Background

Entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus live in symbiosis with soil dwelling nematodes of the genera Steinernema or Heterorhabditis, respectively (Goodrich-Blair and Clarke, 2007; Chaston et al., 2011). They are able to propagate without their nematode host and can be easily cultivated in the laboratory using standard media. Their genome harbors a rich diversity for natural products (NPs) with potential activities as antibiotics, antifungals, and cytotoxic or signaling functions. These NPs are often produced by non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and mixed NRPS-PKS hybrids, encoded by biosynthetic gene clusters (BGCs) (H. B. Bode, 2009; Shi and Bode, 2018; Shi et al., 2022).

The access to this promising source of bioactive compounds is possible via different approaches: either by optimizing the culture conditions for enhanced NP production, by heterologous expression or promoter activation of the BGC of interest, or chemical synthesis of the NP derived thereof (H. B. Bode et al., 2002; Biggins et al., 2011; Biggins et al., 2014; E. Bode et al., 2017). All these methods have their limitations: chemical synthesis might be impossible, difficult, or too time consuming, requiring profound knowledge of the NP structure as a starting point. Heterologous production of the desired NP is dependent on building blocks that might only be present in the natural host, on the size of the BGC to be cloned into the heterologous host, on its toxicity, or on special transport systems or modifying enzymes (Brachmann et al., 2012; Nollmann et al., 2015). Isolation of natural products from crude extracts from wildtype (WT) bacteria might be hindered by low production titers, regulatory elements that silence the expression of BGCs, and/or sophisticated culture conditions that have to be established. A promoter activation of desired gene clusters in WT strains might result in NP overproduction that facilitates the isolation of the compound for bioactivity testing and structure elucidation, but still requires NP isolation from crude extracts. The easyPACId (easy Promoter Activation and Compound Identification) approach circumvents all these limitations.

easyPACId describes a simple and fast enrichment and preparation of selected NPs for bioactivity screening just from the crude extract of a bacterial culture of an induced ∆hfq_pCEP-gene_xyz easyPACId mutant. It avoids laborious steps often encountered during NP isolation from WT crude extracts and is not hindered by limitations concerning heterologous expression or chemical synthesis. The targeted activation via the P_BAD promoter (Guzman et al., 1995) in a clean ∆hfq background serves as an excellent tool to enable production of only desired NPs or NP classes derived from single BGCs (Tobias et al., 2017; Valverde, 2017; E. Bode et al., 2019). The RNA chaperone Hfq is widespread in proteobacteria. It has a pleiotropic effect on gene expression by mediating the interaction of small non-coding RNAs (sRNA) and mRNAs, and thereby can activate or suppress translation (Vogel and Luisi, 2011). For NP-producing bacteria like Xenorhabdus, Photorhabdus, or Pseudomonas, a ∆hfq mutant has lost the ability to produce all or most NPs or shows a severely reduced NP amount. Consequently, the chromatogram of crude extracts from a ∆hfq mutant analyzed via HPLC/MS shows no NP-derived signals anymore (clean background) compared to HPLC/MS chromatograms of the corresponding WT where all NPs are produced, and their corresponding signals show a typical pattern for the strain (Figure 1a).

Briefly, the experiment starts via detection of potential BGCs encoding NRPSs, PKS, hybrids thereof, or other BGC classes, via antiSMASH analysis (or based on other bioinformatic tools) (Medema et al., 2011) in the genome of selected NP producers, here exemplified for the genera Photorhabdus and Xenorhabdus. By deletion of the gene hfq, encoding the so-called RNA-chaperone Hfq, a mutant is generated, which is deficient in NP production (Tobias et al., 2017). By exchange of the natural promoter against the L-arabinose inducible promoter P_BAD (Guzman et al., 1995), encoded on the conjugatable vector pCEP via homologous integration (E. Bode et al., 2015), the selective activation of the targeted BGC in a ∆hfq background is now possible (Figure 1). Cell-free culture supernatants or even whole cultures from respective ∆hfq_pCEP-gene_xyz can be freeze dried and dissolved in water for subsequent testing on various target organisms (E. Bode et al., 2015 and 2019; Gulsen et al., 2022). Often, the selected compounds are overproduced and can be easily isolated for further investigations like structure elucidation.

This simple and beneficial method of gaining potentially bioactive compounds can be used to activate natural product biosynthesis gene clusters, not only in Xenorhabdus and Photorhabdus bacteria. It has been applied already to Pseudomonas and is likely to work in any other proteobacteria that encode Hfq, like Serratia or Vibrio (Thoma and Schobert, 2009; Hmelo et al., 2015). Besides Hfq itself, other regulative components of the hfq-sRNA regulon like ArcZ (Neubacher et al., 2020) or even other global regulators might also be useful to generate NP non-producing phenotypes in the described or other Gram-negative bacteria.

Figure 1. Schematic overview of the easyPACId workflow