The role of the phosphopantetheinyltransferase enzyme, PswP, in the biosynthesis of antimicrobial secondary metabolites by <em>Serratia marcescens </em>Db10

Phosphopantetheinyltransferase (PPTase) enzymes fulfil essential roles in primary and secondary metabolism in prokaryotes, archaea and eukaryotes. PPTase enzymes catalyse the essential modification of the carrier protein domain of fatty acid synthases, polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs). In bacteria and fungi, NRPS and PKS enzymes are often responsible for the biosynthesis of secondary metabolites with clinically relevant properties; these secondary metabolites include a variety of antimicrobial peptides. We have previously shown that in the Gram-negative bacterium Serratia marcescens Db10, the PPTase enzyme PswP is essential for the biosynthesis of an NRPS-PKS dependent antibiotic called althiomycin. In this work we utilize bioinformatic analyses to classify PswP as belonging to the F/KES subfamily of Sfp type PPTases and to putatively identify additional NRPS substrates of PswP, in addition to the althiomycin NRPS-PKS, in Ser. marcescens Db10. We show that PswP is required for the production of three diffusible metabolites by this organism, each possessing antimicrobial activity against Staphylococcus aureus. Genetic analyses identify the three metabolites as althiomycin, serrawettin W2 and an as-yet-uncharacterized siderophore, which may be related to enterobactin. Our results highlight the use of an individual PPTase enzyme in multiple biosynthetic pathways, each contributing to the ability of Ser. marcescens to inhibit competitor bacteria by the production of antimicrobial secondary metabolites

The insect pathogen Serratia marcescens Db10 uses a hybrid non-ribosomal peptide synthetase-polyketide synthase to produce the antibiotic althiomycin

There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1-alb6. Bioinformatic analysis of the proteins encoded by alb1-6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2-Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism

<i>S. marcescens</i> Db10 is able to inhibit growth of Gram-positive bacteria.

<p><b>A.</b> Activity assays using <i>B. subtilis</i> NCIB3610, <i>S. aureus</i> 113 and <i>M. luteus</i> ATCC4698 as indicator strains, with <i>S. marcescens</i> Db10 or <i>S. marcescens</i> Db10 <i>SMA2290::</i>Tn<i>5</i> (NRS2992) as the producer strains. <b>B.</b> Culture supernatant assays (described in experimental procedures) indicate the diffusible molecule is produced by <i>S. marcescens</i> Db10 in stationary phase. Clearance halo sizes (radius of cleared area) are averages of three replicates; error bars represent standard error of the mean. A representative growth curve is shown for reference.</p

The PPTase encoded by <i>SMA2452</i> is required for althiomycin biosynthesis.

<p><b>A.</b> Assay to assess the antimicrobial activity of <i>S. marcescens</i> Db10, the <i>alb4–5</i> mutant strain (SAN5), the Δ<i>4147</i> mutant strain (SAN96) and the Δ<i>2452</i>::Cml mutant strain (SAN112) against <i>B. subtilis</i> 3610. <b>B.</b> Activity assays were performed using <i>Bacillus subtilis</i> NRS1473 as the indicator lawn. The producer strains are indicated above as: Db10+VC (<i>S. marcescens</i> Db10 pSUPROM); Db10+2452 (<i>S. marcescens</i> Db10 pSAN46); Δ<i>2452</i>::Cml+VC (SAN112 pSUPROM); Δ<i>2452</i>::Cml+<i>2452</i> (SAN112 pSAN46). ‘VC’ represents the empty vector control.</p

Selected Bacterial Strains and Plasmids Used in This Study.

<p>Selected Bacterial Strains and Plasmids Used in This Study.</p

Spectroscopic analysis of althiomycin produced by <i>S. marcescens</i> Db10.

<p><b>A</b>. Comparison of the extracted ion chromatogram at <i>m/z</i> 440.0600 for wild type <i>S. marcescens</i> Db10 (dashed line) and Db10 Δ<i>alb6</i> (solid line). <b>B</b>. Top panel: high resolution mass spectrum of althiomycin detected in culture supernatant of <i>S. marcescens</i> Db10. Bottom panel: simulated mass spectrum for the C₁₆H₁₈N₅O₆S₂⁺ ion. <b>C</b>. Summary of COSY and HBMC NMR correlations observed for althiomycin isolated from <i>S. marcescens</i> Db10.</p

The tailoring enzymes are required for althiomycin production.

