TetR-Type Regulator SLCG_2919 Is a Negative Regulator ofLincomycin Biosynthesis in Streptomyces lincolnensis

Yurong Xu,a,b Meilan Ke,a Jie Li,a Yaqian Tang,a Nian Wang,a Guoqing Tan,a Yansheng Wang,a Ruihua Liu,e Linquan Bai,d

Lixin Zhang,a,c Hang Wu,a Buchang Zhanga

aSchool of Life Sciences, School of Chemistry & Chemical Engineering, Institute of Physical Science and Information Technology, Anhui University, Hefei, ChinabDepartment of Chemical and Chemical Engineering, Hefei Normal University, Hefei, ChinacState Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, ChinadState Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, ChinaeXinyu Pharmaceutical Co. Ltd., Suzhou, China

ABSTRACT Lincomycin A (Lin-A) is a widely used antibacterial antibiotic fermentedby Streptomyces lincolnensis. However, the transcriptional regulatory mechanisms un-derlying lincomycin biosynthesis have seldom been investigated. Here, we first iden-tified a TetR family transcriptional regulator (TFR), SLCG_2919, which negativelymodulates lincomycin biosynthesis in S. lincolnensis LCGL. SLCG_2919 was found tospecifically bind to promoter regions of the lincomycin biosynthetic gene cluster (lincluster), including 25 structural genes, three resistance genes, and one regulatorygene, and to inhibit the transcription of these genes, demonstrating a directly regu-latory role in lincomycin biosynthesis. Furthermore, we found that SLCG_2919 wasnot autoregulated, but directly repressed its adjacent gene, SLCG_2920, which en-codes an ATP/GTP binding protein whose overexpression increased resistanceagainst lincomycin and Lin-A yields in S. lincolnensis. The precise SLCG_2919 bindingsite within the promoter region of SLCG_2920 was determined by a DNase I foot-printing assay and by electrophoretic mobility shift assays (EMSAs) based on basesubstitution mutagenesis, with the internal 10-nucleotide (nt) AT-rich sequence (AAATTATTTA) shown to be essential for SLCG_2919 binding. Our findings indicate thatSLCG_2919 is a negative regulator for controlling lincomycin biosynthesis in S. lincol-nensis. The present study improves our understanding of molecular regulation forlincomycin biosynthesis.

IMPORTANCE TetR family transcriptional regulators (TFRs) are generally found toregulate diverse cellular processes in bacteria, especially antibiotic biosynthesis inStreptomyces species. However, knowledge of their function in lincomycin biosynthe-sis in S. lincolnensis remains unknown. The present study provides a new insight intothe regulation of lincomycin biosynthesis through a TFR, SLCG_2919, that directlymodulates lincomycin production and resistance. Intriguingly, SLCG_2919 and its ad-joining gene, SLCG_2920, which encodes an ATP/GTP binding protein, were exten-sively distributed in diverse Streptomyces species. In addition, we revealed a new TFRbinding motif, in which SLCG_2919 binds to the promoter region of SLCG_2920, de-pendent on the intervening AT-rich sequence rather than on the flanking invertedrepeats found in the binding sites of other TFRs. These insights into transcriptionalregulation of lincomycin biosynthesis by SLCG_2919 will be valuable in paving theway for genetic engineering of regulatory elements in Streptomyces species to im-prove antibiotic production.

KEYWORDS lincomycin, SLCG_2919, Streptomyces lincolnensis, TetR familytranscriptional regulator

Citation Xu Y, Ke M, Li J, Tang Y, Wang N, TanG, Wang Y, Liu R, Bai L, Zhang L, Wu H, Zhang B.2019. TetR-type regulator SLCG_2919 is anegative regulator of lincomycin biosynthesisin Streptomyces lincolnensis. Appl EnvironMicrobiol 85:e02091-18. https://doi.org/10.1128/AEM.02091-18.

Editor Harold L. Drake, University of Bayreuth

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Hang Wu,wuhang@ahu.edu.cn, or Buchang Zhang,zhbc@ahu.edu.cn.

Received 28 August 2018Accepted 1 October 2018

Accepted manuscript posted online 19October 2018Published

GENETICS AND MOLECULAR BIOLOGY

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Streptomyces lincolnensis is a Gram-positive actinomycete that is generally utilized forthe industrial-scale production of a lincosamide antibiotic, lincomycin A (Lin-A),

which consists of an �-methylthiolincosaminide (MTL) and an amino acid derivative,propylproline (PPL) (1). Lin-A and its semisynthetic derivative clindamycin are usedclinically for the treatment of infective diseases caused by Gram-positive bacteria (2).Clindamycin can also be used for the treatment of protozoal diseases, e.g., malaria (2).The lincomycin biosynthetic gene cluster (lin cluster) contains 29 genes that encodeproteins for biosynthesis, resistance, and regulation, spanning over 35 kb of DNA in S.lincolnensis (3, 4). In recent years, genetic and biochemical strategies have beenperformed to explore the molecular mechanisms of lincomycin biosynthesis (5–7).Through heterologous expression, key gene inactivation, and in vitro combinatorialbiosynthesis, some lincosamide derivatives with relevant bioactivities have been ob-tained in the engineered strains (7, 8).

Recently, Meng et al. confirmed that the ABC1 transporter gene and the lincomycinexport gene, lmrA, were transcriptionally activated by the global regulator GlnR withnitrate supplementation (9). Subsequently, Hou et al. showed that LmbU from the lincluster served as a cryptic cluster-situated regulator (CSR), promoting lincomycinproduction (10). Yet, the transcriptional regulatory mechanisms underlying lincomycinbiosynthesis remain obscure, limiting production improvements for Lin-A.

