Synteruptor: mining genomic islands for non-classical specialized metabolite gene clusters.

Microbial specialized metabolite biosynthetic gene clusters (SMBGCs) are a formidable source of natural products of pharmaceutical interest. With the multiplication of genomic data available, very efficient bioinformatic tools for automatic SMBGC detection have been developed. Nevertheless, most of these tools identify SMBGCs based on sequence similarity with enzymes typically involved in specialised metabolism and thus may miss SMBGCs coding for undercharacterised enzymes. Here we present Synteruptor (https://bioi2.i2bc.paris-saclay.fr/synteruptor), a program that identifies genomic islands, known to be enriched in SMBGCs, in the genomes of closely related species. With this tool, we identified a SMBGC in the genome of Streptomyces ambofaciens ATCC23877, undetected by antiSMASH versions prior to antiSMASH 5, and experimentally demonstrated that it directs the biosynthesis of two metabolites, one of which was identified as sphydrofuran. Synteruptor is also a valuable resource for the delineation of individual SMBGCs within antiSMASH regions that may encompass multiple clusters, and for refining the boundaries of these SMBGCs.

Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways.

Iron is essential to many biological processes but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways in the producing organism. Although the biological role(s) of these new derivatives remain to be elucidated, they may confer advantages in terms of metal chelation in the competitive soil environment due to the additional bidentate hydroxamic functional groups. The metabolites may also find application in various fields including biotechnology, bioremediation, and immuno-PET imaging.IMPORTANCEIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload and in diagnostic imaging, as well as in biotechnology, including as agents for biocontrol of pathogens and bioremediation. In this study, we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens-the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance, 89Zr-based immuno-PET imaging) and other applications of metal chelators.

Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins.

The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combine multiple, state-of-the-art approaches to reprogram the stambomycin PKS by deleting seven internal modules. One system produces the target 37-membered mini-stambomycin metabolites - a reduction in chain length of 14 carbons relative to the 51-membered parental compounds - but also substantial quantities of shunt metabolites. Our data also support an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain of the PKS. The mini-stambomycin yields are reduced relative to wild type, likely reflecting the poor tolerance of the modules downstream of the modified interfaces to the non-native substrates. Overall, we identify factors contributing to the productivity of engineered whole assembly lines, but our findings also highlight the need for further research to increase production titers.

Dynamics of the compartmentalized Streptomyces chromosome during metabolic differentiation.

Bacteria of the genus Streptomyces are prolific producers of specialized metabolites, including antibiotics. The linear chromosome includes a central region harboring core genes, as well as extremities enriched in specialized metabolite biosynthetic gene clusters. Here, we show that chromosome structure in Streptomyces ambofaciens correlates with genetic compartmentalization during exponential phase. Conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends tend to be transcriptionally quiescent compartments with different structural features. The onset of metabolic differentiation is accompanied by a rearrangement of chromosome architecture, from a rather 'open' to a 'closed' conformation, in which highly expressed specialized metabolite biosynthetic genes form new boundaries. Thus, our results indicate that the linear chromosome of S. ambofaciens is partitioned into structurally distinct entities, suggesting a link between chromosome folding, gene expression and genome evolution.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Inhibitions Dominate but Stimulations and Growth Rescues Are Not Rare Among Bacterial Isolates from Grains of Forest Soil.

