A tunable and reversible thermo-inducible bio-switch for streptomycetes

doi:10.21203/rs.3.rs-4838944/v1

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A tunable and reversible thermo-inducible bio-switch for streptomycetes

https://doi.org/10.21203/rs.3.rs-4838944/v1

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Programmable control of bacterial gene expression is of great interest for both applied and academic research. This is particularly true for Gram-positive bacteria of the genus Streptomyces, major producers of prodigious natural products. Despite a few inducible regulatory systems have been developed for use in Streptomyces, there is an increasing pursuit to augment the toolkit of high-performance induction systems. We herein report a robust and reversible thermo-inducible bio-switch, referred to as StrepT-switch. The bio-switch enables tunable and bidirectional control of gene expression with temperature as stimulation input. StrepT-switch has been proved successful for highly efficient CRISPR/Cas9-mediated genome engineering, as well as programmable control of antibiotic production and morphology differentiation. The versatility of the device has also been exemplified by thermal induction of a site-specific relaxase ZouA-mediated DNA amplification for overproduction of actinorhodin, a blue pigmented polyketide antibiotic. This study demonstrates the exploration a temperature-sensing module and exemplifies its versatility for programmable control of various target genes in Streptomyces species.

Biological sciences/Microbiology/Applied microbiology

Biological sciences/Microbiology/Industrial microbiology/Metabolic engineering

Biological sciences/Biological techniques/Microbiology techniques

RheA repressor

thermal-inducible bio-switch

CRISPR/Cas9

Streptomyces

Streptomyces is a unique genus in the phylum Actinobacteria of Gram-positive bacteria. Unlike most bacteria, Streptomyces species exhibit a multicellular life cycle strikingly similar to that of filamentous fungi. Notably, they are best-known for their prolific capability to produce numerous bioactive natural products. Over the past few decades, a plethora of genetic tools have been developed for Streptomyces. Among them, there are only a few number of induction systems available for use in Streptomyces¹. Typically, they are based upon transcriptional regulators, which can sense a specific chemical signal and activate target gene expression. Examples include regulatory systems with an array of chemical stimuli, such as antibiotics, sugars and other chemicals². However, the existing inducible systems have certain limitations, such as toxicity associated with antibiotic inducers, utilization of sugars as nutrients, and a lack of reversibility with all chemical inducers. To address these issues, it is imperative to devise high-performance induction system by exploring alternative stimuli.

When exposed to a sudden temperature upshift, microorganisms respond by rapidly inducing the expression of various heat shock proteins (HSPs), including molecular chaperones and proteases. In bacteria, transcriptional regulation of heat-shock genes can be either positive or negative. Positive regulation exploits alternative sigma factors to redirect RNA polymerase to heat shock gene promoters, while negative regulation relies on dedicated transcriptional repressors³. In Streptomyces species, the synthesis of different HSPs is under the control of different regulatory networks³. Of note is that the regulator RheA represses the expression of divergently oriented hsp18 gene, which encodes a small heat shock protein⁴. The RheA binding region covers the − 10 box and transcription start site (TSS) of the rheA promoter, suggesting that RheA synthesis is negatively autoregulated. At lower temperature, RheA exerts its regulatory function by binding directly to its operator (rheO) embedded within the intergenic region between rheA and hsp18. At high temperature, RheA was able to sense temperature changes and to relieve its repression on hsp18 expression. Of interest is that the temperature-induced derepression by RheA is reversible⁴.

To fulfill the need for robust, tunable, and reversible control of gene expression, we created two highly responsive thermo-inducible genetic systems, referred to as TRS01 and TRS02. The regulatory systems are based upon the thermo-sensing RheA repressor and the hsp18 promoter. Both TRS01 and TRS02 exhibit a high level of induced reporter activity and almost no leaky expression in the model Streptomyces species. TRS02 allows tunable and reversible control of target gene expression with alternating temperature stimuli. Thus, the thermo-inducible bio-switch was designated as StrepT-switch. The device has been proved successful for highly efficient CRISPR/Cas9-mediated genome engineering, as well as programmable control of antibiotic production and morphology differentiation. The utilization of this system has also been exemplified by thermal induction of ZouA-mediated amplification of actinorhodin gene cluster. This study demonstrates the exploration a temperature-sensing module and exemplifies the versatility of this system for programmable control on expression of various genes of interest. Our study augments the toolkit of inducible regulatory systems that are crucial for fine-tuning of gene expression in Streptomyces, a major source of medically important natural products.

