Phosphopantetheinyl Transferase Research Articles

Characterization of lpaH2 gene corresponding to lipopeptide synthesis in Bacillus amyloliquefaciens HAB-2

BackgroundBacillus spp. have prominent ability to suppress plant pathogens and corresponding diseases. Previous analyses of Bacillus spp. revealed numerous gene clusters involved in nonribosomal synthesis of cyclic lipopeptides with distinct antimicrobial action. The 4′-phosphopantetheinyl transferase (PPTase) encoded by sfp gene is a key factor in lipopeptide synthesis in Bacillus spp. In previous study, B. amyloliquefaciens strain HAB-2 was found to inhibit a broad range of plant pathogens, which was attributed to its secondary metabolite lipopeptide.ResultsA sfp homologue lpaH2 which encoded phosphopantetheinyl transferase but shared 71% sequence similarity was detected in strain HAB-2. Disruption of lpaH2 gene resulted in losing the ability of strain HAB-2 to produce lipopeptide, as well as antifungal and hemolytic activities. When lpaH2 replaced sfp gene of B. subtilis strain 168, a non-lipopeptide producer, the genetically engineered strain 168 could produced lipopeptides and recovered antifungal activity. Quantitative PCR assays indicated that, the expression level of lpaH2 in B. subtilis 168 strain decrease to 0.27-fold compared that of the wild type B. amyloliquefaciens strain HAB-2.ConclusionFew studies have reported about lpa gene which can replace sfp gene in the different species. Taken together, our study showed for the first time that lpaH2 from B. amyloliquefaciens could replace sfp gene.

Only Acyl Carrier Protein 1 (AcpP1) Functions in Pseudomonas aeruginosa Fatty Acid Synthesis.

The genome of Pseudomonas aeruginosa contains three open reading frames, PA2966, PA1869, and PA3334, which encode putative acyl carrier proteins, AcpP1, AcpP2, and AcpP3, respectively. In this study, we found that, although these apo-ACPs were successfully phosphopantetheinylated by P. aeruginosa phosphopantetheinyl transferase (PcpS) and all holo-forms of these proteins could be acylated by Vibrio harveyi acyl-ACP synthetase (AasS), only AcpP1 could be used as a substrate for the synthesis of fatty acids, catalyzed by P. aeruginosa cell free extracts in vitro, and only acpP1 gene could restore growth in the Escherichia coli acpP mutant strain CY1877. And P. aeruginosa acpP1 could not be deleted, while disruption of acpP2 or acpP3 in the P. aeruginosa genome allowed mutant strains to grow as well as the wild type strain. These findings confirmed that only P. aeruginosa AcpP1 functions in fatty acid biosynthesis, and that acpP2 and acpP3 do not play roles in the fatty acid synthetic pathway. Moreover, disruption of acpP2 and acpP3 did not affect the ability of P. aeruginosa to produce N-acylhomoserine lactones (AHL), but replacement of P. aeruginosa acpP1 with E. coli acpP caused P. aeruginosa to reduce the production of AHL molecules, which indicated that neither P. aeruginosa AcpP2 nor AcpP3 can act as a substrate for synthesis of AHL molecules in vivo. Furthermore, replacement of acpP1 with E. coli acpP reduced the ability of P. aeruginosa to produce some exo-products and abolished swarming motility in P. aeruginosa.

A phylogenetic and evolutionary analysis of antimycin biosynthesis.

