Faecalibacterium duncaniae as a novel next generation probiotic against influenza.

The gut-lung axis is critical during viral respiratory infections such as influenza. Gut dysbiosis during infection translates into a massive drop of microbially produced short-chain fatty acids (SCFAs). Among them, butyrate is important during influenza suggesting that microbiome-based therapeutics targeting butyrate might hold promises. The butyrate-producing bacterium Faecalibacterium duncaniae (formerly referred to as F. prausnitzii) is an emerging probiotic with several health-promoting characteristics. To investigate the potential effects of F. duncaniae on influenza outcomes, mice were gavaged with live F. duncaniae (A2-165 or I-4574 strains) five days before infection. Supplementation of F. duncaniae was associated with less severe disease, a lower pulmonary viral load, and lower levels of lung inflammation. F. duncaniae supplementation impacted on gut dysbiosis induced by infection, as assessed by 16S rRNA sequencing. Interestingly, F. duncaniae administration was associated with a recovery in levels of SCFAs (including butyrate) in infected animals. The live form of F. duncaniae was more potent that the pasteurized form in improving influenza outcomes. Lastly, F. duncaniae partially protected against secondary (systemic) bacterial infection. We conclude that F. duncaniae might serve as a novel next generation probiotic against acute viral respiratory diseases.

Faecalibacterium duncaniae A2-165 regulates the expression of butyrate synthesis, ferrous iron uptake, and stress-response genes based on acetate consumption.

The promising next-generation probiotic Faecalibacterium prausnitzii is one of the most abundant acetate-consuming, butyrate-producing bacteria in the healthy human gut. Yet, little is known about how acetate availability affects this bacterium's gene expression strategies. Here, we investigated the effect of acetate on temporal changes in the transcriptome of F. duncaniae A2-165 cultures using RNA sequencing. We compared gene expression patterns between two growth phases (early stationary vs. late exponential) and two acetate levels (low: 3 mM vs. high: 23 mM). Only in low-acetate conditions, a general stress response was activated. In high-acetate conditions, there was greater expression of genes related to butyrate synthesis and to the importation of B vitamins and iron. Specifically, expression was strongly activated in the case of the feoAABC operon, which encodes a FeoB ferrous iron transporter, but not in the case of the feoAB gene, which encodes a second putative FeoAB transporter. Moreover, excess ferrous iron repressed feoB expression but not feoAB. Lastly, FeoB but not FeoAB peptides from strain A2-165 were found in abundance in a healthy human fecal metaproteome. In conclusion, we characterized two early-stationary transcriptomes based on acetate consumption and this work highlights the regulation of feoB expression in F. duncaniae A2-165.

Faecalibacterium: a bacterial genus with promising human health applications.

In humans, many diseases are associated with alterations in gut microbiota, namely increases or decreases in the abundance of specific bacterial groups. One example is the genus Faecalibacterium. Numerous studies have underscored that low levels of Faecalibacterium are correlated with inflammatory conditions, with inflammatory bowel disease (IBD) in the forefront. Its representation is also diminished in the case of several diseases, including colorectal cancer (CRC), dermatitis, and depression. Additionally, the relative presence of this genus is considered to reflect, at least in part, intestinal health status because Faecalibacterium is frequently present at reduced levels in individuals with gastrointestinal diseases or disorders. In this review, we first thoroughly describe updates to the taxonomy of Faecalibacterium, which has transformed a single-species taxon to a multispecies taxon over the last decade. We then explore the links discovered between Faecalibacterium abundance and various diseases since the first IBD-focused studies were published. Next, we examine current available strategies for modulating Faecalibacterium levels in the gut. Finally, we summarize the mechanisms underlying the beneficial effects that have been attributed to this genus. Together, epidemiological and experimental data strongly support the use of Faecalibacterium as a next-generation probiotic (NGP) or live biotherapeutic product (LBP).