The TetR family transcriptional regulators (TFRs) are widespread in bacteria, playingimportant roles in a series of diverse processes (11, 12). It was reported that TFRs bindto consensus or apparent palindromic DNA sequences and could regulate the biosyn-thesis of secondary metabolites in actinomycetes (11, 12). It is worthwhile to note thatgenetic engineering of TFR elements has been shown to increase the yields of valuableantibiotics in industrial settings (13–15). Nonetheless, distinct from research related toTFRs for the biosynthesis of avermectin and erythromycin (13, 15–19), insights into theregulatory role of TFRs in lincomycin biosynthesis have not been reported. In thegenome of S. lincolnensis LCGL, a derivative of S. lincolnensis LC-G with artificialsynthetic 4�attB�C31 (20), we identified 123 putative TFRs based on BLAST analysiswith Pfam PF00440 (TetR_N) and the genome annotation of S. lincolnensis LC-G.Through in vivo and in vitro evidence, we identify a TFR (SLCG_2919) that regulateslincomycin biosynthesis in S. lincolnensis.

RESULTSSLCG_2919 negatively regulates lincomycin biosynthesis. To search for potential

TFRs associated with lincomycin production, we inactivated a number of TFRs in strainLCGL and successively obtained these desired mutants by thiostrepton resistancescreening and PCR confirmation (Fig. 1A and B, with the currently studied SLCG_2919strain as an example). By shake-flask fermentation and ultraperformance liquid chro-matography (UPLC) analysis, we found that the ΔSLCGL_2919 mutant displayed ahigher yield of Lin-A than its parental strain LCGL, with �25% improvement, and wasselected for further investigation.

According to the available genome sequence of S. lincolnensis LC-G, SLCG_2919consists of 210 amino acids with a molecular mass of approximately 24 kDa. Thelocations of SLCG_2919 and its adjacent genes on the chromosome are shown inFig. 1A. SLCG_2919 is adjoined with genes encoding a putative ATP/GTP bindingprotein and a phosphotransferase (Fig. 1A), but they are not cotranscriptional (Fig.S1). Furthermore, complementation of the SLCG_2919 gene in the ΔSLCGL_2919mutant showed approximate recovery of Lin-A production (Fig. 1C), suggesting thatSLCG_2919 negatively regulates lincomycin biosynthesis in S. lincolnensis. To furtherconfirm the negatively regulatory role of SLCG_2919 in lincomycin production, pIB139-2919, as well as pIB139, was introduced into strain LCGL. Results showed that the Lin-Ayield of LCGL/pIB139-2919 was 15% lower than that of the control LCGL/pIB139(P � 0.01) (Fig. 1C).

TFRs often play much broader roles, such as in antibiotic production and inmorphological differentiation (21, 22), so we compared LCGL and the ΔSLCGL_2919

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mutant in cell growth and morphological differentiation. The results showed that theΔSLCGL_2919 mutant and the parental strain, LCGL, had similar growth rates in yeast-malt-glucose (YMG) culture, as measured by mycelium dry weight, and had similarsporulation rates on MGM agar medium (Fig. S2), indicating that SLCG_2919 was notinvolved in cell growth and morphological differentiation of S. lincolnensis.

To examine the widespread use of SLCG_2919 as a negative regulator for lincomycinbiosynthesis in S. lincolnensis, SLCG_2919 was deleted in the high-yield strain S. lincol-nensis LA219X (20). As expected, Lin-A production of the obtained S. lincolnensisΔSLA219X_2919 mutant (2.89 g/liter) was 15% higher than that of LA129X (2.51 g/liter)when cultured in 30 ml of industrial fermentation medium for 7 days (Fig. 1D).

Determination of transcription units found in the lin cluster. Peschke et al. (3)had shown eight individual transcription units (lmrA, lmrB, lmrC, lmbC, lmbD, lmbK,lmbW, and lmbA-B1-B2) in the lin cluster of S. lincolnensis. In this study, we confirmedthe above eight units and determined the remaining transcription units in the lincluster, including one monocistronic transcription unit (lmbE) and four multicistronic

FIG 1 SLCG_2919 negatively regulates lincomycin production in S. lincolnensis LCGL. (A) Schematic deletion of SLCG_2919 byhomologous recombination in S. lincolnensis LCGL. (B) PCR confirmation of the SLCG_2919 deletion mutant by the primers 2919-P5and 2919-P6. Lane M, 5,000-bp DNA ladder; lane 1, the positive control, 1,500 bp amplified from pKC1139-Δ2919; lane 2, the negativecontrol, 450 bp amplified from LCGL; lane 3, 1,500 bp amplified from the ΔSLCGL_2919 mutant. (C) Lin-A production of S. lincolnensisLCGL and its derivatives. (D) Lin-A production of S. lincolnensis LA219X and ΔSLA219X_2919. Mean values of three replicates are shown,with the standard deviation indicated by error bars. *, P � 0.05; **, P � 0.01.

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transcription units (lmbR-S-O-P-Z-N-M-L, lmbJ-IH-G-F, lmbV-T-Q, and lmbU-Y-X) (Fig. 2Aand B). These results established a foundation for investigating the effects of regulatoryfactors on the lin cluster.