Streptomyces species and other Actinobacteria are ubiquitous in diverse environments worldwide and are the source of, or inspiration for, the majority of antibiotics. The genomic era has enhanced biosynthetic understanding of these valuable chemical entities and has also provided a window into the diversity and distribution of natural product biosynthetic gene clusters. Antimycin is an inhibitor of mitochondrial cytochrome c reductase and more recently was shown to inhibit Bcl-2/Bcl-XL-related anti-apoptotic proteins commonly overproduced by cancerous cells. Here we identify 73 putative antimycin biosynthetic gene clusters (BGCs) in publicly available genome sequences of Actinobacteria and classify them based on the presence or absence of cluster-situated genes antP and antQ, which encode a kynureninase and a phosphopantetheinyl transferase (PPTase), respectively. The majority of BGCs possess either both antP and antQ (L-form) or neither (S-form), while a minority of them lack either antP or antQ (IQ- or IP-form, respectively). We also evaluate the biogeographical distribution and phylogenetic relationships of antimycin producers and BGCs. We show that antimycin BGCs occur on five of the seven continents and are frequently isolated from plants and other higher organisms. We also provide evidence for two distinct phylogenetic clades of antimycin producers and gene clusters, which delineate S-form from L- and I-form BGCs. Finally, our findings suggest that the ancestral antimycin producer harboured an L-form gene cluster which was primarily propagated by vertical transmission and subsequently diversified into S-, IQ- and IP-form biosynthetic pathways.

Cyanobacterial Sfp-type phosphopantetheinyl transferases functionalize carrier proteins of diverse biosynthetic pathways

Cyanobacteria produce structurally and functionally diverse polyketides, nonribosomal peptides and their hybrids. Sfp-type phosphopantetheinyl transferases (PPTases) are essential to the production of these compounds via functionalizing carrier proteins (CPs) of biosynthetic megaenzymes. However, cyanobacterial Sfp-type PPTases remain poorly characterized, posing a significant barrier to the exploitation of cyanobacteria for biotechnological and biomedical applications. Herein, we describe the detailed characterization of multiple cyanobacterial Sfp-type PPTases that were rationally selected. Biochemical characterization of these enzymes along with the prototypic enzyme Sfp from Bacillus subtilis demonstrated their varying specificities toward 11 recombinant CPs of different types of biosynthetic pathways from cyanobacterial and Streptomyces strains. Kinetic analysis further indicated that PPTases possess the higher binding affinity and catalytic efficiency toward their cognate CPs in comparison with noncognate substrates. Moreover, when chromosomally replacing the native PPTase gene of Synechocystis sp. PCC6803, two selected cyanobacterial PPTases and Sfp supported the growth of resulted mutants. Cell lysates of the cyanobacterial mutants further functionalized recombinant CP substrates. Collectively, these studies reveal the versatile catalysis of selected cyanobacterial PPTases and provide new tools to synthesize cyanobacterial natural products using in vitro and in vivo synthetic biology approaches.

The Lactococcus lactis KF147 nonribosomal peptide synthetase/polyketide synthase system confers resistance to oxidative stress during growth on plant leaf tissue lysate.

Strains of Lactococcus lactis isolated from plant tissues possess adaptations that support their survival and growth in plant‐associated microbial habitats. We previously demonstrated that genes coding for a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) system involved in production of an uncharacterized secondary metabolite are specifically induced in L. lactis KF147 during growth on plant tissues. Notably, this NRPS/PKS has only been identified in plant‐isolated strains of L. lactis. Here, we show that the L. lactis KF147 NRPS/PKS genes have homologs in certain Streptococcus mutans isolates and the genetic organization of the NRPS/PKS locus is conserved among L. lactis strains. Using an L. lactis KF147 mutant deficient in synthesis of NrpC, a 4′‐phosphopantetheinyl transferase, we found that the NRPS/PKS system improves L. lactis during growth under oxidative conditions in Arapidopsis thaliana leaf lysate. The NRPS/PKS system also improves tolerance of L. lactis to reactive oxygen species and specifically H2O2 and superoxide radicals in culture medium. These findings indicate that this secondary metabolite provides a novel mechanism for reactive oxygen species detoxification not previously known for this species.

A Chemoproteomics Approach to Investigate Phosphopantetheine Transferase Activity at the Cellular Level.

