcription of five VM biosynthetic genes (bkdA/bkdB and virA/virB/virC) was not influenced by the vms mutations. This result suggested the involvement of another regulator for their expression, which is coordinated with expression of other virginiamycin biosynthetic genes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). VmsS is among the SARP-family proteins that are frequently present in biosynthetic gene clusters of Streptomyces origin and that regulate the production of corresponding secondary metabolites. VmsT resembles response regulators of two-component systems, but belongs to a minor group of response regulators that lack the conserved amino acid residues necessary for phosphorylation and do not possess a neighbouring cognate sensor kinase gene. Gene disruption analysis clearly demonstrated that VmsS is necessary for the production of both VM and VS, whereas VmsT is only necessary for the production of VM. These findings, taken together with demonstration of the presence of another SARP-family gene, vmsR, in the right-hand region of the virginiamycin biosynthetic gene cluster and its involvement in the production of both VM and VS, suggest that the three regulators form a hierarchical network to ensure the synchronized production of virginiamycin.
The loss of vmsT expression in the vmsR disruptant indicated that vmsT is under the control of VmsR. Surprisingly, no transcriptional changes were observed in the vmsT disruptant among the 33 genes in the 77 kb region examined, although VM production was lost completely; these observations suggested that VmsT controls VM biosynthetic gene(s) located outside the 77 kb region of interest, and these genes might be associated with the last condensation step of VM biosynthesis (proline condensation), as the only missing module for VM biosynthesis in this 77 kb region is that for proline condensation (Pulsawat et al., 2007). Defective VM production in the vmsR disruptant appears to have been primarily due to the lack of vmsT transcription, because almost all of the VM structural genes in the 77 kb region, with the exception of virN and virM, showed similar levels of expression among the vmsR and vmsT disruptants, and the wild-type strain. The reduced expression of virN and virM, encoding putative post-polyketide modification enzymes for VM biosynthesis, in the vmsR disruptant may additionally have been responsible for the loss of VM production in the vmsR disruptant.
To date, only a few response regulators, referred to as ‘pseudo-response regulators’, have been examined in terms of their regulatory functions in the control of secondary metabolism; these regulators possess DNA-binding ability for the regulation of gene expression, but are incapable of phosphorylation. Representative proteins are RedZ of the undecylprodigiosin pathway from S. coelicolor A3(2) (Guthrie et al., 1998) and DnrN of the daunorubicin pathway from S. peucetius (Furuya & Hutchinson, 1996). RedZ lacks an Asp residue for phosphorylation, a Tyr residue as a rotameric residue, and a Lys residue for formation of the ‘acid pocket’ binding site. Similarly, DnrN lacks Asp and Thr residues, which are key amino acid residues for phosphorylation. In both cases, the pseudo-response regulators are upper-level regulators in the signalling cascade, and they activate the transcription of the SARP-family genes redD and dnrI, which in turn directly activate the transcription of biosynthetic genes for undecylprodigiosin and daunorubicin, respectively. However, in the case of virginiamycin biosynthesis, the SARP-family gene vmsR controls the expression of vmsT, the pseudo-response regulator gene for VM production in S. virginiae. To the best of our knowledge, this hierarchical direction from SARP-family protein to pseudo-response regulator is the first case of such directionality in the Streptomyces regulatory cascade of secondary metabolism.
In the vmsR disruptant, vmsS expression was severely reduced, although the basal level expression of vmsS appeared to be maintained in the absence of vmsR (Fig. 5b). These findings were in clear contrast to the requirement of vmsR for the expression of vmsT. In the vmsS disruptant, defective in terms of both VM and VS biosynthesis, levels of transcription of eight VM biosynthetic genes (virD to virK) were markedly lower than in the wild-type strain, and the transcription of three VS biosynthetic genes (visB, visC and visD) was abolished, suggesting that VmsS acts as a major transcriptional activator of these 11 genes. Similar loss of visB/visC/visD transcription was observed in the vmsR disruptant, which may indicate that the baseline level of VmsS production is insufficient to activate the expression of these three genes. The SARP-family proteins activate the transcription of their target genes by binding to heptameric direct repeats in the cognate promoters (Arias et al., 1999; Sheldon et al., 2002; Tanaka et al., 2007). As regards the target sequence of VmsS for the expression of visB/visC/visD, it was of note that two direct repeats composed of a heptameric sequence (GTGTCAG or GTGTCAA, with intervals of 4 bp) were present in the –35 region of visC, and an identical sequence (GTGTCAA) was present around the –35 region of visB. Because these genes formed two bicistronic operons in the opposite direction (i.e. the visB-visA operon and the visC-visD operon), the intergenic region between visB and visC is the most plausible candidate to serve as the VmsS target. The constitutive expression of vmsS in the vmsR-disruptant conferred the increased transcription of six VS structural genes, including visA, visB, visC and visD. This result strongly supported that the transcription of the four VS biosynthetic genes (visA to visD) is directly activated by VmsS, expression of which is under the control of VmsR, although in vitro experiments will still be needed to clarify the regulatory mechanism.