SLCG_2919 directly represses the transcription of structural genes in the lincluster. To investigate the regulatory mode of SLCG_2919, we assessed the influenceof SLCG_2919 on the expression of structural genes found in the lin cluster by reversetranscription-quantitative PCR (qRT-PCR). The results showed that the transcriptionallevels of the structural genes in the ΔSLCGL_2919 mutant, including lmbA, lmbC, lmbE,lmbG, lmbK, lmbR, lmbV, and lmbW, were 1.6-, 3.7-, 3.2-, 2.5-, 2.6-, 22.0-, 4.3-, and 2-foldhigher, respectively, than those of LCGL (Fig. 3A). Only the transcriptional level of lmbDwas not obviously influenced. These results indicated that SLCG_2919 had a negativeeffect on lincomycin biosynthesis by repressing the transcription of the structuralgenes.

To examine whether SLCG_2919 might directly regulate the transcription of struc-tural genes in lincomycin biosynthesis, we expressed His6-tagged SLCG_2919 in Esch-erichia coli BL21(DE3) (Fig. S3) and examined its affinity with the promoter regions oflmbA, lmbC-lmbD, lmbE, lmbJ-lmbK, lmbR, and lmbV-lmbW. As detected by electropho-retic mobility shift assays (EMSAs), mobility shifts were obviously detected upon theaddition of different concentrations of His6-SLCG_2919 (Fig. 3B). When 20-fold unla-beled probes were added into the reaction system, they dramatically competed withlabeled probes for binding to His6-SLCG_2919 (Fig. 3B). As a negative control, anonspecific DNA, poly(dI-dC), was used to compete with labeled probe, and the shiftedband did not disappear, thereby indicating that SLCG_2919 bound specifically to theabove promoter regions. These findings revealed that SLCG_2919 directly represseslincomycin biosynthetic structural genes in S. lincolnensis.

SLCG_2919 directly represses transcription of resistance genes in the lin clus-ter. In the lin cluster, there are three lincomycin resistance genes, lmrA, lmrB, and lmrC,with ImrA encoding a proton-dependent lincomycin transporter, ImrB encoding a 23SrRNA adenine(2058)-N-MTase, and ImrC encoding a member of the ABC transporterfamily (3, 23). We found that when SLCG_2919 was inactivated in LCGL, transcriptionallevels of the three resistance genes were enhanced by 6-, 1.8-, and 2.5-fold, respectively(Fig. 4A). The results from EMSAs showed that His6-SLCG_2919 could specifically bindto the promoter regions of lmrA, lmrB, and lmrC (Fig. 4B, C and D), indicating thatSLCG_2919 also directly represses the three resistance genes in the lin cluster from S.lincolnensis.

FIG 2 Determination of transcription units in the lin cluster. (A) The validation of the transcription units of the lin cluster by fourteencross-spaced primers (Table S1). Lane M, 5,000-bp DNA ladder; lanes 1 to 14, the PCR products from cDNA by fourteen cross-spacedprimers. (B) Diagram of the transcription units of the lin cluster. Bent arrow, transcription site and direction. The numbers in the circlesindicate locations of fourteen cross-spaced primers.

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SLCG_2919 directly represses transcription of the CSR gene lmbU. The functionof a positive cluster-situated regulator (CSR), LmbU, from the lincomycin biosyntheticpathway was recently described (10). To find a relationship between SLCG_2919 andLmbU, we compared transcriptional levels of lmbU between LCGL and the ΔSLCGL_2919mutant. Results showed that lmbU was transcriptionally increased by 8-fold in theΔSLCGL_2919 mutant in comparison to the level in LCGL (Fig. 5A). Since the distance ofthe intergenic region between lmbU and lmrC is 757 nucleotides (nt), we designed twoDNA fragments with no overlapped sequence, PlmbU1 (372 nt) and PlmbU2 (385 nt), forEMSAs. The results revealed that His6-SLCG_2919 bound to either PlmbU1 or PlmbU2 (Fig.5B). Therefore, these data indicated that SLCG_2919 directly represses the expression ofthe regulatory gene lmbU in S. lincolnensis.

SLCG_2919 directly represses transcription of its adjacent gene, SLCG_2920. Itwas previously found that TFRs generally have significant effects on their adjacentgenes (11). To investigate whether SLCG_2919 directly binds to its adjacent genes andto its own promoter regions, His6-SLCG_2919 was individually used to bind with the

FIG 3 SLCG_2919 directly represses transcription of the lin cluster structural genes. (A) Quantitative transcriptionlevels of lmbA, lmbC, lmbD, lmbE, lmbG, lmbK, lmbR, lmbV, and lmbW in LCGL and the ΔSLCGL_2919 mutant culturedfor 24 h in fermentation medium. Mean values of three replicates are shown, with the standard deviation indicatedby error bars. *, P � 0.05. (B) EMSAs of His6-SLCG_2919 with the promoter regions of lmbA, lmbC-lmbD, lmbE,lmbJ-lmbK, lmbR, and lmbV-lmbW. Each lane contained 150 ng of DNA probes. S, unlabeled specific probe (20-fold)was added; N, nonspecific probe poly(dI-dC) (20-fold) was added.

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promoter regions of SLCG_2918, SLCG_2919, and SLCG_2920. As shown in Fig. 6A, agel-based band shift was detected at 50 nM His6-SLCG_2919, with the promoter regionof SLCG_2920. However, SLCG_2919 could not bind to the promoter region ofSLCG_2918 or to its own promoter region, even when it reached the concentration of2 �M (data not shown).

