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Auxin/Indole-3-Acetic Acid (Aux/IAA) genes are involved in auxin signaling pathway and play an important role in plant growth and development. However, many studies focus on Aux/IAA gene families and much less known in Bletilla striata. In this study, a total of 27 Aux/IAA genes (BsIAA1-27) were cloned from the transcriptome of Bletilla striata. Based on a phylogenetic analysis of the Aux/IAA protein sequences from B. striata, Arabidopsis thaliana and Dendrobium officinale, the Aux/IAA genes of B. striata (BsIAAs) were categorized into 2 subfamilies and 9 groups. While BsIAAs were more closer to those of D. officinale compared to A. thaliana. EST-SSR marker mining test showed that 4 markers could be stably amplified with obvious polymorphisms among 4 landraces. Our results suggested that BsIAAs were involved in the process of tuber development and provided insights into functional roles of Aux/IAA genes in B. striata and other plants., Graphical Abstract In this paper, we identified the 27 members of the Aux/IAA gene family which can be divided into 2 groups and 9 subfamilies. The similar branches of Aux/IAA proteins had the same or similar motifs, such as genes between BsIAA10 and BsIAA12, BsIAA11 and BsIAA13, BsIAA18 and BsIAA20, indicating that Aux/IAA proteins were conserved. Among them, BsIAAs were more closer to those of D. officinale compared to A. thaliana.

In Streptomyces, antibiotic biosynthesis is triggered in phosphate limitation that is usually correlated with energetic stress. Polyphosphates constitute an important reservoir of phosphate and energy and a better understanding of their role in the regulation of antibiotic biosynthesis is of crucial importance. We previously characterized a gene, SLI_4384/ppk, encoding a polyphosphate kinase, whose disruption greatly enhanced the weak antibiotic production of Streptomyces lividans. In the condition of energetic stress, Ppk utilizes polyP as phosphate and energy donor, to generate ATP from ADP. In this paper, we established that ppk is co-transcribed with its two downstream genes, SLI_4383, encoding a phosin called PptA possessing a CHAD domain constituting a polyphosphate binding module and SLI_4382 encoding a nudix hydrolase. The expression of the ppk/pptA/SLI_4382 operon was shown to be under the positive control of the two-component system PhoR/PhoP and thus mainly expressed in condition of phosphate limitation. However, pptA and SLI_4382 can also be transcribed alone from their own promoter. The deletion of pptA resulted into earlier and stronger actinorhodin production and lower lipid content than the disruption of ppk, whereas the deletion of SLI_4382 had no obvious phenotypical consequences. The disruption of ppk was shown to have a polar effect on the expression of pptA, suggesting that the phenotype of the ppk mutant might be linked, at least in part, to the weak expression of pptA in this strain. Interestingly, the expression of phoR/phoP and that of the genes of the pho regulon involved in phosphate supply or saving were strongly up-regulated in pptA and ppk mutants, revealing that both mutants suffer from phosphate stress. Considering the presence of a polyphosphate binding module in PptA, but absence of similarities between PptA and known exo-polyphosphatases, we proposed that PptA constitutes an accessory factor for exopolyphosphatases or general phosphatases involved in the degradation of polyphosphates into phosphate.

BackgroundBletilla striata has been widely used in the pharmacology industry. To effectively produce the secondary metabolites through suspension cultured cells of B. striata, it is important to exploring the full-length transcriptome data and the genes related to cell growth and chemical producing of all culture stages. We applied a combination of Real-Time Sequencing of Single Molecule (SMRT) and second-generation sequencing (SGS) to generate the complete and full-length transcriptome of B. striata suspension cultured cells.MethodsThe B. striata transcriptome was formed in de novo way by using PacBio isoform sequencing (Iso-Seq) on a pooled RNA sample derived from 23 samples of 10 culture stages, to explore the potential for capturing full-length transcript isoforms. All unigenes were obtained after splicing, assembling, and clustering, and corrected by the SGS results. The obtained unigenes were compared with the databases, and the functions were annotated and classified.Results and conclusionsA total of 100,276 high-quality full-length transcripts were obtained, with an average length of 2530 bp and an N50 of 3302 bp. About 52% of total sequences were annotated against the Gene Ontology, 53,316 unigenes were hit by KOG annotations and divided into 26 functional categories, 80,020 unigenes were mapped by KEGG annotations and clustered into 363 pathways. Furthermore, 15,133 long-chain non-coding RNAs (lncRNAs) were detected. And 68,996 coding sequences were identified based on SSR analysis, among which 31 pairs of primers selected at random were amplified and obtained stable bands. In conclusion, our results provide new full-length transcriptome data and genetic resources for identifying growth and metabolism-related genes, which provide a solid foundation for further research on its growth regulation mechanisms and genetic engineering breeding mechanisms of B. striata.