Construction of high-performance thermo-inducible modules

Inducible gene expression systems typically comprise two main components: a transcriptional regulator (TF) and a cognate target promoter harbouring TF-specific DNA-binding sequences. To construct thermo-inducible modules, the coding region of rheA with the noncoding intergenic region between rheA and hsp18 was assembled with the promoter-less neomycin phosphotransferase gene (neo). It is worth noting that the promoters of rheA and hsp18 are located in a divergent orientation, and the RheA DNA-binding sequences (RheA operator, rheO) is embedded within the intergenic region (Fig. 1a and Supplementary Fig. 1). The construct was transferred into S. albidoflavus J1074 to obtain J1074-RS01-neo. For comparison, J1074-neo and J1074-P_hrdB-neo were included to serve as negative control and positive control, respectively (Fig. 1a). J1074-neo contains the promoter-less neo cassette, while J1074-P_hrdB-neo contains the neo cassette driven by the constitutive hrdB promoter². J1074-RS01-neo was then cultivated, along with J1074-neo and J1074-P_hrdB-neo, on minimal medium (MM) agar plates supplemented with increasing concentrations of kanamycin. As seen here, J1074-neo displayed resistance to kanamycin at 1.0 µg/mL, whereas it became sensitive at 2.5 µg/mL of kanamycin (Fig. 1b). The phenomenon is consistent with our previous findings that S. albidoflavus J1074 exhibited a low level of intrinsic kanamycin resistance². When cultivated at 28 °C, J1074-RS01-neo exhibited kanamycin resistance up to 25.0 µg/mL, suggesting that the native hsp18 promoter exhibited a relatively high level of leaky expression (Fig. 1b). To mitigate leakiness, TRS01 was generated by replacing the native promoter of rheA with the constitutive hrdB promoter, and the tfd terminator was included to avoid interference from unwanted transcriptional readthrough (Fig. S1). Moreover, an additional rheO was inserted immediately after the transcription start site (TSS) of hsp18 promoter in TRS01 to generate TRS02 (Supplementary Fig. 1). Transfer of the two constructs into S. albidoflavus J1074 generated J1074-TRS01-neo and J1074-TRS02-neo, respectively (Fig. 1a). When cultivated at 28 °C, both J1074-TRS01-neo and J1074-TRS02-neo can grow on MM agar plates containing at 2.5 µg/mL kanamycin. However, both strains are unable to tolerate 5.0 µg/mL or higher kanamycin concentrations (Fig. 1b), suggesting that a strong promoter driven repressor is an effective way to minimize leaky expression. When cultivated at 37 °C, J1074-TRS01-neo and J1074-TRS02-neo exhibited a high level of expression similar to that of the constitutive hrdB promoter, with kanamycin resistance up to 50 µg/mL (Fig. 1b). Next, electrophoretic mobility shift assays (EMSAs) were performed to examine protein-DNA interactions. For this purpose, recombinant RheA protein (∼25.4 kDa, calculated mass) was purified from E. coli BL21 (DE3) harboring pET28a::rheA (Supplementary Fig. 2). Of interest is that one shift band was observed for the original RS01 probe in a RheA concentration dependent manner, while two shifted bands for the RS02 probe with two copies of rheO was noticed at a lower RheA protein concentration. With an increase in protein concentration, the abundance of the upper band was increased obviously accompanied by disappearance of the lower band (Supplementary Fig. 3). Taken together, the results suggest that TRS01 and TRS02 exhibit tight modulation at 28 °C and a high level of induced gene expression at 37 °C in S. albidoflavus J1074.

