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Mycothiol maintains the homeostasis and signalling of nitric oxide in Streptomyces coelicolor A3(2) M145

Abstract

Background

Previous studies have revealed a nitric oxide (NO) metabolic cycle in which NO, nitrate (NO3), and nitrite (NO2) circulate. The NO produced in this cycle serves as a signalling molecule that regulates actinorhodin (ACT) production via the DevS/DevR NO-dependent two-component system (TCS) in Streptomyces coelicolor A3(2) M145. However, the mechanisms involved in the regulation of NO signalling in S. coelicolor have not yet been elucidated. Mycothiol (MSH), a thiol molecule produced by Actinomyces, is involved in the defence mechanisms against oxidative stress. Therefore, this study focused on the correlation between intracellular NO and MSH levels.

Results

To investigate the interaction of MSH with endogenously produced NO, we generated an S. coelicolor A3(2) strain deficient in MSH biosynthesis. This mutant strain exhibited a decrease in low-molecular-weight S-nitrosothiols and intracellular NO levels during culture compared to those of the wild-type strain. Moreover, the mutant strain exhibited reduced activity of the DevS/DevR TCS, a regulator of NO homeostasis and ACT production, from the early stage of culture, along with a decrease in ACT production compared to those of the wild-type strain.

Conclusions

This study suggests that MSH maintains intracellular NO homeostasis by forming S-nitrosomycothiol, which induces NO signalling. Finally, we propose a metabolic model in which MSH from endogenously produced NO facilitates the maintenance of both NO homeostasis and signalling in S. coelicolor A3(2) M145.

Peer Review reports

Background

Nitric oxide (NO) is a highly reactive and diffusible molecule that can easily permeate the cell membrane [1]. Since Palmer first reported its activity in 1987, NO has been recognised as a signalling molecule that regulates various physiological functions in most organisms [2]. NO mediates cell-to-cell communication and regulates several metabolic pathways by interacting with transition metals (e.g., Fe and Cu [3]), thiol groups [4], free-radical oxygen [5], and unsaturated fatty acids [6]. However, increased levels of NO exert cytotoxic effects owing to its high reactivity. NO reacts with O2•− to form peroxynitrite (ONOO), which leads to intracellular dysregulation due to the oxidation of a wide range of cellular molecules such as proteins, lipids, and nucleic acids [7]. Thus, cells must strictly regulate the internal NO concentration to ensure their viability.

Several studies have reported mechanisms involved in the regulation of intracellular NO levels. NO production by enzymes such as nitric oxide synthase and nitrite reductase, which reduce nitrite to NO, depends on the transient expression of genes [8, 9]. To protect cells from NO toxicity, flavohaemoglobin (Fhb) and nitric oxide reductase eliminate cellular NO by converting it into nitrate and nitrous oxide, respectively [10, 11]. Additionally, low-molecular-weight (LMW) thiol compounds such as glutathione (GSH) and thionein modulate the NO concentration by generating S-nitrosothiol [12, 13]. These small molecules can therefore protect cells against nitrosylative stress and regulate the S-nitrosylation of proteins and their corresponding functions.

Streptomyces species are gram-positive soil-dwelling filamentous bacteria that exhibit a complex life cycle similar to that of fungi. These bacteria produce many commercially available secondary metabolites, including anticancer agents [14], agrochemicals [15], and antibiotics [16]. Recently, we reported an NO metabolic cycle in which NO, nitrate, and nitrite are circulated by Fhb and nitrate reductase (Nar) in Streptomyces coelicolor A3(2) M145; the NO produced in this cycle serves as a signalling molecule to regulate morphological differentiation and secondary metabolism in bacteria [17]. In addition, the production of NO and actinorhodin (ACT; a blue-pigmented antibiotic) were demonstrated to be regulated by the NO-dependent two-component regulatory system (TCS) DevS/DevR in Streptomyces coelicolor A3(2) M145 [18]. The sensor protein DevS binds NO and phosphorylates DevR, a cognate response regulator. Phosphorylated DevR then activates the expression of its own genes (devR, narG2, and actII-ORF4). In addition, DevS senses NO elevation during cell growth and inactivates its autophosphorylation activity, thereby inhibiting the NO synthesis pathway. Thus, DevS/R functions as an autoregulatory system to maintain NO homeostasis in S. coelicolor A3(2) M145 [18].

