Exploiting a Global Regulator for Small Molecule Discovery in Photorhabdus luminescens

Abstract

Bacterially-produced small molecules demonstrate a remarkable range of structural and functional diversity and include some of our most useful biological probes and therapeutic agents. Annotations of bacterial genomes reveal a large gap between the number of known small molecules and the number of biosynthetic genes/loci that could produce such small molecules, a gap that most likely originates from tight regulatory control by the producing organism. This study coupled a global transcriptional regulator, HexA, to secondary metabolite production in Photorhabdus luminescens, a member of the Gammaproteobacteria that participates in a complex symbiosis with nematode worms and insect larvae. HexA is a LysR-type transcriptional repressor, and knocking it out to create a P. luminescens ΔhexA mutant led to dramatic upregulation of biosynthesized small molecules. Use of this mutant expanded a family of stilbene-derived small molecules, which were known to play important roles in the symbiosis, from three members to at least nine members.Photorhabdus luminescens, a Gammaproteobacterium, uses a functionally diverse suite of secondary metabolites to participate in a complex symbiosis with nematode worms (Heterorhabditis spp.) and insect larvae. The bacteria persist quietly in the guts of infective juvenile (IJ) nematodes that hunt insect larvae. When a worm succeeds in entering its prey's circulatory system (hemolymph), it regurgitates the bacteria, which then proceed to make toxins that kill the larva, proteases and esterases that liquefy the larva's interior, signals that cause the IJ worms to become reproducing adults, molecules that counter insect defense mechanisms, and molecules that protect their prey from competing bacteria and fungi. Some of the small molecules produced by P. luminescens have been identified (1), but despite efforts in many laboratories, these known small molecules represent only a small fraction of the bacteria's metabolic potential. The sequenced P. luminescens genome contains at least 33 genes in 20 loci that encode proteins similar to polyketide synthases, nonribosomal peptide synthetases, and β-lactam-producing enzymes (2). The genomic potential for secondary metabolism seen in P. luminescens rivals members of the Streptomyces genus, the most productive antibiotic-producing bacterial genus (3).To access these uncharacterized small molecules, we searched for the molecular signals and their targets that control P. luminescens metabolism. Recently we reported that the bacteria respond to the high concentrations of L-proline in insect hemolymph by initiating a profound upregulation of secondary metabolite production (4). L-proline enhances the production of small molecules known to be involved in antibiosis, insect virulence, and nematode mutualism along with many structurally and functionally uncharacterized † Corresponding author, jon_clardy@hms.harvard.edu. * These authors contributed equally to this work. molecules. L-proline acts both as an osmoprotectant in the high solute concentrat...

Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster

“…HexA is a LysR-type transcriptional repressor that regulates stilbene production and participates in the insect pathogen, nematode mutualist transition (Joyce and Clarke, 2003; Kontnik et al, 2010), and global transcriptomic analysis indicates that pepteridine genes are upregulated in Δ hexA/hfq strains (Tobias et al, 2016). We cultivated the wild-type and mutant strains in a culture medium based on the high concentrations of free proteinogenic amino acids found in insect hemolymph (Crawford et al, 2010).…”

“…Allelic-exchange mutagenesis was used to excise the pepteridine genetic locus in P. luminescens TT01 ( pluCDS3313775R-plu2799 )(Crawford et al, 2010; Kontnik et al, 2010). Approximately 1.5 kB of both upstream and downstream genomic sequences were amplified using the primer pairs Up-F and Up-R ; and Dwn-F and Dwn-R , respectively.…”

Heterogeneous regulation of bacterial natural product biosynthesis via a novel transcription factor

“…A global regulator that mediates phenotypic heterogeneity in Photorhabdus is HexA, which was identified as global transcriptional regulator and repressor of secondary metabolism [29, 30]. The influence of HexA on symbiosis was valued when exploring that 2° cells of P. temperata restore the ability to support nematode development upon inactivation of hexA .…”

Merging chemical ecology with bacterial genome mining for secondary metabolite discovery

“…There are far fewer examples, however, in which natural product chemists examined the chemical potential of regulatory mutants directly connected to bacterial lifestyle decisions. In P. luminescens , for example, insertional inactivation of hexA , the repressor previously identified to be involved in regulating primary form characteristics [66], led to an upregulation of previously described and new bacterial stilbenes [71]. A newly identified dihydrostilbene derivative ( 31 ) was shown to undergo oxidation in aerobic conditions to form the major aromatic stilbene ( 34 ) ( Fig.…”

“…3 ). Such easily oxidized metabolites could assist in overcoming oxidative stress from the insect's innate immune system [71]. L-2,5-Dihydrophenylalanine ( 30 ), an antimetabolite of phenylalanine biosynthesis, was identified in P. luminescens cultures and shown to be the substrate for dihydrostilbene in isotopic feeding studies [25].…”