To explore whether SLCG_2919 also regulates the transcription of its adjacent geneSLCG_2920, encoding a putative ATP/GTP binding protein, qRT-PCR was performedwith RNA isolated from S. lincolnensis LCGL and from the ΔSLCGL_2919 mutant culturedfor 24 h in fermentation medium. The transcriptional level of SLCG_2920 in the

FIG 4 SLCG_2919 directly represses transcription of resistance genes in the lin cluster. (A) Quantitative transcrip-tional levels of lmrA, lmrB, and lmrC in LCGL and the ΔSLCGL_2919 mutant cultured for 24 h in fermentationmedium. (B to D) EMSAs of His6-SLCG_2919 with the promoter regions of lmrA (B), lmrB (C), and lmrC (D). Each lanecontained 150 ng of DNA probes. S, unlabeled specific probe (20-fold) was added; N, nonspecific probe poly(dI-dC)(20-fold) was added. *, P � 0.05; **, P � 0.01.

FIG 5 SLCG_2919 directly represses transcription of the CSR gene lmbU. (A) Quantitative transcriptionallevel of lmbU in LCGL and the ΔSLCGL_2919 mutant cultured for 24 h in fermentation medium. (B) EMSAsof His6-SLCG_2919 with the promoter regions of lmbU (PlmbU1 and PlmbU2). Each lane contained 150 ng ofDNA probes. S, unlabeled specific probe (20-fold) was added; N, nonspecific probe poly(dI-dC) (20-fold) wasadded. **, P � 0.01.

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ΔSLCGL_2919 mutant exhibited a 2-fold increase compared with that in LCGL (Fig. 6B).Taken together, these results demonstrated that SLCG_2919 directly represses thetranscription of SLCG_2920.

Overexpression of SLCG_2920 increases Lin-A production by improving linco-mycin resistance in S. lincolnensis LCGL. Since an ATP/GTP binding protein is likelycoupled with a transmembrane protein that may be involved in antibiotic resistance(24, 25), we investigated lincomycin resistance by overexpressing SLCG_2920 in LCGL.As shown in Fig. 7A, when lincomycin concentrations reached 350 �g/ml, LCGL/pIB139-2920 could grow normally, while the growth of LCGL/pIB139 was inhibited, suggestingthat overexpression of SLCG_2920 effectively improved the resistance against lincomy-cin in S. lincolnensis LCGL. Meanwhile, LCGL/pIB139-2920 showed 14% improvement inthe production of Lin-A compared with that of LCGL/pIB139 (P � 0.05) (Fig. 7B),indicating that SLCG_2920 positively affects Lin-A production through improving lin-comycin resistance.

AT-rich sequence in the promoter region of SLCG_2920 is the precise site ofSLCG_2919 binding. To precisely determine the SLCG_2919 binding site in thepromoter region of SLCG_2920, a DNase I footprinting assay was performed using6-carboxyfluorescein (FAM)-labeled DNA fragments. We found that there was only oneSLCG_2919 binding site, a 24-nt sequence (ACCGAGT-AAATTATTTA-ACTCGGT) thatincluded identical 7-nt inverted repeats separated by an internal 10-nt AT-rich se-quence (Fig. 8A and B). To confirm the importance of the 24-nt sequence forSLCG_2919 binding, EMSAs were performed, with SLCG_2919 binding to the originalDNA fragment P1 and to mutated DNA fragment P1m without the above 24-nt

FIG 6 SLCG_2919 directly represses transcription of its adjacent gene. (A) EMSA of His6-SLCG_2919 withthe promoter region of SLCG_2920. Each lane contained 150 ng of DNA probes. S, unlabeled specificprobe (20-fold) was added; N, nonspecific probe poly(dI-dC) (20-fold) was added. (B) Quantitativetranscriptional level of SLCG_2920 in LCGL and the ΔSLCGL_2919 mutant cultured for 24 h in fermentationmedium.

FIG 7 Overexpression of SLCG_2920 increases Lin-A resistance and Lin-A production in S. lincolnensis LCGL. (A) Resistance assay ofLCGL/pIB139 and LCGL/pIB139-2920 strains against Lin-A. (B) Lin-A production of S. lincolnensis LCGL/pIB139 and LCGL/pIB139-2920. *,P � 0.05.

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sequence. In contrast to P1, P1m did not show the band shift (Fig. 8B). The bindingsequence of SLCG_2919 is located in the upstream region of SLCG_2920, extendingfrom nucleotide �17 to �6 with respect to the first nucleotide of the predictedtranscriptional start site of SLCG_2920 (Fig. 8C).

FIG 8 Analysis of the precise SLCG_2919 binding site. (A) Determination of SLCG_2919 binding site in the promoter region of SLCG_2920 by DNase I footprintingassay. Top fluorogram shows control reaction without protein. Protection regions were acquired with increasing concentrations (0.5 �M and 0.75 �M) ofHis6-SLCG_2919 protein. (B) EMSAs of SLCG_2919 binding to P1 and P1m (lacking 24-nt sequence). Underlining indicates inverted repeats. (C) Nucleotidesequences of SLCG_2920 promoter region and SLCG_2919 binding site. Bigger black font, SLCG_2920 transcription start site (TSS); black dotted boxes, putative�10 and �35 regions and start codon; underlining, SLCG_2919 binding site. (D) The different base substitution mutagenesis of the binding site (24 nt). (E)EMSAs of SLCG_2919 binding to the fragment P1 (wild type [WT]) and mutated fragments P2, P3, P4, and P5.