Colorimetric aptasensor for detection of Bacillus cytotoxicus spores in milk and ready-to-use food.

The high incidence of foodborne diseases caused by pathogenic bacteria raises concerns worldwide and imposes considerable public healthcare challenges. This is especially observed with dormant spores of Bacilli, which can often survive treatments used by the food industry to kill growing bacteria. The early and rapid detection of bacterial spores is essential to ensure food safety. Commercial availability of such a test will present a high potential for food sector. We present a point-of-need colorimetric assay for detection of Bacillus cytotoxicus spores in food. The detection principle is based on spore-enhanced peroxidase-like catalytic activity of gold nanoparticles. The sensing platform consists of a microtube containing gold nanoparticles (AuNPs), and magnetic particles (MPs), both conjugated with specific aptamer BAS6R that recognize B. cytotoxicus spores. Upon the addition of the sample, spores were determined as present by the enhanced color change of the solution, due to the oxidation of tetramethylbenidine (TMB) with H2O2. The assay was evaluated by the naked eye (on/off) and quantitatively with use of a spectrophotometer. BAS6R@AuNPs aptasensor coupled to BAS6R@MPs proved to be highly sensitive, achieving the naked-eye limit of detection as low as 102 cfu/mL in water and milk, and 104 cfu/mL in mashed potatoes. Moreover, discrimination between spores of B. cytotoxicus and B. subtilis as well as bacterial vegetative cells was achieved in contaminated food samples, providing a good selectivity. This work provides a promising proof of concept for the development of instrument-free, low-cost and rapid assay for Bacillus cytotoxicus spore detection, which is able to compete in sensitivity with conventional costly and time-consuming laboratory analyses.

The Tolerance of Gut Commensal Faecalibacterium to Oxidative Stress Is Strain Dependent and Relies on Detoxifying Enzymes.

Obligate anaerobic bacteria in genus Faecalibacterium are among the most dominant taxa in the colon of healthy individuals and contribute to intestinal homeostasis. A decline in the abundance of this genus is associated with the occurrence of various gastrointestinal disorders, including inflammatory bowel diseases. In the colon, these diseases are accompanied by an imbalance between the generation and elimination of reactive oxygen species (ROS), and oxidative stress is closely linked to disruptions in anaerobiosis. In this work, we explored the impact of oxidative stress on several strains of faecalibacteria. An in silico analysis of complete genomes of faecalibacteria revealed the presence of genes encoding O2- and/or ROS-detoxifying enzymes, including flavodiiron proteins, rubrerythrins, reverse rubrerythrins, superoxide reductases, and alkyl peroxidase. However, the presence and the number of these detoxification systems varied greatly among faecalibacteria. These results were confirmed by O2 stress survival tests, in which we found that strains differed widely in their sensitivity. We showed the protective role of cysteine, which limited the production of extracellular O2•- and improved the survival of Faecalibacterium longum L2-6 under high O2 tension. In the strain F. longum L2-6, we observed that the expression of genes encoding detoxifying enzymes was upregulated in the response to O2 or H2O2 stress but with different patterns of regulation. Based on these results, we propose a first model of the gene regulatory network involved in the response to oxidative stress in F. longum L2-6. IMPORTANCE Commensal bacteria in the genus Faecalibacterium have been proposed for use as next-generation probiotics, but efforts to cultivate and exploit the potential of these strains have been limited by their sensitivity to O2. More broadly, little is known about how commensal and health-associated bacterial species in the human microbiome respond to the oxidative stress that occurs as a result of inflammation in the colon. In this work, we provide insights regarding the genes that encode potential mechanisms of protection against O2 or ROS stress in faecalibacteria, which may facilitate future advances in work with these important bacteria.

Exploring Disease Management and Control through Pathogen Diagnostics and One Health Initiative: A Concise Review.

