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Characterization of a Hybrid Nonribosomal Peptide– Carbohydrate Biosynthetic Pathway in Photorhabdus luminescens Corey E. Perez, and Jason M. Crawford Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01120 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019

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Biochemistry

Characterization of a Hybrid Nonribosomal Peptide– Carbohydrate Biosynthetic Pathway in Photorhabdus luminescens Corey E. Perez‡, § and Jason M. Crawford*,‡,§,∥ ‡

Department of Chemistry, Yale University, New Haven, CT 06520, USA Chemical Biology Institute, Yale University, West Haven, CT 06516, USA ∥Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06536, USA §

Supporting Information Placeholder ABSTRACT: Advances in genome sequencing and analysis have afforded a trove of “orphan” bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydroβ-D-N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathwaytargeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new “glycoaminoacid” metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl-unit derived from the NRPS machinery onto the free 1,6anhydro-β-D-N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.

Microbial natural products are a prized resource as drugs and drug leads owing to their evolutionarily-optimized origins for target binding.1, 2 Their scaffolds are also harnessed in the design of molecular probes, where they aid in illuminating new biological phenomena. In bacteria and fungi, the biocatalysts responsible for natural product production are most often encoded on a contiguous stretch of the genome termed a biosynthetic gene cluster, which is thought to facilitate transcriptional regulation and expression.3-5 These clusters are readily found within genomic islands as a result of horizontal gene transfer and are functionally maintained when they confer a fitness advantage to the host in its ecological niche. As microbial genome sequence information continues to expand, so, too, does the probability of novel biocatalyst discovery and characterization.6, 7 This mounting capacity is reflected in the growing number of hypothetical proteins, which represent a

source of enzymes with the potential to catalyze novel biotransformations, encoded in this sequence space.8 The gammaproteobacterial entomopathogen Photorhabdus luminescens is known for its largely unexplored biosynthetic capabilities.8-11 This biosynthetic power likely arises from its complex ecological niche in which it is mutualistic with its Heterorhabditis nematode host and pathogenic with the nematode’s larval prey. To adapt to this lifestyle, the bacteria engage in stochastic phase variation.12 During active infections, P. luminescens predominantly resides in its pathogenic form (P-form). In this state, it produces various molecules with diverse functions, including insect virulence factors, nematode growth factors, signaling molecules, immunomodulators, and antimicrobials, among others.8, 11 P. luminescens represses these pathways in its mutualistic form (M-form) that colonizes specific cells in the nematode’s intestinal tract, facilitating vertical transmission to nematode progeny.12-15 Given its multipartite symbiotic lifestyle, bacteria belonging to the Photorhabdus genus encode a variety of important bioactive products, including small molecules that have been developed into drugs such as tapinarof16 for the autoimmune disorder psoriasis and pristinamycin10, 17, 18 for the treatment of bacterial infections. We previously identified an atypical orphan biosynthetic gene cluster in P. luminescens that possessed machineries with similarities to carbohydrate and distant nonribosomal peptide synthesis genes (Figure 1A).19 While lacking conservation among closely related species, portions of this pathway are shared among the related entomopathogens Photorhabdus temperata ssp. temperata Meg1 and Photorhabdus asymbiotica ssp. australis, which is also a known human pathogen20, 21 (Figures 1 and S1). This pathway deviates from architectures of known glycopeptide and NRPScarbohydrate biosynthetic pathways, such as the capuramycin,22, 23 C-1027,24 and mannopeptimycin pathways,25 among many others.26-29 Pathway reconstruction, heterologous expression, and metabolite characterization demonstrated that the gene cluster was responsible for the production of a small family of carbohydrates, featuring a 1,6-anhydro-β-D-N-acetyl-glucosamine moiety. However, genetic analysis revealed that only two genes, plu2409 and plu2411, were responsible for the formation of these products, and the NRPS components remained undefined. Biochemical studies of these proteins demonstrated that the glycosyltransferase Plu2409 utilized UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) to anabolically generate chitin-type products that the hypothetical protein Plu2411, functioning as a lytic transglycosylase, then