Tuning the bio-switch with a physiological range of temperatures

To examine the dynamic range of the thermo-sensing modules, TRS02 was selected to drive the expression of the reporter gene gusA, which encodes the β-glucuronidase enzyme (Fig. 2a). For comparison, the promoter-less gusA and gusA driven by the constitutive hrdB promoter were included to serve as negative control and positive control, respectively⁵. The resulting strain J1074-TRS02-gusA, J1074-P_hrdB-gusA and J1074-gusA were cultivated on R2 agar plates for visualization of the characteristic blue color of GusA activity. Six different temperatures, covering a physiological range from 26 to 37 °C, were used for cultivation of these recombinant strains. A rise in cultivation temperature stimulates gusA expression, which in turn results in a gradual increase in blue color intensity. A vivid blue color was observed when J1074-TRS02-gusA was incubated at 34 and 37 °C (Fig. 2b). The color intensity of J1074-TRS02-gusA is similar to that of the positive control J1074-P_hrdB-gusA. To quantify GusA activity, the strains were inoculated in liquid R5A and cultivated at six different temperatures. A gradual induction of GusA activity was observed with an increase in the cultivation temperature, and a full induction of gusA expression was observed at 37 °C when cultivated for 24 h or 34 °C when cultivated for 48 h (Fig. 2c). The regulatory system shows sharp thermal transitions with a dynamic range of ∼62-fold induction.

To investigate the performance of TRS02 in other Streptomyces species, the construct was also transferred into the model organisms S. coelicolor M1146 and S. lividans TK24 to obtain M1146-TRS02-gusA and TK24-TRS02-gusA, respectively. An obvious induction of GusA activity was observed with M1146-TRS02-gusA (Supplementary Fig. 4a). It should be noted that the strain exhibited a low level of leaky expression as judged by the light blue color of colonies at lower incubation temperatures. TK24-TRS02-gusA exhibited a high level of induced GusA activity with no leaky expression (Supplementary Fig. 4b). Thus, we established a high-performance thermo-inducible system exhibiting low leakiness and high maximal expression. The results suggested that TRS02 provides a switch-like control of gene expression with temperature as stimulation input, and the temperature-sensing module is functional in three model Streptomyces species.

The thermal bio-switch enables bidirectional control of gene expression

As mentioned earlier, existing induction systems have certain limitations. The main disadvantage is a lack of reversibility associated with chemical inducible systems. In terms of reversibility, temperature has superiority over chemical inducers, as it can be easily adjusted. Thus, TRS02 was used to drive the expression of enhanced green fluorescent protein (eGFP). The resulting J1074-TRS02-eGFP was cultivated in liquid R5A with incubation temperatures switched back and forth between alternating temperatures. No eGFP expression was detected when the culture was initially kept at 28 °C for 12 h. An obvious eGFP expression was observed after the culture was shifted to 34 °C (Fig. 3). Reversibility can be achieved when the culture was switched back to 28 °C, followed by fluoresce activation at 37 °C and then fluoresce quenching at 28 °C (Fig. 3). Of note is that residual fluorescent signals were observed with J1074-TRS02-eGFP in the second and third round cultivations at 28 °C. Considering J1074-eGFP, a derivative of S. albidoflavus J1074 containing a promoter-less eGFP, also displayed low intensity of fluorescent signals, we speculate that the fluorescent signals in J1074-TRS02-eGFP may arise from a combination of background signals and the residual eGFP left from the preceding activation event. Our results demonstrated that the thermal bio-switch, designated as StrepT-switch thereafter, enables bidirectional control of gene expression.

The bio-switch facilitates highly efficient CRISPR/Cas9-mediated genome engineering