The "One Health" initiative is a critical strategy that recognizes the interconnectedness between human, animal, and environmental health in the spread and containment of infectious pathogens. With the ease of global transportation, transboundary disease outbreaks pose a significant threat to food safety and security, endangering public health and having a negative economic impact. Traditional diagnostic techniques based on genotypic and phenotypic analyses are expensive, time-consuming, and cannot be translated into point-of-care tools, hindering effective disease management and control. However, with advancements in molecular methods, biosensors, and new generation sequencing, rapid and reliable diagnostics are now available. This review provides a comprehensive insight into emergent viral and bacterial pathogens and antimicrobial resistance, highlighting the importance of "One Health" in connecting detection and effective treatment. By emphasizing the symbiotic relationship between human and animal health, this paper underscores the critical role of "One Health" initiatives in preventing and controlling infectious diseases.

Impact of Dietary Arachidonic Acid on Gut Microbiota Composition and Gut-Brain Axis in Male BALB/C Mice.

Although arachidonic acid (ARA) is the precursor of the majority of eicosanoids, its influence as a food component on health is not well known. Therefore, we investigated its impact on the gut microbiota and gut-brain axis. Groups of male BALB/c mice were fed either a standard diet containing 5% lipids (Std-ARA) or 15%-lipid diets without ARA (HL-ARA) or with 1% ARA (HL + ARA) for 9 weeks. Fatty acid profiles of all three diets were the same. The HL-ARA diet favored the growth of Bifidobacterium pseudolongum contrary to the HL + ARA diet that favored the pro-inflammatory Escherichia-Shigella genus in fecal microbiota. Dietary ARA intake induced 4- and 15-fold colic overexpression of the pro-inflammatory markers IL-1ß and CD40, respectively, without affecting those of TNFα and adiponectin. In the brain, dietary ARA intake led to moderate overexpression of GFAP in the hippocampus and cortex. Both the hyperlipidic diets reduced IL-6 and IL-12 in the brain. For the first time, it was shown that dietary ARA altered the gut microbiota, led to low-grade colic inflammation, and induced astrogliosis in the brain. Further work is necessary to determine the involved mechanisms.

Gene co-expression network analysis of the human gut commensal bacterium Faecalibacterium prausnitzii in R-Shiny.

Faecalibacterium prausnitzii is abundant in the healthy human intestinal microbiota, and the absence or scarcity of this bacterium has been linked with inflammatory diseases and metabolic disorders. F. prausnitzii thus shows promise as a next-generation probiotic for use in restoring the balance of the gut microbial flora and, due to its strong anti-inflammatory properties, for the treatment of certain pathological conditions. However, very little information is available about gene function and regulation in this species. Here, we utilized a systems biology approach-weighted gene co-expression network analysis (WGCNA)-to analyze gene expression in three publicly available RNAseq datasets from F. prausnitzii strain A2-165, all obtained in different laboratory conditions. The co-expression network was then subdivided into 24 co-expression gene modules. A subsequent enrichment analysis revealed that these modules are associated with different kinds of biological processes, such as arginine, histidine, cobalamin, or fatty acid metabolism as well as bacteriophage function, molecular chaperones, stress response, or SOS response. Some genes appeared to be associated with mechanisms of protection against oxidative stress and could be essential for F. prausnitzii's adaptation and survival under anaerobic laboratory conditions. Hub and bottleneck genes were identified by analyses of intramodular connectivity and betweenness, respectively; this highlighted the high connectivity of genes located on mobile genetic elements, which could promote the genetic evolution of F. prausnitzii within its ecological niche. This study provides the first exploration of the complex regulatory networks in F. prausnitzii, and all of the "omics" data are available online for exploration through a graphical interface at https://shiny.migale.inrae.fr/app/faeprau.

Lactococcus lactis engineered to deliver hCAP18 cDNA alleviates DNBS-induced colitis in C57BL/6 mice by promoting IL17A and IL10 cytokine expression.

