AdpAlin, a Pleiotropic Transcriptional Regulator, Is Involved in the Cascade Regulation of Lincomycin Biosynthesis in Streptomyces lincolnensis

Lincomycin is one of the most important antibiotics in clinical practice. To further understand the regulatory mechanism on lincomycin biosynthesis, we investigated a pleiotropic transcriptional regulator AdpAlin in the lincomycin producer Streptomyces lincolnensis NRRL 2936. Deletion of adpAlin (which generated ΔadpAlin) interrupted lincomycin biosynthesis and impaired the morphological differentiation. We also found that putative AdpA binding sites were unusually scattered in the promoters of all the 8 putative operons in the lincomycin biosynthetic gene cluster (BGC). In ΔadpAlin, transcript levels of structural genes in 8 putative operons were decreased with varying degrees, and electrophoretic mobility shift assays (EMSAs) confirmed that AdpAlin activated the overall putative operons via directly binding to their promoter regions. Thus, we speculated that the entire lincomycin biosynthesis is under the control of AdpAlin. Besides, AdpAlin participated in lincomycin biosynthesis by binding to the promoter of lmbU which encoded a cluster sited regulator (CSR) LmbU of lincomycin biosynthesis. Results of qRT-PCR and catechol dioxygenase activity assay showed that AdpAlin activated the transcription of lmbU. In addition, AdpAlin activated the transcription of the bldA by binding to its promoter, suggesting that AdpAlin indirectly participated in lincomycin biosynthesis and morphological differentiation. Uncommon but understandable, AdpAlin auto-activated its own transcription via binding to its own promoter region. In conclusion, we provided a molecular mechanism around the effect of AdpAlin on lincomycin biosynthesis in S. lincolnensis, and revealed a cascade regulation of lincomycin biosynthesis by AdpAlin, LmbU, and BldA.


INTRODUCTION
Lincomycin is a naturally occurring antibiotic isolated from soil sample, and it was first introduced into clinical practice in 1963 (Macleod et al., 1964). Lincomycin and its derivatives belong to lincosamide antibiotics and exhibit biological activities against anaerobic and some protozoal infections by inhibiting protein synthesis in sensitive targets (Spizek and Rezanka, 2017).
Clindamycin is a semi-synthetic chlorinated derivative of lincomycin and it is marked by being one of the 20 most important antibiotics (Spizek and Rezanka, 2004a). Given the extensive clinical application of lincomycin, multiple attempts have been taken into industrial practice to increase the production yields of lincomycin or to optimize the products (Spizek and Rezanka, 2004b;Li et al., 2007). Genetic manipulations are also adopted as complement to enhance the production of lincomycin (Pang et al., 2015;Xu et al., 2018). Though the pathway of lincomycin biosynthesis was assembled mainly within the recent 10 years (Neusser et al., 1998;Novotna et al., 2004;Sasaki et al., 2012;Lin et al., 2014;Pang et al., 2015;Zhao et al., 2015;Jiraskova et al., 2016), studies on the regulation mechanism of lincomycin biosynthesis are prompted quite slowly. Thus, various methods have limited effect on yield improvement of lincomycin.
Biosynthesis of antibiotics is controlled by elaborate regulatory mechanisms. Hormone-like signaling molecules, for example γ-butyrolactone (Takano et al., 2000;Hsiao et al., 2009;Du et al., 2011), serve as stimuli that interact with their receptor proteins to prelude the secondary metabolism (Niu et al., 2016). Global regulators and/or pleiotropic regulators then deliver these signals to CSRs which directly control the onset of antibiotic biosynthesis. In addition, researches on the secondary metabolism in Streptomyces is expanded to the posttranscriptional regulation. For example, BldA, a rare tRNA in Streptomyces, has significant importance on morphological differentiation and antibiotic biosynthesis (Hackl and Bechthold, 2015). As for lincomycin biosynthesis, only limited researches contribute to decipher the regulatory network. Lu et al. (2008) found that LmbU contributes to lincomycin biosynthesis. Hou et al. (2018aHou et al. ( , 2019 demonstrated that LmbU, as a CSR, positively regulates lincomycin biosynthesis by controlling the transcription of lmbA, lmbC, lmbJ, lmbK, lmbW, and lmbU itself, and subsequently solved the subtle mechanism of LmbU regulon. Besides, Hou et al. (2018b) also found that BldA functions as a global regulator on both morphological differentiation and lincomycin biosynthesis at the level of translation, and genes lmbB2, lmbY, and lmbU, which all contain TTA rare codon, get involved in the regulon. Meng et al. (2017) revealed the regulatory network between nitrate metabolism and lincomycin biosynthesis where GlnR activates the transcription of lmrA, the lincomycin exporter gene. Lately, a TetR-type regulator SLCG_2919 has been identified as a repressor of lincomycin biosynthesis that controls the transcription of lmbA, lmbC, lmbE, lmbG, lmbK, lmbR, lmbV, and lmbW . However, to complete the regulatory network of lincomycin biosynthesis, there are lots of gaps remained.