<p><b>A</b>. Assay to assess antimicrobial activity of <i>S. marcescens</i> Db10 and the <i>alb4–5</i> mutant strain (SAN5) against <i>B. subtilis</i> 3610. <b>B.</b> Visualisation of proteins corresponding to the predicted size of Alb4 and Alb5. Total cellular protein from Db10 (wild type <i>S. marcescens</i> Db10), Δ<i>alb3</i> (SAN4) or Δ<i>alb4</i>–<i>5</i> (SAN5) mutants was isolated from cultures grown to stationary phase and separated by SDS-PAGE, followed by staining with Coomassie blue. <b>C–E.</b> Activity assays were performed using <i>Bacillus subtilis</i> NRS1473 as the indicator lawn. The producer strains used are indicated above as: Db10+VC (<i>S. marcescens</i> Db10 pSUPROM); Db10+<i>alb2</i> (<i>S. marcescens</i> Db10 pSAN2); Δ<i>alb2</i>+VC (SAN3 pSUPROM); Δ<i>alb2</i>+<i>alb2</i> (SAN3 pSAN2); Db10+<i>alb3</i> (<i>S. marcescens</i> Db10 pSAN3);Δ<i>alb3_51–262</i>+VC (SAN88 pSUPROM); Δ<i>alb3_51–262</i>+<i>alb3</i> (SAN88 pSAN3); Db10+<i>alb6</i> (<i>S. marcescens</i> Db10 pSAN38); Δ<i>alb6</i>+VC (SAN60 pSUPROM); Δ<i>alb6</i>+<i>alb6</i> (SAN60 pSAN38). ‘VC’ represents the empty vector control.</p

The <i>alb1</i> gene encodes an althiomycin resistance determinant.

<p><b>A</b>. Activity assays using <i>B. subtilis</i> NRS1473 as the indicator lawn for the producer strains: Db10+VC (<i>S. marcescens</i> Db10 pSUPROM); Db10+<i>alb1</i> (<i>S. marcescens</i> Db10 pSAN1); Δ<i>alb1</i>+VC (SAN2 pSUPROM); Δ<i>alb1</i>+<i>alb1</i> (SAN2 pSAN1) <b>B.</b> RT-PCR analysis of <i>alb1</i>–<i>alb6</i> transcript levels in each of the <i>alb</i> mutant strains. The template used in the PCR reaction is indicated above the gels as: Db10 (wild type <i>S. marcescens</i> Db10 cDNA); Δ1 (SAN2(Δ<i>alb1</i>) cDNA); Δ2 (SAN2 (Δ<i>alb2</i>) cDNA); Δ3 (SAN4 (Δ<i>alb3</i>) cDNA); Δ4–5 (SAN5 (Δ<i>alb4–5</i>) cDNA); Δ6 (SAN60 (Δ<i>alb6</i>) cDNA). Reactions were performed in the presence (+) or absence (−) of reverse transcriptase. Con. represents <i>S. marcescens</i> Db10 genomic DNA as a positive control (+) and water as a negative control (−). The primer pairs used to amplify a product internal to a particular gene are indicated to the right of each gel. Twenty five cycles of PCR amplification were used. <b>C.</b> Activity assays using <i>B. subtilis</i> 3610 as the indicator lawn and <i>S. marcescens</i> ATCC274 as the producer strain. <b>D.</b> Activity assays using Db10 (<i>S. marcescens</i> Db10 pSUPROM), Db10 Δ<i>alb4–5</i> (SAN5 pSUPROM) and Db10 P_T5-<i>alb1–6</i> (SAN100 pSUPROM) as the producer strain and using ATCC274+VC (<i>S. marcescens</i> 274 pSUPROM) and ATCC274+<i>alb1</i> (<i>S. marcescens</i> 274 pSAN1) as the indicator lawn. ‘VC’ represents the empty vector control.</p

The 4-methoxy-3-pyrrolin-2-one moiety of althiomycin is shared by other bioactive natural products.

<p>The position of the 4-methoxy-3-pyrrolin-2-one moiety in althiomycin is highlighted by a broken box and this moiety is depicted in grey within the other molecules.</p