Control of mRNA translation by dynamic ribosome modification.

Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions as a rapid-response mechanism that tunes global gene translation in response to environmental signals.

Stuffed Methyltransferase Catalyzes the Penultimate Step of Pyochelin Biosynthesis.

Nonribosomal peptide synthetases use tailoring domains to incorporate chemical diversity into the final natural product. A structurally unique set of tailoring domains are found to be stuffed within adenylation domains and have only recently begun to be characterized. PchF is the NRPS termination module in pyochelin biosynthesis and includes a stuffed methyltransferase domain responsible for S-adenosylmethionine (AdoMet)-dependent N-methylation. Recent studies of stuffed methyltransferase domains propose a model in which methylation occurs on amino acids after adenylation and thiolation rather than after condensation to the nascent peptide chain. Herein, we characterize the adenylation and stuffed methyltransferase didomain of PchF through the synthesis and use of substrate analogues, steady-state kinetics, and onium chalcogen effects. We provide evidence that methylation occurs through an SN2 reaction after thiolation, condensation, cyclization, and reduction of the module substrate cysteine and is the penultimate step in pyochelin biosynthesis.

Probing the limits of interrupted adenylation domains by engineering a trifunctional enzyme capable of adenylation, N-, and S-methylation.

The adenylation (A) domains found in nonribosomal peptide synthetases (NRPSs) exhibit tremendous plasticity. Some A domains have been shown to display the ability to contain within them the catalytic portion of an auxiliary domain, most commonly that of a methyltransferase (M) enzyme. This unique feature of A domains interrupted by M domains allows them to possess bifunctionality, where they can both adenylate and methylate an amino acid substrate. Additionally, these types of inserted M domains are able to selectively carry out either backbone or side chain methylation of amino acids. Interruptions with M domains are naturally found to occur either between the a2-a3 or the a8-a9 of the ten conserved motifs of A domains. Herein, we set out to answer the following question: Can one A domain support two different M domain interruptions occurring in two different locations (a2-a3 and a8-a9) of the A domain and possess the ability to adenylate an amino acid and methylate it on both its side chain and backbone? To answer this question we added a backbone methylating M3S domain from TioS(A3aM3SA3b) between the a8-a9 region of a mono-interrupted A domain, TioN(AaMNAb), that already contained a side chain methylating MN domain between its a2-a3 region. We evaluated the di-interrupted A domain TioN(AMNAM3SA) with a series of radiometric and mass spectrometry assays and found that this engineered enzyme was indeed capable of all three activities. These findings show that production of an active trifunctional di-interrupted A domain is possible and represents an exciting new avenue for future nonribosomal peptide (NRP) derivatization.

Expanding tryptophan-containing cyclodipeptide synthase spectrum by identification of nine members from Streptomyces strains.

Cyclodipeptide synthases (CDPSs) comprise normally 200-300 amino acid residues and are mainly found in bacteria. They hijack aminoacyl-tRNAs from the ribosomal machinery for cyclodipeptide formation. In this study, nine new CDPS genes from eight Streptomyces strains were cloned into pET28a vector and expressed in Escherichia coli. Structural elucidation of the isolated products led to the identification of one cyclo-L-Trp-L-Leu, two cyclo-L-Trp-L-Pro, and three cyclo-L-Trp-L-Trp synthases. Other three CDPSs produce cyclo-L-Trp-L-Ala or cyclo-L-Trp-L-Tyr as the major cyclodipeptide. Total product yields of 46 to 211 mg/L E. coli culture were obtained. Our findings represent rare examples of CDPS family derived from actinobacteria that form various tryptophan-containing cyclodipeptides. Furthermore, this study highlights the potential of the microbial machinery for tryptophan-containing cyclodipeptide biosynthesis and provides valid experimental basis for further combination of these CDPS genes with other modification genes in synthetic biology.