With its antimicrobial and immunomodulating properties, the cathelicidin (LL37) plays an important role in innate immune system. Here, we attempted to alleviate chemically induced colitis using a lactococci strain that either directly expressed the precursor to LL37, hCAP18 (LL-pSEC:hCAP18), or delivered hCAP18 cDNA to host cells under the control of the cytomegalovirus promoter (LL-Probi-H1:hCAP18). We also investigated whether the alleviation of symptoms could be explained through modification of the gut microbiota by hCAP18. Mice were administered daily doses of LL-pSEC:hCAP18 or LL-Probi-H1:hCAP18. On day 7, colitis was induced by DNBS. During autopsy, we assessed macroscopic tissue damage in the colon and collected tissue samples for the characterization of inflammation markers and histological analysis. Feces were collected at day 7 for 16S DNA sequencing. We also performed a fecal transplant experiment in which mice underwent colon washing and received feces from Lactococcus lactis-treated mice before DNBS-colitis induction. Treatment with LL-Probi-H1:hCAP18 reduced the severity of colitis symptoms. The protective effects were accompanied by increased levels of IL17A and IL10 in mesenteric lymph node cells. L. lactis administration altered the abundance of Lachnospiraceae and Muribaculaceae. However, fecal transplant from L. lactis-treated mice did not improve DNBS-induced symptoms in recipient mice.

Description of a Newly Isolated Blautia faecis Strain and Its Benefit in Mouse Models of Post-Influenza Secondary Enteric and Pulmonary Infections.

The expanding knowledge on the systemic influence of the human microbiome suggests that fecal samples are underexploited sources of new beneficial strains for extra-intestinal health. We have recently shown that acetate, a main circulating microbiota-derived molecule, reduces the deleterious effects of pulmonary Streptococcus pneumoniae and enteric Salmonella enterica serovar Typhimurium bacterial post-influenza superinfections. Considering the beneficial and broad effects of acetate, we intended to isolate a commensal strain, producing acetate and potentially exploitable in the context of respiratory infections. We designed successive steps to select intestinal commensals that are extremely oxygen-sensitive, cultivable after a freezing process, without a proinflammatory effect on IL-8 induction, and producing acetate. We have identified the Blautia faecis DSM33383 strain, which decreased the TNFα-induced production of IL-8 by the intestinal epithelial cell line HT-29. The beneficial effect of this bacterial strain was further studied in two preclinical models of post-influenza Streptococcus pneumoniae (S.p) and Salmonella enterica serovar Typhimurium (S.t) superinfection. The intragastrical administration of Blautia faecis DSM33383 led to protection in influenza-infected mice suffering from an S.p. and, to a lesser extent, from an S.t secondary infection. Altogether, this study showed that Blautia faecis DSM33383 could be a promising candidate for preventive management of respiratory infectious diseases.

Intraspecific Diversity of Microbial Anti-Inflammatory Molecule (MAM) from Faecalibacterium prausnitzii.

The commensal bacterium Faecalibacterium prausnitzii has unique anti-inflammatory properties, at least some of which have been attributed to its production of MAM, the Microbial Anti-inflammatory Molecule. Previous phylogenetic studies of F. prausnitzii strains have revealed the existence of various phylogroups. In this work, we address the question of whether MAMs from different phylogroups display distinct anti-inflammatory properties. We first performed wide-scale identification, classification, and phylogenetic analysis of MAM-like proteins encoded in different genomes of F. prausnitzii. When combined with a gene context analysis, this approach distinguished at least 10 distinct clusters of MAMs, providing evidence for functional diversity within this protein. We then selected 11 MAMs from various clusters and evaluated their anti-inflammatory capacities in vitro. A wide range of anti-inflammatory activity was detected. MAM from the M21/2 strain had the highest inhibitory effect (96% inhibition), while MAM from reference strain A2-165 demonstrated only 56% inhibition, and MAM from strain CNCM4541 was almost inactive. These results were confirmed in vivo in murine models of acute and chronic colitis. This study provides insights into the family of MAM proteins and generates clues regarding the choice of F. prausnitzii strains as probiotics for use in targeting chronic inflammatory diseases.

