TetR family regulator AbrT controls lincomycin production and morphological development in Streptomyces lincolnensis

Abstract

Background

The TetR family of transcriptional regulators (TFRs), serving as crucial regulators of diverse cellular processes, undergo conformational changes induced by small-molecule ligands, which either inhibit or activate them to modulate target gene expression. Some ligands of TFRs in actinomycetes and their regulatory effects have been identified and studied; however, regulatory mechanisms of the TetR family in the lincomycin-producing Streptomyces lincolnensis remain poorly understood.

Results

In this study, we found that AbrT (SLCG_1979), a TetR family regulator, plays a pivotal role in regulating lincomycin production and morphological development in S. lincolnensis. Deletion of abrT gene resulted in increased lincomycin A (Lin-A) production, but delayed mycelium formation and sporulation on solid media. AbrT directly or indirectly repressed the expression of lincomycin biosynthetic (lin) cluster genes and activated that of the morphological developmental genes amfC, whiB, and ftsZ. We demonstrated that AbrT bound to two motifs (5′-CGCGTACTCGTA-3′ and 5′-CGTACGATAGCT-3′) present in the bidirectional promoter between abrT and SLCG_1980 genes. This consequently repressed abrT itself and its adjacent gene SLCG_1980 that encodes an arabinose efflux permease. D-arabinose, not naturally occurring as L-arabinose, was identified as the effector molecule of AbrT, reducing its binding affinity to abrT-SLCG_1980 intergenic region. Furthermore, based on functional analysis of the AbrT homologue in Saccharopolyspora erythraea, we inferred that the TetR family regulator AbrT may play an important role in regulating secondary metabolism in actinomycetes.

Conclusions

AbrT functions as a regulator for governing lincomycin production and morphological development of S. lincolnensis. Our findings demonstrated that D-arabinose acts as a ligand of AbrT to mediate the regulation of lincomycin biosynthesis in S. lincolnensis. Our findings provide novel insights into ligand-mediated regulation in antibiotic biosynthesis.

Background

Actinomycetes are prolific producers of secondary metabolites, making them valuable sources for natural drug development [1, 2]. Given the prevalence of drug-resistant bacteria, the development of new drugs and high-yield strains are the primary goals in the exploitation of actinomycete drug resources [3]. Innovative metabolic engineering and synthetic biology tools have facilitated the discovery and overproduction of natural products in actinomycetes [4, 5]. Engineering modifications of actinomycetes predominantly involve combining genetic and metabolic engineering techniques with various strategies, such as increasing or decreasing the expression of regulatory factors, enhancing precursor supply, reducing competitive pathways, and boosting the quantity and activity of rate-limiting enzymes [6, 7]. Therefore, understanding the intricate metabolic networks in actinomycetes is crucial for optimising antibiotic production.

Lincomycin, a lincosamide antibiotic produced by Streptomyces (S) lincolnensis, is mainly used in clinical settings to treat gram-positive bacterial associated infections [8]. Among the lincomycin biosynthetic gene (lin) cluster of S. lincolnensis, 29 genes are organized into 13 transcriptional units, spanning over 35 kb of DNA [9,10,11]. Research on multiple types of transcription factors (TFs) in S. lincolnensis has elucidated the intricate mechanisms underlying the molecular regulation of lincomycin biosynthesis [11,12,13]. The cluster-situated regulatory factor (LmbU) promotes the biosynthesis of lincomycin, while the N-terminal of LmbU includes an auto-inhibitory domain, inhibiting the DNA-binding activity of LmbU [10, 13, 14]. Additionally, LmrC exhibits dual functionality in drug resistance and regulation and can affect the expression of LmbU through antibiotic signal transduction, thereby promoting lincomycin biosynthesis [9]. AdpA positively regulates lincomycin biosynthesis and affects morphological differentiation [15]. The GntR family transcriptional regulator (LcbR1) inhibits lincomycin biosynthesis by indirectly repressing the expression of lin cluster genes [16]. SLCG_7083, another major regulator that features a PAS domain, has been shown to promote glucose utilisation, slow mycelial growth, and affect sporulation in S. lincolnensis [12]. We have previously demonstrated that the TetR family transcriptional regulator (TFR) SLCG_2919 binds to all promoters within the lin cluster to inhibit lincomycin synthesis [11]. The Lrp family regulator (SLCG_ Lrp) can positively regulate lincomycin biosynthesis, with arginine and phenylalanine acting as ligands [17, 18]. While these regulatory factors have been shown to directly or indirectly influence lincomycin biosynthesis and morphological differentiation, a deeper understanding of the regulatory network of lincomycin biosynthesis remains lacking, especially in ligand-mediated molecular regulation.