Phosphopantetheinylation is an essential post-translational protein modification to primary and secondary metabolic pathways that ensures bacterial cell viability and virulence, and it is used in the production of many pharmaceuticals. Traditional methods have not provided a comprehensive understanding of these modifications. By using chemical proteomic probes for adenylation and thiolation domains in nonribosomal peptide synthetases (NRPSs), chemoproteomics has been applied to survey and validate the cellular activity of 4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), which is a potent and selective small-molecule 4'-phosphopantetheinyl transferase (PPTase) inhibitor that attenuates secondary metabolism and viability of bacterial cells. ML267 inhibited Sfp-type PPTase and antagonized phosphopantetheinylation in cells, which resulted in a decrease in phosphopantetheinylated NRPSs and the attenuation of Sfp-PPTase-dependent metabolite production. These results indicate that this chemoproteomics platform should enable a precise interpretation of the cellular activities of Sfp-type PPTase inhibitors.

Identification of biosynthetic gene clusters from metagenomic libraries using PPTase complementation in a Streptomyces host.

The majority of environmental bacteria are not readily cultured in the lab, leaving the natural products they make inaccessible using culture-dependent discovery methods. Cloning and heterologous expression of DNA extracted from environmental samples (environmental DNA, eDNA) provides a means of circumventing this discovery bottleneck. To facilitate the identification of clones containing biosynthetic gene clusters, we developed a model heterologous expression reporter strain Streptomyces albus::bpsA ΔPPTase. This strain carries a 4΄-phosphopantetheinyl transferase (PPTase)-dependent blue pigment synthase A gene, bpsA, in a PPTase deletion background. eDNA clones that express a functional PPTase restore production of the blue pigment, indigoidine. As PPTase genes often occur in biosynthetic gene clusters (BGCs), indigoidine production can be used to identify eDNA clones containing BGCs. We screened a soil eDNA library hosted in S. albus::bpsA ΔPPTase and identified clones containing non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS) and mixed NRPS/PKS biosynthetic gene clusters. One NRPS gene cluster was shown to confer the production of myxochelin A to S. albus::bpsA ΔPPTase.

A Nonredundant Phosphopantetheinyl Transferase, PptA, Is a Novel Antifungal Target That Directs Secondary Metabolite, Siderophore, and Lysine Biosynthesis in Aspergillus fumigatus and Is Critical for Pathogenicity.

ABSTRACTSecondary metabolites are key mediators of virulence for many pathogens. Aspergillus fumigatus produces a vast array of these bioactive molecules, the biosynthesis of which is catalyzed by nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs). Both NRPSs and PKSs harbor carrier domains that are primed for acceptance of secondary metabolic building blocks by a phosphopantetheinyl transferase (P-pant). The A. fumigatus P-pant PptA has been shown to prime the putative NRPS Pes1 in vitro and has an independent role in lysine biosynthesis; however, its role in global secondary metabolism and its impact on virulence has not been described. Here, we demonstrate that PptA has a nonredundant role in the generation of the vast majority of detectable secondary metabolites in A. fumigatus, including the immunomodulator gliotoxin, the siderophores triacetylfusarinine C (TAFC) and ferricrocin (FC), and dihydroxy naphthalene (DHN)-melanin. We show that both the lysine and iron requirements of a pptA null strain exceed those freely available in mammalian tissues and that loss of PptA renders A. fumigatus avirulent in both insect and murine infection models. Since PptA lacks similarity to its mammalian orthologue, we assert that the combined role of this enzyme in both primary and secondary metabolism, encompassing multiple virulence determinants makes it a very promising antifungal drug target candidate. We further exemplify this point with a high-throughput fluorescence polarization assay that we developed to identify chemical inhibitors of PptA function that have antifungal activity.

Engineering Saccharomyces cerevisiae for Efficient Biosynthesis of Fatty Alcohols Based on Enhanced Supply of Free Fatty Acids.