Mycothiol (MSH) is an Actinomycetes-specific LMW thiol compound consisting of an acetylated cysteine linked to glucosamine and inositol [19]. Additionally, MSH acts as an antioxidant buffer by forming mycothione, which is reduced by disulphide reductase [20]. Similar to GSH, MSH reacts with NO to form S-nitrosomycothiol (MSNO) and mitigates reactive nitrogen species-induced damage [21]. Four enzymes [MshA (SCO4204), MshB (SCO5126), MshC (SCO1663), and MshD (SCO4151)] are involved in the MSH biosynthesis pathway in S. coelicolor A3(2) M145 [22]. Although the correlation between LMW thiol compounds and oxidative stress has been extensively studied, the relationship between MSH and intracellular NO signalling in S. coelicolor A3(2) M145 remains unclear. In the present study, we investigated the relationship between MSH and endogenously produced NO in S. coelicolor A3(2) M145 cells.

Results

Effect of MSH deficiency on cell growth

Previous studies have reported that MSH eliminates reactive oxygen and nitrogen species generated in cells [20, 21]. However, the relationship between MSH expression and endogenous NO production remains unclear. A Streptomyces coelicolor A3(2) M145 strain lacking MSH synthesis-related genes (mshA, mshC, and mshD) cannot synthesize MSH [22]. Thus, we deleted mshC by homologous recombination using the plasmid pGM160 (Fig. 1a) to obtain a model strain incapable of MSH biosynthesis.

Fig. 1
figure 1

Effect of mycothiol (MSH) deficiency on colony growth. a Construction of the mshC disruptant strain. The plasmid pGM160, including the 2,000 nucleotides upstream and downstream of mshC flanking an aac(3)IV gene, was transformed into Streptomyces coelicolor A3(2) M145. The double-crossover event is shown schematically. b The colony area of strains M145 (blue bars), ∆mshC (red bars), and ∆mshC::mshC (yellow bars) cultured for the indicated durations. Data are presented as mean and standard deviation (n = 9). ***p < 0.001; NS, not significant (Student’s t-test). c The wet weight of M145 (blue bars), ∆mshC (red bars), and ∆mshC::mshC (yellow bars) strains cultured for the indicated durations. Data are presented as mean and standard deviation values from three independent experiments. ***p < 0.001; *p < 0.05. NS, not significant (Student’s t-test). d The colony area of ∆nar (blue bars) and ∆nar/mshC (red bars) strains cultured for the indicated durations. Data are presented as mean and standard deviation (n = 9) from three independent experiments. e The wet weight of ∆nar (blue bars) and ∆nar/mshC (red bars) strains cultured for the indicated durations. Data are presented as the mean and standard deviation values from three independent experiments. ***p < 0.001; **p < 0.01; NS, not significant (Student’s t-test)

To evaluate the effect of the loss of MSH biosynthesis ability in the cells, we comparatively investigated the colony areas and wet weights of the wild-type (WT) M145 strain and MSH-deficient ∆mshC strain cultured on yeast extract-malt extract-glutamine (YEME-gln) agar medium using ImageJ. Only relatively small differences in colony area and wet weight were found between strains after 72 and 168 h of incubation, respectively (Fig. 1b, c). Furthermore, only slight differences in colony area and wet weight were also found between the nar deletion strain (∆nar strain), with reduced NO production ability, and the nar and mshC double-deletion strains (∆nar/mshC strain) (Fig. 1d, e). These results suggested that MSH has a relatively small effect on the growth of S. coelicolor A3(2) M145, regardless of the presence or absence of endogenously produced NO.

