Similar to the well-documented N-acyl-homoserine lactones (AHLs) in Gram-negative bacteria, hormone-like signaling molecules in the Gram-positive Streptomyces bacteria are diffusible low molecular weight chemical substances that can elicit antibiotic production and/or induce morphological differentiation at nanomolar concentrations (Swift et al., 1994; Takano, 2006). To date, hormone-like signaling molecules identified from Streptomyces are classified into five major classes, including γ-butyrolactones (GBLs), furans, γ-butenolides as well as PI factor and N-methylphenylalanyl-dehydrobutyrine diketopiperazine (MDD) (Niu et al., 2016). GBLs, furans and γ-butenolides are based on five-membered heterocyclic rings containing four carbons and one oxygen, while PI factor and MDD have quite different structures (Recio et al., 2004; Matselyukh et al., 2015). As most recent investigations are largely on GBLs and γ-butenolides, we herein only summarize recent findings on these two important classes. GBLs, the largest group of these signaling molecules, share a characteristic 2,3-disubstituted GBL core skeleton but differ in the length, branching and stereochemistry of the acyl side chain (Takano, 2006). So far, a total of 19 GBLs have been identified in streptomycetes, including the A-factor from S. griseus, eight butanolides (SCB1-8) from Streptomyces coelicolor, five virginiae butanolides (VBs A–E) from Streptomyces virginiae, IM-2 from Streptomyces lavendulae, factor 1 from Streptomyces viridochromogenes, and three Gräfe’s factors from Streptomyces bikinensis and Streptomyces cyaneofuscatus (Figure 1; Niu et al., 2016; Sidda et al., 2016). To gain insight to the distribution of GBLs, an A-factor-deficient mutant of S. griseus was used as an indicator strain to screen against 203 actinomycete strains. Thirty of the strains tested showed distinct A-factor activity, indicating that A-factor was widely distributed among actinomycetes (Hara and Beppu, 1982). Earlier studies identified the butenolide synthase AfsA as a key enzyme for A-factor biosynthesis in S. griseus, and the AfsA homolog ScbA was required for the biosynthesis of the three SCBs in S. coelicolor (Takano et al., 2001; Hsiao et al., 2007). Among the eight butanolides identified in the model organism S. coelicolor, SCB1-3 were identified in the 2000s (Takano et al., 2000; Hsiao et al., 2009). Recently, the engineered strain S. coelicolor M1152 was found to overproduce GBLs SCB1–3 as well as five novel GBLs designated as SCB4–8 (Sidda et al., 2016). The S. coelicolor M1152 is a derivative of S. coelicolor M145 lacking four biosynthetic gene clusters (BGCs) for the biosynthesis of actinorhodin (ACT), undecylprodigiosin (RED), calcium-dependent antibiotic (CDA) and coelimycin. It should be noted that the deletion of the coelimycin gene cluster included scbR2, which encodes a repressor of SCBs biosynthetic gene scbA, and thereby contributed to the overproduction of SCBs in S. coelicolor M1152 (Gomez-Escribano and Bibb, 2012; Sidda et al., 2016). In a BLAST survey, we found one to three afsA-like genes in each of nine Streptomyces genomes (Niu et al., 2016). In another study, BLAST search for homologs of AfsA from S. griseus within the actinobacteria genomes available from public databases showed that AfsA-like proteins are present in most actinomycetes (Ahmed et al., 2017). The widespread distribution of GBLs was further confirmed by using VB- and IM-2-deficient strains as indicators to examine VB and IM-2 distribution. A total of 10 strains of the 40 Streptomyces and 11 endophytic actinomycetes tested produce IM-2, and the same number of strains produced VB active compounds (Thao et al., 2017). These studies reinforce the notion that GBLs are the most common hormone-like signaling molecules among actinomycetes. More efforts should be directed to identifying chemical structures of these signaling molecules.