In recent years, production of fatty acid derivatives has attracted much attention because of their wide range of applications in renewable oleochemicals. Microorganisms such as Saccharomyces cerevisiae provided an ideal cell factory for such chemical synthesis. In this study, an efficient strategy for the synthesis of fatty alcohols based on enhanced supply of free fatty acids (FFAs) was constructed. The FAA1 and FAA4 genes encoding two acyl-CoA synthetases in S. cerevisiae were deleted, resulting in the accumulation of FFAs with carbon chain length from C8 to C18. The coexpression of the carboxylic acid reductase gene (car) from Mycobacterium marinum and the phosphopantetheinyl transferase gene (sfp) from Bacillus subtilis successfully converted the accumulated FFAs into fatty alcohols. The concentration of the total fatty alcohols reached 24.3 mg/L, which is in agreement with that of the accumulated FFAs. To further increase the supply of FFAs, the DGAI encoding the acyl-CoA:diacylglycerol acyltransferase involved in the rate-limiting step of triacylglycerols storage was codeleted with FAA1 and FAA4, and the acyl-CoA thioesterase gene (acot) was expressed together with car and sfp, resulting in an enhanced production of fatty alcohols, the content of which increased to 31.2 mg/L. The results herein demonstrated the efficiency of the engineered pathway for the production of fatty acid derivatives using FFAs as precursors.

Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles.

Single-molecule force spectroscopy greatly benefits from site-specific surface immobilization and specific probing with a functionalized cantilever. Here, we describe a streamlined approach to such experiments by covalently attaching mechanically stable receptors onto proteins of interest (POI) to improve pickup efficiency and specificity. This platform provides improved throughput, allows precise control over the pulling geometry, and allows for multiple constructs to be probed with the same ligand-modified cantilever. We employ two orthogonal enzymatic ligation reactions [sortase and phosphopantetheinyl transferase (Sfp)] to covalently immobilize POI to a pegylated surface and to subsequently ligate the POI to a mechanically stable dockerin domain at the protein’s C-terminus for use as a high-strength pulling handle. Our configuration permits expression and folding of the POI to proceed independently from the mechanically stable receptor used for specific probing and requires only two short terminal peptide sequences (i.e., ybbR-tag and sortase C-tag). We applied this system successfully to proteins expressed using in vitro transcription and translation reactions without a protein purification step and to purified proteins expressed in Escherichia coli.

High iron supply inhibits the synthesis of the genotoxin colibactin by pathogenic Escherichia coli through a non-canonical Fur/RyhB-mediated pathway.

The genotoxin colibactin is a secondary metabolite produced by a variety of pathogenic Enterobacteria, and is associated with colon cancer development and acute systemic infections. The colibactin biosynthesis requires the enzymatic activity of the phosphopantetheinyl transferase ClbA. We recently evidenced that two master regulators of bacterial iron homeostasis, i.e. the ferric uptake regulator (Fur) and the small regulatory non-coding RNA RyhB, were involved in the regulation of the clbA transcription and of the colibactin production. In this study, we investigated the impact of high iron supply on clbA transcription and colibactin production in wild type, ΔryhB, Δfur and ΔryhB Δfur strains. This revealed that high iron resulted in decreased synthesis of the genotoxin colibactin through both pathways dependent and independent of Fur/RyhB. This work highlights the complex regulatory mechanism that controls an important bacterial virulence and carcinogenesis factor by regulators of bacterial iron homeostasis.

Activation of Natural Products Biosynthetic Pathways via a Protein Modification Level Regulation.

Natural products are critical for drug discovery and development; however their discovery is challenged by the wide inactivation or silence of microbial biosynthetic pathways. Currently strategies targeting this problem are mainly concentrated on chromosome dissembling, transcription, and translation-stage regulations as well as chemical stimulation. In this study, we developed a novel approach to awake cryptic/silenced microbial biosynthetic pathways through augmentation of the conserved protein modification step-phosphopantetheinylation of carrier proteins. Overexpression of phosphopantetheinyl transferase (Pptase) genes into 33 Actinomycetes achieved a significantly high activation ratio at which 23 (70%) strains produced new metabolites. Genetic and biochemical studies on the mode-of-action revealed that exogenous PPtases triggered the activation of carrier proteins and subsequent production of metabolites. With this approach we successfully identified five oviedomycin and halichomycin-like compounds from two strains. This study provides a novel approach to efficiently activate cryptic/silenced biosynthetic pathways which will be useful for natural products discovery.