TFRs are widely distributed in bacteria and archaea as crucial regulators controlling diverse cellular processes [19, 20]. These regulators undergo conformational changes induced by small-molecule ligands, thereby inhibiting or promoting them to control target gene expression [21, 22]. TFRs have numerous ligands, including carbohydrates, proteins, fatty acids and their derivatives, and metal ions [20, 23]. Hence, TFRs regulate several physiological processes, from basic metabolism to quorum sensing and secondary metabolism [23]. Some TFR ligands in actinomycetes and their regulatory effects have been identified and investigated [23]. However, the regulatory role of TFR-responsive ligands in controlling lincomycin synthesis in S. lincolnensis remains unknown.

This study reveals the multifaceted role of AbrT (Arabinose-responsive TetR type regulator), a TFR, in the regulation of lincomycin production and morphological differentiation. AbrT acts as a repressor of lincomycin production while acting as an activator to regulate morphological differentiation. AbrT could bind to the bidirectional promoter between acrT and SLCG_1980 to transcriptionally inhibit abrT and SLCG_1980. Then, we identified two motifs within abrT-SLCG_1980 intergenic region, crucial for AbrT recognition. Furthermore, we demonstrated that D-arabinose acts as a ligand of AbrT, reducing its binding affinity to abrT-SLCG_1980 intergenic region. AbrT homologue in the actinomycete Saccharopolyspora erythraea was also found to inhibit the erythromycin production, suggesting that AbrT may play an important role in regulating secondary metabolism in actinomycetes.

Methods

Strains, plasmids, and growth conditions

Table 1 lists the bacterial strains and plasmids used in this study. Escherichia is usually cultured in liquid Luria Bertani (LB) medium or solid LB medium, maintained at 37℃ for approximately 8–12 h [11]. S. lincolnensis LCGL and its derivatives were incubated on a solid MGM medium at 30℃ for 7 days [11]. Sac. erythraea A226 and its derivatives were cultured on a solid R3M medium at 30℃ for sporulation and protoplast regeneration [6]. Cell growth was measured in liquid TSBY medium (3% tryptone soy broth, 0.5% yeast extract, 10.3% sucrose, with/without apramycin or thiostrepton) [17]. Liquid minimal medium (MM) (50% glucose, 0.5 g/l L-asparagine, 0.5 g/l K₂HPO₄, 0.2 g/l MgSO₄·7H₂O, 0.01 g/l FeSO₄·7H₂O) supplemented with 1 mM glutamine, glutamate, NaNO₂ or NaNO₃ was used for the detection of fluorescence reporting system in S. lincolnensis [17]. Yeast Malt Glucose (YMG) medium (10 g/l malt extract, 4 g/l yeast extract, 4 g/l glucose) was used for the determination of S. lincolnensis biomass [11].

Table 1 Bacterial strains and plasmids used in this study

Primers

Supplementary Table S1 lists all primers used in this study.

