Valérie Leclère

NORINE: a database of nonribosomal peptides

Norine is the first database entirely dedicated to nonribosomal peptides (NRPs). In bacteria and fungi, in addition to the traditional ribosomal proteic biosynthesis, an alternative ribosome-independent pathway called NRP synthesis allows peptide production. It is performed by huge protein complexes called nonribosomal peptide synthetases (NRPSs). The molecules synthesized by NRPS contain a high proportion of nonproteogenic amino acids. The primary structure of these peptides is not always linear but often more complex and may contain cycles and branchings. In recent years, NRPs attracted a lot of attention because of their biological activities and pharmacological properties (antibiotic, immunosuppressor, antitumor, etc.). However, few computational resources and tools dedicated to those peptides have been available so far. Norine is focused on NRPs and contains more than 700 entries. The database is freely accessible at http://bioinfo.lifl.fr/norine/. It provides a complete computational tool for systematic study of NRPs in numerous species, and as such, should permit to obtain a better knowledge of these metabolic products and underlying biological mechanisms, and ultimately to contribute to the redesigning of natural products in order to obtain new bioactive compounds for drug discovery.

The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties

The colonizing behaviour and the pellicle formation of Bacillus subtilis strains producing different families of lipopeptides were evaluated under several cultural conditions. The pattern of lipopeptides produced determined the architecture of the colony on a swarming medium as well as the flotation and the thickness of the pellicle formed at the air/liquid interface. The overproduction of mycosubtilin, a lipopeptide of the iturin family, led to increased spreading but had no effect on pellicle formation. A physico-chemical approach was developed to gain an insight into the mode of action of the biosurfactants facilitating the colonization. A relationship between surface tension of the culture medium and spreading of a lipopeptide non-producing strain, B. subtilis 168, was established. Goniometry was used to highlight the modification of the in situ wettability in the area where spreading was enhanced. On a solid medium, co-cultures of a surfactin producing with other strains showed a diffusion ring of the surfactin around the colony. This ring characterized by a higher wettability favoured the propagation of other colonies.

Diversity of Monomers in Nonribosomal Peptides: towards the Prediction of Origin and Biological Activity

Nonribosomal peptides (NRPs) are molecules produced by microorganisms that have a broad spectrum of biological activities and pharmaceutical applications (e.g., antibiotic, immunomodulating, and antitumor activities). One particularity of the NRPs is the biodiversity of their monomers, extending far beyond the 20 proteogenic amino acid residues. Norine, a comprehensive database of NRPs, allowed us to review for the first time the main characteristics of the NRPs and especially their monomer biodiversity. Our analysis highlighted a significant similarity relationship between NRPs synthesized by bacteria and those isolated from metazoa, especially from sponges, supporting the hypothesis that some NRPs isolated from sponges are actually synthesized by symbiotic bacteria rather than by the sponges themselves. A comparison of peptide monomeric compositions as a function of biological activity showed that some monomers are specific to a class of activities. An analysis of the monomer compositions of peptide products predicted from genomic information (metagenomics and high-throughput genome sequencing) or of new peptides detected by mass spectrometry analysis applied to a culture supernatant can provide indications of the origin of a peptide and/or its biological activity.

High-level biosynthesis of the anteiso-C(17) isoform of the antibiotic mycosubtilin in Bacillus subtilis and characterization of its candidacidal activity.

ABSTRACT High-level production (880 mg liter−1) and isolation of the anteiso-C17 isoform of the lipopeptide mycosubtilin produced by a genetically engineered Bacillus subtilis strain are reported. Antifungal activity of this isoform, as determined via culture and fluorometric and cell leakage assays, suggests its potential therapeutic use as an antifungal agent, in particular against Candida spp.

Structure, biosynthesis, and properties of kurstakins, nonribosomal lipopeptides from Bacillus spp.

A new family of lipopeptides produced by Bacillus thuringiensis, the kurstakins, was discovered in 2000 and considered as a biomarker of this species. Kurstakins are lipoheptapeptides displaying antifungal activities against Stachybotrys charatum. Recently, the biosynthesis mechanism, the regulation of this biosynthesis and the potential new properties of kurstakins were described in the literature. In addition, kurstakins were also detected in other species belonging to Bacillus genus such as Bacillus cereus. This mini-review gathers all the information about these promising bioactive molecules.

New linear lipopeptides produced by Pseudomonas cichorii SF1-54 are involved in virulence, swarming motility, and biofilm formation

Pseudomonas cichorii is the causal agent of lettuce midrib rot, characterized by a dark-brown to green-black discoloration of the midrib. Formation of necrotic lesions by several plant-pathogenic pseudomonads is associated with production of phytotoxic lipopeptides, which contribute to virulence. Therefore, the ability of P. cichorii SF1-54 to produce lipopeptides was investigated. A cell-free culture filtrate of SF1-54 showed surfactant, antimicrobial, and phytotoxic activities which are typical for lipopeptides. High-performance liquid chromatography analysis of P. cichorii SF1-54 culture filtrate revealed the presence of seven compounds with lipopeptide characteristics. Two related lipopeptides, named cichofactin A and B, were studied in more detail: they are linear lipopeptides with a decanoic and dodecanoic lipid chain, respectively, connected to the N-terminus of an eight-amino-acid peptide moiety. Both cichofactins are new members of the syringafactin lipopeptide family. Furthermore, two nonribosomal peptide synthethase-encoding genes, cifA and cifB, were identified as responsible for cichofactin biosynthesis. A cifAB deletion mutant no longer produced cichofactins and was impaired in swarming motility but showed enhanced biofilm formation. Upon spray inoculation on lettuce, the cichofactin-deficient mutant caused significantly less rotten midribs than the wild type, indicating that cichofactins are involved in pathogenicity of P. cichorii SF1-54. Further analysis revealed that P. cichorii isolates vary greatly in swarming motility and cichofactin production.