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Figure 1. The Photolose Biosyntetic Pathway. (A) Genome synteny analysis of the photolose biosynthetic pathway from P. luminescens TT01. Regions of protein identity are denoted with darker shades indicating higher identity. The predicted function of the protein products are denoted below the genetic comparison. (B) The proposed biosynthesis of the previously characterized carbohydrate metabolites arising from the photolose biosynthetic pathway. The 1,6-anhydro disaccharide previously characterized by X-ray analysis is presented, although products up to the tetrasaccharide were observed. converted to the observed 1,6-anhydo oligosaccharides (Figure 1B). These molecules share structural similarity to the cell wall recycling product N-acetyl-glucosamine-(1→4)-1,6-anhydro-Nacetyl-b-D-muramic acid; however, any possible parallel roles with cell wall biosynthesis are not yet understood.30 In contrast to canonical cell wall recycling pathways, this pathway encodes the uncharacterized NRPS-like proteins Plu2407, a hypothetical/putative acyl- or peptidyl-carrier protein (PCP), and Plu2408, a putative ligase/adenylation protein.19 Additionally, based on sequence information, an apparent condensation domain was not present in the pathway to couple the NRPS and carbohydrate machineries. Here, employing a top-down protein mass spectrometric assay,31 we characterized the specificity of the putative adenylation protein Plu2408. Plu2408 demonstrated substrate selectivity for glycine and loaded Plu2407, which was confirmed to be a distant peptidyl-carrier protein. To identify glycylfunctionalized metabolites, we employed pathway-targeted molecular networking on the heterologously-expressed gene cluster in conjunction with stable isotope labeling. This analysis revealed a single new glycoaminoacid molecule termed “photolose” (1), which requires all of the carbohydrate and NRPS genes in the operon for its production. Using a hemolymph mimetic medium, we were able to detect 1 along with its previously characterized disaccharide substrate in the native host, while deletion of the pathway abolished production. Hypothetical Plu2410 was also required for the production of 1. Consequently, we evaluated Plu2410 for its ability to bridge the carbohydrate and NRPS components in the pathway. In vitro biochemical assessment of Plu2410, chemical derivatization, and high-resolution tandem mass spectroscopy (MS2) demonstrated that this enzyme functions as an atypical condensation protein to tether the glycyl-moiety

from the peptidyl-carrier protein onto the 1,6-anhydro-β-D-Nacetyl-glucosamine moiety of a free disaccharide substrate. Challenge of wild-type P. luminescens and a pathway mutant with the antimicrobial peptide cecropin A revealed that the photolose biosynthetic pathway confers a significant growth advantage to cecropin A. MATERIALS AND METHODS

General Unless otherwise specified, Photorhadus luminescens and Escherichia coli were maintained on lysogeny broth (LB) agar plates [BD; 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, and 1.5% (w/v) agar] at 30 °C or 37 °C, respectively, with suspension cultures occurring in LB medium [BD; 1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) NaCl] under aerobic conditions at 250 rpm. Kanamycin (American Bioanalytical) and chloramphenicol (American Bioanalytical) were utilized at a concentration of 25 µg/mL when needed, unless stated otherwise. Liquid chromatography/high-resolution quadrupole time-offlight electrospray ionization mass spectrometry (LC-HR-QTOFESI-MS) analyses were conducted using an Agilent iFunnel 6550 QTOF MS fitted with a Dual Agilent Jet Stream (AJS) ESI source coupled to an Agilent 1290 Infinity high performance liquid chromatography (HPLC) system. Microtiter plate assays were

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Biochemistry

quantified using an EnVisionÒ Multilabel Plate Reader (PerkinElmer) employing the monochromator. Detailed protocols for all experimental procedures can be found in the supporting information.

Accession Codes Plu2407, UniProtKB Q7N4D4 Plu2408, UniProtKB Q7N4D3 Plu2410, UniProtKB Q7N4D1

Construction of Strep-tag II N-terminal Fusion Proteins Standard molecular biology, protein expression, and protein purification procedures were used to prepare the epitope fused proteins employed in this study (see supporting information).

Top-down Proteomic Analysis of the PCP, Plu2407 Fifty microliter reactions were assembled containing 10 µM Plu2408, 100 µM Plu2407, and 5 mM of each amino acid (Gly, DL-Ala, DL-Leu, DL-Ile, and DL-Val) in a reaction buffer composed of 100 mM Tris-HCl, 300 mM NaCl, 5 mM ATP, and 1 mM MgCl2. Reactions were performed at both pHRT 7.4 and 8. After incubation for 1 h at 25.0 °C, the reactions were snap-frozen in liquid N2. Reactions were thawed immediately prior to analysis via LC-HR-QTOF-MS and MS2 (see supporting information).

Metabolomic Analysis of pE-Photolose E. coli BAP1 harboring both the previously generated pEReconstructed plasmid,19 herein renamed pE-Photolose, and pRARE2 along with an E. coli BAP1 control strain harboring pET-28a and pRARE2 were grown overnight and then subcultured into M9-glucose medium [10.5 g/L M9 minimal salts, 2 mM MgSO4, 0.1 mM CaCl2, and 0.4 (w/v) glucose] supplemented with kanamycin and chloramphenicol. The cultures were grown to midexponential phase and induced with isopropyl β-D-1thiogalactopyranoside (IPTG) at 0.01 mM. Growth was continued for 48 h at 16 °C. The cells were harvested via centrifugation. The supernatants were decanted and filtered before being frozen and lyophilized. The pellets were resuspended in phosphate buffered saline [PBS, 0.8% (w/v) NaCl, 0.02% (w/v) KCl, 0.144% (w/v) Na2HPO4, and 0.024% (w/v) KH2PO4; pH 7.4] and recollected via centrifugation. The supernatants were decanted, and the pellets were frozen and lyophilized. The lyophilized materials were extracted with methanol. The methanolic extracts were filtered, and the filtrates was dried via centrifugal evaporation (Genevac). The dried residues were resuspended in water and filtered prior to being analyzed via LC-HR-QTOF-MS. See the supporting information for data processing details.