Deficiency of MSH synthesis decreases the intracellular NO level in S. coelicolor A3(2)M145

Because hmpA (encoding Fhb) is regulated by the NO-dependent transcription factor NsrR, the gene expression level is reflective of the intracellular NO concentration [23]. To verify the effect of MSH synthesis deficiency on endogenous NO levels, we examined the expression levels of hmpA in the M145 and ∆mshC strains. The transcription levels of hmpA increased in a time-dependent manner in both strains but were maintained at lower levels in the ∆mshC strain compared to those in the M145 strain (Fig. 2a).

Fig. 2
figure 2

Mycothiol (MSH) gene deletion decreases intracellular nitric oxide (NO) levels. a Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis of the relative expression levels of hmpA in the total RNA extracted from strains M145 (blue bars), ∆mshC (red bars), and ∆mshC::mshC (yellow bars) cultured for the indicated durations. Data are presented as the mean and standard deviation values from six independent experiments. **p < 0.01; *p < 0.05; NS, not significant (Student’s t-test). b Relative mRNA expression levels of devR in the total RNA extracted from strains M145 (blue bars), ∆mshC (red bars), and ∆mshC::mshC (yellow bars) cultured for the indicated durations. Data are presented as the mean and standard deviation values from three independent experiments. **p < 0.01, *p < 0.05 (Student’s t-test). c Relative mRNA expression levels of hmpA in the total RNA extracted from M145 and ∆mshC strains. After the strains were cultured for 72 h, they were treated with 1 ml water (control; blue bars), 100 μM NO2. for 1 h (red bars), or 1 μM NOC5 solution (yellow bars); incubated for 30 min; and subjected to RT-qPCR analysis. Data are presented as mean and standard deviation values from three independent experiments. *p < 0.05; NS, not significant (Dunnett’s test)

DevS/R has been reported to regulate NO synthesis in a dose-dependent manner to maintain NO homeostasis [18]. Therefore, we further examined NO homeostasis in the ∆mshC strain by evaluating the expression of the DevS/R TCS regulon devR. The transcription level of devR in the ∆mshC strain was decreased compared to that in the M145 strain during culture (Fig. 2b). These results suggest that the lack of MSH suppressed NO production in the ∆mshC strain during the early stages of culture. This was supported by two exogenous NO donors inducing the expression of hmpA in the ∆mshC strain (Fig. 2c).

MSH deletion decreases LMW S-nitrosothiol levels

Since previous studies demonstrated that S-nitrosothiols are formed by thiol groups that bind to NO in the presence of electron acceptors in cells [24, 25], we verified LMW S-nitrosothiol levels in the M145, ∆mshC, and ∆nar strains in a time-dependent manner. The LMW S-nitrosothiol levels increased from 96 to 144 h but decreased after 144 h of culture in the M145 strain (Fig. 3). This is consistent with the results of a previous study demonstrating time-dependent changes in NO production in vivo [18]. Intracellular NO levels in S. coelicolor A3(2) M145 increased during the middle phase of culture and decreased during the late phase. In contrast, the LMW S-nitrosothiol levels in the ∆mshC and ∆nar strains were lower than those in the M145 strain between 96 and 144 h (Fig. 3). These results indicated that the amount of LMW S-nitrosothiols is positively correlated with intracellular NO levels in S. coelicolor A3(2) M145.

Fig. 3
figure 3

Defect of mycothiol (MSH) synthesis decreases low-molecular-weight (LMW) S-nitrosothiol (SNO) levels. M145 (blue bars), ∆mshC (red bars), and ∆nar (yellow bars) strains were cultured for the indicated durations. After deproteinisation, SNO was converted to NO2 using Hg.2+ as a catalyst. SNO levels were measured using the Saville–Griess assay. Concentrations below the detection limit (500 nM) were recorded as the detection limit concentration. Data are presented as the mean and standard deviation values from three or four independent experiments. ***p < 0.001; *p < 0.05; NS, not significant (Tukey–Kramer test)

MSH is needed for optimal ACT synthesis

A previous study demonstrated that the DevS/R TCS modulated ACT synthesis by regulating actII-ORF4 expression levels [18]. Since we found that the lack of MSH affected cellular NO levels, ACT production and actII-ORF4 expression levels were examined in the M145 and ∆mshC strains. Minimal ACT production (Fig. 4a) and downregulated actII-ORF4 expression (Fig. 4b) were found in the ∆mshC strain inoculated on YEME-gln agar and cultured for 96 h. These results suggest that the lack of MSH negatively affects NO signalling and ACT production in S. coelicolor A3(2) M145 cells.