International audience; In condition of over-expression, SCO3201, a regulator of the TetR family was previously shown to strongly inhibit antibiotic production and morphological differentiation in Streptomyces coelicolor M145. In order to elucidate the molecular processes underlying this interesting, but poorly understood phenomenon, a comparative analysis of the lipidomes and transcriptomes of the strain over-expressing sco3201 and of the control strain containing the empty plasmid, was carried out. This study revealed that the strain over-expressing sco3201 had a higher triacylglycerol content and a lower phospholipids content than the control strain. This was correlated with up- and down- regulation of some genes involved in fatty acids biosynthesis (fab) and degradation (fad) respectively, indicating a direct or indirect control of the expression of these genes by SCO3201. In some instances, indirect control might involve TetR regulators, whose encoding genes present in close vicinity of genes involved in lipid metabolism, were shown to be differentially expressed in the two strains. Direct interaction of purified His6-SCO3201 with the promoter regions of four of such TetR regulators encoding genes (sco0116, sco0430, sco4167, and sco6792) was demonstrated. Furthermore, fasR (sco2386), encoding the activator of the main fatty acid biosynthetic operon, sco2386-sco2390, has been shown to be an illegitimate positive regulatory target of SCO3201. Altogether our data demonstrated that the sco3201 over-expressing strain accumulates TAG and suggested that degradation of fatty acids was reduced in this strain. This is expected to result into a reduced acetyl-CoA availability that would impair antibiotic biosynthesis either directly or indirectly.

To better explore the application potential of heat shock protein Hsp70s in diverse areas including biomonitoring, a further investigation of the details of the regulatory mechanism governing Hsp70 transcription is required. A transcriptional factor ChGATA-4 that displayed affinity to the ChHsp70 promoter of Crassostrea hongkongensis was isolated and identified by DNA affinity purification as well as mass spectrometry analysis. The ChGATA-4 cDNA is 2162 bp in length and the open reading frame encodes a polypeptide containing 482 amino acids with a conserved zinc finger domain. The over-expression of ChGATA-4 significantly inhibited the expression of ChHsp70 promoter in heterologous HEK293T cells. However, the depletion of ChGATA-4 mRNA by RNAi technique resulted in significant increase of ChHsp70 transcription in oyster hemocytes. The RT-PCR results demonstrated that the transcription of both ChHsp70 and ChGATA-4 were induced by heat, Cd, or NP (Nonyl phenol) stress. This suggested a potential correlation between ChHsp70 and ChGATA-4 in the stress-mediated genetic regulatory cascade. This study demonstrated that ChGATA-4 acts in a negative manner in controlling ChHsp70 transcription in C. hongkongensis and promotes to further understand the mechanisms leading Hsp70 transcription.

