Streptomyces species, as filamentous Gram-positive bacteria, are acknowledged for their advanced secondary metabolism and cellular differentiation entangled with complex regulatory circuitry, which revolves around hormone-like signaling molecules known as gamma-butyrolactones (GBLs) (Khokhlov et al., 1967; Horinouchi and Beppu, 1994; Kong et al., 2019; Jose et al., 2021). GBL-based regulatory circuits follow quorum sensing (QS) principles of cooperative population-dependent gene expression and share an architecture with QS systems based on acyl-homoserine lactones (abbreviated as Acyl-HSLs or AHLs); hence, they are considered to be analogous to the latter (Figure 1; Polkade et al., 2016; Barreales et al., 2020).

AHLs were primarily extracted from Gram-negative bacteria as autoinducers running QS gene regulation circuitry (Eberhard et al., 1981). Historically, extensive reports on the existence of AHLs in various Gram-negative bacterial taxa, such as Proteobacteria (Fuqua and Greenberg, 2002), Cyanobacteria (Sharif et al., 2008), and Bacteroidetes (Huang et al., 2008; Twigg et al., 2014), have led to the general assumption that AHLs and related cellular components (i.e., synthases and receptors) are exclusive to these species (Swift et al., 1994; Parsek and Greenberg, 2000; Miller and Bassler, 2001). However, three noticeable studies (Biswa and Doble, 2013; Bose et al., 2017; de Moura et al., 2021) that reported the production of AHLs in some Gram-positive bacteria undermine this broadly accepted view. Following these findings, the discovery of AHL synthases and receptors’ homologs in a limited number of Streptomyces species further justifies the quest for AHLs in these species (Santos et al., 2012). Moreover, due to the industrial and medical significance of Streptomyces (Khushboo et al., 2022), studying lactone-based signaling circuits and molecules, their evolutionary origin, and the existence of additional relevant circuits and molecules (e.g., AHLs and related circuits) would be of importance. Furthermore, the presence of novel gene regulation mechanisms in Streptomyces as complex prokaryotes demonstrating multicellular behaviors (see Flärdh and Buttner, 2009) will be notable from an evolutionary point of view.

In this study, we have demonstrated the production of AHLs by six well-known Streptomyces species, including S. griseus, S. lavendulae FRI-5, S. clavuligerus, S. nodosus, S. lividans, and S. coelicolor A3(2), via the chromatography/mass-spectrometry method. Afterward, we investigated the presence of AHL synthases homologous to the LuxI enzyme family and AHL receptors homologous to the LuxR receptor family in Streptomyces via in silico methods.

Gene regulatory circuits are of great interest due to their evolutionary, clinical, and industrial significance. Streptomyces, as Gram-positive bacteria, utilize GBL-based gene regulatory circuits to control their noticeable secondary metabolism (e.g., production of antibiotics) and cellular differentiation (Du et al., 2011; Mingyar et al., 2015; Barreales et al., 2020). Notably, GBLs have been only found in Streptomyces and are the sole lactone-based autoinducers in these species. On the other hand, AHLs, structurally similar molecules that also take on similar roles, are explicitly attributed to Gram-negative bacteria such as V. fischeri (Winson et al., 1995). GBL- and AHL-based gene regulatory circuits also share a common architecture and, along with their components (i.e., synthases and receptors), are considered analogous to each other (Figure 1). This study revealed that while the LuxI and AfsA enzyme families synthesize similar molecules and the LuxR and ArpA receptor families regulate gene expression in response to structurally similar molecules, each protein family has distinct protein structures, amino acid sequences, and conserved regions. Hence, in the absence of an evolutionary relationship between GBLs and AHL circuits, we wondered whether Streptomyces possessed AHLs and relevant components.

We found several AHLs produced by Streptomyces species, S. griseus, S. lavendulae FRI-5, S. clavuligerus, S. nodosus, S. lividans, and S. coelicolor A3(2), as Gram-positive bacteria, in addition to GBLs. While some AHLs (i.e., m/z 134, 228, 230, 256, and 376) were common between certain Streptomyces, each species demonstrated a unique AHL profile. The potential AHL compounds could be seen in four clusters when plotted by their m/z and retention time (Figure 2). Each cluster consists of AHLs from at least four Streptomyces species. Moreover, all species possessed at least one compound in the range of m/z 100–200, 200–300, and 300–400, except for S. lavendulae FRI-5 and S. clavuligerus, lacking any in the first and third range, respectively. The diversity of AHLs from different size ranges may be due to distinct properties of AHLs within each range and their specific physiological roles, which requires Streptomyces to possess a set of AHLs with different sizes. For instance, while AHLs with shorter acyl chains (e.g., 3-oxo-C6-HSL) can diffuse passively across the membrane, those with longer and more hydrophobic acyl chains might concentrate in lipid bilayers or require efflux pumps for their accelerated transport (Fuqua et al., 2001; Fuqua and Greenberg, 2002). Moreover, properties of the acyl chain, such as its length, provide specificity to QS systems (Fuqua et al., 2001). Therefore, various AHLs from different ranges might facilitate the regulation of multiple independent gene expressions in a specific species or intra-species and intra-kingdom communications (Shiner et al., 2005; Feng et al., 2014). On the other hand, multiple AHLs and relevant circuitry may also interactively regulate common target genes (Winson et al., 1995).