Representative γ-butenolides include two butenolides (SRB1 and SRB2) from Streptomyces rochei (Arakawa et al., 2012; Arakawa, 2014), the avenolide from Streptomyces avermitilis (Kitani et al., 2011), four butenolides from Streptomyces albus J1074 (Nguyen et al., 2018), and three butenolides (SAB1-3) from Streptomyces ansochromogenes (Wang et al., 2018; Figure 1). Of special note is the avenolide that is necessary for triggering avermectin production in S. avermitilis (Kitani et al., 2011). The gene cluster responsible for avenolide biosynthesis consists of three genes (avaR1-3) that encode GBL receptor homologs, and two genes (aco and cyp17) that encode an acyl-CoA oxidase and a cytochrome P450 hydroxylase, respectively. Studies suggested that both the acyl-CoA oxidase Aco and the cytochrome P450 hydroxylase Cyp17 were required for the enzymatic production of avenolide. Furthermore, Aco/Cyp17 homologs were also found in the genomes of Streptomyces fradiae, Streptomyces ghanaensis, Streptomyces griseoauranticus, and S. albus J1074, suggesting that they have the capacity to produce avenolide or avenolide-like autoregulators (Kitani et al., 2011; Ahmed et al., 2017). Recently, Luzhetskyy and colleagues identified an avenolide-like compound 4 in a genetically modified strain of S. albus 1074 (Ahmed et al., 2017). In another study, Nihira and colleagues investigated the distribution of avenolide by using a S. avermitilis aco disruptant as a biosensor in a collection of 40 Streptomyces and 11 endophytic actinomycetes. They showed that 12 of the 51 actinomycetes strains exhibited avenolide activity with S. albus J1074 showing the highest activity (Thao et al., 2017). Metabolite profiling of a disruptant of the S. albus aco gene led to the identification of the compound 4 along with three other butenolides (compounds 2, 3, and 5). The four avenolide-like compounds showed different levels of avenolide activity in stimulating avermectin production in S. avermitilis (Nguyen et al., 2018). It is interesting to note that the four compounds have been isolated previously from marine-derived Streptomyces strains, though there are no reports on their connection with antibiotic biosynthesis in the native producers (Cho et al., 2001; Strand et al., 2014; Viegelmann et al., 2014; Igarashi et al., 2015). In a recent study, Tan and colleagues identified a putative BGC for autoregulator biosynthesis in S. ansochromogenes (nikkomycin producer). Within the gene cluster, sabA encodes an AfsA-like enzyme, whereas sabP and sabD encode phosphatase and dehydrogenase enzymes, respectively. Heterologous expression of sabAPD in E. coli and Streptomyces led to the identification of three novel butenolides (SAB1, 2, and 3) (Wang et al., 2018). Since there are only limited numbers of natural GBLs and γ-butenolides identified in Streptomyces, these studies provide effective strategies for the discovery of new signaling molecules, which are normally produced in very small quantities. It is noteworthy that several butenolides have also been identified in Streptomyces species (Wang T. et al., 2014; de Oliveira et al., 2019), especially from marine-derived Streptomyces (Cho et al., 2001; Strand et al., 2014; Viegelmann et al., 2014; Igarashi et al., 2015). The roles of these butenolides in their native producers await further investigation.