Fig. 4
figure 4

Mycothiol (MSH) is needed for optimal actinorhodin (ACT) synthesis. a Spores were inoculated onto yeast extract-malt extract-glutamine agar medium with a toothpick and cultured for 96 h at 30 °C. b M145 (blue bar), ∆mshC (red bar), and ∆mshC::mshC (yellow bar) strains were cultured under the same conditions as those in (a). Total RNA was extracted from each strain to measure the relative expression level of actII-ORF4 using reverse transcription-quantitative polymerase chain reaction. Data are presented as mean and standard deviation values from three independent experiments. ***p < 0.001 (Student’s t-test)

Thus, MSH is essential for maintaining intracellular NO homeostasis and for mediating NO signalling in S. coelicolor A3(2) M145.

Discussion

As NO is highly reactive, its cellular concentration must be strictly regulated to exert its physiological functions. The results of the present study indicate that MSH-mediated intracellular NO homeostasis facilitates optimal NO signalling in bacteria. The GSH synthesis pathway has not been detected in Actinomycetes; however, members of this bacterial genus produce MSH as their major redox buffer and coenzyme [26,27,28]. MSH-deficient mutants of Mycobacterium smegmatis exhibit increased sensitivity to exogenous NO [29], reactive oxygen species [30], alkali agents, and antibiotics [31]. Mycobacterium tuberculosis relies on MSH-dependent systems to infect macrophages [32]. Corynebacterium glutamicum metabolises aromatic compounds that require MSH as a coenzyme [33]. Thus, Actinomycetes use MSH to adapt to their environments. In addition, an MSH-defective strain of S. coelicolor A3(2) was reported to exhibit high sensitivity to hydrogen peroxide [34].

While being statistically significant, the differences in cell growth between the M145 and ∆mshC strains remained relatively small during culture. However, the lack of MSH due to mshC deletion disrupted intracellular NO homeostasis. These results suggest that MSH acts not only as a defence molecule against reactive oxygen species but also as a mediator involved in maintaining intracellular NO homeostasis.

Although previous studies have revealed that bacteria endogenously produce NO and possess regulatory mechanisms for NO-mediated metabolism, these are transient adaptive responses to environmental changes [35,36,37,38]. Streptomyces coelicolor A3(2) M145 scavenges endogenously produced NO via MSH, in addition to Fhb-mediated NO metabolism. NO signalling can start gradually as intracellular NO levels overcome the metabolisable levels of both mechanisms. Therefore, S. coelicolor A3(2) M145 possesses an advanced mechanism for regulating intracellular NO homeostasis.

We further found that the ACT production level in the ∆mshC strain was lower than that in the WT strain. The DevS/R activity in the ∆mshC strain was also lower than that in the WT strain. ACT production is regulated by NO signalling via the DevS/R TCS [18]. Therefore, the dysregulation of ACT production in the ∆mshC strain can be explained by the upregulation of intracellular NO-induced DevS/R repression in the absence of MSH.

Thiols bind directly to NO under aerobic conditions [39] or in the presence of electron acceptors such as NAD+, metals, and metalloproteins in vivo [24, 25]. GSH forms S-nitrosoglutathione (GSNO) with the intracellular NO produced by nitric oxide synthase to maintain the life cycle of mammalian cells [40]. GSNO is a stable NO-carrying molecule in both plant and animal cells that regulates protein activity by transferring an NO group to cysteines [41,42,43]. LMW S-nitrosothiols function as donors of NO and nitrosonium ions and are involved in cellular signalling [44, 45]. S-Nitrosothiols are required for cell survival. In particular, GSNO interacts with specific thiol groups of proteins to produce high-molecular-weight S-nitrosothiols in a process called S-transnitrosation [46] and regulates the secondary metabolite content through protein S-nitrosylation [47].