On the other hand, the loss of VM production in the vmsS disruptant could be attributed to the reduced levels of transcription of eight VM biosynthetic genes (virD to virK). Although vmsS transcription was found to be under the control of VmsR, no reduction in the transcription of eight VM biosynthetic genes was observed in the vmsR disruptant, suggesting that the basal level of expression of vmsS is sufficient to maintain the expression of the eight VM biosynthetic genes, and the loss of VM production in the vmsR-disruptant could be attributed primarily to the loss of VmsT (see discussion above).
The results of the present extensive transcriptional analysis strongly suggested that vmsR has two signalling pathways for the regulation of virginiamycin production, namely (i) a vmsS/vmsT-dependent pathway, and (ii) a vmsS/vmsT-independent pathway. Initiated by VB production starting at 10–11 h cultivation, the vmsR/barZ bicistronic transcript became readily detected at 12 h cultivation. At 14 h, most genes in the biosynthetic cluster, including vmsS and vmsT, were expressed to yield virginiamycin, while the transcription of barS2 and barX appears to have slightly decreased by a mechanism or physiological factor that remains unknown at the present time. The disruption of either vmsS or vmsT did not appear to affect the expression of vmsR or genes in the regulatory island, suggesting that vmsS and vmsT are positioned downstream of vmsR in the signalling cascade. This possibility was supported by the finding that vmsS transcription was severely reduced and vmsT transcription abolished in the vmsR disruptant. Thus, vmsR appears to act as the major transcriptional activator of the two regulatory genes, although further investigation will be needed to clarify whether the mechanism is direct or indirect. No heptameric direct repeats were found in the region immediately upstream of vmsS, although such repeats are frequently recognized by SARP-family proteins. However, in the vmsT-virN intergenic region, which would be expected to contain the promoter(s) responsible for controlling the expression of vmsT and the divergently transcribed virN-virM-vmsS tricistronic operon, at least three kinds of direct repeat were found, suggesting that VmsR might regulate the expression of vmsS and vmsT in a coordinated manner via binding to these direct repeats.
In addition to its involvement in the vmsS/vmsT-dependent pathway for the regulation of virginiamycin production, VmsR appears to regulate the expression of genes (e.g. visE, visF, virN and virM) by a pathway that differs from the vmsS/vmsT-dependent pathway, as a loss of transcription of visE and visF, together with a loss of virN and virM (as part of the putative tricistronic virN-virM-vmsS operon) transcription, was observed in the vmsR disruptant, whereas levels of transcription of these genes in the vmsS and vmsT disruptants were similar to those of the wild-type strain. However, as shown in mutant IC108, when constitutively expressing vmsS was introduced into the vmsR disruptant, visE and visF were transcribed at levels higher than those of the parent strain, but the amounts of the transcripts of vmsT, virN and virM were unchanged, indicating that the expression of the three genes (vmsT, virN and virM) was dominated by a vmsS-independent pathway, most likely by vmsR itself. Regarding the transcription of visE and visF, combined data indicated that either VmsR or a sufficient amount of VmsS could drive their transcription. One possibility is that VmsR drives the transcription of a vmsT-visE-visF tricistronic operon (together with the transcription of the virN-virM-vmsS operon in the opposite direction), while VmsS might drive that of a visE-visF bicistronic operon in the absence of intact VmsR, which should be clarified in the future by detailed in vitro analysis. Detailed transcriptional analysis of these three genes would provide information on vmsR- and vmsS-regulatory mechanisms of virginiamycin production.
In general, the biosynthetic gene cluster for a secondary metabolite in streptomycetes possesses either one or two pathway-specific regulators for controlling production. It is of note that three pathway-specific regulators were found to be present in the virginiamycin biosynthetic gene cluster, which is a compressed-interspersing gene cluster (i.e. a so-called ‘supercluster’). These regulators are responsible for the coordinated synthesis of two structurally different compounds of the streptogramin family. In our previous study (Pulsawat et al., 2007), the expression of vmsR, and hence that of vmsS and vmsT, was shown to be under the control of BarA, which is a pivotal regulator for sensing the presence of VB in the environment. These previous observations, taken together with those of the present study, indicate that vms regulatory genes are lower-level regulators in the regulatory cascade of the VB-BarA system responsible for controlling virginiamycin production.
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ACKNOWLEDGEMENTS |
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Edited by: Mark S. Paget
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Received 22 July 2008;
revised 4 January 2009;
accepted 5 January 2009.
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. The numbers indicate the amino acid positions within each sequence. Black boxes indicate positions in the alignment at which the same amino acid is found in at least four of the seven sequences. RedZ (CAA69209), AbsA2 (AAB08053), ChiR (CAB94548) and RamR (NP_733711) are from Streptomyces coelicolor A3(2); CinR (CAD60529) is from Streptomyces cinnamoneus; DnrN (AAD15247) is from Streptomyces peucetius. RedZ is also a member of the pseudo-response regulators, which are not regulated by phosphorylation.