AdpA was previously found to be an A-factor dependent regulator and repressed by ArpA (Kato et al., 2007). AdpA amplifies the A-factor signal and thus participates in morphological differentiation and secondary metabolism. By means of chromatin affinity precipitation (ChAP) and chromatin immunoprecipitation (ChIP), Higo et al. (2012) found that AdpA controls more than 500 genes in Streptomyces griseus. Afterward AdpA is considered to be a regulator of great importance in Streptomyces. In S. chattanoogensis, AdpA ch controls the expression of wblA ch , and thus participates in the regulation of natamycin biosynthesis (Yu et al., 2014). In S. roseosporus, AdpA controls the expression of atrA and indirectly control daptomycin biosynthesis (Mao et al., 2015). In a recent research, AdpA interacts with the twocomponent system PhoRP and both of them contribute to the transcription of atrA (Zheng et al., 2019). Effects of AdpA on antibiotic biosynthesis is always a popular topic. AdpA always serves as an activator of antibiotic biosynthesis, a classic case is that AdpA activates the transcription of strR, which encodes the CSR of streptomycin biosynthesis. Therefore, AdpA regulates streptomycin biosynthesis positively and indirectly . Similarly, for the biosynthesis of grixazone (Higashi et al., 2007), nikkomycin (Pan et al., 2009), and natamycin (Yu et al., 2018), AdpA activates the transcription of CSRs in their BGCs and indirectly regulates antibiotic biosynthesis. On the other hand, AdpA has a negative impact on oviedomycin biosynthesis in S. ansochromogenes by repressing the transcription of CSR (Xu et al., 2017). Very recently, it was reported that AdpA from S. xiamenensis 318 negatively regulates morphological differentiation as well as polycyclic tetramate macrolactams (PTMs) production, while positively regulates xiamenmycin production by activating the transcription of two of the structural genes ximA and ximB (Bu et al., 2019). As we can speculate from previous studies, AdpA typically controls the CSR and/or some structural genes in an antibiotic BGC. So, we scanned the lincomycin BGC and found that putative AdpA binding sites were extraordinarily scattered upstream all of the 8 putative operons with different amounts and locations. Thus, we focused on the pleiotropic regulator AdpA (GenBank accession no. ANS65440.1) and attempted to investigate its regulatory mechanism of lincomycin biosynthesis in Streptomyces lincolnensis in this study.

Deletion and Complementation of AdpA lin
To construct AdpA lin disruption strain adpA in S. lincolnensis, DNA fragments of upstream and downstream region of AdpA lin were amplified separately using primers ad-F1/R1 and ad-F2/R2. Then digested, respectively, with restriction enzyme Hind III/Xba I and BamHI/EcoR I (Thermo Fisher Scientific, United States) and ligated into the E. coli-Streptomyces shuttle plasmid pMJ1 to generate plasmid pADNU. Then E. coli ET12567/pUZ8002 was used to introduce pADNU into S. lincolnensis NRRL 2936 by conjugal transfer (Hou et al., 2018b). As a result of homologous recombination adpA was constructed. DNA sequencing with primers IDad-F1/IDneo-R1 and IDneo-F2/IDad-R2 was adopted for further identification.