De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.

The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.

An external substrate-free blue/white screening system in Escherichia coli.

Since the lacZα-based blue/white screening system was introduced to molecular biology, several different visual reporter systems were developed and used for various purposes in Escherichia coli. A common limit to the existent visual reporter systems is that an extracellular chromogenic substrate has to be added for the visible pigment production. In this study, we developed a new blue/white screening system based on a non-ribosomal peptide synthetase encoded by idgS from Streptomyces and a phosphopantetheinyl transferase encoded by sfp from Bacillus. When IdgS is activated from an apo-form to a holo-form via a posttranslational modification catalyzed by Sfp, it can synthesize a blue pigment indigoidine using L-glutamine, the amino acid abundant in cells, as a substrate. The new blue/white screening system contains a recipient E. coli strain with an optimized idgS gene cassette and a cloning vector harboring an sfp gene with an in-frame insertion of a multiple cloning site close to its N-terminal. We demonstrated that the IdgS/Sfp-based blue/white screening system is a powerful alternative to the lacZα-based screening system, which does not require any external substrate addition.

Production of (S)-2-aminobutyric acid and (S)-2-aminobutanol in Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae (baker’s yeast) has great potential as a whole-cell biocatalyst for multistep synthesis of various organic molecules. To date, however, few examples exist in the literature of the successful biosynthetic production of chemical compounds, in yeast, that do not exist in nature. Considering that more than 30% of all drugs on the market are purely chemical compounds, often produced by harsh synthetic chemistry or with very low yields, novel and environmentally sound production routes are highly desirable. Here, we explore the biosynthetic production of enantiomeric precursors of the anti-tuberculosis and anti-epilepsy drugs ethambutol, brivaracetam, and levetiracetam. To this end, we have generated heterologous biosynthetic pathways leading to the production of (S)-2-aminobutyric acid (ABA) and (S)-2-aminobutanol in baker’s yeast.ResultsWe first designed a two-step heterologous pathway, starting with the endogenous amino acid l-threonine and leading to the production of enantiopure (S)-2-aminobutyric acid. The combination of Bacillus subtilis threonine deaminase and a mutated Escherichia coli glutamate dehydrogenase resulted in the intracellular accumulation of 0.40 mg/L of (S)-2-aminobutyric acid. The combination of a threonine deaminase from Solanum lycopersicum (tomato) with two copies of mutated glutamate dehydrogenase from E. coli resulted in the accumulation of comparable amounts of (S)-2-aminobutyric acid. Additional l-threonine feeding elevated (S)-2-aminobutyric acid production to more than 1.70 mg/L. Removing feedback inhibition of aspartate kinase HOM3, an enzyme involved in threonine biosynthesis in yeast, elevated (S)-2-aminobutyric acid biosynthesis to above 0.49 mg/L in cultures not receiving additional l-threonine. We ultimately extended the pathway from (S)-2-aminobutyric acid to (S)-2-aminobutanol by introducing two reductases and a phosphopantetheinyl transferase. The engineered strains produced up to 1.10 mg/L (S)-2-aminobutanol.ConclusionsOur results demonstrate the biosynthesis of (S)-2-aminobutyric acid and (S)-2-aminobutanol in yeast. To our knowledge this is the first time that the purely synthetic compound (S)-2-aminobutanol has been produced in vivo. This work paves the way to greener and more sustainable production of chemical entities hitherto inaccessible to synthetic biology.

