Substrate Promiscuity of the Triceptide Maturase XncB Leads to Incorporation of Various Amino Acids and Detection of Oxygenated Products.

Triceptides are cyclophane-containing ribosomally synthesized and post-translationally modified peptides. The characteristic cross-links are formed between an aromatic ring to Cß on three-residue Ω1X2X3 motifs (Ω1 = aromatic). Here, we explored the promiscuity of the XYE family triceptide maturase, XncB from Xenorhabdus nematophila DSM 3370. Single amino acid variants were coexpressed with XncB in vivo in Escherichia coli, and we show that a variety of amino acids can be incorporated into the Phe-Gly-Asn cyclophane. Aromatic amino acids at the X3 position were accepted by the enzyme but yielded hydroxylated, rather than the typical cyclophane, products. These studies show that oxygen can be inserted but diverges in the final product formed relative to daropeptide maturases. Finally, truncations of the leader peptide showed that it is necessary for complete modification by XncB.

A Conserved Nonribosomal Peptide Synthetase in Xenorhabdus bovienii Produces Citrulline-Functionalized Lipopeptides.

The entomopathogenic bacterium Xenorhabdus bovienii exists in a mutualistic relationship with nematodes of the genus Steinernema. Free-living infective juveniles of Steinernema prey on insect larvae and regurgitate X. bovienii within the hemocoel of a host larva. X. bovienii subsequently produces a complex array of specialized metabolites and effector proteins that kill the insect and regulate various aspects of the trilateral symbiosis. While Xenorhabdus species are rich producers of secondary metabolites, many of their biosynthetic gene clusters remain uncharacterized. Here, we describe a nonribosomal peptide synthetase (NRPS) identified through comparative genomics analysis that is widely conserved in Xenorhabdus species. Heterologous expression of this NRPS gene from X. bovienii in E. coli led to the discovery of a family of lipo-tripeptides that chromatographically appear as pairs, containing either a C-terminal carboxylic acid or carboxamide. Coexpression of the NRPS with the leupeptin protease inhibitor pathway enhanced production, facilitating isolation and characterization efforts. The new lipo-tripeptides were also detected in wild-type X. bovienii cultures. These metabolites, termed bovienimides, share an uncommon C-terminal d-citrulline residue. The NRPS lacked a dedicated chain termination domain, resulting in product diversification and release from the assembly line through reactions with ammonia, water, or exogenous alcohols.

Cell-Free Synthesis of Natural Compounds from Genomic DNA of Biosynthetic Gene Clusters.

A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding because of the lack of specific functions or substrates in the host cell, the cell's sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. Specifically, we synthesize indigoidine and rhabdopeptides with the in vitro produced multidomain nonribosomal peptide synthetases BpsA and KJ12ABC from the organisms Streptomyces lavendulae and Xenorhabdus KJ12.1, respectively. These in vitro produced proteins are analyzed in yield, post-translational modification and in their ability to synthesize the natural compounds, and compared to recombinantly produced proteins. Our study highlights cell-free PURE system as suitable setting for the characterization of biosynthetic gene clusters that can potentially be harnessed for the rapid engineering of biosynthetic pathways.

Nonribosomal Peptides Produced by Minimal and Engineered Synthetases with Terminal Reductase Domains.

Nonribosomal peptide synthetases (NRPSs) use terminal reductase domains for 2-electron reduction of the enzyme-bound thioester releasing the generated peptides as C-terminal aldehydes. Herein, we reveal the biosynthesis of a pyrazine that originates from an aldehyde-generating minimal NRPS termed ATRed in entomopathogenic Xenorhabdus indica. Reductase domains were also investigated in terms of NRPS engineering and, although no general applicable approach was deduced, we show that they can indeed be used for the production of similar natural and unnatural pyrazinones.

Novel insecticidal chitinase from the insect pathogen Xenorhabdus nematophila.

Xenorhabdus nematophila strain ATCC 19061 is an insect pathogen that produces various protein toxins which intoxicate and kill its larval host. In the present study, we have described the cloning, expression and characterization of a 76-kDa chitinase protein of X. nematophila. A 1.9 kb DNA sequence encoding the chitinase gene was PCR amplified and cloned. Further, the chitinase protein was expressed in Escherichia coli and purified by using affinity chromatography. Two highly conserved domains were identified GH18 and ChiA. The purified chitinase protein showed chitobiosidase activity, ß-N-acetylglucosaminidase and endochitinase activity, when enzyme activity was measured using respective substrates. The purified chitinase protein was found to be orally toxic to the larvae of a major crop pest, Helicoverpa armigera when fed to the larvae mixed with artificial diet. It also had adverse effect on the growth and development of the surviving larvae. Surviving larvae showed 9-fold reduction in weight, as a result the transformation of larvae into pupae was adversely affected. Our results demonstrated that the chitinase protein of X. nematophila has insecticidal property and can prove to be a potent candidate for pest control in plants.