To construct adpA complementation strain adpA:adpA, a DNA fragment covering AdpA lin was amplified by PCR with primers ad-C-F/R and then digested with Nde I/EcoR I (Thermo Fisher Scientific, United States). Then ligated into the corresponding sites of the integrative vector pSET152. The resulting plasmid pADC was introduced into adpA by conjugal transfer and integrated into the chromosome to generate adpA:adpA where the complemented AdpA lin was under the control of the promoter ermE * p. DNA sequencing with primers 152-F/R was adopted for further identification.
All primers used in this study are listed in Supplementary  Table S1, and synthesized by Genewiz (China).

Scanning Electron Microscope (SEM)
Scanning electron microscope assay referred to a previously established method (Hou et al., 2018b) with some optimizations. S. lincolnensis NRRL 2936, adpA, and adpA:adpA were cultured on SMA medium at 28 • C for about 5 days. Equivalent areas of the lawn were harvested and placed in 2.5% glutaraldehyde solution overnight. Dehydrated by vacuum freezing drying and sprayed with platinum by Gatan ALTO 1000E (Gatan, United States). Then observed with Hitachi S-3400N scanning electron microscopy (Hitachi, Japan).

Dry Cell Weight Determination and Lincomycin Bioassay Analysis
Streptomyces lincolnensis NRRL 2936, adpA, and adpA:adpA were inoculated from SMA medium into FM1 at 28 • C with shaking (210 rpm) for 3 days and then inoculated into FM2 and cultured for 6 days at 28 • C with shaking (210 rpm). Precipitate of each sample was harvested every 24 h and dried at 55 • C for 24 h. Then the weights of the dried precipitates represent the dry cell weights. Meanwhile, to analyze the bioassay of lincomycin, supernatant of each sample was harvested at the same time, and previously mentioned method (Pharmacopoeia of the People's Republic of China [PPRC], 1990;Hou et al., 2018a) with some modifications was adopted. M. luteus 28001, used as indicator, was cultured on medium III at 37 • C for 16-18 h and the lawn was washed off with 0.9% NaCl and suspended readily to use. Lincomycin standard solutions (4, 6, 8, 10, 12, 14, and 16 µg/mL) were used for the standard curve and internal control. Diameters of inhibition zone were linearized with the logarithmic values of the concentrations of the lincomycin standard solutions. Concentration of each sample was calculated on the basis of the standard curves. All assays in this section were performed in duplication and standard errors of the mean were calculated. The software GraphPad Prism 7.00 was used to draw the line graph of dry cell weight and histogram of lincomycin bioassay.

RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
Streptomyces lincolnensis NRRL 2936, and adpA cultured on the second day in FM2 medium were used to extract total RNA. Precipitate of samples were ground in liquid nitrogen (Liu et al., 2013) and followed by the method using TRIzol (Thermo Fisher Scientific, United States) (Setinova et al., 2017). After reacting with Recombinant DNase I (Takara, Japan) to remove the trace amount of DNA, 800 ng of RNA samples (analyzed by NanoDrop 2000, Thermo Fisher Scientific, United States) were reverse transcribed to cDNA using Reverse Transcriptase M-MLV (Takara, Japan). SYBR green PCR master mix (ToYoBo, Japan) was used and qRT-PCR was performed in triplication for each transcript. qRT-PCR conditions were mentioned previously (Hou et al., 2018a). To detect the transcript level of AdpA lin targets, primers qbl-F/R, qU-F/R, qA-F/R, qC-F/R, qD-F/R, qJ-F/R, qK-F/R, qV-F/R, and qW-F/R in Supplementary Table S1 were used. And primers qhrdB-F/R were used to detect the transcript level of hrdB which served as an internal control. qRT-PCR was performed with samples in triplication and data were treated with the threshold cycle (2 − C T ) method (Livak and Schmittgen, 2001) and standard errors of the mean were calculated. GraphPad Prism 7.00 was used to draw the histogram of relative expression level of each AdpA lin target.