Methylotrophic yeast Pichia pastoris as a chassis organism for polyketide synthesis via the full citrinin biosynthetic pathway

With the rapid development of synthetic biology, exploring various chassis organisms has become necessary to improve the heterologous biosynthesis of natural products and pharmaceuticals. In this study, we tested the potential of the industrial methylotrophic yeast strain Pichia pastoris for the heterologous synthesis of polyketides. A recombinant P. pastoris GS-pksCT-npgA carrying the Monascus purpureus citrinin polyketide synthase gene pksCT and the Aspergillus nidulans phosphopantetheinyl transferase gene npgA was constructed. Subsequently, a specific compound was isolated and identified as citrinin intermediate trimethylated pentaketide aldehyde. On account of the hypothetic functions of the genes in the citrinin gene cluster, mpl1 encoding serine hydrolase, mpl2 encoding oxygenase, and mpl4 encoding dehydrogenase were gradually expressed. Proteins were also normally expressed, but a new compound was undetected. Basing on the recently reported citrinin gene cluster in Monascus ruber, we obtained two other genes (mpl6 and mpl7) participating in citrinin biosynthesis by genome walking in M. purpureus. Then, we co-transformed intron-removed mpl6 and mpl7 into the P. pastoris strain carrying pksCT, npgA, mpl1, mpl2, and mpl4. All genes were activated by the methanol-induced AOX1 promoter, and a complete biosynthetic pathway of citrinin was assembled. Finally, citrinin was successfully produced under methanol induction in P. pastoris. These results prove that P. pastoris is a promising chassis organism for polyketide production.

Legionella shows a diverse secondary metabolism dependent on a broad spectrum Sfp-type phosphopantetheinyl transferase.

Several members of the genus Legionella cause Legionnaires’ disease, a potentially debilitating form of pneumonia. Studies frequently focus on the abundant number of virulence factors present in this genus. However, what is often overlooked is the role of secondary metabolites from Legionella. Following whole genome sequencing, we assembled and annotated the Legionella parisiensis DSM 19216 genome. Together with 14 other members of the Legionella, we performed comparative genomics and analysed the secondary metabolite potential of each strain. We found that Legionella contains a huge variety of biosynthetic gene clusters (BGCs) that are potentially making a significant number of novel natural products with undefined function. Surprisingly, only a single Sfp-like phosphopantetheinyl transferase is found in all Legionella strains analyzed that might be responsible for the activation of all carrier proteins in primary (fatty acid biosynthesis) and secondary metabolism (polyketide and non-ribosomal peptide synthesis). Using conserved active site motifs, we predict some novel compounds that are probably involved in cell-cell communication, differing to known communication systems. We identify several gene clusters, which may represent novel signaling mechanisms and demonstrate the natural product potential of Legionella.

Disruptions of the genes involved in lysine biosynthesis, iron acquisition, and secondary metabolisms affect virulence and fitness in Metarhizium robertsii

Based on genomic analysis, polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways account for biosynthesis of the majority of the secondary metabolites produced by the entomopathogenic fungus Metarhizium robertsii. To evaluate the contribution of these pathways to M. robertsii fitness and/or virulence, mutants deleted for mrpptA, the Sfp-type 4′ phosphopantetheinyl transferase gene required for their activation were generated. ΔmrpptA strains were deficient in PKS and NRPS activity resulting in colonies that lacked the typical green pigment and failed to produce the nonribosomal peptides (destruxins, serinocylins, and the siderophores ferricrocin and metachelins) as well as the hybrid polyketide-peptides (NG-39x) that are all produced by the wild type (WT) M. robertsii. The ΔmrpptA colonies were also auxotrophic for lysine. Two other mutant strains were generated: ΔmraarA, in which the α-aminoadipate reductase gene critical for lysine biosynthesis was disrupted, and ΔmrsidA, in which the L-ornithine N5-oxygenase gene that is critical for hydroxamate siderophore biosynthesis was disrupted. The phenotypes of these mutants were compared to those of ΔmrpptA to separate effects of the loss of lysine or siderophore production from the overall effect of losing all polyketide and non-ribosomal peptide production. Loss of lysine biosynthesis marginally increased resistance to H2O2 while it had little effect on the sensitivity to the cell wall disruptor sodium dodecyl sulfate (SDS) and no effect on sensitivity to iron deprivation. In contrast, combined loss of metachelin and ferricrocin through the inactivation of mrsidA resulted in mutants that were as hypersensitive or slightly more sensitive to H2O2, iron deprivation, and SDS, and were either identical or marginally higher in ΔmrpptA strains. In contrast to ΔmrpptA, loss of mrsidA did not completely abolish siderophore activity, which suggests the production of one or more non-hydroxamate iron-chelating compounds. Deletion of mrpptA, mrsidA, and mraarA reduced conidium production and conidia of a GFP-tagged ΔmrpptA strain displayed a longer germination delay than WT on insect cuticles, a deficiency that was rescued by lysine supplementation. Compared with WT, ΔmrpptA strains displayed ∼19-fold reduction in virulence against Drosophila suzukii. In contrast, lysine auxotrophy and loss of siderophores accounted for ∼2 and ∼6-fold decreases in virulence, respectively. Deletion of mrpptA had no significant effect on growth inhibition of Bacillus cereus. Our results suggest that PKS and NRPS metabolism plays a significant role in M. robertsii virulence, depresses conidium production, and contributes marginally to resistance to oxidative stress and iron homeostasis, but has no significant antibacterial effect.