Preliminary Results on the Evaluation of the Occurrence of Tetrodotoxin Associated to Marine Vibrio spp. in Bivalves from the Galician Rias (Northwest of Spain).

Tetrodotoxins (TTX) are a potent group of natural neurotoxins putatively produced by symbiotic microorganisms and affecting the aquatic environment. These neurotoxins have been recently found in some species of bivalves and gastropods along the European Coasts (Greece, UK, and The Netherlands) linked to the presence of high concentrations of Vibrio, in particular Vibrio parahaemolyticus. This study is focused on the evaluation of the presence of Vibrio species and TTX in bivalves (mussels, oysters, cockles, clams, scallops, and razor clams) from Galician Rias (northwest of Spain). The detection and isolation of the major Vibrio spp. and other enterobacterial populations have been carried out with the aim of screening for the presence of the pathways genes, poliketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) possibly involved in the biosynthesis of these toxins. Samples containing Vibrio spp. were analyzed by biochemical (API20E-galery) and genetic tests (PCR-RT). These samples were then screened for TTX toxicity by a neuroblastoma cell-based assay (N2a) and the presence of TTX was further confirmed by LC-MS/MS. TTX was detected in two infaunal samples. This is the first confirmation of the presence of TTX in bivalve molluscs from the Galician Rias.

Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens.

Covering: up to 2017.Natural products are important secondary metabolites produced by bacterial and fungal species that play important roles in cellular growth and signaling, nutrient acquisition, intra- and interspecies communication, and virulence. A subset of natural products is produced by nonribosomal peptide synthetases (NRPSs), a family of large, modular enzymes that function in an assembly line fashion. Because of the pharmaceutical activity of many NRPS products, much effort has gone into the exploration of their biosynthetic pathways and the diverse products they make. Many interesting NRPS pathways have been identified and characterized from both terrestrial and marine bacterial sources. Recently, several NRPS pathways in human commensal bacterial species have been identified that produce molecules with antibiotic activity, suggesting another source of interesting NRPS pathways may be the commensal and pathogenic bacteria that live on the human body. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) have been identified as a significant cause of human bacterial infections that are frequently multidrug resistant. The emerging resistance profile of these organisms has prompted calls from multiple international agencies to identify novel antibacterial targets and develop new approaches to treat infections from ESKAPE pathogens. Each of these species contains several NRPS biosynthetic gene clusters. While some have been well characterized and produce known natural products with important biological roles in microbial physiology, others have yet to be investigated. This review catalogs the NRPS pathways of ESKAPE pathogens. The exploration of novel NRPS products may lead to a better understanding of the chemical communication used by human pathogens and potentially to the discovery of novel therapeutic approaches.

Genomic and functional features of the biosurfactant producing Bacillus sp. AM13.

Genomic studies provide deeper insights into secondary metabolites produced by diverse bacterial communities, residing in various environmental niches. This study aims to understand the potential of a biosurfactant producing Bacillus sp. AM13, isolated from soil. An integrated approach of genomic and chemical analysis was employed to characterize the antibacterial lipopeptide produced by the strain AM13. Genome analysis revealed that strain AM13 harbors a nonribosomal peptide synthetase (NRPS) cluster; highly similar with known biosynthetic gene clusters from surfactin family: lichenysin (85 %) and surfactin (78 %). These findings were substantiated with supplementary experiments of oil displacement assay and surface tension measurements, confirming the biosurfactant production. Further investigation using LCMS approach exhibited similarity of the biomolecule with biosurfactants of the surfactin family. Our consolidated effort of functional genomics provided chemical as well as genetic leads for understanding the biochemical characteristics of the bioactive compound.

Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.

Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations.

Differences in the purification and solution properties of PurC gene products from Streptococcus pneumoniae and Bacillus anthracis.