Artificial Splitting of a Non-Ribosomal Peptide Synthetase by Inserting Natural Docking Domains.

The interaction in multisubunit non-ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit-to-subunit interaction. We introduced natural docking domains into the three-module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC-ESI-MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS-PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split-module cases compared to the full-length wild-type enzyme.

A New Docking Domain Type in the Peptide-Antimicrobial-Xenorhabdus Peptide Producing Nonribosomal Peptide Synthetase from Xenorhabdus bovienii.

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of different natural products from amino acid precursors. In contrast to single protein NRPS, the NRPS of the bacterium Xenorhabdus bovienii producing the peptide-antimicrobial-Xenorhabdus (PAX) peptide consists of three individual proteins (PaxA/B/C), which interact with each other noncovalently in a linear fashion. The specific interactions between the three different proteins in this NRPS system are mediated by short C- and N-terminal docking domains (C/NDDs). Here, we investigate the structural basis for the specific interaction between the CDD from the protein PaxB and the NDD from PaxC. The isolated DD peptides feature transient α-helical conformations in the absence of the respective DD partner. Isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) titration experiments showed that the two isolated DDs bind to each other and form a structurally well-defined complex with a dissociation constant in the micromolar range as is typical for many DD interactions. Artificial linking of this DD pair via a flexible glycine-serine (GS) linker enabled us to solve the structure of the DD complex by NMR spectroscopy. In the complex, the two DDs interact with each other by forming a three helix bundle arranged in an overall coiled-coil motif. Key interacting residues were identified in mutagenesis experiments. Overall, our structure of the PaxB CDD/PaxC NDD complex represents an architecturally new type of DD interaction motif.

Metabolic engineering of Bacillus subtilis for production of para-aminobenzoic acid - unexpected importance of carbon source is an advantage for space application.

High-strength polymers, such as aramid fibres, are important materials in space technology. To obtain these materials in remote locations, such as Mars, biological production is of interest. The aromatic polymer precursor para-aminobenzoic acid (pABA) can be derived from the shikimate pathway through metabolic engineering of Bacillus subtilis, an organism suited for space synthetic biology. Our engineering strategy included repair of the defective indole-3-glycerol phosphate synthase (trpC), knockout of one chorismate mutase isozyme (aroH) and overexpression of the aminodeoxychorismate synthase (pabAB) and aminodeoxychorismate lyase (pabC) from the bacteria Corynebacterium callunae and Xenorhabdus bovienii respectively. Further, a fusion-protein enzyme (pabABC) was created for channelling of the carbon flux. Using adaptive evolution, mutants of the production strain, able to metabolize xylose, were created, to explore and compare pABA production capacity from different carbon sources. Rather than the efficiency of the substrate or performance of the biochemical pathway, the product toxicity, which was strongly dependent on the pH, appeared to be the overall limiting factor. The highest titre achieved in shake flasks was 3.22 g l-1 with a carbon yield of 12.4% [C-mol/C-mol] from an amino sugar. This promises suitability of the system for in situ resource utilization (ISRU) in space biotechnology, where feedstocks that can be derived from cyanobacterial cell lysate play a role.

Structure, Biosynthesis, and Occurrence of Bacterial Pyrrolizidine Alkaloids.

Pyrrolizidine alkaloids (PAs) are widespread plant natural products with potent toxicity and bioactivity. Herein, the identification of bacterial PAs from entomopathogenic bacteria using differential analysis by 2D NMR spectroscopy (DANS) and mass spectrometry is described. Their biosynthesis was elucidated to involve a non-ribosomal peptide synthetase. The occurrence of these biosynthesis gene clusters in Gram-negative and Gram-positive bacteria indicates an important biological function in bacteria.

Examining Reaction Specificity in PvcB, a Source of Diversity in Isonitrile-Containing Natural Products.

Many bacteria produce isonitrile-containing natural products that are derived from aromatic amino acids. The synthetic clusters that control biosynthesis most commonly encode two enzymes, designated PvcA and PvcB, as well as additional enzymes that direct synthesis of the natural product. The PvcA enzyme installs the isonitrile moiety at the amino group of either tyrosine or tryptophan, as dictated by the particular pathway. The common pathway intermediate produced by PvcA is directed toward different ultimate products by PvcB, a member of the family of Fe(2+), α-ketoglutarate-dependent oxygenases. To continue our investigation of the structural and functional properties of the isonitrile biosynthetic pathways, we present here a study of the PvcB homologues from three organisms. Two pathways, derived from Pseudomonas aeruginosa and Xenorhabdus nematophila, produce known products. A third PvcB homologue from Erwinia amylovora is part of an uncharacterized pathway. Our results demonstrate the diversity of reactions catalyzed. Although all PvcB enzymes catalyze the hydroxylation of the tyrosine isonitrile substrate, the elimination of the hydroxyl in Pseudomonas and Erwinia is driven by deprotonation at Cα, resulting in the initial production of an unsaturated tyrosine isonitrile product that then cyclizes to a coumarin derivative. PvcB from Xenorhabdus, in contrast, catalyzes the same oxygenation, but loss of the hydroxyl group is accompanied by decarboxylation of the intermediate. Steady-state kinetic analysis of the three reactions and a docking model for the binding of the tyrosine isonitrile substrate in the PvcB active site highlight subtle differences between the PvcB homologues.