Stable Isotope Labeling Studies The pET-28a or pE-Photolose plasmid along with pRARE2 were cotransformed into chemically competent E. coli BAP1 ΔglyA via standard heat shock procedures. The strains were grown in LB supplemented with glycine overnight and subsequently subcultured into a defined minimal medium in which the glycine content was replaced with universally labeled glycine (U-13C-1,2Gly), or, alternatively, with 1:1 labeled:unlabeled glycine. Growth, processing, and analysis proceeded as described in “Metabolomic Analysis of pE-Photolose” (see supporting information).

analyzed as described in “Metabolomic Analysis of pEPhotolose” except only M9-glucose medium was utilized and only the processed pellets were analyzed (see supporting information).

Assessment of Photolose Production in P. luminescens P. luminescens TTO1, locked P-form, locked M-form, and P. luminescens TTO1 Δplu2407-2411 were grown in LB overnight before being subcultured into Galleria mellonella hemolymph mimetic medium32, 33 plus sodium chloride (HMMNa, see Supporting Table S6 for composition). The cultures were grown for two days at 30 °C and 250 rpm before being clarified via centrifugation. The supernatant was decanted and filtered. The pellet was resuspended in a volume of PBS equal to the initial starting culture and recollected via centrifugation. This step was repeated with fresh PBS, and the supernatant was decanted after harvesting the pellet. Both the supernatants and pellets were frozen and lyophilized, and subsequently extracted with methanol. The methanolic extracts were then filtered and dried via centrifugal evaporation (Genevac). The dried residue was resuspended in water and filtered prior to LC-HR-QTOF-MS analysis (see supporting information).

In vitro Generation of Photolose Fifty microliter reactions were prepared as described in “Topdown Proteomic Analysis of the PCP, Plu2407”; however, each reaction contained a single amino acid along with 10 uM Plu2408, 10 uM Plu2407, 10 uM Plu2410, and 0.5 mM disaccharide. Reactions were incubated at 25.0 °C for 1 h before being snap-frozen in liquid N2 and lyophilized. The dried residues were extracted with methanol and analyzed via LC-HR-QTOF-MS. All reactions were performed in triplicate (see supporting information).

Structural Characterization of Photolose Peracetylation of photolose was achieved by suspending the dried methanolic extracts from 30 mL cultures of both P. luminescens TT01 and P. luminescens TT01 Dplu2407-2411 in 2 mL of acetic anhydride and 1 mL of pyridine under a nitrogen atmosphere. The reactions were allowed to proceed for ~4 h, until all material was solubilized, and were then dried via centrifugal evaporation. The dried reactions were resuspended in methanol and subsequently analyzed by LC-HR-QTOF-MS and MS2 (see supporting information).

Assessment of Cecropin A Resistance Wild-type P. luminescens TT01 and P. luminescens TT01 Dplu2407-2411 were grown overnight in LB and subsequently subcultured into HMMNa with outgrowth occurring overnight. These cultures were subcultured into HMMNa a final time and grown to OD600 = 1. Cultures were then diluted 1:2000 into HMMNa immediately before use. Cecropin A (Bachem) was dissolved in HMMNa to a concentration of 0.333 mg/mL. A twofold dilution series was prepared in HMMNa in the wells of sterile polypropylene 96-well plates (100 µL cecropin A solution per well; high concentration 0.333 mg/mL – prior to cell addition). One hundred microliters of the diluted culture were then added to each well (final cecropin A high concentration, 0.167 mg/mL). The plates were sealed with gas permeable seals and incubated at 30 C for 18 h. Growth was then assessed via OD600 (see supporting information).