LMW S-nitrosothiols were detected in the WT M145 strain. In contrast, LMW S-nitrosothiols were almost undetectable in the ∆mshC strain. This suggests that MSH interacts with endogenously produced NO and regulates the intracellular NO concentration through MSNO formation; however, we did not identify MSNO in the present study. In the future, it will be important to elucidate the mechanisms by which LMW S-nitrosothiols, including MSH, affect NO metabolism in actinomycetes.

The LMW S-nitrosothiol levels were not significantly different between the M145 and ∆mshC strains at 192 h, suggesting that the amount of other LMW thiols increased instead of MSH and that these thiols were involved in maintaining intracellular NO levels in the late stages of culture. This is consistent with the results of a previous study, which demonstrated that the amounts of other thiol compounds increased in an MSH-deficient strain of S. coelicolor A3(2) [34]. However, the levels of LMW S-nitrosothiols in the mshC strain were lower than those in the M145 strain at both 96 and 144 h. MSH deficiency inactivated the DevS/R TCS from the early stages, indicating that MSH is the main LMW thiol for maintaining NO homeostasis in the early stages of culture, which is necessary for signalling secondary metabolism in S. coelicolor A3(2) M145. Actinomycetes synthesise ergothioneine, an LMW thiol produced by Actinobacteria, in addition to MSH [48]. Thus, to understand the homeostatic regulation of NO in Actinomycetes, the roles of other thiols in NO homeostasis must be considered.

This study suggests that intracellular NO homeostasis, which must be strictly maintained for optimal secondary metabolism, depends on MSH, a novel component that regulates NO signalling. MSH deficiency decreased ACT production. Elucidation of the regulatory mechanism of secondary metabolism will contribute to the efficient improvement of fermentation products. Additionally, M. tuberculosis has a similar form of NO metabolism [49, 50]. Therefore, identifying the role of MSH in maintaining intracellular NO homeostasis will enable the development of clinical therapeutic strategies against M. tuberculosis infections. Future studies should focus on the regulation of NO homeostasis through various mechanisms in several organisms, which can provide novel perspectives on NO biology and improvement of human health.

Conclusions

This study suggests that MSH regulates intracellular NO homeostasis by promoting MSNO formation. MSH-mediated maintenance of NO homeostasis, which is dependent on intracellular NO levels, is important for ACT production in S. coelicolor A3(2) M145 under NO-producing conditions (Fig. 5).

Fig. 5
figure 5

Proposed model of intracellular nitric oxide (NO) homeostasis in Streptomyces coelicolor A3(2) M145. Flavohaemoglobin (Fhb) and nitrate reductase (Nar) mediate the circulation of NO, NO3, and NO2.. DevS/R is activated or inactivated depending on intracellular NO levels. NO directly regulates actinorhodin synthesis through the DevS/R two-component regulatory system, which modulates actII-ORF4 expression. DevR also regulates nar2 expression and NO production. Intracellular NO homeostasis is maintained through Fhb metabolism and its interaction with mycothiol (MSH) (S-nitrosylation) to form S-nitrosomycothiol (MSNO)

Methods

Bacterial strains, plasmids, and culture conditions

The S. coelicolor A3(2) M145 parental strain and all mutants used in this study are listed in Table 1. To induce sporulation, the strains were cultured on mannitol soya flour agar (2% mannitol, 2% soya flour, and 2% agar) at 30 °C. Additionally, the strains were incubated for each experiment in YEME-gln agar medium (0.3% yeast extract, 0.3% malt extract, 0.5% bacto-peptone, 1% glucose, 50 mM l-glutamine, and 2% agar, pH 7.2) at 30 °C. As NO easily diffuses and affects strains grown in the same incubator, the ∆nar and ∆nar/mshC strains were incubated independently. Detailed culture conditions are described in the figure legends. Escherichia coli DH5α and E. coli HST04 dam/dcm (TaKaRa Bio) strains were used as hosts for cloning and cultured in Luria–Bertani broth (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) at 37 °C.