Soil is a complex environment made of multiple microhabitats in which a wide variety of microorganisms co-exist and interact to form dynamic communities. While the abiotic factors that regulate the structure of these communities are now quite well documented, our knowledge of how bacteria interact with each other within these communities is still insufficient. Literature reveals so far contradictory results and is mainly focused on antagonistic interactions. To start filling this gap, we isolated 35 different bacterial isolates from grains of soil assuming that, at this scale, these bacteria would have been likely interacting in their natural habitat. We tested pairwise interactions between all isolates from each grain and scored positive and negative interactions. We compared the effects of simultaneous versus delayed co-inoculations, allowing or not to a strain to modify first its environment. One hundred fifty-seven interactions, either positive or negative, were recorded among the 525 possible one's. Members of the Bacillus subtilis, Pseudomonas and Streptomyces genera were responsible for most inhibitions, while positive interactions occurred between isolates of the Bacillales order and only in delayed inoculation conditions. Antagonist isolates had broad spectral abilities to acquire nutrients from organic and inorganic matter, while inhibited isolates tended to have little potentials. Despite an overall domination of antagonistic interactions (87%), a third of the isolates were able to stimulate or rescue the growth of other isolates, suggesting that cooperation between bacteria may be underestimated.

Molecular Dynamics to Elucidate the DNA-Binding Activity of AlpZ, a Member of the Gamma-Butyrolactone Receptor Family in Streptomyces ambofaciens.

Signaling molecule receptors play a central role in quorum sensing and in the coordination onset of specialized metabolite biosynthesis in Streptomyces due to their dual function in signal detection and gene expression control through DNA-binding in the promoter region of their target genes. In Streptomyces ambofaciens the alp biosynthetic gene cluster includes the signaling molecule receptor AlpZ that negatively regulates through a complex regulatory cascade the expression of key genes involved in the kinamycin antibiotic production until its cognate ligand, a yet unidentified signaling molecule, prompts its release from target promoters. Here we use an original molecular dynamics method to evaluate the DNA-binding properties of AlpZ to its target DNA sequence and the impact the signaling molecule has on the interaction. It is the first time this approach is used to characterize a regulator from the γ-butyrolactone receptor family. The observed KD in the nanomolar range indicates that AlpZ-DNA constitute a particularly stable complex. The signaling molecule ably disturbs this binding while kinamycin has no effect on the activity of AlpZ. Regulator size was determined and found to be considerably large regarding protein sequence, indicating that AlpZ regulates gene expression by binding the DNA as a homodimer, and structural modeling comparison with closely related γ-butyrolactone receptors supports this conclusion.

N-acylation of L-amino acids in aqueous media: Evaluation of the catalytic performances of Streptomyces ambofaciens aminoacylases.

N-acylated amino acids are widely used as surfactants and/or actives in cosmetics and household formulations. Their industrial production is based on the use of the Schotten-Baumann chemical and unselective reaction. Faced to the growing demand for greener production processes, selective enzymatic synthesis in more environment-friendly conditions starts to be considered as a potential alternative. This study concerns the use of the aminoacylases from Streptomyces ambofaciens to selectively catalyse aminoacid acylation reaction by fatty acids in aqueous medium. The results demonstrated that, when using undecylenoic acid as acyl donor, these aminoacylases properly catalyse the acylation of 14 of the 20 proteogenic l-amino acids tested on their α amino group with a great variability depending on the nature of the amino acid (polar or not, positively/negatively charged, aromatic or not…). More precisely, the following 9 amino acids were shown to be preferentially acylated by S. ambofaciens aminoacylases as follows: lysine > arginine > leucine > methionine > phenylalanine > valine > cysteine > isoleucine > threonine. Different fatty acids were used as acyl donors and, in most cases, the fatty acid length influenced the conversion yield. The kinetic study of α-lauroy-lysine synthesis showed a positive influence of lysine concentration with Vmax and Km of 3.7 mM/h and 76 mM, respectively. Besides, the lauric acid had an inhibitory effect on the reaction with Ki of 70 mM. The addition of cobalt to the reaction medium led to a more than six-fold increase of the reaction rate. These results, achieved with the aminoacylases from S. ambofaciens represent an improved enzyme-based N-acylated amino acids production in order to provide an alternative way to the Schotten-Baumann chemical reaction currently used in the industry.

Draft Whole-Genome Shotgun Sequence of Streptomyces sp. Strain ETH9427.