4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide synthetase (PurC) is a key enzyme in the de novo purine biosynthetic pathway of bacteria and an ideal target pathway for the discovery of antimicrobials. Bacillus anthracis (Ba) and Streptococcus pneumoniae (Sp) are two of the bacteria shown to be severe detriments to public health. To be able to carry out the experimentation that leads to drug discovery, high yields of pure soluble recombinant protein must first be obtained. We studied two recombinant PurC proteins from B. anthracis and S. pneumoniae, using Escherichia coli as the host cells. These two proteins, with very similar amino acid sequences, exhibit very different solution properties, leading to a large difference in yields during protein purification under the same conditions. The yield for SpPurC (>50mG per gram of cells) is ten times greater than that for BaPurC (<5mG per gram of cells). The BaPurC samples in solution consisted of oligomers and dimers, with dimers as its functional form. Comparing the yields of dimers, SpPurC is 25 times greater than that for BaPurC (∼2mG per gram of cell). Our studies suggest that the difference in exposed hydrophobic surface area is responsible for the difference in yields under the same conditions.

MreB and MurG as scaffolds for the cytoplasmic steps of peptidoglycan biosynthesis.

Peptidoglycan is a major determinant of cell shape in bacteria, and its biosynthesis involves the concerted action of cytoplasmic, membrane-associated and periplasmic enzymes. Within the cytoplasm, Mur enzymes catalyse the first steps leading to peptidoglycan precursor biosynthesis, and have been suggested as being part of a multicomponent complex that could also involve the transglycosylase MurG and the cytoskeletal protein MreB. In order to initialize the characterization of a potential Mur interaction network, we purified MurD, MurE, MurF, MurG and MreB from Thermotoga maritima and characterized their interactions using membrane blotting and surface plasmon resonance. MurD, MurE and MurF all recognize MurG and MreB, but not each other, while the two latter proteins interact. In addition, we solved the crystal structures of MurD, MurE and MurF, which indicate that their C-termini display high conformational flexibilities. The differences in Mur conformations could be important parameters for the stability of an intracytoplasmic murein biosynthesis complex.

[Activity of the SrfAC-A domain from Bacillus subtilis fmbj].

OBJECTIVE: We studied the A domain of surfactin synthase in vitro to obtain new surfactin analogues. METHODS: We cloned the srfAC-A gene from Bacillus subtilis fmbj by PCR, and constructed a recombinant expression vector named pET-23a-srfAC-A. Furthermore, the SrfAC-A domain was expressed in E. coli BL21 (DE3) and purified by Ni-NTA agarose column. Then the activity of srfAC-A domain was detected. RESULTS: The srfAC-A domain had specificity towards Ile, but almost no activity to other amino acids. CONCLUSION: The independent A domain from surfactin synthase had selectivity to specific amino acids in vitro.

A detailed biochemical characterization of phosphopantothenate synthetase, a novel enzyme involved in coenzyme A biosynthesis in the Archaea.

We have previously reported that the majority of the archaea utilize a novel pathway for coenzyme A biosynthesis (CoA). Bacteria/eukaryotes commonly use pantothenate synthetase and pantothenate kinase to convert pantoate to 4'-phosphopantothenate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, two novel enzymes specific to the archaea, pantoate kinase and phosphopantothenate synthetase, are responsible for this conversion. Here, we examined the enzymatic properties of the archaeal phosphopantothenate synthetase, which catalyzes the ATP-dependent condensation of 4-phosphopantoate and ß-alanine. The activation energy of the phosphopantothenate synthetase reaction was 82.3 kJ mol(-1). In terms of substrate specificity toward nucleoside triphosphates, the enzyme displayed a strict preference for ATP. Among several amine substrates, activity was detected with ß-alanine, but not with γ-aminobutyrate, glycine nor aspartate. The phosphopantothenate synthetase reaction followed Michaelis-Menten kinetics toward ß-alanine, whereas substrate inhibition was observed with 4-phosphopantoate and ATP. Feedback inhibition by CoA/acetyl-CoA and product inhibition by 4'-phosphopantothenate were not observed. By contrast, the other archaeal enzyme pantoate kinase displayed product inhibition by 4-phosphopantoate in a non-competitive manner. Based on our results, we discuss the regulation of CoA biosynthesis in the archaea.