Table 1 Strains used in this study

Construction of the ∆mshC and complementation strains

Two fragments corresponding to the upstream and downstream regions of mshC (sco1663) and a fragment corresponding to the apramycin-resistance cassette [51] flanked by flippase recognition target sites were polymerase chain reaction (PCR)-amplified with PrimeSTAR GXL DNA polymerase (TaKaRa Bio) using specific primer sets (Table 2), along with the introduction of EcoRI and HindIII restriction sites. The two fragments were ligated into the SmaI-digested pUC19 plasmid using Ligation High Ver. 2 (TOYOBO). The two products were transformed into E. coli DH5α competent cells (TaKaRa Bio). The plasmids were purified from E. coli cells, digested with EcoRI and HindIII, and the two fragments were ligated into the EcoR I-digested pKU450 plasmid using Ligation High Ver. 2. The resulting plasmids were transformed into E. coli DH5α competent cells. The plasmids were then purified. After digestion of the apramycin-resistance cassettes and plasmids with HindIII, the two fragments were ligated using Ligation High Ver. 2. The resulting products were transformed into E. coli DH5α competent cells. The cosmids were purified, digested with EcoRI, and ligated with EcoRI-digested pGM160 using Ligation High Ver. 2. The resulting construct was introduced into E. coli HST04 dam/dcm competent cells (TaKaRa Bio) to obtain non-methylated cosmids. Purified cosmids were introduced into the S. coelicolor A3(2) M145 parental strain or each mutant. The drug-resistant recombinant strains were screened. Genomic DNA was checked for DNA recombination by PCR with the appropriate primer sets (Table 2).

Table 2 Primers used in this study

The pKU460 plasmid was used for genetic complementation of the knockout mutants. The complementary cassette was obtained by PCR using the complementary primer set (Table 2) and ligated to the SmaI-digested pUC19 plasmid using Ligation High Ver. 2. The ligation product was introduced into E. coli DH5α competent cells, and the cosmid pUC19-mshC was purified and ligated with pKU460. The ligation product pKU460-mshC was introduced into E. coli HST04 dam/dcm competent cells to obtain a non-methylated cosmid. The obtained cosmid was then introduced into the ∆mshC strain. The complementary mutant was verified by PCR using the appropriate primer sets (Table 2) and a complementation study.

Measurement of colony areas

Spores were incubated on the YEME-gln agar medium at intervals of 1.8 cm (total of nine spots, 3 × 3 squares) using a sterilised toothpick. After incubating at 30 °C, a line of colonies (three colonies) including the central colony were photographed under a microscope (OLYMPUS) at a 7 × multiplier, and then the nine colony areas for three plates were measured using ImageJ (Version 1.53t, 24 August 2022 upgrade).

Measurement of cell wet weight

Spores were incubated on YEME-gln agar medium at intervals of 1.8 cm (total of nine spots, 3 × 3 squares) using a sterilised toothpick. After incubating at 30 °C, all colonies were collected in a microtube while carefully removing the agar medium with a toothpick and then the cell wet weight was measured using a precision balance.

S-nitrosothiol quantification

Spores were incubated on YEME-gln agar medium at intervals of 1.8 cm (total of nine spots, 3 × 3 squares) using a sterilised toothpick. After incubating at 30 °C, all colonies were collected in a 10 ml conical tube while carefully removing the agar medium with a toothpick.