Catechol Dioxygenase Activity Analysis
DNA fragment covering reporter gene xylTE was amplified by PCR with primers xyl-F/R. Promoters of different AdpA lin targets were amplified separately by PCR with primers , and Wp-xyl-F/R (for lmbWp, from −456 to +4). In respect to bldAp, +1 represents the start point of mature bldA. As for other promoters, +1 represents the translation starting point of the genes controlled by them. Promoter fragment and xylTE fragment were inserted into Pvu II site of the integrative vector pSET152 using Super Efficiency Fast Seamless Cloning kits (DoGene, China) (Hou et al., 2018b) to construct reporter plasmids pADPX, pBLPX, pUPX, pAPX, pCPX, pDPX, pJPX, pKPX, pVPX, pWPX, and pADPX2. Then introduced into S. lincolnensis NRRL 2936 or adpA respectively, to investigate the effects of AdpA lin on these targets. Referred to the method optimized by Hou et al. (2018b), Catechol dioxygenase activity analysis was carried out in triplication. Standard errors of the mean were calculated and the software GraphPad Prism 7.00 was used to draw the histogram of catechol dioxygenase activity.

Electrophoretic Mobility Shift Assays (EMSAs)
The AdpA lin gene was amplified by PCR with primers ad-C-F/R and digested with Nde I/EcoR I. DNA fragment was cloned into corresponding sites of pET-28a (+) vector (Novagen, United States), and the resulting plasmid pADH was transformed into E. coli BL21 (DE3). Overexpression and purification of recombinant protein refer to the procedures described previously (Hou et al., 2018a). According to our experience, DNA probes with length around 200 bp are appropriate for EMSAs with  Supplementary Table S1 were used to amplify DNA probes with putative AdpA binding sites, and primers nad-F/R were used to amplify DNA probe with no AdpA binding site as a negative control. Genes lmbV and lmbW share the same DNA probe. For the second round, amplified DNA fragments are used as templates with primer Biotin-linker * to harvest DNA probes with biotin labeled at 5 terminal. EMSAs were performed as previously described (Liao et al., 2015) using chemiluminescent EMSA kits (Beyotime Biotechnology, China). AdpA lin of different concentrations (0, 1.6, 3.2, and/or 6.4 µM, respectively) interacted with 2.5 nM biotin labeled DNA probe in binding buffer TGB [20 mM Tris-HCl (Shize, China), 5% glycerol (Titan, China), and 0.1% BSA (Sangon, China), pH 7.5], and 200-folds excess of unlabeled probes were added as competitive assays.

AdpA lin Positively Regulates Both Lincomycin Biosynthesis and Morphological Differentiation in S. lincolnensis
It has been shown that AraC/XylS family regulators control various metabolic pathways including antibiotic biosynthesis (Ibarra et al., 2008). There are about 30 AraC/XylS family regulators in S. lincolnensis, among which, AdpA is the most famous one. Based on this, we investigated the effects of AdpA lin on lincomycin biosynthesis and attempted to propose some innovative idea on this classic regulator. Alignment of AdpA from 26 Streptomyces species (Supplementary  Figures S1A,B) showed that AdpA retained the conserved N-terminal ThiJ/PfpI/DJ-1-like (also referenced as GATase-1) dimerization domain and C-terminal AraC/XylS-type DNAbinding domain (DBD) . The first 340 amino acids possessed an ortholog with over 90% identities, and the main diversity occurred at the tail end of C-terminus with a length of no more than 90 amino acids, after the conserved DNA binding domain (Supplementary Figure S1A). To infer the evolutionary history of AdpA, phylogenetic analysis was performed using a maximum likelihood method. The results showed that AdpA lin possessed an ortholog with 89% amino acid identity to AdpA sg , and thus we classified AdpA lin to be one member of the AraC/XylS family.
To investigate the effects of AdpA lin on lincomycin biosynthesis, AdpA lin null mutant was constructed and named as adpA. Lincomycin biosynthesis was significantly influenced by the non-functional AdpA lin . In medium FM2, lincomycin started to be produced on the second day in WT, reached a maximum bioassay of 30.10 µg/mL between the second and the fourth day, and maintained thereafter. However, bioassay of lincomycin in adpA remained undetectable throughout the entire 6 days ( Figure 1A). Furthermore, the lawns of adpA on SMA medium exhibited a bald phenotype distinct from WT ( Figure 1B). Deletion of AdpA lin blocked the sporulation and caused long, extended aerial hyphae when detected by SEM ( Figure 1C). Complementation of AdpA lin in adpA strain ( adpA:adpA) restored both lincomycin biosynthesis and sporulation as expected (Figure 1) though lincomycin production in complemented strain did not restore to WT level, probably due to using the promoter ermE * p. Moreover, the biomasses of WT, adpA, and adpA:adpA were measured at all the four detected days. The data showed that biomasses of the three strains have no significant differences at days 1 and 6, while at day 2, adpA had decreased biomass compared to WT and adpA:adpA, and at day 4, adpA:adpA had increased biomass compared to WT and adpA. These data suggested that AdpA lin is an important regulator of lincomycin biosynthesis in S. lincolnensis.