The draft genome of Streptomyces sp. strain ETH9427 was sequenced and assembled into three large scaffolds, a 7.745-Mb linear chromosome with terminal inverted repeats of 201 kb and two probable extrachromosomal elements. Thirty-two biosynthetic gene clusters (BGCs) were identified, out of which four are duplicated in the terminal inverted repeats.

Comparative Genomics among Closely Related Streptomyces Strains Revealed Specialized Metabolite Biosynthetic Gene Cluster Diversity.

Specialized metabolites are of great interest due to their possible industrial and clinical applications. The increasing number of antimicrobial resistant infectious agents is a major health threat and therefore, the discovery of chemical diversity and new antimicrobials is crucial. Extensive genomic data from Streptomyces spp. confirm their production potential and great importance. Genome sequencing of the same species strains indicates that specialized metabolite biosynthetic gene cluster (SMBGC) diversity is not exhausted, and instead, a pool of novel specialized metabolites still exists. Here, we analyze the genome sequence data from six phylogenetically close Streptomyces strains. The results reveal that the closer strains are phylogenetically, the number of shared gene clusters is higher. Eight specialized metabolites comprise the core metabolome, although some strains have only six core gene clusters. The number of conserved gene clusters common between the isolated strains and their closest phylogenetic counterparts varies from nine to 23 SMBGCs. However, the analysis of these phylogenetic relationships is not affected by the acquisition of gene clusters, probably by horizontal gene transfer events, as each strain also harbors strain-specific SMBGCs. Between one and 15 strain-specific gene clusters were identified, of which up to six gene clusters in a single strain are unknown and have no identifiable orthologs in other species, attesting to the existing SMBGC novelty at the strain level.

Diversity and antimicrobial activities of Streptomyces isolates from Fetzara Lake, north eastern Algeria.

A total of 125 Streptomyces strains were isolated from an Algerian wetland (Fetzara Lake) and characterized by growth on different culture media. Phylogenetic analyses were carried out by 16S rRNA sequence comparison after PCR amplification using universal primers. Antibacterial bioassays performed by the agar diffusion method enabled us to retain 33 Streptomyces isolates for their activity against two Gram-positive bacteria (Bacillus subtilis and Micrococcus luteus) and one Gram-negative bacteria (Escherichia coli). Among them, six isolates inhibited all three indicator strains. Antibacterial compounds were then extracted from the solid culture media with ethanol and ethyl acetate as organic solvents. The minimal inhibitory concentration (% v/v) of the extracts was evaluated by a standardized broth dilution method against different clinical-resistant bacterial isolates and Candida albicans. The most active crude extracts were selected for further characterization by chromatographic analysis (RP-HPLC).

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α-position and peptides on N-terminal position.

The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N-α or ε-acetyl-L-lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε-position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N-acylation on the α-position of the lysine or of the amino-acid in N-terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N-α-acetyl-L-lysine could be attributed to the product of SAM23877_1734 gene.

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking.

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS; http://gnps.ucsd.edu), an open-access knowledge base for community-wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS, crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of 'living data' through continuous reanalysis of deposited data.

Role of secondary metabolites in the interaction between Pseudomonas fluorescens and soil microorganisms under iron-limited conditions.

Microorganisms can be versatile in their interactions with each other, being variously beneficial, neutral or antagonistic in their effect. Although this versatility has been observed among many microorganisms and in many environments, little is known regarding the mechanisms leading to these changes in behavior. In the present work, we analyzed the mechanism by which the soil bacterium Pseudomonas fluorescens BBc6R8 shifts from stimulating the growth of the ectomycorrhizal fungus Laccaria bicolor S238N to killing the fungus. We show that among the three secondary metabolites produced by the bacterial strain-the siderophores enantio-pyochelin and pyoverdine, and the biosurfactant viscosin-the siderophores are mainly responsible for the antagonistic activity of the bacterium under iron-limited conditions. While the bacterial strain continues to produce beneficial factors, their effects are overridden by the action of their siderophores. This antagonistic activity of the strain P. fluorescens BBC6R8 in iron-depleted environments is not restricted to its influence on L. bicolor, since it was also seen to inhibit the growth of the actinomycete Streptomyces ambofaciens ATCC23877. We show that the strain P. fluorescens BBc6R8 uses different strategies to acquire iron, depending on certain biotic and abiotic factors.