Biosynthesis of the insecticidal xenocyloins in Xenorhabdus bovienii.

The biosynthesis gene cluster for the production of xenocyloins was identified in the entomopathogenic bacterium Xenorhabdus bovienii SS-2004, and their biosynthesis was elucidated by heterologous expression and in vitro characterization of the enzymes. XclA is an S-selective ThDP-dependent acyloin-like condensation enzyme, and XclB and XclC are examples of the still-rare acylating ketosynthases that catalyze the acylation of the XclA-derived initial xenocyloins with acetyl-, propionyl-, or malonyl-CoA, thereby resulting in the formation of further xenocyloin derivatives. All xenocyloins were produced mainly by the more virulent primary variant of X. bovienii and showed activity against insect hemocytes thus contributing to the overall virulence of X. bovienii against insects.

Extracellular novel metalloprotease from Xenorhabdus indica and its potential as an insecticidal agent.

Proteases produced by Xenorhabdus are known to play a significant role in virulence leading to insect mortality. The present study was undertaken to purify and characterize protease from Xenorhabdus indica, an endosymbiont of nematode Steinernema thermophilum, and to decipher its role in insect mortality and its efficacy to control Helicoverpa armigera. A set of 10 strains of Xenorhabdus isolated from different regions of India were screened for protease activity on the basis of zone of clearing on gelatin agar plates. One potent strain of Xenorhabdus indica was selected for the production of protease, and the highest production (1,552 U/ml) was observed at 15-18 h of incubation at 28°C in soya casein digest broth. The extracellular protease was purified from culture supernatant using ammonium sulfate precipitation and ion-exchange chromatography. The enzyme was further characterized by SDS-PAGE and zymography, which confirmed the purity of the protein and its molecular mass was found to be ~52 kDa. Further MALDI-TOF/TOF analysis and effect of metal chelating agent 1,10-phenanthrolin study revealed the nature of the purified protease as a secreted alkaline metalloprotease. The bioefficacy of the purified protease was also tested against cotton bollworm (Helicoverpa armigera) and resulted in 67.9 ± 0.64% mortality within one week. This purified protease has the potential to be developed as a natural insecticidal agent against a broad range of agriculturally important insects.

Enzymatic characterization of a serralysin-like metalloprotease from the entomopathogen bacterium, Xenorhabdus.

We investigated the enzymatic properties of a serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest k(cat)/K(M) value. The pH profile of activity, similarly to other serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu(2+) and Co(2+) ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn(2+) was inhibitory (both are unusual among serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn(2+), Cu(2+) and Co(2+) up to 90-200% of its original value, while Zn(2+) was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the serralysins contain Asn.

Metabolically engineered soybean seed with enhanced threonine levels: biochemical characterization and seed-specific expression of lysine-insensitive variants of aspartate kinases from the enteric bacterium Xenorhabdus bovienii.

Threonine (Thr) is one of a few limiting essential amino acids (EAAs) in the animal feed industry, and its level in feed rations can impact production of important meat sources, such as swine and poultry. Threonine as well as EAAs lysine (Lys) and methionine (Met) are all synthesized via the aspartate family pathway. Here, we report a successful strategy to produce high free threonine soybean seed via identification of a feedback-resistant aspartate kinase (AK) enzyme that can be over-expressed in developing soybean seed. Towards this goal, we have purified and biochemically characterized AK from the enteric bacterium Xenorhabdus bovienii (Xb). Site-directed mutagenesis of XbAK identified two key regulatory residues Glu-257 and Thr-359 involved in lysine inhibition. Three feedback-resistant alleles, XbAK_T359I, XbAK_E257K and XbAK_E257K/T359I, have been generated. This study is the first to kinetically characterize the XbAK enzyme and provide biochemical and transgenic evidence that Glu-257 near the catalytic site is a critical residue for the allosteric regulation of AK. Furthermore, seed-specific expression of the feedback-resistant XbAK_T359I or XbAK_E257K allele results in increases of free Thr levels of up to 100-fold in R(1) soybean seed when compared to wild-type. Expression of feedback-sensitive wild-type AK did not substantially impact seed Thr content. In addition to high Thr, transgenic seed also showed substantial increases in other major free amino acid (FAA) levels, resulting in an up to 3.5-fold increase in the total FAA content. The transgenic seed was normal in appearance and germinated well under greenhouse conditions.