AdpA lin Directly Activates Transcription of the Structural Genes in the lmb Cluster
AdpA regulates more than 500 genes in S. griseus (Higo et al., 2012), and AdpA binding motifs have been well studied in other Streptomyces species such as S. griseus (Yamazaki et al., 2004), S. coelicolor (Kim et al., 2005), and S. lividans (Guyet et al., 2013). Additionally, Ming et al. have solved the complex structure of AdpA-DBD and target DNA in S. griseus (Yao et al., 2013). AdpA binding site is recognized as 5 -TGGCSNGWWY-3 (where S is G or C, W is A or T, Y is T or C, and N is any nucleotide), and G at position 2 and C at position 4 are more highly conserved than the other nucleotides in this motif (Yao et al., 2013). Lincomycin BGC was named as the lmb cluster, and the gene organization was shown in Supplementary Figure S2A. The lmb cluster contains 8 putative operons and the first genes of them are lmbA, lmbC, lmbD, lmbJ, lmbK, lmbV, lmbW, and lmbU, respectively. We looked into the nucleic acid sequence of the lmb cluster and searched with the conserved AdpA binding sequence, and no more than 3 mismatches in the last 3 bp were allowed. We found that putative AdpA binding sites were scattered in the upstream region of all the 8 putative operons (Supplementary  Figures S2B-F). The numbers of putative AdpA binding sites in the upstream of lmbA, lmbC, lmbD, lmbJ, lmbK, lmbV, lmbW, and lmbU are 1, 6, 1, 3, 3, 2, 2, and 10, respectively. Therefore, we speculated that the entire biosynthesis process of lincomycin might be under the control of AdpA lin .
qRT-PCR analysis showed that the transcript level of lmbA, lmbC, lmbD, lmbJ, lmbV, and lmbW dramatically decreased in adpA with fold changes 129.41, 43.64, 60.30, 215.29, 11.95, and 301.20, respectively (Figure 2A). It was suggested that in adpA, lincomycin biosynthesis was blocked because of the decreased expression of structural genes lmbA, lmbC, lmbD, lmbJ, lmbV, and lmbW. Due to the low transcript level of lmbK (data not shown), we failed to calculate the relative expression in both WT and adpA. To investigate the regulation between AdpA lin and lmbK, we cloned the 895 bp of DNA sequences upstream from lmbK translation starting site (TSS) and constructed reporting plasmid for catechol dioxygenase activity assay. The results demonstrated that AdpA lin got involved in regulating the transcription of lmbK ( Figure 2B). Besides, transcriptions of lmbA, lmbC, lmbD, lmbJ, lmbV, and lmbW were activated by AdpA lin in catechol dioxygenase activity assay as well ( Figure 2B). These data suggested that AdpA lin has primary effects on the structural genes in the lmb cluster.
Then we carried out EMSAs to investigate the interplay between AdpA lin and promotors of above lmb structural genes. DNA fragments containing putative AdpA binding sites were labeled with biotin and incubated with purified AdpA lin . Results of EMSAs showed that AdpA lin interacted with all of the 8 promoter regions containing putative AdpA binding sites ( Figure 2C). DNA probe lmbJp-A served as part of the promoter regions of both lmbJ and lmbK, and similarly, DNA probe lmbVp-A served as the promoter regions of both lmbV and lmbW. For promoters containing more than one AdpA binding sites, such as genes lmbC, lmbJ, lmbK, lmbV, and lmbW, AdpA lin interacted with different putative AdpA binding sites and generated different forms of complexes ( Figure 2C). DNA fragment without AdpA binding site, i.e., probe-neg, could not interact with AdpA lin (Supplementary Figure S3). To confirm the exact binding sites of AdpA lin with promoters, we deleted the putative AdpA binding sites in lmbAp-A, lmbJp-A and lmbKp-B, and EMSAs showed AdpA lin no longer interacted with these DNA probes (Supplementary Figure S4). Thus, we speculated that AdpA lin activates all of the 8 promoters in the lmb cluster by directly binding to putative AdpA binding sites.