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TFRs generally form homodimeric structures and bind to consensus or apparentpalindromic DNA sequences (11, 26). To distinguish the importance of the invertedrepeats from the AT-rich sequence in the promoter region of SLCG_2920, we con-structed DNA fragments with different base substitutions in the 24-nt sequence, withDNA fragment P2 mutated in the left inverted repeat, DNA fragment P3 in the AT-richsequence, DNA fragment P4 in the right inverted repeat, and DNA fragment P5 in bothinverted repeats (Fig. 8D). Unexpectedly, DNA fragments P2, P4, and P5 showed thesame band shift as DNA fragment P1 at a 0.1 �M concentration of SLCG_2919, whileDNA fragment P3 did not exhibit the binding activity (Fig. 8E). These findings demon-strated that the internal AT-rich (AAATTATTTA) sequence, but not the flanking invertedrepeats, was indispensable for SLCG_2919 binding.

DISCUSSION

As a large and important family of one-component signal transduction systems,TFRs are widely associated with metabolism, antibiotic production, quorum sensing,and multidrug resistance (15, 27). In the present study, we discovered that SLCG_2919can regulate lincomycin biosynthesis by repressing the transcription of lincomycinbiosynthetic structural genes, resistance genes, and a regulatory gene in the lin cluster,as well as by repressing its adjacent gene, SLCG_2920, which encodes an ATP/GTPbinding protein. We are not aware of any other TFR besides SLCG_2919 in S.lincolnensis.

Based on genome context analysis, Ahn et al. (28) classified TFRs into three groups.The first group is divergently oriented to one of its neighboring genes, which can beused to predict a regulatory relationship between the two genes. The second group islikely to be cotranscribed with its upstream or downstream neighboring gene whenseparated by 35 bp or less, usually known to be autoregulatory or to regulate theexpression of its cotranscribed genes. The third group lacks a defined relationship withits adjacent genes. To date, little is known about the third group of TFRs. In our study,we have demonstrated that SLCG_2919 is not cotranscribed with its neighboring genes(Fig. S1), so it belongs to the third group, which directly represses transcription of itsadjacent gene, SLCG_2920. This report provides the evidence for further understandingof the third group of TFRs.

SLCG_2919 is located at approximately 3.5 Mb in the chromosome of S. lincolnensisLC-G and is not closely positioned with the lin cluster (GenBank accession no.CP022744; nt 290041 to 322634, 0.3 Mb). In addition, SLCG_2919 was here proved tobind to all promoter regions of the lin cluster, implying that SLCG_2919 remotelyexhibited a directly regulatory role in lincomycin biosynthesis. Although SLCG_2919bound specifically to the promoter region of lmbC-lmbD, only lmbC was differentiallytranscribed upon SLCG_2919 inactivation. We suppose that an unknown regulator andSLCG_2919 might collaboratively or competitively modulate the transcription of lmbD.Recently, the CSR LmbU was shown to directly activate expression of the lmbA andlmbW genes and indirectly stimulate the expression of lmbC and lmbJ, while indirectlyrepressing the expression of lmbK and lmbU (10). These findings implied a morecomplex transcriptional regulatory mechanism of lincomycin biosynthesis than previ-ously expected.

Members of the TetR family of regulators can repress genes whose products areinvolved in multidrug resistance (12). SLCG_2920, a putative ATP/GTP binding protein,is likely related to an ABC transporter that may be involved in antibiotic resistance andproduction (24, 25). In this study, overexpression of the SLCG_2920 gene effectivelyincreased the resistance against lincomycin and the yield of Lin-A in S. lincolnensis.Therefore, we speculate that SLCG_2920 might be involved in exporting lincomycinalong with a transmembrane protein and thus reduce the feedback inhibition onlincomycin production inside the cell. A similar phenomenon was also detected inStreptomyces chattanoogensis, in which multiple transporters were identified to beinvolved in the export of natamycin (29).

Besides the CSR LmbU (10), SLCG_2919 was another regulator participating in

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lincomycin biosynthesis. SLCG_2919 not only regulated genes in the lin cluster, but alsoregulated its adjacent gene, SLCG_2920, suggesting that SLCG_2919 might controlLin-A biosynthesis in multiple ways. By BLASTP analysis, we found that SLCG_2919homologs were broadly distributed in typical antibiotic-producing Streptomyces spe-cies, such as SCO4194 from Streptomyces coelicolor A3(2) (74.9% identity), SAV_4017from Streptomyces avermitillis MA-4680 (79.1% identity), SGR_3980 from Streptomycesgriseus IFO13350 (70.6% identity), etc. (Fig. S4A). More importantly, the cassette ofSLCG_2919 and its adjoining gene, SLCG_2920, which encodes an ATP/GTP bindingprotein, mostly exists in those Streptomyces species (Fig. S4B). So far, these TFRs havenot yet been functionally investigated, potentially demonstrating a novel transcrip-tional regulatory paradigm for antibiotic biosynthesis in Streptomyces species.