The transmission of chemical signals starts with the binding of an autoregulator to its specific receptor protein, that usually belongs to the TetR family of transcriptional regulators. It is not uncommon that many Streptomyces genomes contain multiple genes for ArpA-like GBL receptors. Examples include ScbR, ScbR2, CprA and CprB in S. coelicolor, JadR2 and JadR3 in Streptomyces venezuelae, and AvaR1, AvaR2, and AvaR3 in S. avermitilis, SabR1 and SabR2 in S. ansochromogenes. It is noteworthy that most genes encoding these GBL receptors are closely linked both to each other and to the afsA-like genes, except that cprA and cprB are not linked to an afsA-like gene and therefore referred to as “orphan GBL receptor” genes (Onaka et al., 1998). To date, only a few identified receptors have been shown to interact with endogenous GBL molecules. In the model organism S. coelicolor, ScbR was characterized as a genuine receptor of SCBs (Takano et al., 2005; Gomez-Escribano et al., 2012). ScbR binds the scbR-scbA intergenic region to repress its own expression, while interacting with ScbA to activate scbA expression in response to SCBs (Takano et al., 2001). Another known target of ScbR is kasO, the CSR activator of the coelimycin biosynthetic pathway (Takano et al., 2005). In S. venezuelae, JadR3 was identified as a genuine receptor of SVB1, a GBL identical in structure to SCB3 of S. coelicolor. The SVB1 receptor JadR3 has dual activation and repression effects on jadomycin biosynthesis (Zou et al., 2014; Niu et al., 2016). In S. ansochromogenes, SabR1, the cognate receptor of SABs, was shown to repress the expression of sabA, sabR1, sabR2 and cprC by binding directly to their promoter regions. Interestingly, CprC, a homolog of CprA and CprB in S. coelicolor, was identified as an activator of the pleiotropic regulatory gene adpA, which in turn stimulated nikkomycin biosynthesis via activating the transcriptional initiation of the CSR activator gene sanG and biosynthetic genes of the sanF-X operon (Figure 2; Pan et al., 2009; Wang et al., 2018). Of the three GBL receptors in S. avermitilis, AvaR1 acts as an avenolide receptor to control avenolide and avermectin production by directly repressing the transcription of aco, aveR, its own gene and the other two GBL receptor homologous genes (avaR2 and avaR3) (Figure 3; Zhu et al., 2017). ScbR2, JadR2, AvaR2 and SabR2 were identified as pseudo GBL receptors (Xu et al., 2010; Zhu et al., 2016; Wang et al., 2018), and the roles of these pseudo GBL receptors in the regulation of antibiotic biosynthesis will be discussed in a subsequent section. AvaR3 contains an extra 75-amino acid stretch that is not present in typical GBL receptors, and promotes avermectin production through a yet-unknown regulatory mechanism (Miyamoto et al., 2011). It is interesting to note that AvaR3 may represent a new subgroup of GBL receptors, and therefore further studies are needed to reveal its role in avermectin production.

The first line of antibiotics as autoregulators came from studies on the roles of two atypical response regulators (ARRs) in antibiotic biosynthesis. The CSR activator JadR1, an OmpR-type ARR of S. venezuelae, activates the expression of jadomycin B (JdB) biosynthetic genes in the presence of a low concentration of JdB and its homologs, but high JdB concentrations cause the dissociation of JadR1 from its target promoters. Thus, JdB interacts with JadR1 directly in a dose-dependent manner to control JdB production in a feedback regulatory mechanism (Wang et al., 2009). Similarly, a NarL-type ARR RedZ was shown to promote RED biosynthesis in S. coelicolor by activating the CSR activator gene redD, and the DNA-binding activity of RedZ to its target gene was released by adding RED (Wang et al., 2009). Other than the end-product, studies also suggest that biosynthetic intermediates of antibiotics can also serve as autoregulatory molecules to modulate their own biosynthesis. For example, the TetR family regulator JadR^∗ can respond to early jadomycin intermediates [2,3-dehydro-UWM6 (DHU) and dehydrorabelomycin (DHR)] and release its repression on jadY transcription to ensure the timely supply of cofactors for JadG to convert DHR to the late intermediate jadomycin A (JdA) in jadomycin biosynthesis (Zhang et al., 2013).