Structural pattern matching of nonribosomal peptides

BackgroundNonribosomal peptides (NRPs), bioactive secondary metabolites produced by many microorganisms, show a broad range of important biological activities (e.g. antibiotics, immunosuppressants, antitumor agents). NRPs are mainly composed of amino acids but their primary structure is not always linear and can contain cycles or branchings. Furthermore, there are several hundred different monomers that can be incorporated into NRPs. The NORINE database, the first resource entirely dedicated to NRPs, currently stores more than 700 NRPs annotated with their monomeric peptide structure encoded by undirected labeled graphs. This opens a way to a systematic analysis of structural patterns occurring in NRPs. Such studies can investigate the functional role of some monomeric chains, or analyse NRPs that have been computationally predicted from the synthetase protein sequence. A basic operation in such analyses is the search for a given structural pattern in the database.ResultsWe developed an efficient method that allows for a quick search for a structural pattern in the NORINE database. The method identifies all peptides containing a pattern substructure of a given size. This amounts to solving a variant of the maximum common subgraph problem on pattern and peptide graphs, which is done by computing cliques in an appropriate compatibility graph.ConclusionThe method has been incorporated into the NORINE database, available at http://bioinfo.lifl.fr/norine. Less than one second is needed to search for a pattern in the entire database.

Norine, the knowledgebase dedicated to non-ribosomal peptides, is now open to crowdsourcing

Since its creation in 2006, Norine remains the unique knowledgebase dedicated to non-ribosomal peptides (NRPs). These secondary metabolites, produced by bacteria and fungi, harbor diverse interesting biological activities (such as antibiotic, antitumor, siderophore or surfactant) directly related to the diversity of their structures. The Norine team goal is to collect the NRPs and provide tools to analyze them efficiently. We have developed a user-friendly interface and dedicated tools to provide a complete bioinformatics platform. The knowledgebase gathers abundant and valuable annotations on more than 1100 NRPs. To increase the quantity of described NRPs and improve the quality of associated annotations, we are now opening Norine to crowdsourcing. We believe that contributors from the scientific community are the best experts to annotate the NRPs they work on. We have developed MyNorine to facilitate the submission of new NRPs or modifications of stored ones. This article presents MyNorine and other novelties of Norine interface released since the first publication. Norine is freely accessible from the following URL: http://bioinfo.lifl.fr/NRP.

Bioinformatics and molecular approaches to detect NRPS genes involved in the biosynthesis of kurstakin from Bacillus thuringiensis

Degenerated primers designed for the detection by polymerase chain reaction of nonribosomal peptide synthetases (NRPS) genes involved in the biosynthesis of lipopeptides were used on genomic DNA from a new isolate of Bacillus thuringiensis CIP 110220. Primers dedicated to surfactin and bacillomycin detection amplified sequences corresponding respectively to the surfactin synthetase operon and to a gene belonging to a new NRPS operon identified in the genome of B. thuringiensis serovar pondicheriensis BSCG 4BA1. A bioinformatics analysis of this operon led to the prediction of an NRPS constituted of seven modules beginning with a condensation starter domain and which could be involved in the biosynthesis of a heptalipopeptide similar to kurstakin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) performed on whole cells of B. thuringiensis CIP 110220 confirmed the production of kurstakin by this strain. The kurstakin operon was thus used to design a new set of degenerated primers specifically to detect kurstakin genes. These primers were used to screen kurstakin producers in a collection of nine B. thuringiensis strains isolated from different areas in Algeria and two from the Pasteur Institute collection. For eight among the 11 tested strains, the amplified fragment matched with an operon similar to the kurstakin operon and found in the newly sequenced genome of Bacillus cereus or B. thuringiensis serovar pulsiensis, kurstaki, and thuringiensis. Kurstakin production was detected by MALDI-ToF-MS on whole cells for six strains. This production was compared with the spreading of the strains and their antimicrobial activity. Only the spreading can be correlated with the kurstakin production.

Temperature dependence of mycosubtilin homologue production in Bacillus subtilis ATCC6633.

Bacillus subtilis ATCC6633 produces mycosubtilin, a non-ribosomally synthesized lipopeptide of the iturin family which presents antagonistic activities toward various phytopathogens. Different homologues with fatty acid moiety varying from C15 to C17 are usually co-produced, with their biological activities increasing with the number of carbons in the fatty acid chain. In the present report, we highlight that growth temperature modulates both the extent of mycosubtilin production and the relative abundance of the different homologues. A 30-fold increase in mycosubtilin production was observed when the temperature was decreased from 37 degrees C to 25 degrees C for both strain ATCC6633 and its derivative BBG100, a constitutive mycosubtilin overproducer. However, no significant difference in either the expression of the mycosubtilin synthetase encoding genes or in the intracellular synthetase concentration could be found, suggesting that the observed phenotype originated from a higher mycosubtilin synthetase turnover at lower temperature. We also point out that lower growth temperature leads to an increased proportion of odd-numbered fatty acid homologues as a consequence of de novo synthesis of C17 anteiso fatty acid following cell adaptation to low temperatures.