AdpA lin Directly Activates Transcription of the CSR Gene lmbU
LmbU was recently reported by Hou et al. (2018aHou et al. ( , 2019 to be a novel transcriptional regulator cited in the lmb cluster and positively regulate lincomycin biosynthesis. Here, we investigated the regulatory relationship between AdpA lin and lmbU. We analyzed the 770 bp of promoter region upstream from lmbU TSS, and found 10 putative AdpA binding sites where two of them are overlapped (Supplementary Figure  S2F). Relative expression of lmbU significantly decreased by 26.26 folds in adpA compared with WT ( Figure 3A). We cloned the 770 bp of DNA sequences upstream from lmbU TSS to construct reporter plasmid for catechol dioxygenase assay. In accordance with qRT-PCR results, catechol dioxygenase assay demonstrated that AdpA lin remarkably activated lmbU promoter ( Figure 3B). As displayed in Supplementary Figure  S2F, lmbUp-A, lmbUp-B, and lmbUp-C are three DNA probes containing putative AdpA binding sites in the promoter region of lmbU. EMSAs indicated that AdpA lin directly bound to lmbUp-A, lmbUp-B, and lmbUp-C, separately ( Figure 3C). Then, the putative AdpA binding site in lmbUp-B was deleted, and EMSA showed that AdpA lin could not bind to this DNA probe (Supplementary Figure S4), suggesting that AdpA lin activates the transcription of lmbU by directly binding to the lmbU promoter and thus gets involved in the activation of lincomycin biosynthesis.
AdpA lin Directly Activates the Expression of the Global Regulator Gene bldA As mentioned above, deletion of AdpA lin in S. lincolnensis not only blocked lincomycin biosynthesis, but also significantly impaired the generation of spores (Figures 1B,C), which suggested that AdpA lin had complicated connections with genes outside the lmb cluster. Hou et al. (2018b) previously identified that BldA regulates morphological differentiation and lincomycin biosynthesis in S. lincolnensis. We analyzed the 799 bp of promoter region upstream from the mature bldA, three putative AdpA binding sites were found ( Figure 4A). Relative expression of bldA exhibited a 7.29-fold decreased in adpA compared with WT ( Figure 4B). The 799 bp of DNA sequences upstream from the mature bldA was cloned to construct reporter plasmid for catechol dioxygenase activity assay. And the results indicated that AdpA lin significantly activates the transcription of bldA in vivo ( Figure 4C). BldAp-A and bldAp-B are two DNA probes containing putative AdpA binding sites marked in Figure 4A. We performed EMSAs of AdpA lin with bldAp-A and bldAp-B separately, and as displayed in Figure 4D, AdpA lin directly bound to DNA fragments containing putative AdpA binding sites in vitro. Thus, we speculated that AdpA lin participates in the lincomycin biosynthesis in S. lincolnensis through activating the transcription of bldA, and indirectly mediates the morphological differentiation.
Since AdpA has been identified to regulate the transcription of bldA in S. griseus (Higo et al., 2011), we further analyzed the bldA promoter from S. lincolnensis and S. griseus. The data showed that putative AdpA binding site and its flanking sequence in bldAp-A was highly conserved (Supplementary Figure S5A), and EMSA of AdpA lin with bldAp-A confirmed the binding ( Figure 4D). However, the sequences upstream bldAp-A are various between the two species, which contains the AdpA binding sites in S. griseus, but not in S. lincolnensis (Supplementary Figure  S5A). Further EMSA demonstrated that AdpA lin cannot bind to this fragment bldAp-N (Supplementary Figure S5B), confirming that it is not a functional AdpA binding site in S. lincolnensis. Moreover, putative AdpA binding site in bldAp-B, which has not Solid triangles point to the bands of probes and hollow triangles point to the complexes of AdpA lin and probes. Concentrations of AdpA lin are 0, 1.6, 3.2, 6.4, 6.4, and 6.4 µM, respectively. Competitive assays were carried out with a 200-fold excess of unlabeled specific probe lmbUp-A, lmbUp-B, or lmbUp-B (lane S) or with a 200-fold excess of unlabeled non-specific probe probe-neg (lane N). * * * P < 0.001 and * * P < 0.01, respectively. been studied before, was confirmed to be a AdpA binding site in S. lincolnensis (Supplementary Figure S5C).

