Sylvie Lautru

Diclofenac (DCF) is a widely detected pharmaceutical whose persistence and biologically active transformation products raise environmental concern. This study evaluated DCF tolerance and removal by actinobacterial strains and integrated chemical performance with plant- and earthworm-based ecotoxicological responses. Phenotypic screening at 1, 5, and 10 g L⁻¹ DCF revealed strain- and concentration-dependent responses in growth, sporulation, and pigment production, with Streptomyces sp. E5N50 and Streptomyces sp. Z2 selected for further study. In liquid assays, after 5 days of incubation, only low biomass production was observed when 50 mg L⁻¹ DCF was supplied without glucose, relative to glucose-containing controls. E5N50 showed modest DCF removal (approximately 23–26%), while Z2 achieved 90.8% in glucose-supplemented medium and about 70% when glucose and ammonium were supplied. A seven-day time-course study with Streptomyces sp. Z2 showed that DCF removal continued after biomass had reached its maximum, suggesting that the process was not exclusively growth-associated. Ecotoxicological responses were organism- and endpoint-dependent. The glucose + DCF + Z2 treatment, associated with the highest DCF removal, significantly increased tomato radicle elongation, whereas glucose + ammonium + DCF + Z2 produced the most favorable Eisenia fetida response, with significantly reduced body-mass loss and increased coelomocyte abundance despite the lower DCF removal removalobserved in this treatment. Overall, Z2 exhibited potential for DCF removal under defined laboratory liquid-culture conditions, and the combined chemical and biological assessment highlighted that parent-compound removal and ecotoxicological improvement provide complementary measures of remediation performance.

Streptomyces bacteria are renowned for their intricate life cycle and prolific production of specialized metabolites, including antibiotics. Their linear chromosome is spatially compartmentalized: the central region contains highly conserved and expressed genes, while the terminal regions harbor less conserved, poorly expressed sequences, often rich in specialized metabolite biosynthetic gene clusters. To investigate the relationship between genome architecture and gene expression, we relocated the congocidine antibiotic biosynthetic gene cluster (CGC) from its native terminal position to the central compartment in Streptomyces ambofaciens . This relocation enhanced CGC transcription compared to its original terminal location, both in antisense orientation during exponential growth and in sense orientation after metabolic differentiation, resulting in 50% increase in congocidine production. At the 3D-level, transcription-induced domains formed at both the relocated and native CGC sites, creating sharp boundaries at a larger scale. Notably, the formation of such a boundary in the central compartment during the early stationary phase did not disrupt inter-arm contacts or affect neighboring gene expression. These results indicate that relocating a terminal cluster to the central chromosomal compartment provides a more favorable environment for transcription without altering chromosome compaction in the stationary phase, offering a promising strategy to enhance antibiotic production in the native host.

Phages are a valuable resource for the genetic engineering of Streptomyces antibiotic-producing bacteria. Indeed, a few integrative vectors based on phage integrase are available to insert transgenes at specific genomic loci. Chromosome conformation captures previously demonstrated that the Streptomyces linear chromosome is organized in two spatial compartments: the central compartment encompassing the most conserved and highly expressed genes in exponential phase, and the terminal compartments enriched in poorly conserved sequences including specialized metabolite biosynthetic gene clusters. This study introduces a new integrative tool based on a recently described phage, Samy, which specifically targets the terminal compartment of its native host chromosome. Samy is related to PhiC31 phage and, like the latter, encodes a serine integrase. Whereas PhiC31 targets a site generally located near the origin of replication, the Samy integration site is one of the farthest known attB sites from it. We demonstrated that the Samy integrase efficiently mediates the specific integration of a non-replicating plasmid in six Streptomyces strains from distinct clades. Bioinformatic analyses revealed that the Samy- att B site is rather conserved and located in the terminal compartment of most Streptomyces chromosomes. Finally, heterologous expression of the albonoursin biosynthetic gene cluster from the Samy-, PhiC31-, and R4- attB sites yields quantitatively equivalent levels of production, though qualitative differences were observed. Altogether, these results demonstrate that the att-int Samy system expands Streptomyces genetic engineering tools by enabling targeted integration in the terminal chromosomal compartment. Key points: • Samy-based integrative vectors are new tools for engineering Streptomyces strains. • They target the terminal compartment, farthest from the origin in most strains. • They facilitate efficient heterologous production of the albonoursin antibiotic.