A temporary immersion bioreactor system (TIBS) has been explored as a possible way for Bletilla striata pseudobulbs enlargement and accumulation of polysaccharides under a fed-batch culture method. The culture was established in TIBS to optimize feeding parameters for polysaccharides accumulation and pseudobulb enlargement under controlled conditions, included 1/2 MS, 40 g/L sucrose; 60 g/L potato lixivium; 0.5 mg/L NAA and an immersion rate of 3 min at every 6-hour interval. Results showed that the combined feeding and elicitation strategy (2% sucrose and 0.25 mmol/L Me-JA) was suitable for the enlargement of pseudobulbs (diameter: 6.78 ± 0.16 mm) and the biosynthesis of polysaccharides (14.49 ± 0.45 mg/g). The optimum plant growth (plant height: 123.97 ± 5.42 mm, leaf width: 7.78 ± 0.32 mm and stem diameter: 2.10 ± 0.07 mm) was obtained by the inoculation of nitrogen feed source of NO3−: NH4+ with a 3:1 ratio. At this stage, the corresponding B. striata pseudobulb enlargement and polysaccharide content were decreased to 6.1 ± 0.2 mm and 8.51 ± 0.1 mg/g, respectively, while the highest pseudobulb enlargement (7.21 ± 0.17 mm) and polysaccharide content (12.94 ± 0.35 mg/g) were optimized when inoculated the NO3−: NH4+ with molar ratio of a 2:2. In conclusion, the optimal carbon and nitrogen source supplements effectively induced the pseudobulb enlargement and polysaccharides accumulation. Overall, the present combination of TIBS and feeding strategy constitutes advancement for the industrial production of pseudobulbs as well as polysaccharides, as comparison with the conventional process.

Gnaq, one of Guanine nucleotide-binding protein α subunits, was isolated from cellular nucleus extracts of oyster Crassostrea hongkongensis gills with biotin-labeled ChHsc70 promoter by means of DNA-affinity purification, and preliminarily identified with mass spectrometry analysis. ChGnaq mRNA depletion by RNAi technique led to clear reduction in ChHsc70 mRNA expression of C. hongkongensis hemocytes. Correspondently, ChGnaq over-expression in heterologous HEK293T cells correlated with elevated expression activation of ChHsc70 promoter. Quantitative real time PCR analysis showed that both ChHsc70 and ChGnaq transcriptions were responsive to external physical/chemical stresses by heat, CdCl2 and NP. This suggested a plausible association between ChHsc70 and ChGnaq in the stress-induced genetic regulatory pathway. This study discovered a positively regulatory role of ChGnaq in controlling ChHsc70 transcription of C. hongkongensis, and conduced to a better understanding of the regulatory mechanisms in control of Hsc70 transcription.

Bletilla striata is a plant from the Orchidaceae family that has been employed as a traditional Chinese medicine (TCM) for thousands of years in China. Here, we briefly review the published studies of the last 30 years that were related to chemical constituents, pharmacologic activities, and clinical applications of B. striata. Approximately 158 compounds have been extracted from B. striata tubers with clarified molecular structures that were classified as glucosides, bibenzyls, phenanthrenes, quinones, biphenanthrenes, dihydrophenanthrenes, anthocyanins, steroids, triterpenoids, and phenolic acids. These chemicals support the pharmacological properties of hemostasis and wound healing, and also exhibit anti-oxidation, anti-cancer, anti-viral, and anti-bacterial activities. Additionally, various clinical trials conducted on B. striata have demonstrated its marked activities as an embolizing and mucosa-protective agent, and its application for use in novel biomaterials, quality control, and toxicology. It also has been widely used as a constituent of many preparations in TCM formulations, but because there are insufficient studies on its clinical properties, its efficacy and safety cannot be established from a scientific point of view. We hope that this review will provide reference for further research and development of this unique plant.

HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed by EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl2 stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70. This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression.

A 717-bp 5′-flanking regulatory sequence of the metallothionein gene in Crassostrea hongkongensis (ChMT) was isolated using anchored polymerase chain reaction. Several regulatory elements embedded in the flanking sequence, including the central promoter region, five putative metal-responsive elements (MRE), and an octamer-binding site (OBS), were analyzed using online prediction software. Then, the effects of element-based promoter truncations in the upstream regulatory region of ChMT on the metal stress response were examined. The expression levels of the promoters of various lengths were determined individually using a luciferase reporter gene in HEK293T cells. Transfection assays revealed that MRE1- and MRE5-containing DNA fragments inhibited transcription, whereas MRE4-containing DNA sequences activated promoter expression under both metal-stressed and unstressed conditions. A 39-bp DNA fragment containing the OBS was shown to respond positively to metal stress induced by cadmium and copper, ...