Iron Acquisition Pathways as Targets for Antitubercular Drugs

Tuberculosis nowadays ranks as the second leading cause of death from an infectious disease worldwide. In the last twenty years, this disease has again started to spread mainly for the appearance of multi-drug resistant forms. Therefore, new targets are needed to address the growing emergence of bacterial resistance and for antitubercular drug development. Efficient iron acquisition is crucial for the pathogenesis of Mycobacterium tuberculosis, because it serves as cofactor in many essential biological processes, including DNA biosynthesis and cellular respiration. Bacteria acquire iron chelating non-heme iron from the host using the siderophore mycobactins and carboxymycobactins and by the uptake of heme iron released by damaged red blood cells, through several acquisition systems. Drug discovery focused its efforts on the inhibition of MbtI and MbtA, which are are two enzymes involved in the mycobactin biosynthetic pathway. In particular, MbtI inhibitors have been studied in vitro, while MbtA inhibitors showed activity also in infected mice. Another class of compounds, MmpL3 inhibitors, showed antitubercular activity in vitro and in vivo, but their mechanism of action seems to be off-target. Some compounds inhibiting 4'-phosphopantetheinyl transferase were discovered but not tested on in vivo assays. The available data reported in this study based on inhibitors and gene deletion studies, suggest that targeting iron acquisition systems could be considered a promising antitubercular strategy. Due to their redundancy, the relative importance of each pathway for Mycobacterium tuberculosis survival has still to be determined. Thus, in vivo studies with new, potent and specific inhibitors are needed to highlight target selection.

Biochemical pathways supporting beta-lactam biosynthesis in the springtail Folsomia candida.

ABSTRACTRecently, an active set of beta-lactam biosynthesis genes was reported in the genome of the arthropod springtail Folsomia candida (Collembola). Evidence was provided that these genes were acquired through horizontal gene transfer. However, successful integration of fungal- or bacterial-derived beta-lactam biosynthesis into the metabolism of an animal requires the beta-lactam precursor L-α-aminoadipic acid and a phosphopantetheinyl transferase for activation of the first enzyme of the pathway, δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS). In this study, we characterized these supporting pathways and their transcriptional regulation in F. candida. We identified one phosphopantetheinyl transferase and three pathways for L-α-aminoadipic acid production, distinct from the pathways utilized by microorganisms. We found that after heat shock, the phosphopantetheinyl transferase was co-regulated with ACVS, confirming its role in activating ACVS. Two of the three L-α-aminoadipic acid production pathways were downregulated, while PIPOX, an enzyme participating in the pipecolate pathway, was slightly co-regulated with ACVS. This indicates that L-α-aminoadipic acid may not be a limiting factor in beta-lactam biosynthesis in F. candida, in contrast to microorganisms. In conclusion, we show that all components for L-α-aminoadipic acid synthesis are present and transcriptionally active in F. candida. This demonstrates how springtails could have recruited native enzymes to integrate a beta-lactam biosynthesis pathway into their metabolism after horizontal gene transfer.