Evaluation of Genetic Contributions to Photolose Production

RESSULTS AND DISCUSSION

E. coli BAP1 / pRARE2 strains independently harboring plasmids bearing deletions of each gene in the locus were grown and

As plu2408 was predicted to encode an NRPS-like adenylation protein, canonically responsible for the selection of a cognate


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Figure 2. Top-down Proteomic Analysis Reveals Adenylation Domain Substrate Specificity. The deconvoluted mass spectra corresponding to holo-Plu2407 (A) and holo-Plu2407+Gly (B) are presented. The appendage of glycine onto the phosphopantetheinyl arm of Plu2407 was validated using MS2 resulting in pantetheine ejection ions for holo-Plu2407 (C; obs. 261.1260 m/z, calc. 261.1267 m/z) and holoPlu2407+Gly (D; obs. 318.1479 m/z, calc. 318.1482 m/z) that correspond to their respective predicted masses within 10 ppm. In A-D, stars indicate peaks of interest. (E) A schematic of the selectivity of the adenylation protein, Plu2408, is presented. amino acid, adenylate formation, and loading of the amino acid onto the putative carrier protein Plu2407, we first assessed the adenylation protein amino acid substrate selectivity using NRPSpredictor2.19, 34, 35 This analysis suggested that hydrophobicaliphatic amino acids were candidate substrates of the enzyme (Table S2). To probe this selectivity in vitro, Plu2407 and Plu2408 were both cloned as N-terminal Strep-tag II fusion proteins, overexpressed, and purified (Figure S2). These proteins were then utilized in in vitro biochemical reactions containing an amino acid mixture comprised of the predicted amino acids (Gly, Ala, Val, Leu, and Ile). Top-down protein MS analysis of these reactions and deconvolution of the acquired spectra yielded predominant masses of 11135.8 and 11192.7 amu corresponding to the predicted masses of holo-Plu2407 and holo-Plu2407+Gly, respectively, less the N-terminal formyl-methionine residue (Figure 2A and B, respectively). This study supported the substrate selectivity of the adenylation protein and confirmed that Plu2407 was a carrier protein despite having a divergent primary sequence. To confirm the identity of the amino acid appended onto the phosphopantetheine arm of Plu2407, MS2 studies were performed.31 The pantetheine ejection ions observed for holoPlu2407 and holo-Plu2407+Gly corresponded to their respective predicted masses within 3 and 1 ppm, respectively (holo-Plu2407: obs. 261.1260 m/z, calc. 261.1267 m/z; holo-Plu2407+Gly: obs.

318.1479 m/z, calc. 318.1482 m/z) (Figure 2C and D, respectively), confirming that Plu2408 selectively loads Gly onto Plu2407 in our in vitro assays (Figure 2E). Utilizing the amino acid selectivity of the adenylation protein Plu2408 as a guide, we next sought to identify new metabolites functionalized by glycine. To this end, we performed pathwaytargeted molecular networking to identify metabolites arising from the pathway36-38 (Figure 3A). The resulting molecular network consisted of two regions, one of which harbored a molecular ion corresponding to the 1,6-anhydro disaccharide characterized in our previous study by X-ray analysis. This ion is directly connected to a molecular ion with an exact mass matching that of a protonated, glycyl-functionalized 1,6-anhydro disaccharide. To confirm that this molecular ion was modified with glycine, an E. coli BAP1 glycine auxotroph was generated via l-Red recombineering39, 40 and stable-isotope feeding studies were performed using universally labeled 1,2-13C-Gly. LC-HR-QTOF-MS analysis of culture extracts confirmed the incorporation of the isotopic label (Figure S3). We next set out to characterize the enzyme responsible for the condensation of glycine onto the 1,6-anhydro disaccharide core. LC-HR-QTOF-MS analysis was performed on individual gene knockouts of every gene in the biosynthetic pathway (Figure 3B).


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Biochemistry

Figure 3. Cellular and In Vitro Assessment of Photolose Production. (A) The molecular network of pathway-dependent metabolites in cells heterologously expressing the pathway is presented. Nodes are colored corresponding to parent mass intensity with the more abundant metabolites being darker red. The thickness of edges between nodes indicates greater MS2 spectral similarities. The hexagonal node represents the one molecular feature confirmed through stable isotope labeling to incorporate glycine. (B) The genetic determinants of photolose production were assessed though heterologous production. Extracted ion chromatograms within 10 ppm of the protonated mass of photolose (464.1875 Da) are presented (tR = 2.84 min). (C) The ability of the putative condensation protein, Plu2410, to catalyze the coupling of glycine onto the 1,6-anhydro disaccharide was evaluated. Absolute configuration of the authentic disaccharide substrate was previously confirmed through X-ray analysis. Extracted ion chromatograms corresponding to 464.1875 Da ± 10 ppm are presented. A coinjection of the in vitro reaction product with that derived from heterologous expression is shown in (D), indicating identical product formations. (E) The ability of wild-type P. luminescens TT01 to produce photolose was evaluated under biosynthetic pathway stimulatory conditions. Production was compared against genetically locked P-form and locked M-form, as well as the pathway mutant. Extracted ion chromatograms corresponding to photolose (464.1875 Da ± 10 ppm) are shown (tR = 3.27 min). Photolose exhibits a slightly shifted retention time in this analysis relative to the other panels as a different column was used for this experiment (Figure S5). This analysis demonstrated that plu2407-plu2411 were required for the production of the molecule of interest. Of this set, plu2410 was the only gene which remained uncharacterized and was therefore predicted to be responsible for the condensation reaction. Other genes present in the genomic island (plu2403-2406 and plu2412-2413) were shown to be dispensable for photolose production, defining the gene cluster boundaries. Given the genetic organization (Figure S1) and the results of our knockout analysis, it appeared that at least two distinct operons are present in the genomic island. Genome synteny analysis revealed that the genetic regions comprising plu2403-2406 and plu2412-2413 are more