Many TFRs generally form homodimeric structures and bind to consensus or appar-ent palindromic DNA sequences (26). However, we found an unusual motif in theSLCG_2919 binding sequence, in which an AT-rich sequence (AAATTATTTA), rather thana common palindromic DNA sequence, was the most important for TFR binding. Inaddition, PREDetector analysis (http://www.montefiore.ulg.ac.be/~hiard/PreDetector/PreDetector.php) of the AT-rich sequence (AAATTATTTA) in the genome of S. lincol-nensis LC-G revealed that only partial SLCG_2919 binding sites were predicted withinthe lin cluster genes’ promoter regions (lmbJ-lmbK, lmbV-lmbW, lmrA, lmrB, lmrC, andlmbU) (Fig. S5). We suspect that there exist different binding models in other lin clusterpromoter regions (lmbA, lmbC-lmbD, lmbE, and lmbR) for SLCG_2919 regulation, whichneeds to be further investigated.

Based on the present data, we propose a model for the SLCG_2919-mediatedtranscriptional regulatory network (Fig. 9). SLCG_2919 exerts its negative regulatoryeffect on lincomycin production in at least three ways, as follows: (i) directly repressingtranscription of lincomycin biosynthetic structural genes, (ii) directly repressing expres-sion of resistance genes inside or outside the lin cluster, and (iii) directly controlling theCSR gene lmbU.

MATERIALS AND METHODSStrains, plasmids, and growth conditions. All strains and plasmids used in this study are listed in

Table 1. E. coli was cultured in Luria Bertani (LB) medium at 37°C, supplemented with appropriateantibiotics as required, with shaking at 220 rpm (30). Liquid TSBY medium (3% tryptone soya broth, 0.5%

FIG 9 Proposed model of the SLCG_2919-mediated regulatory network in S. lincolnensis. SLCG_2919 exerts regulatory effects on lincomycin biosynthesis byinteracting with the promoter regions of structural genes, resistance genes, or the CSR gene. Solid arrows, activation; bars, repression; solid lines, direct control;dashed lines, indirect control.

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yeast extract, and 10.3% sucrose, with/without apramycin or thiostrepton) was used for DNA extraction(31). S. lincolnensis LCGL and its derivatives were grown on MGM (2% soluble starch, 0.5% soybean flour,0.1% KNO3, 0.05% NaCl, 0.05% MgSO4, 0.05% K2HPO4, 0.001% FeSO4, and 2% agar, with/withoutapramycin or thiostrepton) for sporulation. Spores were isolated and stored in 20% glycerol at �80°C.Liquid SM medium (0.4% yeast extract, 0.4% tryptone soya broth, 1% glucose, 0.05 g/liter MgSO4,0.2 g/liter KH2PO4, and 0.4 g/liter K2HPO4) was used for S. lincolnensis protoplast preparation (32).

Gene inactivation, complementation, and overexpression. The plasmid pKC1139-Δ2919, with aninternal 450-bp deletion of SLCG_2919, was constructed in two steps. First, with the genomic DNA ofLCGL as the template, two 1.7-kb fragments flanking SLCG_2919 were respectively amplified by PCRusing two primer pairs, 2919-P1/2919-P2 and 2919-P3/2919-P4 (Table S1), cleaved by HindIII/XbaI andKpnI/EcoRI, and ligated into the corresponding sites of pUCTSR (33), yielding pUCTSRΔ2919. Second, the4.8-kb DNA fragment was cleaved with EcoRI/HindIII from pUCTSRΔ2919 and ligated into the same siteof pKC1139 (34), generating pKC1139-Δ2919. By polyethylene glycol (PEG)-mediated protoplast trans-formation, pKC1139-Δ2919 was transformed into S. lincolnensis LCGL. The transformants resistant tothiostrepton but sensitive to apramycin were selected as the ΔSLCGL_2919 double-crossover strain andconfirmed by PCR amplification, using the primers 2919-P5 and 2919-P6 (Table S1).

For the complementation of SLCG_2919 in the ΔSLCGL_2919 mutant, SLCG_2919 was amplified fromthe genomic DNA of S. lincolnensis by PCR, using the primers 2919-P7 and 2919-P8 (Table S1). The PCRproduct was digested with NdeI/XbaI and inserted into the corresponding sites of pIB139, generatingpIB139-2919. Then, pIB139-2919, as well as pIB139, was successively transformed into the ΔSLCGL_2919mutant, and the ΔSLCGL_2919/pIB139-2919 complementation strain, as well as the ΔSLCGL_2919/pIB139control strain, was obtained by apramycin resistance screening and further confirmed by PCR amplifi-cation with the primers apr-P1 and apr-P2 (Table S1). Furthermore, pIB139 and pIB139-2919 were alsotransformed into LCGL, generating the control strain LCGL/pIB139 and overexpressed strain LCGL/pIB139-2919, respectively.

For overexpression of SLCG_2920 in LCGL, a 2,031-bp DNA fragment containing a full-lengthSLCG_2920 was amplified with the primers 2920-F and 2920-R (Table S1). Then, the NdeI/XbaI-digestedfragment was inserted into the corresponding sites of pIB139, and the constructed pIB139-2920 wastransformed into LCGL to obtain the overexpressed strain LCGL/pIB139-2920.

In accordance with above procedures, we constructed the ΔSLA219X_2919 mutant using a high-yieldstrain, S. lincolnensis LA219X.