Gene inactivation, complementation, and overexpression

Genomic DNA from S. lincolnensis LCGL was used as a template for PCR amplification using primers abrT-P1/P2 and abrT-P3/P4 to generate a 1504 bp upstream and 1515 bp downstream fragment, respectively. Subsequently, the plasmid pUCTSR and upstream fragment of abrT were digested with the restriction enzymes HindIII and XbaI, followed by ligation to obtain plasmid pUCTSR-abrT-up, confirmed by enzyme digestion. Plasmid pUCTSR-abrT-up and the downstream fragment of abrT were digested with the restriction enzymes EcoRI and KpnI, followed by ligation. The resulting plasmid pUCTSR-ΔabrT was verified through PCR and enzyme digestion. The plasmid pUCTSR-ΔabrT was digested with HindIII and EcoRI and cloned into pKC1139 to obtain pKC1139-ΔabrT. Finally, pKC1139-ΔabrT was introduced into the protoplasts of S. lincolnensis LCGL. By the homologous chromosomic recombination, a 552-nt fragment of the abrT gene was replaced by thiostrepton resistance gene (tsr) in S. lincolnensis LCGL. The desired thiostrepton-resistant mutant, named ΔabrT, was confirmed by PCR using the primers abrT-P5/P6. The mutant strain LA219XΔabrT was constructed in the high-yielding strain S. lincolnensis LA219X using the same method described above.

abrT was amplified using PCR and the genomic DNA of S. lincolnensis LCGL as a template. The amplified fragment was inserted between the NdeI and XbaI restriction sites of pIB139 to construct complementation plasmid pIB139-abrT with PermE* promoter. Subsequently, ΔabrT was transformed with pIB139 and pIB139-abrT using PEG-mediated protoplast transformation. Initial screening was conducted using apramycin, and PCR identification targeting the apramycin resistance gene was performed to obtain the empty vector control strain ΔabrT/pIB139 and the complementation strain ΔabrT/pIB139-abrT. Using the method described above, pIB139-abrT was transformed into S. lincolnensis LCGL, and the constitutive expression strain LCGL/pIB139-abrT of abrT was identified using PCR.

Sac. erythraea mutants were constructed as previously described [23]. Using Sac. erythraea A226 genomic DNA as a template, two 1.5-kb DNA fragments flanking the SACE_5812 were amplified with PCR using the primer pairs 5812-P1/P2 and 5812-P3/P4. The two PCR products were respectively digested with HindIII/XbaI and KpnI/EcoRI, and ligated into the corresponding sites of pUCTSR to obtain pUCTSR-5812 [23]. The pUCTSR-5812 plasmid was then introduced into the Sac. erythraea A226. A 354 bp fragment within SACE_5812 was replaced by tsr through homologous recombination of linear fragments in A226. Confirmation of the desired thiostrepton-resistant mutant, named ΔSACE_5812, was achieved using the primers 5812-P5/P6 through PCR. Similarly, the ΔSACE_5812/pIB5812 complementation strain and A226/pIB1395812 constitutive expression strain were obtained.

Antibiotic fermentation and measurement

An agar piece of approximately 1 cm³ sample of S. lincolnensis LCGL and its derivatives, grown on spore-producing medium MGM, was taken and inoculated into seed medium. After culturing at 30℃ and 240 rpm for 48 h, the sample was transferred to 30 mL of fermentation medium at a 10% inoculation rate and cultured at 30℃ and 240 rpm for 7 days. Upon fermentation completion, 2 mL of the bacterial suspension was transferred to an Eppendorf tube and centrifuged at 12,000 rpm for 10 min. Subsequently, 200 µL of the supernatant was transferred to a new Eppendorf tube, and 800 µL of anhydrous ethanol was added and mixed. The mixture was allowed to stand for 5 min and centrifuged at 12,000 rpm for 10 min. The supernatant was then collected and filtered through a 0.22 μm organic filter membrane. Prepared filtrate liquid samples were subjected to High-performance liquid chromatography (HPLC) to detect Lin-A production using an Extend-C18 chromatographic column (5 μm, 150 × 4.6 mm). The mobile phase for equilibration comprised 60% methanol and 40% 5 mmol/L ammonium acetate (pH 9.0), with a flow rate of 0.5 mL/min and a detection wavelength of 214 nm. Flask fermentation by Sac. erythraea A226 and its derivatives were prepared as described [23].

The mutant ΔabrT and original strain LCGL were inoculated into YMG medium, respectively, and fermented at 30℃ for 7 days. During the fermentation period, the samples were weighed daily to measure their biomass [17].