highly conserved than the photolose operon (plu2407-2411) itself across multiple related species (Figure S1). To test whether Plu2410 was responsible for catalyzing the condensation of Gly onto the 1,6-anhydro disaccharide, in vitro biochemical reactions were performed. Plu2410 was cloned with an N-terminal Strep-tag II and purified. In vitro reactions were assembled containing 10 µM Plu2407, 10 µM Plu2408, 10 µM Plu2410, 25 mM equimolar amino acid mixture (Gly, Ala, Val, Leu, and Ile), and 0.5 mM of authentic 1,6-anhydro disaccharide in a buffer comprised of 100 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM ATP, and 1 mM MgCl2. Reactions were incubated at


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Figure 4. Photolose Characterization and Its Proposed Structure and Biosynthesis. MS2 spectra of peracetylated photolose (A) and its 1,6anhydro disaccharide precursor (B) are presented. Black diamonds represent the precursor ion. Key ions are highlighted with their putative structures. (C) Photolose is proposed to be synthesized though the convergence of carbohydrate and NRPS machineries. Plu2411 is predicted to anabolically generate the chitin substrate upon which Plu2409 acts to generate the 1,6-anhydro disaccharide precursor. Concurrently, the adenylation protein, Plu2408, selects and loads glycine onto the carrier protein Plu2407. The action of the novel condensation protein, Plu2410, serves to couple the glycyl moiety onto the free 1,6-anhydro disaccharide substrate yielding photolose. 25.0 °C for one hour before being quenched. LC-HR-QTOF-MS analysis of methanolic extracts of the lyophilized reactions confirmed that Plu2410 is responsible for the condensation of Gly onto the disaccharide core (Figure 3C and D). Additionally, to support our top-down proteomic analysis, the in vitro reactions were analyzed for products in which one (or more) of the other amino acids present in the mixture was appended onto the 1,6 anhydro substrate (data not shown). No additional products were identified confirming that the adenylation protein, Plu2408, is specific for Gly. While we had previously not been able to detect the carbohydrate precursors of photolose in the native host, P. luminescens TT01,19 we reassessed its production capability utilizing an alter-

native hemolymph mimetic medium which serves to reproduce the amino acid composition of G. mellonella larval circulatory fluid.32, 33 Cultures of wild-type P. luminescens, as well as M- and P-form genetically phase-locked strains,12 were grown at 30 mL scale for two days. The supernatants were clarified via centrifugation and residual cellular material was removed via passage through a 0.22 µm filter. The pellets were washed twice with PBS to remove residual supernatant. The supernatants and pellets were lyophilized, extracted with methanol, and analyzed by LC-HRQTOF-MS. Under these stimulatory conditions, we were able to detect photolose in P. luminescens in all three genetic backgrounds (Figure 3E). Deletion of the pathway via markerless allelic-exchange mutagenesis abolished photolose production in the native producer (Figure 3E, red trace). The ability of Photorhab-