Fermentation and UPLC analysis of Lin-A production. S. lincolnensis LCGL and its derivatives weregrown on MGM for sporulation (with appropriate antibiotics for recombinant strains). A 1-ml aliquot ofspore suspension (�1 � 107 CFU/ml) was inoculated into a 250-ml flask containing 30 ml of the seed

TABLE 1 Bacterial strains and plasmids used in this study

Strain or plasmid Description Reference or source

StrainsE. coli

DH5� F recA lacZM15 30BL21(DE3) F� ompT hsdSB(rB

�mB�) dcm gal �(DE3) Novagen

S. lincolnensisLC-G CGMCC7.209, a lincomycin producer Xinyu Pharmaceutical

Co., Ltd.LCGL LC-G derivative with artificial integrated attB�C31 site 20ΔSLCGL_2919 LCGL derivative with SLCG_2919 deleted This studyΔSLCGL_2919/pIB139 ΔSLCGL_2919 strain carrying pIB139 This studyΔSLCGL_2919/pIB139-2919 ΔSLCGL_2919 strain carrying pIB139-SLCG_2919 This studyLCGL/pIB139 LCGL carrying pIB139 This studyLCGL/pIB139-2919 LCGL carrying pIB139-SLCG_2919 This studyLCGL/pIB139-2920 LCGL carrying pIB139-SLCG_2920 This studyLA219 A lincomycin high-yield strain Xinyu Pharmaceutical

Co., Ltd.LA219X LA219 derivative with artificial integrated attB�C31 site 20ΔSLA219X_2919 LA219X derivative with SLCG_2919 deleted This study

PlasmidspUCTSR pUC18 derivative containing a 1.36-kb fragment of a thiostrepton resistance gene in

BamHI/SmaI sites33

pUCTSRΔ2919 pUCTSR derivative containing two 1.5-kb fragments, the upstream and downstreamregions of SLCG_2919

This study

pKC1139 ori (pSG5), aac(3)-IV, lacZ 34pKC1139-Δ2919 pKC1139 derivative for SLCG_2919 deletion This studypIB139 �C31 attP-int locus, acc(3)-IV, oriT, PermE* promoter 13pIB139-2919 pIB139 carrying an extra SLCG_2919 for gene complementation This studypIB139-2920 pIB139 carrying an extra SLCG_2920 for gene complementation This studypET28a T7 promoter, His tag, kan NovagenpET28a-2919 pET28a-derived plasmid carrying SLCG_2919 This study

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medium [2% soluble starch, 1% glucose, 1% soybean flour, 3% cream corn, 0.15% (NH4)2SO4, and 0.4%CaCO3 for culture, with/without apramycin] at 30°C with shaking at 240 rpm for 2 days. A 2-ml aliquot ofseed culture was transferred into 30 ml fermentation medium [10% glucose, 2% soybean flour, 0.15%cream corn, 0.8% NaNO3, 0.5% NaCl, 0.6% (NH4)2SO4, 0.03% K2HPO4, and 0.8% CaCO3, with/withoutapramycin]. All fermentation cultures were incubated at 30°C and 240 rpm for 7 days. After fermentation,200 �l supernatant of fermentation broth was mixed with 800 �l ethanol and centrifuged at 12,000 rpmfor 10 min to remove the residue. Subsequently, lincomycin samples extracted from those liquidfermentation cultures were quantified by a Waters H13CHA 394G UPLC system on an Extend-C18 column(5 �m, 150 � 4.6 mm; Agilent), which was equilibrated with 60% methyl alcohol and 40% 5 mM ammo-nium acetate (pH 9.0). An isocratic program was carried out at a flow rate of 0.4 ml/min. The productswere monitored at 214 nm (Fig. S6).

Determination of transcription units of the lin cluster. Total RNA was isolated from S. lincolnensisLCGL after 24 h of growth in fermentation liquid medium using an RNA extraction/purification kit (SBS),and the RNA concentration was determined using a microplate reader (BioTek). Isolated RNA (500 ng)was treated with DNase I (MBI Fermentas), and reverse transcription was performed using a cDNAsynthesis kit (MBI Fermentas). Fourteen primer pairs were designed to amplify intergenic regions in thelin cluster (Table S1). The desired fragments were amplified using cDNA of LCGL as a template with theabove-mentioned primer pairs.

In order to verify whether SLCG_2918, SLCG_2919, and SLCG_2920 were cotranscribed, PCR wasperformed by using cDNA of LCGL as a template with 2918-2919-P1/2918-2919-P2 and 2919-2920-P1/2919-2920-P2 primers (Table S1).

Transcriptional analysis by qRT-PCR. The relative transcriptional levels of structural genes (lmbA,lmbC, lmbD, lmbE, lmbG, lmbK, lmbR, lmbV, and lmbW), resistance genes (lmrA, lmrB, and lmrC), aregulatory gene (lmbU), and SLCG_2920 were determined by qRT-PCR analyses. Specific primers weredesigned as listed in Table S1. In accordance with the above procedures, cDNAs were achieved from RNAsamples (1 �g) after the DNase I treatment and reverse transcription. Then cDNAs were diluted 10-foldas the templates for qRT-PCR. The reaction volume of 20 �l is composed of 0.5 �l per primer (0.25 mM),10 �l Maxima SYBR green/Rox qPCR master mix (MBI Fermentas), 2 �l diluted cDNA template (120 ng),and 7 �l RNase-free water. qRT-PCR was performed on the Applied Biosystems QuantStudio 6 Flexsystem with Maxima SYBR green/ROX qPCR master mix (MBI Fermentas). The reaction protocol consistedof 95°C for 10 min, followed by 40 cycles of 95°C for 19 s and 60°C for 35 s. The melting curve wasinserted, ramping from 65°C to 95°C (increment 0.5°C/5 s), to verify specificity of primer amplificationbased on the presence of a single and sharp peak. The rpoD gene in S. lincolnensis was used as theinternal control, and relative transcription was quantified using a comparative cycle threshold method(35).