Based on the mass-spectrometry analyses of standard AHLs conducted here and data available in other reports (Rodrigues et al., 2022), the chemical structure of some AHLs could be predicted. For instance, a molecular ion peak at m/z 256 in S. lavendulae FRI-5 and S. clavuligerus could be predicted as C10-HSL. Moreover, m/z 228 found in S. nodosus and S. lividans and m/z 324 in S. lividans match C8-HSL and Oxo-C14:1-HSL, respectively. However, further studies such as nuclear magnetic resonance (NMR) analysis are strongly suggested to illuminate the chemical structure of other masses, as they may not consist of a simple acyl chain, such as p-coumaroyl homoserine lactone (Schaefer et al., 2008).

In light of the production of AHLs by Streptomyces, these species were evaluated for harboring LuxI and LuxR homologs to further complete the scheme of AHL signaling circuits in Streptomyces. The protein–protein BLAST of V. fischeri’s LuxI and LuxR against the Streptomyces non-redundant protein sequences database resulted in limited homologs of LuxI and LuxR in a handful of Streptomyces species. However, the HMMsearch using LuxI- and LuxR-specific HMMs resulted in multiple AHL synthases and receptors homologous to LuxI and LuxR in 2336 unique Streptomyces. Previous investigations on the existence of LuxI and LuxR homologs in Actinobacteria only resulted in a single potential LuxI homolog in S. sviceus (SSEG_02829) (Santos et al., 2012) and some LuxR proteins in a limited number of Streptomyces species, including S. avermitilis MA-4680, S. coelicolor A3(2), and S. griseus subsp. griseus NBRC 13350 (Santos et al., 2012). Wet-lab studies on the LuxR family regulator in Streptomyces sp. SN593, although with other ligands (β-carboline), has proved the functional role of these receptors in gene regulation and secondary metabolism (Panthee et al., 2020).

Interestingly, many Streptomyces species were found to carry more than one AHL synthase homologous to LuxI. Similar to most of the Streptomyces covered here, the six Streptomyces of interest in this study possessed between four and seven LuxI homologs. Harboring multiple AHL synthases could be in line with the ability of these species to produce multiple AHLs of different size ranges. We were not able to establish a positive correlation between the copy number of LuxI homologs and the number of AHLs produced by the six Streptomyces of interest, yet we are not surprised due to the imperfection of our methods and the possibility of the role of substrate pools in the combination and abundance of produced AHLs. Regarding the AHL receptors, their high abundance could be interpreted in the context of Streptomyces’ rich secondary metabolism and the need for their physiological regulation activities or their inter- and intra-domain interactions and communications with other species. In spite of supporting evidence, it is worth noting that this data should be considered preliminary, and further investigations are suggested to shed light on the origins of these proteins, their structure, the existence of domains of interest, functions, and their physiological and ecological roles.

AHLs have also been reported in a few other Gram-positive bacteria, i.e., Salinispora (Bose et al., 2017), Exiguobacterium (Biswa and Doble, 2013), and Bacillus (de Moura et al., 2021). However, the discovery of AHLs in Streptomyces species is accompanied by consequential implications for exploring the evolution of signaling circuits and their role in cellular complexity and multicellularity, in addition to benefiting the industry and the clinic. Moreover, while previous studies only reported a few AHLs (Oxo-C10-HSL and Oxo-C12-HSL in Salinispora and Oxo-C8-HSL in Exiguobacterium) limited to a single size range, we demonstrated that each Streptomyces possesses a handful of AHLs from different size ranges, which is more compatible with the physiological complexity of these organisms. Consequently, these findings expand our understanding of lactone-based gene regulation systems in Gram-positive bacteria, especially Streptomyces. Therefore, AHLs might also be involved in regulating secondary metabolites and differentiation in Streptomyces, similar to the role of GBLs. While AHLs might regulate specific target genes, they might also interfere with the GBL–receptor interaction by competing with GBLs to occupy the receptor.

Production of AHLs by Streptomyces species as Gram-positive bacteria stands against previous knowledge suggesting the exclusivity of AHLs to Gram-negative bacterial species (Azimi et al., 2020). Following our observations, further investigations are required to confirm the existence of AHLs, their synthases and receptors, and the function of AHL-based gene regulation systems in Gram-positive bacteria, specifically in the Actinomycetota phylum and Streptomyces species, due to their pharmaceutical and evolutionary significance (Salwan and Sharma, 2020).

AHLs are generally attributed to Gram-negative bacteria as the mediators of QS. Contrarily, six Streptomyces species, S. griseus, S. lavendulae FRI-5, S. clavuligerus, S. nodosus, S. lividans TK-64, and S. coelicolor A3(2), as Gram-positive bacteria, have been found to produce various AHLs, whereas only GBLs have been previously discovered in these species. Furthermore, this study has demonstrated that Streptomyces species potentially possess the enzymes necessary for the synthesis of AHLs and the receptors mediating their effects. These results might suggest the potential presence of an additional regulatory and signaling circuit based on AHLs in Streptomyces. Furthermore, AHLs and related gene regulatory systems might be more prevalent in the prokaryotic domain than previously assumed, and they might play a role in the regulation of secondary metabolites (e.g., antibiotics), leading to significant industrial and clinical value.

The LC-MS/MS raw data presented in the study is deposited in the figshare repository, accession number 10.6084/m9.figshare.25112672. The data used and produced during in-silico studies is provided in the Supplementary material section. Further inquiries can be directed to the corresponding author.

AS-N: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing, Validation. ST: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Data curation, Visualization. GA: Resources, Supervision, Writing – review & editing. JH: Resources, Supervision, Writing – review & editing, Validation.