Bletilla striata is an endangered traditional Chinese medicinal plant with multiple uses and a slow regeneration rate of its germplasm resources. To evaluate the callus growth kinetics and accumulation of secondary metabolites (SMs), a callus suspension culture was proven to be a valuable approach for acquiring high yields of medicinal compounds. An effective callus suspension culture for obtaining B. striata callus growth and its SMs was achieved with the in vitro induction of calluses from B. striata seeds. The callus growth kinetics and accumulation of SMs were analyzed using a mathematical model. The resulting callus growth kinetic model revealed that the growth curves of B. striata suspension-cultured calluses were sigmoidal, indicating changes in the growth of the suspension-cultured calluses. Improved Murashige and Skoog callus growth medium was the most favorable medium for B. striata callus formation, with the highest callus growth occurring during the stationary phase of the cultivation period. Callus growth acceleration started after 7 days and thereafter gradually decreased until day 24 of the cultivation period and reached its highest at day 36 period in both the dry weight and fresh weight analyses. The coelonin concentration peaked during the exponential growth stage and decreased afterward during the stationary stage of the callus suspension culture. The maximum content of coelonin (approximately 0.3323 mg/g callus dry weight) was observed on the 18th day of the cultivation cycle, while dactylorhin A and militarine reached the highest concentrations at day 24, and p-hydroxybenzyl alcohol at day 39. This investigation also laid a foundation for a multimathematical model to better describe the accumulation variation of SMs. The production of SMs showed great specificity during callus growth and development. This research provided a well-organized way to increase the accumulation and production of SMs during the scaled-up biosynthesis of calluses in B. striata callus suspension cultures.

Bletilla striata is an endangered orchid that has been used for millennia as a medicinal herb, in cosmetics and as a horticultural plant. To construct the first nucleotide database for this species and to develop abundant EST-SSR markers for facilitating further studies, various tissues and organs of plants in the main developmental stages were harvested for mRNA isolation and subsequent RNA sequencing. A total of 106,054,784 clean reads were generated by using Illumina paired-end sequencing technology. The reads were assembled into 127,261 unigenes by the Trinity package; the unigenes had an average length of 612 bp and an N50 of 957 bp. Of these unigenes, 67,494 (51.86%) were annotated in a series of databases. Of these annotated unigenes, 41,818 and 24,615 were assigned to gene ontology categories and clusters of orthologous groups, respectively. Additionally, 20,764 (15.96%) unigenes were mapped onto 275 pathways using the KEGG database. In addition, 25,935 high-quality EST-SSR primer pairs were developed from the 15,433 unigenes by MISA mining. To validate the accuracy of the newly designed markers, 87 of 100 randomly selected primers were effectively amplified; 63 of those yielded PCR products of the expected size, and 25 yielded products with significant amounts of polymorphism among the 4 landraces. Furthermore, the transferability test of the 25 polymorphic markers was performed in 6 individuals of two closely related genus Phalaenopsis and dendrobium. Which results showed a total of 5 markers can successfully amplified among these populations. This research provides a comprehensive nucleotide database and lays a solid foundation for functional gene mining and genomic research in B. striata. The developed EST-SSR primers could facilitate phylogenetic studies and breeding.

A Y-box binding protein ChYB-1 was discovered as a ChHsc70 promoter-associated protein in Crassostrea hongkongensis by DNA-affinity purification and mass spectrometry analysis. The overexpression of ChYB-1 in heterologous HEK293T cells led to clear enhancement of ChHsc70 promoter expression, while ChYB-1 depletion correlated with significant reduction of ChHsc70 transcription in the hemocytes of C. hongkongensis. Quantitative Real-time PCR analysis revealed that both ChHsc70 and ChYB-1 were transcriptionally responsive to external chemical or physical stressors. This indicates a plausible correlation between ChHsc70 and ChYB-1 in the genetic regulatory pathway triggered by external stresses. This study presents the first evidence of positive regulator for Hsc70 transcription and contributes to a better understanding of the regulatory mechanisms governing Hsc70 expression.