Complete genome sequence of Streptomyces ambofaciens ATCC 23877, the spiramycin producer.

Streptomyces ambofaciens ATCC23877 is a soil bacterium industrially exploited for the production of the macrolide spiramycin which is used in human medicine as an antibacterial and anti-toxoplasmosis chemical. Its genome consists of a 8.3 Mbp linear chromosome and a 89 kb circular plasmid. The complete genome sequence reported here will enable us to investigate Streptomyces genome evolution and to discover new secondary metabolites with potential applications notably in human medicine.

Identification of Alp1U and Lom6 as epoxy hydrolases and implications for kinamycin and lomaiviticin biosynthesis.

The naturally occurring diazobenzofluorenes, kinamycins, fluostatins and lomaiviticins, possess highly oxygenated A-rings, via which the last forms a dimeric pharmacophore. However, neither the A-ring transformation nor the dimerization mechanisms have been explored thus far. Here we propose a unified biosynthetic logic for the three types of antibiotics and verify one key reaction via detailed genetic and enzymatic experiments. Alp1U and Lom6 from the kinamycin and lomaiviticin biosynthesis, respectively, are shown to catalyse epoxy hydrolysis on a substrate that is obtained by chemical deacetylation of a kinamycin-pathway-derived intermediate. Thus, our study provides the first evidence for the existence of an epoxy intermediate in lomaiviticin biosynthesis. Furthermore, our results suggest that the dimerization in the lomaiviticin biosynthesis proceeds after dehydration of a product generated by Lom6.

Kinamycin biosynthesis employs a conserved pair of oxidases for B-ring contraction.

The biosynthesis of diazobenzofluorene kinamycins requires a hitherto uncharacterized B-ring contraction. Via detailed genetic and enzymatic analyses we unambiguously characterized the conserved pairs of oxidases, AlpJ and AlpK homologs, as nature's machinery for benzofluorenone formation, which paves the way for the investigation of the following diazo assembly.

Pseudomonas fluorescens pirates both ferrioxamine and ferricoelichelin siderophores from Streptomyces ambofaciens.

Iron is essential in many biological processes. However, its bioavailability is reduced in aerobic environments, such as soil. To overcome this limitation, microorganisms have developed different strategies, such as iron chelation by siderophores. Some bacteria have even gained the ability to detect and utilize xenosiderophores, i.e., siderophores produced by other organisms. We illustrate an example of such an interaction between two soil bacteria, Pseudomonas fluorescens strain BBc6R8 and Streptomyces ambofaciens ATCC 23877, which produce the siderophores pyoverdine and enantiopyochelin and the siderophores desferrioxamines B and E and coelichelin, respectively. During pairwise cultures on iron-limiting agar medium, no induction of siderophore synthesis by P. fluorescens BBc6R8 was observed in the presence of S. ambofaciens ATCC 23877. Cocultures with a Streptomyces mutant strain that produced either coelichelin or desferrioxamines, as well as culture in a medium supplemented with desferrioxamine B, resulted in the absence of pyoverdine production; however, culture with a double mutant deficient in desferrioxamines and coelichelin production did not. This strongly suggests that P. fluorescens BBbc6R8 utilizes the ferrioxamines and ferricoelichelin produced by S. ambofaciens as xenosiderophores and therefore no longer activates the production of its own siderophores. A screening of a library of P. fluorescens BBc6R8 mutants highlighted the involvement of the TonB-dependent receptor FoxA in this process: the expression of foxA and genes involved in the regulation of its biosynthesis was induced in the presence of S. ambofaciens. In a competitive environment, such as soil, siderophore piracy could well be one of the driving forces that determine the outcome of microbial competition.