Streptomyces sp. MC1, a bacterium isolated from an environment contaminated with organic and inorganic pollutants, can reduce chromium and degrade lindane, making it a promising candidate for bioremediation. However, a major challenge in bioremediation trials is monitoring bacteria survival in soil. To assess the survival of Streptomyces sp. MC1 during bioremediation, we introduced fluorescence tagging and a selectable marker into this strain by intergeneric conjugation from Escherichia coli. Conjugation assays were performed using two E. coli strains (ET12567/pUZ8002 or S17-1) and Streptomyces sp. MC1 (spores or mycelium). The integrative plasmid pSC001, carrying a gene encoding the monomeric green fluorescent protein (mGFP), was used. Various donor and recipient concentrations were tested and the presence of MgCl₂ or CaCl₂ during conjugation was also evaluated. Optimal conditions included low concentrations of both Streptomyces sp. MC1 spores and E. coli S17-1, with MgCl₂ in the medium. Exconjugants were analyzed, confirming plasmid site-specific integration and mGFP expression. In bioremediation assays with soils co-contaminated with Cr(VI) and lindane, fluorescence-tagged Streptomyces sp. MC1 successfully demonstrated survival over 28 days. Our results, combined with the availability of the Streptomyces sp. MC1 genome sequence, will facilitate further characterization of this strain’s features and accelerate its development for bioremediation applications.

Vanilla flavour is widely used in various industries and is the most broadly used flavouring agent in the food industry. The demand for this flavour is, therefore, extremely high, yet vanilla bean extracts can only meet about 1% of the overall demand. Vanillin, the main constituent of vanilla flavour, can easily be obtained through chemical synthesis. Nonetheless, consumer demands for natural products and environmentally friendly industrial processes drive the development of biotechnological approaches for its production. Some microorganisms can naturally produce vanillin when fed with various substrates, including eugenol, isoeugenol, and ferulic acid. The characterisation of the genes and enzymes involved in these bioconversion pathways, as well as progress in the understanding of vanillin biosynthesis in Vanilla orchids, allowed the development of genetic engineering and synthetic biology approaches to increase vanillin production in naturally vanillin-producing microorganisms, or to implement novel vanillin biosynthetic pathways in microbial chassis. This review summarises and discusses these genetic engineering and synthetic biology approaches for the microbial production of vanillin.

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.

Microbial specialised 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 under characterised 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 earlier versions of antiSMASH, 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.

Streptomyces are renowned for their prolific production of specialized metabolites with applications in medicine and agriculture. These multicellular bacteria present a sophisticated developmental cycle, and play a key role in soil ecology. Little is known about Streptomyces -phage interactions and the impact of phages on Streptomyces physiology. In this study, we investigated the conditions governing the expression and production of ‘Samy’, a prophage found in Streptomyces ambofaciens ATCC 23877. This siphoprophage is produced simultaneously with the activation of other mobile genetic elements. We show that Samy production increases bacterial dispersal under in vitro stress conditions. Altogether, we unveiled a new property of a bacteriophage infection that it is closely linked to the multicellular community life of Streptomyces bacteria. Graphical summary: IMPORTANCE: Streptomyces are multicellular bacteria producing valuable metabolites, including antibiotics, with applications in medicine and agriculture. In this study, we characterized a novel temperate phage, named Samy, and its impact on bacteria physiology. Remarkably, the presence and production of Samy increases bacterial dispersal under in vitro stress conditions. This constitutes an emerging property associated with bacteriophage infection that might enhance the spread of the species. Our study reveals a new aspect of bacteriophage infection in the context of multicellular aggregate dynamics.

Understanding the mechanisms of regulation of specialized metabolite production can have important implications both at the level of specialized metabolism study (expression of silent gene clusters) and the biotechnological level (increase of the production of a metabolite of interest). We report here a study on the regulation of the biosynthesis of a metabolite from the pyrrolamide family, congocidine.

Streptomyces are among the most prolific bacterial producers of specialized metabolites, including antibiotics. The linear chromosome is partitioned into a central region harboring core genes and two extremities enriched in specialized metabolite biosynthetic gene clusters (SMBGCs). The molecular mechanisms governing structure and function of these compartmentalized genomes remain mostly unknown. Here we show that in exponential phase, chromosome structure correlates with genetic compartmentalization: conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends are transcriptionally, largely quiescent compartments with different structural features. Onset of metabolic differentiation is accompanied by remodeling of chromosome architecture from an ‘open’ to a rather ‘closed’ conformation, in which the SMBGCs are expressed forming new boundaries. Altogether, our results reveal that S. ambofaciens’ linear chromosome is partitioned into structurally distinct entities, indicating a link between chromosome folding, gene expression and genome evolution.