ChPur-α, a purine-rich element-binding protein, was discovered showing affinity to the ChHsc70 promoter in Crassostrea hongkongensis by DNA affinity purification and mass spectrometry analysis. Direct interaction between purified ChPur-α and the ChHsc70 promoter region was demonstrated by electrophoretic mobility shift assay in vitro. ChPur-α reduction led to clear enhancements of ChHsc70 transcription in the hemocytes of C. hongkongensis. Consistently, ChPur-α overexpression in heterologous HEK293T cells correlated with repressive phenotype in ChHsc70 promoter expression. ChHsc70 transcription was responsive to heat shock or CdCl2 stress by RT-PCR, signifying an inducible feature of ChHsc70 transcription by physical/chemical stress despite its constitutive nature. ChPur-α transcription was also induced by the two stressors. This indicates a plausible association between ChHsc70 and ChPur-α in the stress-induced genetic regulatory pathway. This study discovered a negatively regulatory role of ChPur-α in controlling ChHsc70 transcription in C. hongkongensis, and contributed to better understanding the regulatory mechanisms in control of Hsc70 transcription.

ChHP1BP3, a chromatin complex-related protein known with dynamic features, was identified as a ChHsp70 promoter-associated factor in Crassostrea hongkongensis by DNA-affinity purification and mass spectrometry analysis. Direct interaction between purified ChHP1BP3 and the ChHsp70 promoter region was demonstrated using EMSA. ChHp1bp3 depletion led to clear enhancements in ChHsp70 mRNA expression in C. hongkongensis hemocytes. However, ChHp1bp3 overexpression in heterologous HEK293T cells correlated with fluctuations in ChHsp70 transcription. Quantitative RT-PCR analysis showed that both ChHsp70 and ChHp1bp3 transcription were responsive to external physical/chemical stresses by heat, CdCl2 and NP. This indicated a plausible correlation between ChHsp70 and ChHp1bp3 in the stress-induced genetic regulatory pathway. While, the distinctive ChHp1bp3 expression patterns upon physical and chemical stresses suggest that the mechanisms that mediate ChHp1bp3 induction might be stress-specific. This study discovered a novel role for HP1BP3 as a negative regulator in controlling Hsp70 transcription in C. hongkongensis, and contributed to better understanding the complex regulatory mechanisms governing Hsp70 transcription.

Regulators of the WhiB-like (wbl) family are playing important role in the complex regulation of metabolic and morphological differentiation in Streptomyces. In this study, we investigated the role of wblI, a member of this family, in the regulation of secondary metabolite production in Streptomyces lividans. The over-expression of wblI was correlated with an enhanced biosynthesis of undecylprodigiosin and actinorhodin and with a reduction of the biosynthesis of yCPK and of the grey spore pigment encoded by the whiE locus. Five regulatory targets of WblI were identified using in vitro formaldehyde crosslinking and confirmed by EMSA and qRT-PCR. These included the promoter regions of wblI itself, two genes of the ACT cluster (actVA3 and the intergenic region between the divergently orientated genes actII-1 and actII-2) and that of wblA, another member of the Wbl family. Quantitative RT-PCR analysis indicated that the expression of actVA3 encoding a protein of unknown function as well as that of actII-1, a TetR regulator repressing the expression of actII-2, encoding the ACT transporter, were down regulated in the WblI over-expressing strain. Consistently the expression of the transporter actII-2 was up-regulated. The expression of WblA, that is known to have a negative impact on ACT biosynthesis, was strongly down regulated in the WblI over-expressing strain. These data are consistent with the positive impact that WblI over-expression has on ACT biosynthesis. The latter might result from direct activation of ACT biosynthesis and export and from repression of the expression of WblA, a likely indirect, repressor of ACT biosynthesis.

A bacterium identified as Burkholderia sp. HD05 isolated from Baimang River sediment was determined to have strong antifungal activity towards the hyphal growth as well as spore germination of pathogenic Saprolegnia sp. The antifungal substance was secreted into the supernatant and was thermo-stable. The active substance was purified from the HD05 fermentation broth by silica gel column chromatography and preparative HPLC. The nature of the active agent was identified as 2-pyrrolidone-5-carboxylic acid, a derivative of glutamic acid, by EI-MS and nuclear magnetic resonance spectrum (1H NMR, 13C NMR). The application of HD05 culture broth to the fish rearing water at 0.2% effectively prevented the saprolegiasis progression and a 53.3% decrease of the Saprolegnia infection rate of grass carp was reached. The promising potential of HD05 development as a biocontrol agent for controlling Saprolegnia sp. infections in aquaculture was discussed.