S-nitrosothiol was measured using the Griess assay [52]. Cells were lysed in extraction buffer (0.1 M Tris–HCl, pH 7.2) on ice using sonication. The lysate was centrifuged at 20,000 × g and 4 °C for 15 min. Protein concentrations were measured using Bradford Protein assay kit (BIO-RAD) [53]. To precipitate proteins, the cell-free extract was incubated with acetone at − 20 °C for 15 min. The samples were then centrifuged at 20,000 × g and 4 °C for 10 min. Acetone was removed using an evaporator. The sample was incubated with an equivalent volume of buffer A (0.5 M HCl, 1% sulphanilamide) or buffer B (0.5 M HCl, 1% sulphanilamide, 0.2% HgCl2) for 15 min at 20–25 °C, followed by incubation with an equal volume of Griess reagent [0.5 M HCl, 0.02% N-(1-naphtyl)-ethylenediamine dihydrochloride] for 15 min. S-nitrosothiol was quantified by measuring the absorbance of the reaction mixture at 540 nm. A standard curve was constructed using different concentrations (0, 1, 5, 10, 15, and 20 μM) of GSNO in 50 μM NaNO2. Concentrations below the detection limit (500 nM) [54].

RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)

Spores were inoculated on YEME-gln agar medium at intervals of 1.8 cm (total of nine spots, 3 × 3 squares) using a sterilised toothpick. After incubating at 30 °C, all colonies were collected in a microtube while carefully removing the agar medium with a toothpick. Total RNA was purified from cells using the ReliaPrep RNA Cell Miniprep System (Promega). The isolated RNA was reverse-transcribed into complementary DNA using ReverTra Ace RT-qPCR Master Mix with gDNA Remover (TOYOBO), which was used as a template for qPCR analysis. The primers used for RT-qPCR analysis are listed in Table 2. qPCR was performed using the Thermal Cycler Dice Real-Time System (TaKaRa Bio). The PCR mixture (20 μL) contained 1 μg cDNA, 10 pmol of an appropriate primer set (Table 2), and THUNDERBIRD SYBR qPCR Mix (TOYOBO) or KOD SYBR qPCR Mix (TOYOBO). The expression levels of the target genes were normalised to those of hrdB, a housekeeping gene.

ACT production and RNA extraction

The strains were inoculated in a triangular shape in the YEME-GLN medium. The ACT-derived blue pigment was visualised using a camera (Canon). Total RNA was extracted from the cells using the ReliaPrep RNA Cell Miniprep System (Promega) by the same technique as described above.

Statistical analysis and reproducibility

All bars in figures represent the mean values obtained from at least three independent biological experiments and error bars represent the standard deviation. The detailed sample number (n) is provided in the legend of each figure. Differences in means between strains or conditions were analysed using Student’s t-test, Dunnett’s test, or the Tukey–Kramer test, depending on the comparison items. Differences were considered statistically significant at p < 0.05.

Availability of data and materials

The dataset supporting the conclusions of this study is included in the article and an additional file (Additional file 1). All data will be made available from the corresponding author upon request.

Abbreviations

ACT:

Actinorhodin

Fhb:

Flavohaemoglobin

GSH:

Glutathione

GSNO:

S-nitrosoglutathione

LMW:

Low molecular weight

MSH:

Mycothiol

MSNO:

S-nitrosomycothiol

Nar:

Nitrate reductase

NO:

Nitric oxide

PCR:

Polymerase chain reaction

RT-qPCR:

Reverse transcription-quantitative polymerase chain reaction

TCS:

Two-component system

WT:

Wild type

YEME-gln:

Yeast extract-malt extract-glutamine

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This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (C; Grant No. 20K05811) and a research project from the Tokyo University of Agriculture.

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T.Y., Y.S.,M.K, S.H., S.I., S.Y., and Y.S. designed the study. T.Y.,M.K and Y.S. carried out experiments and analyzed data. T.Y. prepared the manuscript. All authors approved final draft.

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Correspondence to Yasuyuki Sasaki.

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Yoshizumi, T., Shibui, Y., Kogo, M. et al. Mycothiol maintains the homeostasis and signalling of nitric oxide in Streptomyces coelicolor A3(2) M145. BMC Microbiol 23, 285 (2023). https://doi.org/10.1186/s12866-023-03036-z

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