AdpA lin Positively Autoregulates Its Own Transcription via Directly Binding to Its Own Promoter
We carried out a detailed analysis of 610 bp of upstream DNA sequences from AdpA lin TSS and found 6 putative AdpA lin binding sites where 4 of them are overlapped in pairs ( Figure 5A). To investigate the transcriptional regulation between AdpA lin and its own transcription, we cloned the 610 bp of DNA sequence upstream from AdpA lin TSS to construct a reporter plasmid for catechol dioxygenase activity assay. The results revealed that AdpA lin slightly activated its own transcription in vivo ( Figure 5B). As displayed in Figure 5A, adpAp-A and adpAp-B are two DNA probes containing putative AdpA lin binding sites, and the results of EMSAs verified that AdpA lin directly interacted with the promoter region of AdpA lin in vitro ( Figure 5C).
AdpA also has been identified to autoregulate its own transcription in S. griseus , then we compared AdpA lin promoter from S. lincolnensis with adpA promoter from S. griseus. In S. lincolnensis, the putative AdpA binding site and its flanking sequence in adpAp-A was high conserved (Supplementary Figure S6A) and the result of EMSA verified the binding (Figure 5C). However, the nucleic acid sequences upstream adpAp-A were highly diverse (Supplementary Figure  S6A). Then we designed two probes, adpAp-B-1 and adpAp-B-2, which contain the unconserved AdpA binding motifs from S. griseus  and putative AdpA binding sites from S. lincolnensis, respectively. The results of EMSAs showed that AdpA lin can bind to adpAp-B-2, but not to adpAp-B-1 (Supplementary Figures S6B,C), indicating that two new AdpA binding sites are found in S. lincolnensis, and the differences between the two probes may allow them to respond to different regulatory mechanisms.

DISCUSSION
In this study, we elucidated the effect of AdpA lin on lincomycin biosynthesis and morphological differentiation. There are 8 putative operons in the lmb cluster. Before this study, none of a regulator has been identified to directly bind to all the eight promoters in the lmb cluster and have an overall impact on the entire lincomycin biosynthesis progress. By deciphering the regulations on the 8 putative operons, we attempted to envision the transcriptional regulatory network on lincomycin biosynthesis. Lincomycin biosynthesis contains three main parts: formation of α-methylthiolincosaminide (MTL), formation of propylproline (PPL), and condensation and final methylation (Supplementary Figure S2A). AdpA lin activates lmbKp and lmbVp, therefore we speculate that AdpA lin directly activates the transcription of lmbK, lmbR, lmbO, and lmbN, so that AdpA lin positively regulates the biosynthesis of MTL structure (Sasaki et al., 2012;Lin et al., 2014). AdpA lin activates the transcription of lmbAp, lmbWp, and lmbUp, which means lmbB1, lmbB2, lmbX and lmbY are also activated by AdpA lin , thus we speculated that AdpA lin directly regulates the biosynthesis of PPL structure (Novotna et al., 2004(Novotna et al., , 2013Pang et al., 2015;Jiraskova et al., 2016). In addition, AdpA lin activates the transcription lmbCp, lmbDp, lmbJp, and lmbVp, which means transcriptions of lmbT, lmbE, lmbF, and lmbG are activated by AdpA lin (Hola et al., 2003;Zhao et al., 2015;Kadlcik et al., 2017;Zhong et al., 2017;Zhang et al., 2018). Therefore, we inferred that AdpA lin functions as a primary activator of lincomycin biosynthesis and regulates the entire biosynthetic process. And this is the first case that AdpA directly activates the transcription of the overall structural genes in such a complicated antibiotic biosynthetic gene cluster (BGC). In addition, EMSAs of AdpA lin with targets showed that AdpA lin binds to different binding sites with different affinities (Figure 2C). Based on these results, some strategies of genetic manipulations may be proposed for hyper-production of lincomycin, such as mutation, deletion or addition of AdpA binding sites in the promoter regions of target genes.