Isolation and characterization of culturable endophytic actinobacteria associated with Artemisia annua L.

Endophytic actinobacteria isolated from Artemisia annua were characterized and evaluated for their bioactivities. A total of 228 isolates representing at least 19 different genera of actinobacteria were obtained and several of them seemed to be novel taxa. An evaluation of antimicrobial activity showed that more isolates possessed activity towards plant pathogens than activity against other pathogenic bacteria or yeasts. High frequencies of PCR amplification were obtained for type I polyketide synthases (PKS-I, 21.1%), type II polyketide synthases (PKS-II, 45.2%) and nonribosomal peptide synthetases (NRPS, 32.5%). The results of herbicidal activity screening indicated that 19 out of 117 samples of fermentation broths completely inhibited the germination of Echinochloa crusgalli. This study indicated that endophytic actinobacteria associated with A. annua are abundant and have potentially beneficial and diverse bioactivities which should be pursued for their biotechnical promise.

[Rapid cloning and functional characterization of hypericin synthase gene].

Hypericin, a red-colored naphtodianthrone, is a natural product synthesized in the medicinal plant Hypericum perforatum, commonly known as St. John's wort. Hypericin has attracted a growing attention of the pharmaceutical industry because of its potential application to various therapies, including the treatment of depression and remarkable antiviral and photodynamic activities, hyp-1 gene encodes for phenolic coupling protein which catalyzes in vitro direct and specific conversion of emodin to hypericin which, however, has not formed common opinion so far. Six pairs of primers specific to hyp-1 gene were synthesized. The rapid cloning of hyp-1 gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pET32ahyp containing hyp-1 gene was constructed and was transformed into E. coli to induce heterologous expression. SDS-PAGE and Western blot results showed the recombinant Hyp-1 protein was expressed successfully in E. coli. The soluble fraction was used to test the function of the recombinant Hyp-1. Hypericin was detected by LC-MS/MS with emodin as a substrate under in vitro conditions. The above results corroborated the Hyp-1 function, a confusing question, which lay a material foundation for the synthesis of hypericin by synthetic biotechnology.

Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.

Structure elucidation and biosynthesis of fuscachelins, peptide siderophores from the moderate thermophile Thermobifida fusca.

Bacteria belonging to the order Actinomycetales have proven to be an important source of biologically active and often therapeutically useful natural products. The characterization of orphan biosynthetic gene clusters is an emerging and valuable approach to the discovery of novel small molecules. Analysis of the recently sequenced genome of the thermophilic actinomycete Thermobifida fusca revealed an orphan nonribosomal peptide biosynthetic gene cluster coding for an unknown siderophore natural product. T. fusca is a model organism for the study of thermostable cellulases and is a major degrader of plant cell walls. Here, we report the isolation and structure elucidation of the fuscachelins, siderophore natural products produced by T. fusca. In addition, we report the purification and biochemical characterization of the termination module of the nonribosomal peptide synthetase. Biochemical analysis of adenylation domain specificity supports the assignment of this gene cluster as the producer of the fuscachelin siderophores. The proposed nonribosomal peptide biosynthetic pathway exhibits several atypical features, including a macrocyclizing thioesterase that produces a 10-membered cyclic depsipeptide and a nonlinear assembly line, resulting in the unique heterodimeric architecture of the siderophore natural product.

Substrate-induced closing of the active site revealed by the crystal structure of pantothenate synthetase from Staphylococcus aureus.