Total RNA extraction and quantitative real-time PCR (qRT‐PCR)

RNA was extracted from S. lincolnensis, and gene expression was measured as described by Xu et al. [17].

Protein expression and purification

The genomic DNA from S. lincolnensis LCGL was used as a template, and the abrT fragment was amplified using PCR and primers abrT-P7 and abrT-P8. After digestion with NdeI and HindIII, the abrT fragment was cloned into the pET28a vector, generating the protein expression vector pET28a-abrT. This vector was then introduced into Escherichia coli (E. coli) BL21 (DE3) cells. The transformed strain was cultured in 50 mL of liquid LB medium at 37 °C until its optical density at 600 nm (OD₆₀₀) reached 0.4–0.6. Subsequently, IPTG was added to a final concentration of 0.5 mM, and expression was induced for 20 h at 16 °C. After induction, the strain was briefly cooled on ice and centrifuged at 6000 rpm for 10 min. The collected cells were resuspended in a lysis buffer (20 mM Tris, 500 mM NaCl, pH8.0) and disrupted via ultrasonication. The lysate was centrifuged at 4000 rpm for 30 min, and the His₆-tagged AbrT protein in the supernatant was recovered using a Ni²⁺-nitrilotriacetic acid (NTA) spin column (Bio-Rad). The AbrT protein was subsequently eluted from the column with 500 mM imidazole. The purity of the fusion protein was assessed via SDS-PAGE, and the protein concentration was determined using a BCA assay.

Electrophoretic mobility shift assays (EMSAs)

EMSAs for AbrT and SACE_5812 protein were conducted as previously described [17]. To investigate potential effectors of AbrT, D-arabinose and L-arabinose were separately added to AbrT protein and probe DNA in a 20 µL reaction mixture, respectively. The samples were then analysed through PAGE, as described by Xu et al. [17].

DNase I footprinting assay

To identify the binding site of AbrT in the intergenic region between abrT and SLCG_1980, we performed a DNase I footprint analysis following the methods outlined by Xu et al. [11].

Fluorescence reporting system experiment

To construct the reporter plasmid, we amplified the abrT-SLCG_1980-int fragment using the LCGL genome as a template. The resulting fragments were cleaved using HindIII and XbaI. Simultaneously, egfp fragments were amplified using the pUPW-EGFP plasmid as a template and digested with XbaI and BamHI. Subsequently, the cleaved abrT and egfp fragments were cloned into the pKC1139 plasmid, resulting in the fluorescent reporter system control plasmid, pKC-DE. The plasmid pKC-AbrT-DE was obtained using the same method. These plasmids were used to transform E. coli DH5α to obtain the corresponding strains. In a 48-well cell culture plate containing 500 µL LB, 5 µL of transformed strains were inoculated. After adding ligands at various concentration gradients, the bacteria were cultured at 37 °C until the OD₆₀₀ reached 0.8–1.0. The optical density (OD) values of the bacterial solution at 600 nm were measured using a multifunctional microplate reader, and the fluorescence signal values of the corresponding samples were detected using an excitation filter at 485 nm and an emission filter at 535 nm on a high-end SpectraMax Paradigm.

Similarly, the plasmids pKC-DE and pKC-AbrT-DE were transformed into the S. lincolnensis ΔabrT to obtain the corresponding strains. These transformed strains were then transferred to a 24-well cell culture plate containing 1 mL MM medium with different concentration ligands and cultured until their OD₆₀₀ reached 1.2–1.3. The detection was conducted using the method described above.

Statistical analysis

All data in this study were collected in triplicate and are presented as means ± standard deviation [29]. Significance was analysed using an unpaired two-tailed Student’s t-test, with *p < 0.05, ** p < 0.01, and ***p < 0.001.