It is well documented that CRISPR/Cas9 shows high toxicity to the host, thus limiting its application in many Streptomyces strains⁶. To demonstrate the utility of our bio-switch, StrepT-switch was used for flexible control of the CRISPR/Cas9-mediated genome editing tools. First, StrepT-switch was used for CRISPR/Cas9-mediated target knock-in to activate the expression of indC. The indC gene was identified within a small gene cluster encoding the blue pigment indigoidine⁷. Under routine laboratory conditions, the gene cluster remained silent in S. albidoflavus J1074 as no pigment formation was observed. A previous study found that substitution of the native indC promoter with constitutive promoter led to the activation of the gene cluster⁷. For this purpose, the tipA promoter of Cas9 was replaced with StrepT-switch for knock-in of the constitutive kasO* promoter proceeding the coding region of indC (Fig. 4a). When introduced into S. albidoflavus J1074, there is only a few colonies observed on MS agar plates with the construct containing tipA promoter-driven Cas9 (Fig. 4b). This observation is in agreement with the fact that the tipA promoter exhibits a high level of leaky expression even in the absence of the thiostrepton inducer. In contrast, more transformants were obtained with the construct containing StrepT-switch-driven Cas9, indicating that StrepT-switch ensures almost no expression of Cas9 and avoid its toxicity when the transformants were maintained at 28 °C. Once we obtained the transformants, they can be passed on MS agar and then incubate at 34 °C to activation Cas9 expression for CRISPR/Cas9-mediated target knock-in (Fig. 4c). A similar strategy was used for CRISPR/Cas9-mediated target knock-out. An obvious high efficiency was observed with knock-out of approximate 48 kb of the daptomycin gene cluster from the chromosome of Streptomyces roseosporus (Supplementary Fig. 5). StrepT-switch-driven Cas9 has also been used successfully for knock-out of target genes in the industrial Streptomyces strains (Data not shown), while no transformants were obtained with tipA-driven Cas9, suggesting that StrepT-switch is particularly useful for strains with low DNA transformation efficiency. The results showed that StrepT-switch improved the DNA transformation efficiency by reducing the toxicity of Cas9 at lower temperature, and thus greatly increased the chance to obtain CRISPR/Cas9-mediated knock-in or knock-out mutants.

Programmable control of antibiotic production and morphology differentiation via thermo-inducible CRISPRi

To demonstrate the utility of our thermal bio-switch, StrepT-switch was used to repress gene expression via CRISPR interference (CRISPRi) in a programmable manner. In previous studies, the dCas9 gene, a catalytically dead variant of Streptococcus pyogenes Cas9, was controlled either by the thiostrepton-inducible tipA promoter or the constitutive ermE* promoter^{2, 8, 9}. For programmable control of gene repression, a tight regulatory expression of dCas9 is highly preferred. To this end, pSET152::dCas9-actI1 was selected as it has been used for efficient repression of actinorhodin (ACT) production by targeting actI-ORF1⁸. Thus, the expression of dCas9 was subjected to the control of StrepT-switch rather than the constitutive ermE* promoter (Fig. 5a). The resulting construct was then introduced into S. coelicolor M145 to obtain M145- StrepT-switch-dCas9-actI1. For comparison, M145-dCas9-actI1 was included to serve as a positive control. Unlike S. coelicolor M145, which could produce the blue pigment ACT and the red pigment undecylprodigiosins (RED), M145-dCas9-actI1 lost the ability to produce ACT on R5MS agar plates regardless of the incubation temperature (Fig. 5b). M145-StrepT-switch-dCas9-actI1 retained the ability to produce ACT when cultivated at 28 °C. However, it lost the ability to produce ACT and retained the ability to produce RED when cultivated at 37 °C (Fig. 5b). The same strategy was also used for flexible control of gene expression involved in morphology differentiation. Thermal inducible repression of ftsZ resulted in aerial hyphae that lack both cross-walls and sporulation septa in S. venezuelae (Fig. 5c). These results suggest that StrepT-switch can be used for programmable control of gene expression via CRISPRi based upon the thermo-inducible expression of dCas9.

Induction of ZouA-dependent amplification of act gene cluster multiplies antibiotic yield