The development of the Gram-positive genus Streptomyces is characterized by a complex morphological differentiation process thought to be triggered by conditions of nutritional limitation that often correlate with high cell density (41). This process includes the arising of an aerial mycelium from the vegetative mycelium and then the differentiation of the tips of the aerial hyphae into spores (10, 15). This process is coupled to a metabolic differentiation that correlates with the production of a wide range of pharmaceutically important secondary metabolites, including antibacterial, anticancer, and immunosuppressive drugs (8, 10). The genes responsible for the biosynthesis of these secondary metabolites are clustered in the genome and coordinately regulated by pathway-specific transcriptional regulators (1, 4, 18, 19, 62, 70). The expression of these specific regulators linked to the biosynthetic pathways is directly or indirectly controlled either by positive pleiotropic regulators, such as AfsR2 (69), AfsS (29), AtrA (65), PtpA (67), PkaD (68), and the two-component systems AfsK/AfsR (66) and EcrA1/EcrA2 (39), by negative regulators, including the two-component systems AbsA1/AbsA2 (44), PhoR/PhoP (55), and CutR/CutS (9), or by enzymatic systems, such as Ppk (16). The pleiotropic regulators, thought to sense a variety of extracellular or intracellular signals related to nutriment availability, cell crowding, or energy shortage, are necessary to trigger the necessary metabolic adjustments to adapt to these conditions (27, 43), whereas the ppk gene is thought to act as an ATP-regenerating enzyme (54). Streptomyces coelicolor A3(2) is usually used as the reference strain to study morphological and metabolic differentiation in relation with antibiotic biosynthesis (10, 15). S. coelicolor has long been known to produce four major antibiotics, actinorhodin (ACT) (40), undecylprodigiosin (RED) (13), methylenomycin (MMY) (36), and calcium-dependent antibiotic (CDA) (21), and recently, two novel ones were characterized, CPK, a putative type I polyketide (17, 50), and albaflavenone, a sesquiterpene antibiotic (72). ACT is a secondary metabolite of the polyketide family that is strongly produced by S. coelicolor but only weakly produced by its close relative, Streptomyces lividans. However, S. lividans has the genetic capability to produce this compound since the weak production of ACT could be stimulated by various genetic manipulations that include the overexpression of the pathway-specific activator gene actII-ORF4 (7), the afsR and afsR2 (now afsS) genes (35), the rep gene (42), and the phosphotyrosine protein phosphatase-encoding gene ptpA (67). The inactivation of other genes, such as ppk (11), or mutations in the rpoB gene (25) or in the ribosomal protein S12 (23) are also leading to an enhancement of ACT production, indicating that ACT production was subjected to complex positive and negative controls. Furthermore, the extracellular addition of the signal molecule S-adenosylmethionine (34) or of ATP (38) or the replacement of glucose by glycerol in a minimal medium (35) was also shown to enhance ACT production in S. lividans. However, a systemic understanding of the regulation of antibiotic biosynthesis and in particular of the molecular basis of the very different abilities of S. lividans and S. coelicolor to produce ACT is still lacking. In order to tackle this question, we hypothesized that a major negative regulator of ACT production might be present in S. lividans but absent or nonfunctional in S. coelicolor. In an attempt to get this putative negative regulator, a genomic library of S. lividans DNA was constructed into the high-copy-number replicative plasmid pIJ702 (33) and introduced into S. coelicolor. An unique white transformant (ACT−) was obtained. This phenotype was shown to result from the overexpression of a regulator of the TetR family (equivalent to SCO3201 of S. coelicolor). The consequences of the overexpression and of the deletion of SCO3201 on the metabolic and morphological differentiation processes of S. coelicolor and S. lividans (wild type [wt] and ppk mutant) were assessed. The ability of SCO3201 to negatively regulate its own transcription was demonstrated by both in vitro and in vivo approaches, and the sequence of its operator site located in its own promoter region was determined. Sequences related to the SCO3201 operator site were found in the promoter region of scbA, a gene encoding a butyrolactone-synthesizing enzyme (61). These sequences were shown to be part of the binding site of ScbR, a TetR family regulator, positively controlling scbA expression, as SCO3201 does when overexpressed.