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Biochemistry

dus to glycylate 1,6-anhydro tri- and tetra-saccharides was also assessed. Extracted ion chromatograms of the calculated protonated masses for glycylated 1,6-anhydro tri- and tetra-saccharides failed to exhibit any novel peaks relative to the pathway deletion control (data not shown), indicating Plu2410 selectivity for the disaccharide substrate. The ability of the di-, tri-, and tetrasaccharide products to become glycylated at multiple positions was similarly evaluated; however, no new masses beyond 1 were identified (data not shown). Interestingly, the M- and P-forms demonstrated markedly different titers for the production of 1 under the conditions of our experiment. This difference is reflected in the expression levels of the genes comprising the photolose pathway where the pathway is downregulated in the M-form relative to the P-form (log2 expression ratio (M-form/P-form) plu2407: -2.61; plu2408: -2.07; plu2409: -3.06; plu2410: -5.13; and plu2411: -2.93) during exponential growth when grown in LB supplemented with pyruvate at 28 °C.12 Moreover, unlike in our E. coli heterologous expression system where photolose is predominantly secreted (Figure S4), in the native host, photolose is strictly associated with the cell pellet. This suggests that either E. coli possesses a transporter which P. luminescens lacks capable of translocating this molecule to the extracellular space, or that P. luminescens harbors machinery which is capable of utilizing photolose as a substrate, potentially incorporating it into the cell wall. Having characterized the biosynthetic route to photolose, we next sought to provide structural support of the new molecule, 1. As the glycoaminoacid is highly polar and the glycyl ester is subject to hydrolysis during isolation, attempts at scale up and isolation from the supernatant of E. coli overexpressing the pathway or from in vitro biochemical reactions proved challenging. We therefore employed MS2 to assign structural connectivities. The MS2 spectrum of photolose demonstrates a fragment ion at 76.8389 m/z corresponding to protonated Gly, suggesting that Gly is incorporated via an ester linkage (Figure S7). Based on structural homology to known 1,6-anhydro sugars, we hypothesized that glycine would be appended at the 3-OH of the 1,6-anhydro sugar, the only site capable of ester formation on this residue, which was supported by MS2 analysis of the product. However, due to the potential ions formed in the gas phase during collision induced dissociation, particularly the possibility of 1,6-anhydro formation on the GlcNAc residue, extracts from the pellets of 30 mL P. luminescens cultures were also peracetylated utilizing pyridine and acetic anhydride prior to MS2 analysis to eliminate potential gasphase 1,6-anhydro formation. Comparison of the fragmentation pattern of peracetylated photolose with that of the peracetylated precursor 1,6-anhydro disaccharide reveals a major ion 330.1186 m/z in both fragmentation spectra which represents the peracetylated GlcNAc oxocarbenium fragment (Figure 4A and B). This ion validates the lack of modification at the GlcNAc residue. One new major ion at 303.1193 m/z (Figure 4A) corresponds to the glycyl-functionalized 1,6-anhydro moiety strongly supporting glycyl functionalization at the 3-OH of the 1,6-anhydro moiety and the structural assignment of photolose. Muropeptides generated during cell wall recycling, with which photolose shares structural similarities, are known to elicit immune responses41-43 as well as serve as bacterial cytosolic effectors of b-lactamase induction.44-46 We therefore postulated that photolose might play a role in modulating immune response during larval infection conferring enhanced virulence. To assess this hypothesis, we performed an insecticidal assay using wild-type P. luminescens and the corresponding photolose pathway mutant in a G. mellonella model system. Death of the larvae was monitored over a 36 h period until all larvae in the experimental sample were

killed. Larval survival was indistinguishable between the wildtype and mutant infection groups (Figure S8). While no difference was observed in this model, it is possible that the pathway’s contribution to virulence is masked by the functional redundancy of numerous toxins and virulence factors known to be produced by P. luminescens during infections. During insect infection, one canonical immune response is the upregulation of antimicrobial peptides (AMPs).47-49 For P. luminescens, resisting this defense mechanism is one component of its ability to propagate and ultimately cause septicemia. One general route of AMP, specifically cationic AMP, resistance among Gram-negative bacteria is surface modification.50-52 Through this process, the negatively charged lipopolysaccharide is modified, typically with cationic moieties, to modulate the surface charge thereby aiding cationic AMP repulsion.50-52 We postulated that if P. luminescens possessed a pathway by which photolose could be incorporated into the LPS, physically or covalently, it might serve an analogous function given the primary amine present in the structure. To test this hypothesis, we subjected wild-type P. luminescens and the pathway mutant to a minimal inhibitory concentration screen using cecropin A, a known insect AMP (Figure S9). This analysis revealed that the pathway does significantly contribute to cecropin A resistance (IC50-WT = 2.524 mg/mL; IC50D = 1.669 mg/mL) by a yet to be determined mechanism (Figure 5). These data are also biologically consistent with the upregulation of the photolose pathway in the pathogenic P-form variant relative the M-form.

Figure 5. Cecropin A Treatment. The growth of wild-type P. luminescens (black) and the corresponding pathway deletion mutant (grey) under 2.604 mg/mL of cecropin A are shown. Error bars represent the standard deviation. ***, P ≤ 0.001 CONCLUSION Through the application of mass spectral and biochemical techniques, we were able to characterize the NRPS-like enzymes present in an atypical NRPS-carbohydrate biosynthetic pathway, harbored by the bacterium P. luminescens. The amino acid substrate specificity of the putative adenylation protein Plu2408 was established using top-down proteomics and informed pathwaytargeted metabolomic and stable isotope labeling studies. These metabolomic analyses revealed a novel pathway-dependent metabolite, photolose, that represents, to the best of our knowledge, the first simple glycoaminoacid secondary metabolite. Genetic deletions confirmed the molecule’s dependence on both the carbohydrate and NRPS machineries present in the pathway and through a process of elimination suggested Plu2410 functioned as a condensation protein. In vitro assessment of this putative condensation protein supported its role in catalyzing the appendage of glycine onto the 1,6-anhydro disaccharide core. MS2 studies of the free metabolite and its peracetylated product strongly supported the structural connectivity, establishing the location of the glycyl appendage at the 3-OH of the anhydro sugar moiety. Owing to