Results and discussion

AbrT negatively regulates lincomycin biosynthesis

AbrT contains 714 nucleotides (nt) and encodes a TetR family protein consisting of 237 amino acids with an unresolved regulatory function in S. lincolnensis. To elucidate the relationship between AbrT and lincomycin production, we disrupted the abrT gene with tsr replacement in S. lincolnensis LCGL (Fig. 1a) and confirmed the resulting mutant ΔabrT using PCR (Fig. 1b). Fermentation and HPLC analyses revealed that lincomycin A (Lin-A) production in ΔabrT was approximately 28% higher than that in LCGL (Fig. 1c). The Lin-A production could be lowered by complementing the ΔabrT mutant with pIB139-abrT, confirming that the increased Lin-A production was attributed to abrT gene deletion (Fig. 1c). Furthermore, we analysed the growth curve of LCGL and ΔabrT strains in fermentation medium by measuring mycelium dry weight and found that abrT deletion had no significant effect on cell growth (Fig. 1d). These findings indicate that AbrT negatively regulates Lin-A production in S. lincolnensis.

To examine the role of AbrT as a negative regulator of lincomycin production in S. lincolnensis, abrT was deleted from the high-yield strain S. lincolnensis LA219X. When cultured in a 30 mL industrial fermentation medium for 7 days, the resulting S. lincolnensis LA219XΔabrT (2.91 g/L) exhibited a 16% higher increment in Lin-A production higher than that of LA 219X (2.52 g/L) (Fig. 1e).

Fig. 1

AbrT negatively regulates lincomycin production in S. lincolnensis. (a) Schematic representation of abrT gene deletion via homologous recombination in S. lincolnensis LCGL. (b) PCR confirmation of the abrT gene deletion mutant using primers abrT-P5/P6. Lanes: M, 5000 bp DNA ladder; 1, positive control, 1400 bp amplified from pKC1139ΔabrT; 2, negative control, 550 bp amplified from LCGL; 3, sample, 1400 bp amplified from mutant ΔabrT. (c) The Lin-A production in LCGL, ΔabrT, ΔabrT/pIB139, and ΔabrT/pIB139-abrT. (d) Growth curves of LCGL and ΔabrT in the liquid fermentation medium, with dry weights of mycelia measured. (e) The Lin-A production in LA219X and LA219X/ΔabrT. Mean values of three replicates are shown, with the standard deviation indicated by error bars. *p < 0.05, **p < 0.01

AbrT represses the lin cluster genes

To explore the regulatory role of AbrT, we utilised qRT-PCR to compare the transcripts of genes within the lin cluster between LCGL and ΔabrT at 24 h and 48 h. The endogenous rpoD gene, which exhibits a constant transcriptional level in LCGL and its derivatives, was used as an internal control to normalize samples. At 24 h, only the transcription levels of structural gene lmbA, bi-functional gene lmrC, and regulatory gene lmbU in ΔabrT were 1.7-, 1.5-, and 1.4-fold higher, respectively, than those in LCGL (Fig. 2a). While at 48 h, structural genes lmbC, lmbE, lmbG, lmbV, resistance gene lmrA, and lmrC in ΔabrT were 1.5-, 1.4-, 3.8-, 1.5-, 1.8-, and 1.4-fold higher, respectively, than those of LCGL (Fig. 2a). These findings indicate that disrupting abrT gene increased Lin-A production by enhancing the transcription of lin genes.

To determine whether AbrT could directly regulate the transcription of these genes, we expressed His₆-tagged AbrT protein in E. coli BL21 (DE3) (Fig. S1) and examined its affinities with the promoter regions of lmbA, lmbC-lmbD, lmbE, lmbJ-lmbK, lmbV-lmbW, lmrA, lmrC, and lmbU. EMSAs showed that the purified His₆-tagged AbrT bound to the promoter regions of lmrC and lmbU when different concentrations of His₆-AbrT were added (Fig. 2b), These findings indicate that AbrT directly or indirectly repressed five structural genes (lmbA, lmbC, lmbE, lmbG, and lmbV), one resistance gene (lmrA), one bifunctional gene (lmrC), and one regulatory gene (lmbU) in S. lincolnensis.

