gSELEX-based exploiting of potentially new targets of SLCG_2919
To establish the SLCG_2919-mediated regulatory network, we employed the previously improved gSELEX method to identify target sequences of SLCG_2919. Genomic DNA of S. lincolnensis LCGL was initially digested with Sau3AI to generate < 1 kb DNA fragments (Fig. S1A). After two rounds of SELEX, DNA fragments potentially binding to SLCG_2919 were cloned into the T vector for sequencing (Fig. S1B). When the SLCG_2919 concentration was 2 µM, four independent clones were obtained and sequenced (Table S2). DNA segments corresponding to the promoter regions of SLCG_6675, SLCG_4123–4124, SLCG_6579, and SLCG_0139–0140 were captured at least twice. According to the genome annotation of S. lincolnensis, SLCG_6675, SLCG_0139, SLCG_0140, SLCG_6579, SLCG_4123, and SLCG_4124 encode anthranilate synthase, LysR family transcriptional regulator, beta-lactamase, cytochrome P450, bifunctional DNA primase/polymerase, and magnesium or magnesium-dependent protein phosphatase, respectively. In summary, these results imply that SLCG_2919 participates in the control of primary metabolism, transcriptional regulation, oxidoreduction, and anabolism for lincomycin biosynthesis.
Slcg_2919 Binds Specifically To Four Captured Dna Fragments
To identify the four DNA fragments potentially interacting with SLCG_2919, we expressed His6-tagged SLCG_2919 in E. coli BL21 (DE3), as previously described (Xu et al. 2019), and tested its DNA-binding affinity for the promoter regions of SLCG_6675, SLCG_4123–4124, SLCG_0139–0140, and SLCG_6579. Because the length of P4123 − 4124 is 711 nt, we designed two DNA fragments P4123 − 4124−1 (356 nt) and P4123 − 4124−2 (355 nt), without overlapping sequences. Results from the EMSAs showed notable shifts when different amounts of His6-SLCG_2919 were added. Then, 50-fold unlabelled probes or poly dIdC were individually added to the reaction system to evaluate the binding specificities. We found that these labelled probes were able to pull down shifted bands, whereas poly dIdC were not (Fig. 1A). This indicated that SLCG_2919 binds specifically to the above DNA fragments.
Using the defined AT-rich binding site of SLCG_2919 (Xu et al. 2019), we scanned the AT-rich motif in the genome of S. lincolnensis LC-G using PREDetector (http://www.montefiore.ulg.ac.be/~hiard/PreDetector/PreDetector.php). Five DNA fragments were predicted within the promoter regions of the target genes (Fig. 1B). In addition, we used the motif-finding program MEME (http://meme-suite.org/) to evaluate the predicted sequences of targeted genes and a conserved SLCG_2919-binding motif (atTcgT, a: T, C; t: T, G; c: C, G; g: A, C) was identified (Fig. 1B).
SLCG_2919 disruption increases the transcription of its target genes
To confirm whether SLCG_2919 transcriptionally regulates the above-mentioned target genes, we used RT-qPCR to compare the transcription of these genes between LCGL and ΔSLCGL_2919. The results showed that the transcriptional levels of SLCG_6675, SLCG_0139, SLCG_0140, SLCG_6579, SLCG_4123, and SLCG_4124 increased by 3.3-, 4.2-, 3.2-, 2.5-, 4.6-, and 2.2-fold, respectively, in ΔSLCGL_2919 compared to those in LCGL (Fig. 2). It could be concluded that SLCG_2919 acts as a negative regulator to modulate transcriptional expression of the six new targeted genes.
New Targets Of Slcg_2919 Positively Correlate With Lincomycin Yield
To investigate the relevance of the aforementioned target genes to lincomycin production, SLCG_6675, SLCG_0139, SLCG_0140, SLCG_6579, SLCG_4123, and SLCG_4124 were individually disrupted with tsr replacement in LCGL, and the corresponding mutants were obtained and confirmed by PCR analyses (Fig. 3A and B). As shown in Fig. 3C, the mutants ΔSLCGL_6675, ΔSLCGL_0139, ΔSLCGL_0140, ΔSLCGL_6579, ΔSLCGL_4123, and ΔSLCGL_4124 displayed 24%, 20%, 10%, 26%, 24%, and 26% reductions, respectively, in Lin-A yield compared with the parental strain LCGL. Thus, our findings indicated that the six new target genes of SLCG_2919 had a positive effect on lincomycin production.