A previous study identified a ZouA-dependent DNA amplification system in S. kanamyceticus¹⁰. The ZouA amplification system consists of three genetic elements, a site-specific relaxase ZouA, and two oriT-like recombination sites RsA and RsB. It has been used successfully for amplification of the act gene cluster in S. coelicolor MT1110¹⁰, bleomycin in Streptomyces verticillus¹¹, and spinosad in S. coelicolor M1146¹². To expand the utility of StrepT-switch, the regulatory system was used to drive the expression of zouA. For this purpose, the act gene cluster was engineered with flanking sequences, including RsA, RsB, and a neo cassette (Fig. 6a). The engineered gene cluster was transferred into S. coelicolor M1146 to obtain M1146-actAB. Incorporation of StrepT-switch-driven zouA generated M1146-actAB-StrepT-switch-zouA. Twelve conjugates were randomly streaked on R5MS agar plates containing 50 µg/mL kanamycin (generations one, G1), and then passaged an additional seven times (generations two–eight; G2–G8) on R5MS agar plates supplemented with increasing concentrations of kanamycin (100–1200 µg/mL) (Fig. 6b). Three strains with more intense blue color were inoculated into R5MS liquid media for ACT quantification. For comparison, the empty vector pSET152 was introduced into M1146-actAB, and the resulting strain M1146-actAB-pSET152 was included to serve as a negative control. A significant increase in ACT production was observed with M1146-actAB-StrepT-switch-zouA, suggesting that ZouA-dependent DNA amplification multiplies ACT yield.

The bio-switch is functional in E. coli

To broaden the utility of the thermal bio-switch, the intact rheA was positioned under the control of T7 promoter, and StrepT-switch was used to drive the expression of neo reporter gene in E. coli. Ideally, at lower temperature, IPTG-induced RheA will repress the expression of the neo cassette, and the cells are unable to grow in the presence of kanamycin. When cultivated at higher temperature, IPTG-induced RehA will relieve its repression on the neo cassette, and the cells grow well in the presence of kanamycin. Unfortunately, E. coli cells grow normally when cultivated either at 28 °C or 37 °C (Supplementary Fig. 6). We speculate that this observation may arise from codon bias correlated with the expression of rheA in E. coli. To address this issue, a codon-optimized version of rheA was synthesized and the corresponding bio-switch was designated as EcoT-switch. When cultivated at 28 °C, the cells are unable to grow in the presence of kanamycin. However, when cultivated at 37 °C, they grow normally in the presence of kanamycin (Supplementary Fig. 6). The results suggested that the codon-optimized version of rheA retain its regulatory function and maintain its ability of thermal stimuli response. Thus, the thermal bio-switch can be adapted for further use in E. coli.

Effective induction systems are highly desirable for diverse applications in synthetic biology and biotechnology research. Temperature is a unique input signal for flexible control of gene expression in synthetic biotechnological applications. Temperature control is characterized with low cost, easy operability, good dispersity, and reversibility. Over the past few decades, an extended suite of thermal induction systems have been developed for the model organism E. coli^{13, 14, 15, 16}. Existing temperature-dependent regulators include microbial heat shock factors, membrane-associated proteins, RNA thermometers and transcriptional repressors. The most commonly used transcriptional repressors include a temperature-dependent transcriptional repressor TipA from the large virulence-associated plasmid of Salmonella typhimurium and a temperature-sensitive variant of the bacteriophage λ repressor cI (mutant cI⁸⁵⁷) ¹⁶. In a previous study, a temperature-Inducible expression system has been developed for Streptomyces species¹⁷. The system is based upon a mutated repressor TraR and its target promoter P_tra from plasmid pSN22 of Streptomyces nigrifaciens. Of note is that P_tra precedes the traA-traB-spdB (tra) operon, which is essential for conjugative transfer of pSN22. The temperature-Inducible expression system has been used for controllable production of malate dehydrogenase (MDH) of Thermus flavus in S. lividans¹⁷. However, the utilities of this inducible system in metabolic pathway engineering have not been investigated. In this study, we report the exploration of a thermal inducible bio-switch that enables tunable and bidirectional control of various genes of interest. We demonstrate the versatility of this thermal bio-switch in diverse applications, such as highly efficient CRISPR/Cas9-mediated genome engineering, programmable control of antibiotic production and morphology differentiation, as well as thermal induction of ZouA-mediated DNA amplification for multiplication of actinorhodin production.