Fig. 2

AbrT-mediated regulation to the lin cluster genes. (a) Quantitative transcription levels of lin cluster genes in LCGL and ΔabrT cultured for 24 and 48 h in the fermentation medium. Mean values of three replicates are shown, with the standard deviation indicated by error bars. *p < 0.05, **p < 0.01. (b) EMSAs show the interaction of His₆-AbrT with the promoter regions of lmrC (20 nM DNA probes), lmbU (20 nM DNA probes), and lmbE (40 nM DNA probes)

AbrT is involved in the morphological differentiation of S. lincolnensis

Previous studies have shown that the deletion of bldA [30], bldD [31], adpA [15], and ramR [32] delays aerial mycelium formation and sporulation in S. lincolnensis. To determine AbrT contributions to morphological differentiation, we inoculated LCGL and ΔabrT on spore-producing culture medium for 48, 72, and 96 h. The ΔabrT exhibited a markedly delayed formation of mycelia and spores compared with the LCGL. This morphological phenotype was restored in ΔabrT/pIB139-abrT (Fig. 3a). These findings indicate that knocking out abrT retards aerial mycelium formation and sporulation in S. lincolnensis.

Therefore, we conducted qRT-PCR for several key genes involved in morphological differentiation, including amfC (SLCG_2911, encoding an aerial mycelium formation protein) [33], whiB (SLCG_3418, encoding a sporulation regulatory protein) [34] and ftsZ (SLCG_6003, encoding a cell division protein) [35]. The transcriptional levels of ftsZ, whiB, and amfC were lower in ΔabrT than in LCGL (Fig. 3b). Furthermore, we observed direct binding of AbrT to the promoter regions of these three genes (Fig. 3c), suggesting that AbrT positively regulates morphological differentiation by directly activating the transcription of ftsZ, whiB, and amfC. Collectively, these findings indicate that AbrT exhibits opposing regulatory effects, inhibiting lincomycin biosynthesis while stimulating morphological development in S. lincolnensis.

Fig. 3

AbrT positively regulates S. lincolnensis morphological differentiation. (a) Morphological observation of the original strain LCGL, ΔabrT, ΔabrT/pIB139, and ΔabrT/pIB139-abrT on MGM medium for 48, 72, and 96 h. (b) qRT-PCR analysis of the genes ftsZ, whiB, and amfC involved in morphological differentiation in LCGL and ΔabrT cultured for 24 and 48 h in a fermentation medium. Mean values of three replicates are shown, with the standard deviation indicated by error bars. *p < 0.05, **p < 0.01. (c) EMSAs of AbrT binding to the probes P_ftsZ (25 nM), P_whiB (60 nM), and P_amfC (40 nM) represent the promoters of ftsZ, whiB, and amfC, respectively

AbrT represses the transcription of abrT and its adjacent gene SLCG_1980

Following the regulatory paradigm of SLCG_2919 [11], we predicted that AbrT may regulate the expression of its gene and its adjacent divergently transcribed gene, SLCG_1980, which encodes an arabinose efflux permease. We assessed the transcription levels of abrT and SLCG_1980 using qRT-PCR. Analysis of the remaining 100-nt 5’-region of the abrT gene revealed a 16-fold increase in abrT transcription in ΔabrT compared with in LCGL at 24 h (Fig. 4a). Similarly, the expression level of SLCG_1980 in ΔabrT strikingly increased by 500-fold at 24 h. At 48 h, the expression levels of abrT and SLCG_1980 increased by 8-fold and 17-fold, respectively (Fig. 4a). These findings indicate that AbrT acts as a repressor of its gene and the adjacent gene SLCG_1980.

EMSA demonstrated a clearly shifted band formed when AbrT was incubated with abrT-SLCG_1980 intergenic region (abrT-SLCG_1980-int), confirming direct binding of AbrT to abrT-SLCG_1980 intergenic region (Fig. 4b). Previous studies and bioinformatics analyses (http://www.fruitfly.org/seq_tools/promoter.html) predicted that the DNA fragment between abrT and SLCG_1980 is a bidirectional promoter controlling the expression of abrT and SLCG_1980 (Fig. S2).