Proteolytic enzyme production by strains of the insect pathogen xenorhabdus and characterization of an early-log-phase-secreted protease as a potential virulence factor.

As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.

Examination of Xenorhabdus nematophila lipases in pathogenic and mutualistic host interactions reveals a role for xlpA in nematode progeny production.

Xenorhabdus nematophila is a gammaproteobacterium and broad-host-range insect pathogen. It is also a symbiont of Steinernema carpocapsae, the nematode vector that transports the bacterium between insect hosts. X. nematophila produces several secreted enzymes, including hemolysins, lipases, and proteases, which are thought to contribute to virulence or nutrient acquisition for the bacterium and its nematode host in vivo. X. nematophila has two lipase activities with distinct in vitro specificities for Tween and lecithin. The gene encoding the Tween-specific lipase, xlpA, has been identified and is not required for X. nematophila virulence in one insect host, the tobacco hornworm Manduca sexta. However, the gene encoding the lecithin-specific lipase activity is not currently known. Here, we identify X. nematophila estA, a gene encoding a putative lecithinase, and show that an estA mutant lacks in vitro lipase activity against lecithin but has wild-type virulence in Manduca sexta. X. nematophila secondary-form phenotypic variants have higher in vitro lecithinase activity and estA transcript levels than do primary-form variants, and estA transcription is negatively regulated by NilR, a repressor of nematode colonization factors. We establish a role for xlpA, but not estA, in supporting production of nematode progeny during growth in Galleria mellonella insects. Future research is aimed at characterizing the biological roles of estA and xlpA in other insect hosts.

Isolation and characterization of Xenorhabdus nematophila transposon insertion mutants defective in lipase activity against Tween.

We identified Xenorhabdus nematophila transposon mutants with defects in lipase activity. One of the mutations, in yigL, a conserved gene of unknown function, resulted in attenuated virulence against Manduca sexta insects. We discuss possible connections between lipase production, YigL, and specific metabolic pathways.

Pyrimidine nucleoside salvage confers an advantage to Xenorhabdus nematophila in its host interactions.

Xenorhabdus nematophila is a mutualist of entomopathogenic nematodes and a pathogen of insects. To begin to examine the role of pyrimidine salvage in nutrient exchange between X. nematophila and its hosts, we identified and mutated an X. nematophila tdk homologue. X. nematophila tdk mutant strains had reduced virulence toward Manduca sexta insects and a competitive defect for nematode colonization in plate-based assays. Provision of a wild-type tdk allele in trans corrected the defects of the mutant strain. As in Escherichia coli, X. nematophila tdk encodes a deoxythymidine kinase, which converts salvaged deoxythymidine and deoxyuridine nucleosides to their respective nucleotide forms. Thus, nucleoside salvage may confer a competitive advantage to X. nematophila in the nematode intestine and be important for normal entomopathogenicity.

Analysis of Xenorhabdus nematophila metabolic mutants yields insight into stages of Steinernema carpocapsae nematode intestinal colonization.

Xenorhabdus nematophila colonizes the intestinal tract of infective-juvenile (IJ) stage Steinernema carpocapsae nematodes. During colonization, X. nematophila multiplies within the lumen of a discrete region of the IJ intestine termed the vesicle. To begin to understand bacterial nutritional requirements during multiplication in the IJ vesicle, we analysed the colonization behaviour of several X. nematophila metabolic mutants, including amino acid and vitamin auxotrophs. X. nematophila mutants defective for para-aminobenzoate, pyridoxine or l-threonine biosynthesis exhibit substantially decreased colonization of IJs (0.1-50% of wild-type colonization). Analysis of gfp-labelled variants revealed that those mutant cells that can colonize the IJ vesicle differ noticeably from wild-type X. nematophila. One aberrant colonization phenotype exhibited by the metabolic mutants tested, but not wild-type X. nematophila, is a spherical shape indicative of apparently non-viable X. nematophila cells within the vesicle. Because these spherical cells appear to have initiated colonization but failed to proliferate, we term this type of colonization 'abortive'. In a portion of IJs grown on para-aminobenzoate auxotrophs, X. nematophila does not exhibit abortive colonization but rather reduced growth and filamentous cell morphology. Several mutants with defects in other amino acid, vitamin and nutrient metabolism pathways colonize IJs to wild-type levels suggesting that the IJ vesicle is replete with respect to a number of nutrients.

Identification and functional characterization of a Xenorhabdus nematophila oligopeptide permease.

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA(1), oppA(2), or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.