Prophylactic Faecalibacterium prausnitzii treatment prevents the acute breakdown of colonic epithelial barrier in a preclinical model of pelvic radiation disease.

Every year, millions of people around the world benefit from radiation therapy to treat cancers localized in the pelvic area. Damage to healthy tissue in the radiation field can cause undesirable toxic effects leading to gastrointestinal complications called pelvic radiation disease. A change in the composition and/or function of the microbiota could contribute to radiation-induced gastrointestinal toxicity. In this study, we tested the prophylactic effect of a new generation of probiotic like Faecalibacterium prausnitzii (F. prausnitzii) on acute radiation-induced colonic lesions. Experiments were carried out in a preclinical model of pelvic radiation disease. Rats were locally irradiated at 29 Gray in the colon resulting in colonic epithelial barrier rupture. Three days before the irradiation and up to 3 d after the irradiation, the F. prausnitzii A2-165 strain was administered daily (intragastrically) to test its putative protective effects. Results showed that prophylactic F. prausnitzii treatment limits radiation-induced para-cellular hyperpermeability, as well as the infiltration of neutrophils (MPO+ cells) in the colonic mucosa. Moreover, F. prausnitzii treatment reduced the severity of the morphological change of crypts, but also preserved the pool of Sox-9+ stem/progenitor cells, the proliferating epithelial PCNA+ crypt cells and the Dclk1+/IL-25+ differentiated epithelial tuft cells. The benefit of F. prausnitzii was associated with increased production of IL-18 by colonic crypt epithelial cells. Thus, F. prausnitzii treatment protected the epithelial colonic barrier from colorectal irradiation. New-generation probiotics may be promising prophylactic treatments to reduce acute side effects in patients treated with radiation therapy and may improve their quality of life.

Synthesis and antibacterial activity of iron manganite (FeMnO3) particles against the environmental bacterium Bacillus subtilis.

Nanocrystalline iron manganite powder was synthesized using the sol-gel combustion process, with glycine as fuel. It was further calcined at 900 °C for 8 h, resulting in the formation of a loose cubic FeMnO3 powder with a small specific surface area, net-like structure and plate-like particles as confirmed by XRD, N2 physisorption, FESEM and TEM analyses. The metal ion release was studied by ICP-OES and showed that less than 10 ppb of Fe or Mn ions were released by leaching in water, but 0.36 ppm Fe and 3.69 ppm Mn was found in LB (Luria-Bertani) bacterial medium. The generation of reactive oxygen species (ROS) was monitored in distilled water and bacterial medium and showed that FeMnO3 particles do not generate O2˙- ions with or without UV irradiation, but synthesize H2O2 and show an antioxidative effect. Besides the higher stability of FeMnO3 particles in aqueous solution they showed an inhibitory effect on Bacillus subtilis growth in LB medium even at low concentrations (0.01 mg ml-1), but not in BHI medium even at 1 mg ml-1. This study points out that the mechanism of antibacterial action of engineered metal oxides needs continued investigation and specific experimental controls.

Bacillus subtilis Regulators MntR and Zur Participate in Redox Cycling, Antibiotic Sensitivity, and Cell Wall Plasticity.