Inspired by studies on the regulation of jadomycin biosynthesis in S. venezuelae, more antibiotics and their biosynthetic intermediates have been found to serve as autoregulatory molecules to modulate their own biosynthesis in many other streptomycetes. For illustration purposes, we herein only highlight several recent findings. For example, ChlF1, a TetR family regulator involved in chlorothricin biosynthesis of Streptomyces antibioticus, was found to repress the transcription of chlF1, chlG (encoding a major facilitator superfamily transporter), and chlK (encoding a type II thioesterase) but activates chlJ (encoding acyl-CoA carboxyl transferase) by binding to their promoter regions. However, ChlF1 was disassociated from its target promoters in the presence of chlorothricin and its biosynthetic intermediates (demethyl salicyloyl chlorothricin and deschloro-chlorothricin), which directly interacted with the regulator in a concentration-dependent manner (Li et al., 2016). In another study, CalR3, a TetR family regulator involved in calcimycin biosynthesis of Streptomyces chartreusis NRRL 3882, was found to repress the transcription of calR3 and its adjacent calT (encoding a putative transmembrane efflux pump protein of the MMPL family) by binding to their promoter regions. Similarly, both calcimycin and its biosynthetic intermediate (cezomycin) can act as ligands to dissociate CalR3 from its target promoters in a concentration-dependent manner (Gou et al., 2017; Wu et al., 2018). Other than TetR family regulators, Streptomyces antibiotic regulatory proteins (SARPs) family regulator was also found to regulate antibiotic biosynthesis in response to antibiotics and their biosynthetic intermediates. For example, NosP was found to regulate nosiheptide production in response to both the end-product nosiheptide (NOS) and biosynthetic intermediate (NOS-AC) (Li et al., 2018). It should be noted that such findings are not limited to the genus Streptomyces. One example comes from the actinomycete Amycolatopsis mediterranei, in which rifamycin B (the end-product of rifamycin biosynthesis) serves as an extracellular signaling molecule to regulate rifamycin export in a feedback mechanism (Lei et al., 2018). Another study showed that 2,4-diacetylphloroglucinol and its biosynthetic intermediate could serve as autoregulatory ligands to regulate its own biosynthesis in Pseudomonas fluorescens (Yan et al., 2017). These studies suggested that antibiotics and their biosynthetic intermediates could function as autoregulators to regulate their own biosynthesis, mainly through the modulation of the binding activity of CSRs to their target genes within their cognate gene clusters.

Earlier studies suggest that CSRs of one biosynthetic pathway can also control the biosynthesis of disparate antibiotic biosynthetic pathways. For example, RedZ, CSR of the red gene cluster, modulates the production of RED as well as that of ACT and CDA (Huang et al., 2005). In another study, the candicidin CSR FscRI was found to control the biosynthesis of candicidin as well as antimycin, the product of a disparate BGC in Streptomyces albidoflavus S4 (previously known as Streptomyces albus S4) (McLean et al., 2016; Li et al., 2019). Similar observations were also made with coordinated production of geldanamycin and elaiophylin by GdmRIII in Streptomyces autolyticus CGMCC0516 (Jiang et al., 2017), production of cephamycin C and clavulanic acid by CcaR in Streptomyces clavuligerus (Santamarta et al., 2002), and production of jadomycin and chloramphenicol by JadR1 in S. venezuelae (Wang et al., 2009). Recent years see growing evidence that antibiotics can also act as autoregulators regulating other biosynthetic pathways. The first examples to be described involve pseudo GBL receptors. In S. coelicolor, ScbR2, represses the same target, kasO, as the SCBs-interactive ScbR. Although scbR2 is linked to the gene cluster for coelimycin biosynthesis, ScbR2 also responds to the endogenous antibiotics ACT and RED, and thereby regulates the production of these antibiotics (Xu et al., 2010). In S. venezuelae, JadR2, a close homolog of ScbR2, modulates JdB biosynthesis via direct repression of jadR1. This repression is sensitive to the jadomycin end products. Likewise, although jadR2 is linked to the jad gene cluster for jadomycin biosynthesis, JadR2 could also bind to the endogenous antibiotic chloramphenicol, the product of the distant cml biosynthetic gene cluster (Xu et al., 2010). These studies suggest that CSRs of one biosynthetic pathway can also control disparate antibiotic biosynthetic pathways in response to different antibiotics. The pseudo GBL receptor AvaR2 of S. avermitilis was as an important repressor of avermectin and avenolide biosynthesis. It directly repressed transcription of aveR, aco, its own gene and the other two GBL receptor homologous genes (avaR1 and avaR3) (Figure 3; Zhu et al., 2016). Interestingly, DNA-binding activity of AvaR2 can be modulated by endogenous avenolide in a concentration-dependent manner, indicating that avenolide serves as ligands for both the genuine GBL receptor AvaR1 and the pseudo GBL receptor AvaR2. Furthermore, DNA-binding activity of AvaR2 can also be modulated by exogenous antibiotics JdB and aminoglycoside antibiotics such as apramycin, hygromycin B, kanamycin and streptomycin (Zhu et al., 2016). It would be interesting to examine if JdB from S. venezuelae can serve as signals to modulate avermectin production in S. avermitilis under physiological conditions.