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photolose’s structural similarity to established cell wall recycling components, which are known to elicit an immune response, we hypothesized that this molecule might play a role in modulating virulence in insect larvae; however, no effect was observed under the conditions of our invertebrate animal model experiment. In an attempt to deconstruct the pathway’s probable role in immune regulation, we demonstrated that the pathway aids in cecropin A antimicrobial peptide resistance, which is consistent with higher observed production and expression in the pathogenic P-form variant. Future experiments exploring the potential for cell wall incorporation,53 b-lactamase induction,44-46 and immune response43, 54, 55 may help shed light on further functional roles of this novel glycoaminoacid metabolite.

(4) Penn, K., Jenkins, C., Nett, M., Udwary, D. W., Gontang, E. A., McGlinchey, R. P., Foster, B., Lapidus, A., Podell, S., Allen, E. E., Moore, B. S., and Jensen, P. R. (2009) Genomic islands link secondary metabolism to functional adaptation in marine Actinobacteria. ISME J 3, 1193-1203.

ASSOCIATED CONTENT

(8) Vizcaino, M. I., Guo, X., and Crawford, J. M. (2014) Merging chemical ecology with bacterial genome mining for secondary metabolite discovery. J Ind Microbiol Biotechnol 41, 285-299.

Supporting Information Supplemental

materials,

methods,

and

figures.

(PDF)

The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION

Corresponding Author

(5) Ziemert, N., Lechner, A., Wietz, M., Millán-Aguiñaga, N., Chavarria, K. L., and Jensen, P. R. (2014) Diversity and evolution of secondary metabolism in the marine actinomycete genus Salinispora. Proc Natl Acad Sci U S A 111, E1130-1139. (6)

Winter, J. M., Behnken, S., and Hertweck, C. (2011) Genomics-

inspired discovery of natural products. Curr Opin Chem Biol 15, 22-31.

(7) Bachmann, B. O., Van Lanen, S. G., and Baltz, R. H. (2014) Microbial genome mining for accelerated natural products discovery: is a renaissance in the making? J Ind Microbiol Biotechnol 41, 175-184.

(9) Duchaud, E., Rusniok, C., Frangeul, L., Buchrieser, C., Givaudan, A., Taourit, S., Bocs, S., Boursaux-Eude, C., Chandler, M., Charles, J. F., Dassa, E., Derose, R., Derzelle, S., Freyssinet, G., Gaudriault, S., Medigue, C., Lanois, A., Powell, K., Siguier, P., Vincent, R., Wingate, V., Zouine, M., Glaser, P., Boemare, N., Danchin, A., and Kunst, F. (2003) The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens. Nat Biotechnol 21, 1307-1313. (10) Tobias, N. J., Mishra, B., Gupta, D. K., Sharma, R., Thines, M., Stinear, T. P., and Bode, H. B. (2016) Genome comparisons provide insights into the role of secondary metabolites in the pathogenic phase of the Photorhabdus life cycle. BMC Genomics 17, 537.

*E-mail: [email protected]

ORCID Corey E. Perez: 0000-0002-7911-0696 Jason M. Crawford: 0000-0002-7583-1242

(11) Shi, Y. M., and Bode, H. B. (2018) Chemical language and warfare of bacterial natural products in bacteria-nematode-insect interactions. Nat Prod Rep 35, 309-335.

Author Contributions CEP and JMC designed experiments, analyzed the data, and wrote the manuscript. CEP performed experiments.

Funding Sources Financial support from the National Institutes of Health (1DP2CA186575), the Burroughs Wellcome Fund (1016720), the Camille & Henry Dreyfus Foundation (TC-17-011), and Yale University is gratefully acknowledged. ACKNOWLEDGMENT We thank Dr. Xun Guo for his preliminary efforts and previous isolation of authentic oligosaccharides utilized in this study.

(12) Somvanshi, V. S., Sloup, R. E., Crawford, J. M., Martin, A. R., Heidt, A. J., Kim, K. S., Clardy, J., and Ciche, T. A. (2012) A single promoter inversion switches Photorhabdus between pathogenic and mutualistic states. Science 337, 88-93. (13) Clarke, D. J. (2008) Photorhabdus: a model for the analysis of pathogenicity and mutualism. Cell Microbiol 10, 2159-2167. (14)

Waterfield, N. R., Ciche, T., and Clarke, D. (2009) Photorhab-

dus and a host of hosts. Annu Rev Microbiol 63, 557-574.