Fig. 4

AbrT represses the transcription of abrT and its adjacent gene SLCG_1980. (a) Transcriptional levels of abrT and SLCG_1980 in LCGL and ΔabrT assessed using qRT-PCR analysis at 24 and 48 h. Mean values of three replicates are presented, with the standard deviation indicated by error bars. ***p < 0.001. (b) EMSA demonstrating the binding of AbrT to abrT-SLCG_1980-int. Each lane contained 50 nM DNA probes

Determination of AbrT-binding sites within abrT-SLCG_1980 intergenic region

To determine the AbrT binding site in the promoter regions of abrT and SLCG_1980, we conducted a DNase I footprinting assay using Fam-labelled DNA fragments. Two binding sites for AbrT were identified: site a (5′-CGCGTACTCGTA-3′) and site b (5′-CGTACGATAGCT-3′) (Fig. 5a). To verify the importance of these motifs for AbrT binding, EMSAs were performed using the abrT-SLCG_1980-int (N), a single-site mutated DNA fragment (M1 or M2), and a double-site mutated DNA fragment (M3) (Fig. 5b). In contrast to N, probes M1 and M2 displayed only a single shifted band with no shifted band in the gel using probe M3 (Fig. 5c). This indicates that two binding sites are present for AbrT to abrT-SLCG_1980 intergenic region.

Fig. 5

Determination of the precise binding sites of AbrT. (a) DNase I footprinting assay showing AbrT-binding sites within abrT-SLCG_1980 intergenic region. (b) Schematic diagrams of the mutated probes. Probe N, abrT-SLCG_1980-int, served as the control; probes M1 had only site a within the 140 bp DNA fragment mutated; M2 had only site b in the 140 bp DNA fragment mutated; M3 had site a and b within the 128 bp DNA fragment mutated. (c) EMSAs show AbrT binding to probe N and mutated probes M1, M2, and M3

AbrT is responsive to D-arabinose in vitro and in vivo

TFRs have various ligands, including carbohydrates, proteins, fatty acids and their derivatives, and metal ions [20, 23]. Given that SLCG_1980, the target gene of AbrT, encodes an arabinose efflux permease, exploring whether AbrT regulates SLCG_1980 in response to arabinose as an effector is essential. To test this hypothesis, EMSAs were performed with AbrT binding to the abrT-SLCG_1980-int upon exogenous addition of L-arabinose or D-arabinose. The addition of D-arabinose reduced the binding affinity between AbrT and abrT-SLCG_1980-int, suggesting that D-arabinose acts as an effector of AbrT (Fig. 6a).

To determine whether AbrT interacted with D-arabinose in vivo, we constructed a biosensor system in E. coli DH5α following our previous method [6]. This system employed two plasmids: pKC-AbrT-DE, which expressed the reporter egfp gene, and the abrT gene controlled by the bidirectional promoter between abrT and SLCG_1980 genes (P_abrT−1980), and pKC-DE, which solely expressed the reporter egfp gene under the control of P_abrT−1980. (Fig. 6b). Upon the addition of 0.5–100 µM D-arabinose, a gradual increase was observed in green fluorescence in DH5α/pKC-AbrT-DE (Fig. 6c). Meanwhile, the addition of L-arabinose in the same concentration range showed no difference in bioluminescence (Fig. 6d). These findings indicate that similar to its in vitro effects, D-arabinose is an effector of AbrT in vivo.