In recent years, growing evidence suggests that antibiotics, at sub-inhibitory concentrations, can potentiate antibiotic production in multiple Streptomyces species. For example, lincomycin at a sub-inhibitory concentration resulted in an elevated expression of the CSR activator gene actII-ORF4, and therefore increased ACT overproduction in S. coelicolor (Imai et al., 2015). A recent study suggests that lincomycin produced profound changes in gene expression profiles of S. coelicolor (Ishizuka et al., 2018). In another example, a sub-inhibitory concentration of JdB (the angucycline from S. venezuelae) induced early RED production and premature differentiation (formation of sporulating aerial mycelium) in S. coelicolor. Other angucyclines were also found to elicit similar phenotypes (Wang W. et al., 2014). Examples also include perturbation of antibiotics production by ribosome-targeting antibiotics (thiostrepton, spectinomycin, and chloramphenicol) in multiple Streptomyces species (Tanaka et al., 2017; Wang et al., 2017). Furthermore, antibiotics can also be used as chemical elicitors for the discovery of novel natural products. For example, S. lividans 1326 grown in the presence of lincomycin at a sub-inhibitory concentration produced abundant antibacterial compounds that were absent in cells grown in lincomycin-free medium. Some of these antibacterial compounds were revealed as novel congeners of CDA (Imai et al., 2015). In another case, polyether antibiotics, including promomycin and closely related salinomycin, monensin, and nigercin, at sub−inhibitory concentrations elicited antibiotic production in multiple Streptomyces strains (Amano et al., 2010, 2011). These studies suggest that antibiotics have the potential to serve as chemical elicitors for the discovery of novel bioactive natural products.

It is well-known that the cryptic gene clusters represent a treasure trove of new natural products, and various strategies and methods has been devised for the activation of these gene clusters (Liu et al., 2013; Niu et al., 2016; Niu, 2018; Niu and Li, 2019). Of special note is a chemogenetic method referred to as high-throughput elicitor screening approach (HiTES) (Figure 4). In this approach, a reporter gene is inserted into the BGC of interest to allow a rapid read-out for its expression. The resulting reporter strain is then screened against small molecule libraries to identify candidate elicitors (Seyedsayamdost, 2014). This approach was initially applied to activate two cryptic gene clusters in the Gram-negative bacterium Burkholderia thailandensis, leading to the identification of a new malleilactone analog. Interestingly, almost all elicitors identified from a library of 800 compounds were antibiotics (Seyedsayamdost, 2014). Furthermore, Seyedsayamdost and colleagues have employed this approach to identify 14 novel specialized products in S. albus J1074 (Xu et al., 2017). HiTES with S. albus J1074 using a library of ∼500 natural products identified two classical antibiotics (ivermectin and etoposide) as the best elicitors of a cryptic non-ribosomal peptide synthetase (NRPS) gene cluster (Xu et al., 2017). A major limitation of this method is that genetic manipulations and/or molecular biology approaches are required for the insertion of the reporter gene. To circumvent this problem, HiTES is then combined with bioactivity assays or imaging mass spectrometry (IMS) to identify novel antibiotics. In these approaches, a wild-type microorganism is subjected to HiTES. The resulting induced cultures are then screened directly for biological activity or subject to IMS (Figure 4). Application of these modified methods have uncovered two novel specialized products in Streptomyces hiroshimensis (Moon et al., 2019b), and many other novel specialized metabolites in diverse microorganisms (Moon et al., 2019a; Xu et al., 2019). These studies suggest that HiTES represents a promising new avenue for the discovery of novel bioactive natural products in a high-throughput manner.