Upon sudden temperature upshift, microorganisms respond by initiating the heat-shock response, resulting in the prompt production of various heat-shock proteins. As mentioned earlier, the synthesis of different HSPs is under the control of four different regulatory networks in Streptomyces³. For example, the HrcA repressor is responsible for the regulation of the groEL genes. The HspR repressor regulates the dnaK operon and the clpB gene. A third regulator PopR allows tightly regulated expression of the clpP3 and clpP4 operon^{18, 19, 20, 21}. The fourth regulator RheA represses the expression of divergently oriented hsp18 gene, which encodes a small HSP protein⁴. Of note is that three different repressors, HrcA, HspR and RheA, specifically regulate distinct sets of heat shock genes without any cross-regulation and regulons overlap in S. albus. It is well established that RheA exerts its regulatory function by binding directly to its operator (rheO) embedded within the intergenic region between rheA and hsp18. The DNA-binding activity of the RheA repressor is modulated by high temperature to trigger its dissociation from the rheO operator. Circular dichroism (CD) spectroscopy revealed that RheA is able to sense temperature changes and undergo conformational changes, representing a reversible transition between an active and an inactive form⁴. However, the mechanism underlying the change of conformation triggered by temperature remains obscure.

Due to its robustness and flexibility, CRISPR-Cas system has emerged as a versatile tool for genome engineering in many organisms. CRISPR/Cas9-based tools have been extensively used for genome editing in Streptomyces ^{22, 23, 24}. However, Cas9-based editing tools exhibited toxicity in a large number of microorganisms including many Streptomyces species. These tools generally require high DNA transformation efficiency, which is unavailable for many Streptomyces species, thus impeding their broad applications. To address this issue, strategies mainly focus on using alternative Cas nucleases, fine-tuning Cas9 nucleases activity with anti-Cas protein, and mitigating the expression of Cas9 nucleases⁶. Fine-tuning the expression of Cas9 nucleases is a straightforward approach to reduce Cas toxicity while maintaining sufficient double-strand break (DSB) activity for genome engineering. Since the level of Cas9 toxicity varies among different Streptomyces species, it is difficulty to accomplish optimization of the Cas9 expression in a species specific manner. In this study, the expression of Cas9 nucleases was placed under the control of the thermo-inducible bio-switch. Programmable control of gene expression can be achieved via CRISPR-Cas9 mediated genome engineering with easy operability of temperature control. The thermal inducible device greatly improved the DNA transformation efficiency by reducing the toxicity of Cas9 at lower temperature, and thus greatly increased the chance to obtain CRISPR/Cas9-mediated knock-in or knock-out mutants, suggesting that the bio-switch is particularly useful for strains with low DNA transformation efficiency.

In summary, the thermal switch based on temperature-sensing repressor protein enables a tunable and bidirectional modulation of gene expression for dynamic control of metabolic pathways in Streptomyces. This study has demonstrated that the thermal switch has properties superior to the commonly used inducible expression systems. Programmable control of gene expression can be achieved with easy operability of temperature control. Moreover, the thermal bio-switch has also been adapted for use in Gram-negative bacterium E. coli. Taken together, our study expanded the toolkit of versatile induction systems that are crucial for flexible gene expression in the genus Streptomyces, major producers of prodigious natural products.

Bacterial Strains and Culture Conditions

Bacterial strains, plasmids, and primers used in this study were listed in the Supplementary Information. Escherichia coli DH5α was used as a general host strain for routine subcloning. E. coli ET12567 (pUZ8002) was used as a host for transferring DNA from E. coli to Streptomyces by intergeneric conjugation²⁵. E.coli BW25113 (pIJ790) was used for the construction of recombinant plasmids via λ-Red-mediated recombination technology²⁶. For general purposes, Streptomyces strains were grown on mannitol soya flour (MS) agar or in yeast extract-malt extract (YEME) liquid medium²⁵. Other culture media are specified in the following sections.

Assessment of Promoter Strength with Kanamycin Resistance Gene as Reporter

The assay was performed essentially as described previously². In brief, spores of recombinant strains were harvested and resuspended in double-distilled water. The optical density of the spore suspension was measured at 450 nm (OD₄₅₀). Each suspension was adjusted to 1.0 at OD₄₅₀. Serial 10-fold dilutions of each sample were prepared and spotted on minimal medium (MM) with mannitol as the sole carbon source. An increasing concentrations of kanamycin was applied to evaluate kanamycin resistance. The plates were cultivated at different temperatures for 5 days before photograph was taken.