Besides directly participating in lincomycin biosynthesis, as a pleiotropic regulator, AdpA lin controls lincomycin biosynthesis by regulating other transcriptional regulators as well. LmbU has been identified to activate the transcription of lmbA, lmbC, and lmbJ, and repress the transcription of lmbK and lmbU itself (Hou et al., 2018a). In this study, we confirmed that the transcription of lmbU was activated by AdpA lin (Figure 3). As described by Hou et al. (2018b) there is a UUA codon in lmbU, and translation of lmbU is controlled by BldA. Existence of rare codon means very small changes of the tRNA could induce the significant change of protein amount (Chater, 2006). Besides, UUA codons also exist in lmbB2 and lmbY in the lmb cluster, indicating that LmbB2 and LmbY might be important regulatory targets during lincomycin biosynthesis. Furthermore, it has been showed that the adpA gene contains a UUA codon as well, on the other hand, transcription of bldA is regulated by AdpA lin (Figure 4), which may function as a feedback regulatory mechanism to keep the organism in balance. In this study, we speculated that AdpA lin , LmbU, and BldA formed a regulatory cascade that mediate lincomycin biosynthesis in S. lincolnensis (Figure 6). In addition, considering AdpA responds to the GBL-involved cascade regulation (Healy et al., 2009;Tan et al., 2015), bioinformatics analysis was performed and two GBL-signaling systems were found in S. lincolnensis (Figure 6). One system consists of the GBL receptor SLINC_6539 (GenBank accession number ANS68763.1) and biosynthetic enzyme SLINC_6540 (GenBank accession number ANS68764.1) which were highly homology with many receptors and enzymes in Streptomyces, whereas SLINC_6539 and SLINC_6540 had identities with ArpA (47%) and AsfA (70%) in S. griseus, respectively. The other system consists of SLINC_5093 (GenBank accession number WP_067437987.1) and SLINC_5094 (GenBank accession number WP_067437989.1) which were low similarities with other receptors and enzymes, while SLINC_5093 and SLINC_5094 had identities with ArpA (44%) and AsfA (33%) in S. griseus, respectively. But how these two GBL-signaling systems works to affect lincomycin biosynthesis will be needed further research.
In S. griseus, AdpA auto-depresses its own transcription , whereas in S. lincolnensis, AdpA lin has a positive impact on its own transcription ( Figure 5B). Differences of AdpA binding sites in S. lincolnensis and S. griseus indicated that AdpA homologs from different resource have specialized regulatory mechanism on their own transcription. Catechol dioxygenase activity showed that AdpA lin had a negatively effect on the AdpA binding site near the AdpA lin TSS (Data not shown). Thus, we speculated that AdpA lin bound to this putative site and prevented the AdpA lin promotor from being activated.  Frontiers in Microbiology | www.frontiersin.org As for another putative AdpA binding sites, we presumed it might recruit RNA polymerase after interacting with AdpA lin and thus the overall effect of AdpA lin on its own promoter appeared to be positive. In the natamycin producer S. chattanoogensis, AdpA ch was an activator of natamycin biosynthesis, and 6 AdpA binding sites were identified in the scnRI-scnRII intergenic region (Du et al., 2011;Yu et al., 2018). It is notable that although the general effect of AdpA ch on the transcription of scnRI is positive, AdpA binding site A and B serve as repression sites. Thus, we speculated that the varying amounts and locations of AdpA binding sites in the promoter region of AdpA lin targets exhibited different effects and constituted a complicated and subtle regulatory network of AdpA regulons.
In summary, we reported a lesser-known case that AdpA lin interacted with all of the 8 putative operons and activated the transcription of structural genes in the lmb cluster. Furthermore, we deduced AdpA lin , LmbU, and BldA in cascade regulation that controlled lincomycin biosynthesis. Based on these knowledge, more efforts should be devoted to complete the regulatory mechanism of lincomycin biosynthesis and to enhance to production of lincomycin.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the manuscript/Supplementary Files.

AUTHOR CONTRIBUTIONS
YK, BH, and HW designed the experiments. YK, YW, and RW carried out the experiments. YK, BH, XZ, JY, and HW analyzed the data. YK, JY, and HW wrote the manuscript. JY and HZ guided the work. All authors assisted with critical reading of the manuscript.