Expression and purification of SLCG_2919. The DNA fragment containing intact SLCG_2919 wassuccessively obtained by PCR using the primers 2919-P9/2919-P10, listed in Table S1. The PCR productwas digested with NdeI/HindIII and inserted into the corresponding site of pET-28a (Novagen), thenintroduced into E. coli BL21(DE3). The transformed cells were grown in 50 ml LB liquid medium at 37°Cuntil the optical density at 600 nm (OD600) reached 0.4 to 0.6 and then induced with isopropyl-�-D-thiogalactopyranoside (IPTG) at a final concentration of 0.2 mM at 16°C for 20 h. The cells were harvestedby centrifugation (8,000 rpm, 10 min, 4°C), resuspended in lysis buffer (20 mM Tris and 500 mM NaCl, pH8.0) and disrupted by ultrasonication at 4°C. The lysate was centrifuged (12,000 rpm, 30 min, 4°C), and theHis6-tagged SLCG_2919 protein present in the supernatant was recovered using a Ni2�-nitrilotriaceticacid (NTA) spin column (Bio-Rad). SLCG_2919 was eluted from the column with eluting buffer (20 mMTris, 300 mM imidazole, and 500 mM NaCl, pH 8.0). The purified protein was desalted by a molecular sievecolumn (AKTA primePlus) with buffer A (20 mM Tris and 200 mM NaCl, pH 8.0). The concentration ofpurified protein was quantified by bicinchoninic acid (BCA) assays, and the purity was judged bySDS-PAGE analysis.

Electrophoretic mobility shift assays. The EMSAs were performed as reported by Hellman andFried (36). The promoter regions of structural genes (lmbA, lmbC-lmbD, lmbE, lmbJ-lmbK, lmbR, andlmbV-lmbW), resistance genes (lmrA, lmrB, and lmrC), the regulatory gene (lmbU), SLCG_2918, SLCG_2919,and SLCG_2920 were amplified by PCR with their respective primers (Table S1). The binding reactionsystem consisted of 10 mM Tris (pH 7.5), 5 mM MgCl2, 50 mM EDTA, 60 mM KCl, 10 mM dithiothreitol(DTT), 10% glycerol, 150 ng labeled probes, and 0.01 to 2 �M purified His6-tagged SLCG_2919 in a totalvolume of 20 �l. After incubation of the mixture at 30°C for 15 min, the samples were separated on 6%native PAGE gels in ice-cold 1� Tris-acetate-EDTA (TAE) buffer at 50 mA for about 40 min.

Analysis of SLCG_2919 binding site in the promoter region of SLCG_2920 by DNase I foot-printing. A DNase I footprinting assay was carried out as described by Zianni et al. (37). To determinethe binding site of SLCG_2919 in the promoter region of its adjacent gene, SLCG_2920, a 176-bp5=-FAM-labeled fragment was amplified by PCR using primers FAM-2919-P1/FAM-2919-P2. The footprint-ing reaction mixture contained 250 ng labeled DNA fragment, 0.25 to 1 �M His6-SLCG_2919, and bindingbuffer (10 mM Tris [pH 7.5], 5 mM MgCl2, 50 mM EDTA, 60 mM KCl, 10 mM DTT, and 10% glycerol) in atotal volume of 50 �l. After incubation of the mixture at 25°C for 20 min, 5.5 �l DNase I buffer and 2 �lDNase I (1 U/�g; Promega) were added. The mixture was incubated at 25°C for 60 s and terminated byaddition of DNase I stop solution and heating for 10 min at 65°C. DNA samples were analyzed with a3730XL DNA genetic analyzer (Applied Biosystems) after purification, and data analyses were performedusing the GeneMarker software program v2.2.

Lincomycin resistance test. To determine the resistance of LCGL/pIB139 and LCGL/pIB139-2920strains against lincomycin, 10 �l spore suspension of LCGL/pIB139-2920, as well as LCGL/pIB139, was

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inoculated into 5 ml liquid TSBY with various concentrations of Lin-A (0 �g/ml, 300 �g/ml, 350 �g/ml,400 �g/ml, 450 �g/ml, and 500 �g/ml) and cultured at 30°C for 48 h to compare growth.

Statistical analysis. All data in this study were obtained from biological triplicates and shown asmeans � standard deviation of the mean (38). Significance analysis was performed with an unpairedtwo-tailed Student’s t test, with * indicating a P value of �0.05, ** indicating a P value of �0.01, and ***indicating a P value of �0.001.

Ethical standards. This article does not contain any studies with human participants or animalsperformed by any of the authors.

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/

AEM.02091-18.SUPPLEMENTAL FILE 1, PDF file, 0.9 MB.

ACKNOWLEDGMENTSWe are grateful to Michael S. DeMott (Department of Biological Engineering, Mas-

sachusetts Institute of Technology) for critical editing of the manuscript.This work was supported by the National Natural Science Foundation of China

(grants 31300081, 31570074, and 31600064), the Open Project of State Key Laboratoryof Microbial Metabolism from Shanghai Jiao Tong University (grant MMLKF13-05), theOpen Fund for Discipline Construction from Institute of Physical Science and Informa-tion Technology at Anhui University, and the Anhui Provincial Natural Science Foun-dation (grant 1708085QC49).

We declare that we have no competing interests.

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