(15) Somvanshi, V. S., Kaufmann-Daszczuk, B., Kim, K. S., Mallon, S., and Ciche, T. A. (2010) Photorhabdus phase variants express a novel fimbrial locus, mad, essential for symbiosis. Mol Microbiol 77, 10211038. (16) Smith, S. H., Jayawickreme, C., Rickard, D. J., Nicodeme, E., Bui, T., Simmons, C., Coquery, C. M., Neil, J., Pryor, W. M., Mayhew, D., Rajpal, D. K., Creech, K., Furst, S., Lee, J., Wu, D., Rastinejad, F., Willson, T. M., Viviani, F., Morris, D. C., Moore, J. T., and Cote-Sierra, J. (2017) Tapinarof Is a Natural AhR Agonist that Resolves Skin Inflammation in Mice and Humans. Journal of Investigative Dermatology 137, 2110-2119.

ACCESSION CODES Plu2407, UniProtKB Q7N4D4 Plu2408, UniProtKB Q7N4D3 Plu2410, UniProtKB Q7N4D1 REFERENCES

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Figure 1. The Photolose Biosyntetic Pathway. (A) Genome synteny analysis of the photolose biosynthetic pathway from P. luminescens TT01. Regions of protein identity are denoted with darker shades indicating higher identity. The predicted function of the protein products are denoted below the genetic comparison. (B) The proposed biosynthesis of the previously characterized carbohydrate metabolites arising from the photolose biosynthetic pathway. The 1,6-anhydro disaccharide previously characterized by X-ray analysis is presented, although products up to the tetrasaccharide were observed. 177x94mm (300 x 300 DPI)


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Biochemistry

Figure 2. Top-down Proteomic Analysis Reveals Adenylation Domain Substrate Specificity. The deconvoluted mass spectra corresponding to holo-Plu2407 (A) and holo-Plu2407+Gly (B) are presented. The appendage of glycine onto the phosphopantetheinyl arm of Plu2407 was validated using MS2 resulting in pantetheine ejection ions for holo-Plu2407 (C; obs. 261.1260 m/z, calc. 261.1267 m/z) and holo-Plu2407+Gly (D; obs. 318.1479 m/z, calc. 318.1482 m/z) that correspond to their respective predicted masses within 10 ppm. In A-D, stars indicate peaks of interest. (E) A schematic of the selectivity of the adenylation protein, Plu2408, is presented.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Cellular and In Vitro Assessment of Photolose Production. (A) The molecular network of pathwaydependent metabolites in cells heterologously expressing the pathway is presented. Nodes are colored corresponding to parent mass intensity with the more abundant metabolites being darker red. The thickness of edges between nodes indicates greater MS2 spectral similarities. The hexagonal node represents the one molecular feature confirmed through stable isotope labeling to incorporate glycine. (B) The genetic determinants of photolose production were assessed though heterologous production. Extracted ion chromatograms within 10 ppm of the protonated mass of photolose (464.1875 Da) are presented (tR = 2.84 min). (C) The ability of the putative condensation protein, Plu2410, to catalyze the coupling of glycine onto the 1,6-anhydro disaccharide was evaluated. Absolute configuration of the authentic disaccharide substrate was previously confirmed through X-ray analysis. Extracted ion chromatograms corresponding to 464.1875 Da ± 10 ppm are presented. A coinjection of the in vitro reaction product with that derived from heterologous expression is shown in (D), indicating identical product formations. (E) The ability of wild-type P. luminescens TT01 to produce photolose was evaluated under biosynthetic pathway stimulatory conditions. Production was compared against genetically locked P-form and locked M-form, as well as the pathway mutant. Extracted ion chromatograms corresponding to photolose (464.1875 Da ± 10 ppm) are shown (tR = 3.27 min). Photolose exhibits a slightly shifted retention time in this analysis relative to the other panels as a different column was used for this experiment (Figure S5). 177x140mm (300 x 300 DPI)


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Biochemistry

Figure 4. Photolose Characterization and Its Proposed Structure and Biosynthesis. MS2 spectra of peracetylated photolose (A) and its 1,6-anhydro disaccharide precursor (B) are presented. Black diamonds represent the precursor ion. Key ions are highlighted with their putative structures. (C) Photolose is proposed to be synthesized though the convergence of carbohydrate and NRPS machineries. Plu2411 is predicted to anabolically generate the chitin substrate upon which Plu2409 acts to generate the 1,6-anhydro disaccharide precursor. Concurrently, the adenylation protein, Plu2408, selects and loads glycine onto the carrier protein Plu2407. The action of the novel condensation protein, Plu2410, serves to couple the glycyl moiety onto the free 1,6-anhydro disaccharide substrate yielding photolose.


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Figure 5. Cecropin A Treatment. The growth of wild-type P. luminescens (black) and the corresponding pathway deletion mutant (grey) under 2.604 mg/mL of cecropin A are shown. Error bars represent the standard deviation. ***, P ≤ 0.001


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