The fluorescent reporter system in E. coli has demonstrated that, in vivo, D-arabinose acts as the dissociative ligand of AbrT, binding to P_abrT−1980, with a minimum effective concentration for allosteric regulation of 1 µmol/L. However, given that the fluorescence reporter system in heterologous hosts cannot reflect the response of AbrT to ligands under physiological conditions, we performed biofluorescence measurements in S. lincolnensis to explore the physiological state of D-arabinose as a ligand influencing the allosteric regulation of AbrT. To minimise interference of AbrT in S. lincolnensis with the fluorescence reporting system, the two plasmids, pKC-DE and pKC-AbrT-DE, were successively introduced into ΔabrT. Biofluorescence assays revealed that supplementation of external D-arabinose ranging from 0.1 to 5 µM in ΔabrT/pKC-DE did not yield a significant change in fluorescence levels. However, in ΔabrT/pKC-AbrT-DE, the fluorescence intensity showed a positive correlation with increasing D-arabinose levels, consistent with the results from E. coli (Fig. 6e). This suggests that the physiological concentration of D-arabinose in regulating its function in S. lincolnensis is approximately 0.1 µmol/L, with a response sensitivity nearly 10 times greater than observed in E. coli.

Fig. 6

D-arabinose induces the dissociation of AbrT from P_abrT−1980. (a) EMSAs of AbrT with D-arabinose or L-arabinose. All arabinose concentrations are expressed in mM. (b) An illustration of the reporter plasmids. The addition of the respective arabinose effectors affects egfp expression in the reporter system. (c) Detection of relative fluorescence units (RFUs) after adding D-arabinose in E. coli DH5α/pKC-AbrT-DE and DH5α/pKC-DE. (d) Detection of RFUs after adding L-arabinose in E. coli DH5α/pKC-AbrT-DE and DH5α/pKC-DE. (e) Detection of RFUs after adding D-arabinose in ΔabrT/pKC-AbrT-DE and ΔabrT/pKC-DE. The Mean values of three replicates are shown, with standard deviation indicated by error bars. *p < 0.05, **p < 0.01, ***p < 0.001

AbrT homolog plays an important role in the antibiotic biosynthesis

Given the presence of AbrT homologues in antibiotic-producing actinomycetes (Fig. 7a), we explored whether their regulatory functions have commonalities. SACE_5812, a TetR family regulator sharing the highest amino acid identity with AbrT (Fig. 7a), was selected to investigate the erythromycin A (Er-A)-producing actinomycete Sac. erythraea [24]. Genetic experiments involving disruption and complementation of SACE_5812 demonstrated its negative effect on Er-A production (Fig. 7b). Using qRT-PCR and EMSAs, we found that SACE_5812 directly repressed the expression of SACE_5813, eryAI (encoding polyketide synthase I), and ermE (encoding rRNA methyltransferase), suggesting its direct regulation of the erythromycin biosynthesis gene cluster and its adjacent gene in Sac. erythraea A226 (Fig. 7c and d). Those results are consistent with the regulatory function of AbrT in S. lincolnensis. These findings suggest that AbrT and its homologs may play the important role in regulating secondary metabolism in actinomycetes.

Fig. 7

AbrT homologous protein functions in Sac. erythraea. (a) Construction of neighbor-joining distance tree depicting AbrT and its homologs. The tree was constructed using the amino acid sequences of AbrT and its homologs in actinomycetes with MEGA. The percentages represent the identities between AbrT and its homologs. (b) HPLC analyses of Er-A production in A226 and its derivatives. (c) qRT-PCR analyses of ery cluster genes and SLCG_5813 in A226 and ΔSACE_5812 cultured for 24 h. The mean values of three replicates are shown, with the standard deviation indicated by error bars. *p < 0.05, **p < 0.01, ***p < 0.001. (d) EMSAs of SACE_5812 binding to SLCG_5813, eryAI, and ermE promoter

Conclusions

The findings of this study demonstrated the regulatory role of the TetR family protein AbrT in lincomycin production by repressing the expression of lin cluster genes in S. lincolnensis. AbrT directly inhibited the expression of its gene and that of the neighbouring gene, SLCG_1980, through two DNA-binding sequences. Our study reveals that D-arabinose acts as a ligand to modulate lincomycin biosynthesis in S. lincolnensis. Furthermore, functional analysis of an AbrT-homologue protein in Sac. erythraea suggested that AbrT may play an important role in regulating secondary metabolism in actinomycetes. Moreover, AbrT affected mycelial growth and morphological development by directly activating the expression of amfC, whiB, and ftsZ.