GUS Assays

For agar plate-based assays, S. albidoflavus strains were streaked on R2 agar plates containing 30 mg/mL 5-bromo-4-chloro-3-indolyl-β-Dglucoronide (X-gluc). For strains of S. coelicolor and S. lividans, spores of recombinant strains were harvested and resuspended in double-distilled water. The optical density of the spore suspension was measured at 450 nm, and normalized to the same level. A total of 10 µL spore suspension was spotted on R2 agar plates containing 30 mg/mL X-gluc. All plates were kept at different temperatures and photographed after 4 days of incubation. Spectrophotometric GUS assays were performed as described previously²⁷.

Light Microscopy

Sterile coverslips were inserted at an angle of about 45 ° into MM agar plates. Spore suspensions were prepared and 2 µL of each spore suspension was pipetted along the line where the surface of the coverslips met the medium. After incubation at 28 °C or 37 °C for 3 days, the coverslips were withdrawn and fixed with formaldehyde for 30 seconds. After washing with double-distilled water, the coverslips were then covered with crystal violet staining solution. After staining for one minutes, the crystal violet solution was removed by washing with double-distilled water. The coverslips were left dry under a heating lamp, mounted on glass slides, and fixed with a few drops of 50% glycerol prior to observation under a light microscope of Nikon ECLIPSE Ei.

Confocal Laser-Scanning Fluorescence Microscopy Analysis

Spores were inoculated into liquid YEME media and incubated at 28°C on a rotary shaker (220 rpm) for 48 h as seed culture. One milliliter of seed culture was then transferred into 50 mL of R5A liquid media with incubation temperatures switched back and forth between lower and higher temperatures. After cultivation for every 12 h, 1 mL culture was withdrawn and the cells were collected by centrifugation. All samples were washed with double-stilled water to remove residual culture media and then resuspended in 10% glycerol. The suspensions were then observed under Leica SP8 confocal laser-scanning microscope at excitation wavelengths of 488 nm and emission wavelengths of 495∼530 nm. Images were merged using the Leica confocal software.

Measurement of Actinorhodin Production

The quantification of actinorhodin was performed essentially as described previously²⁸. In brief, S. coelicolor M145 derivatives were grown in 50 mL of R5MS at 28 °C. After cultivation, 1 mL culture was withdrawn at different time intervals and treated with KOH (1 N final concentration). The titer of actinorhodin was then calculated by measuring the absorbance at 640 nm.

Competing Interests:

The authors declare no competing interest.

Author Contributions:

L.L., Y.F., S.L., Y. Z, and M.W. performed the experiments. L.L., Y.F., and S.L. analyzed the primary data. Y.L. made contributions for the design and analysis of CRISPR–Cas experiments. G.N. conceived the project, analyzed the data, and wrote the manuscript.

Acknowledgments

This work was supported in part by a grant from the National Key Research and Development Program of China (No. 2023YFD1700700), and a grant from the National Natural Science Foundation of China (No. 32370077). We also thank Prof. Mervyn Bibb (John Innes Centre, Norwich, UK) for providing us S. coelicolor M1146.

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Supplementaryinformation.pdf
Supplementary information
BacterialStrains.xlsx
Dataset 1
Plasmids.xlsx
Dataset 2
Primers.xlsx
Dataset 3

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A tunable and reversible thermo-inducible bio-switch for streptomycetes

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Abstract

Figures

Introduction

Results

Construction of high-performance thermo-inducible modules

Tuning the bio-switch with a physiological range of temperatures

The thermal bio-switch enables bidirectional control of gene expression

The bio-switch facilitates highly efficient CRISPR/Cas9-mediated genome engineering

Discussion

Methods

Bacterial Strains and Culture Conditions

Assessment of Promoter Strength with Kanamycin Resistance Gene as Reporter

GUS Assays

Light Microscopy

Confocal Laser-Scanning Fluorescence Microscopy Analysis

Measurement of Actinorhodin Production

Declarations

Competing Interests:

Author Contributions:

Acknowledgments

References

Additional Declarations

Supplementary Files

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