Pantothenate synthetase (PS, EC 6.3.2.1) is the last enzyme in the pantothenate biosynthesis pathway, a metabolic pathway identified as a potential target for new antimicrobials. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to form pantothenate. Here we report the overexpression, purification, enzyme assay, and tertiary structure of PS from Staphylococcus aureus. PS activity was experimentally confirmed, indicating a k(cat) value comparable to those of enzymes from other organisms. The structures of the apoenzyme and the reaction intermediate (pantoyl adenylate; PA) complex were determined by X-ray crystallography to resolutions of 2.5 and 1.85 A, respectively. Structural analysis indicated that the apoenzyme adopts an open and relatively mobile structure, while the complex structure is closed and entirely rigid. Structural comparison of the apoenzyme and the complex revealed how S. aureus PS undergoes open/close conformational change, and also determined the key interactions with the adenine ring of PA for a hinge bending domain closure. In the complex structure, PA and acetate are bound in the active site. We suggest that the acetate mimics the substrate beta-alanine. Therefore, the complex structure seems to represent a catalytic state poised for in-line nucleophilic attack on PA. These data also offer an alternative strategy for designing novel compounds that selectively inhibit PS activity.

Expression and purification of an adenylation domain from a eukaryotic nonribosomal peptide synthetase: using structural genomics tools for a challenging target.

Nonribosomal peptide synthetases (NRPSs) are large multimodular and multidomain enzymes that are involved in synthesising an array of molecules that are important in human and animal health. NRPSs are found in both bacteria and fungi but most of the research to date has focused on the bacterial enzymes. This is largely due to the technical challenges in producing active fungal NRPSs, which stem from their large size and multidomain nature. In order to target fungal NRPS domains for biochemical and structural characterisation, we tackled this challenge by using the cloning and expression tools of structural genomics to screen the many variables that can influence the expression and purification of proteins. Using these tools we have screened 32 constructs containing 16 different fungal NRPS domains or domain combinations for expression and solubility. Two of these yielded soluble protein with one, the third adenylation domain of the SidN NRPS (SidNA3) from the grass endophyte Neotyphodium lolii, being tractable for purification using Ni-affinity resin. The initial purified protein exhibited poor solution behaviour but optimisation of the expression construct and the buffer conditions used for purification, resulted in stable recombinant protein suitable for biochemical characterisation, crystallisation and structure determination.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SAICAR synthase from Streptococcus suis serotype 2.

Phosphoribosylaminoimidazole-succinocarboxamide synthase (SAICAR synthase) plays an essential role in the de novo biosynthesis of purine nucleotides. In this study, the SAICAR synthase from Streptococcus suis was cloned and overexpressed in Escherichia coli. The subsequent product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.8 A resolution and belonged to space group P2, with unit-cell parameters a=70.2, b=52.2, c=153.9 A, beta=102.8 degrees.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of the putative SAICAR synthetase (PH0239) from Pyrococcus horikoshii OT3.

The study of proteins involved in de novo biosynthesis of purine nucleotides is central in the development of antibiotics and anticancer drugs. In view of this, a protein from the hyperthermophile Pyrococcus horikoshii OT3 was isolated, purified and crystallized using the microbatch method. Its primary structure was found to be similar to that of SAICAR synthetase, which catalyses the seventh step of de novo purine biosynthesis. A diffraction-quality crystal was obtained using Hampton Research Crystal Screen II condition No. 34, consisting of 0.05 M cadmium sulfate hydrate, 0.1 M HEPES buffer pH 7.5 and 1.0 M sodium acetate trihydrate, with 40%(v/v) 1,4-butanediol as an additive. The crystal belonged to space group P3(1), with unit-cell parameters a = b = 95.62, c = 149.13 A. Assuming the presence of a hexamer in the asymmetric unit resulted in a Matthews coefficient (V(M)) of 2.3 A(3) Da(-1), corresponding to a solvent content of about 46%. A detailed study of this protein will yield insights into structural stability at high temperatures and should be highly relevant to the development of antibiotics and anticancer drugs targeting the biosynthesis of purine nucleotides.