The Bacillus subtilis MntR and Zur transcriptional regulators control homeostasis of manganese and zinc, two essential elements required in various cellular processes. In this work, we describe the global impact of mntR and zur deletions at the protein level. Using a comprehensive proteomic approach, we showed that 33 and 55 proteins are differentially abundant in ΔmntR and Δzur cells, respectively, including proteins involved in metal acquisition, translation, central metabolism, and cell wall homeostasis. In addition, both mutants showed modifications in intracellular metal ion pools, with significant Mg2+ accumulation in the ΔmntR mutant. Phenotypic and morphological analyses of ΔmntR and Δzur mutants revealed their high sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress. Mutant strains had a modified cell wall thickness and accumulated lower levels of intracellular reactive oxygen species (ROS) than the wild-type strain. Remarkably, our results highlight an intimate connection between MntR, Zur, antibiotic sensitivity, and cell wall structure.IMPORTANCE Manganese and zinc are essential transition metals involved in many fundamental cellular processes, including protection against external oxidative stress. In Bacillus subtilis, Zur and MntR are key transcriptional regulators of zinc and manganese homeostasis, respectively. In this work, proteome analysis of B. subtilis wild-type, ΔmntR, and Δzur strains provided new insights into bacterial adaptation to deregulation of essential metal ions. Deletions of mntR and zur genes increased bacterial sensitivity to lysozyme, beta-lactam antibiotics, and external oxidative stress and impacted the cell wall thickness. Overall, these findings highlight that Zur and MntR regulatory networks are connected to antibiotic sensitivity and cell wall plasticity.

Exploring the impact of Mg-doped ZnO nanoparticles on a model soil microorganism Bacillus subtilis.

The environmental contamination of soil by metal oxide nanomaterials is a growing global concern because of their potential toxicity. We investigated the effects of Mg doped ZnO (Mg-nZnO) nanoparticles on a model soil microorganism Bacillus subtilis. Mg-nZnO exhibited only a moderate toxic effect on B. subtilis vegetative cells but was able to prevent biofilm formation and destroy already formed biofilms. Similarly, Mg-nZnO (≤1 mg/mL) was moderately toxic towards Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enterica, Saccharomyces cerevisiae and murine macrophages. Engineered Mg-nZnO produced H2O2 and O2•- radicals in solutions of various salt and organic molecule compositions. A quantitative proteomic analysis of B. subtilis membrane proteins showed that Mg-nZnO increased the expression of proteins involved in detoxification of ROS, translation and biofilm formation. Overall, our results suggest that Mg-nZnO released into the environment may hinder the spreading, colonization and biofilm formation by B. subtilis but also induce a mechanism of bacterial adaptation.

Exploring multiple effects of Zn0.15Mg0.85O nanoparticles on Bacillus subtilis and macrophages.

The increasing number of multidrug resistant bacteria raises a serious public-health concern, which is exacerbated by the lack of new antibiotics. Metal oxide nanoparticles are already applied as an antibacterial additive in various products used in everyday life but their modes of action have remained unclear. Moreover, their potential negative effects to human health are still under evaluation. We explored effects of mixed metal oxide Zn0.15Mg0.85O on Bacillus subtilis, as a model bacterial organism, and on murine macrophages. Zn0.15Mg0.85O killed planktonic bacterial cells and prevented biofilm formation by causing membrane damages, oxidative stress and metal ions release. When exposed to a sub-inhibitory amount of Zn0.15Mg0.85O, B. subtilis up-regulates proteins involved in metal ions export, oxidative stress response and maintain of redox homeostasis. Moreover, expression profiles of proteins associated with information processing, metabolism, cell envelope and cell division were prominently changed. Multimode of action of Zn0.15Mg0.85O suggests that no single strategy may provide bacterial resistance. Macrophages tolerated Zn0.15Mg0.85O to some extend by both the primary phagocytosis of nanoparticles and the secondary phagocytosis of damaged cells. Bacterial co-treatment with ciprofloxacin and non-toxic amount of Zn0.15Mg0.85O increased antibiotic activity towards B. subtilis and E. coli.

Revisiting the in vivo GlnR-binding sites at the genome scale in Bacillus subtilis.

BACKGROUND: In Bacillus subtilis, two major transcriptional factors, GlnR and TnrA, are involved in a sophisticated network of adaptive responses to nitrogen availability. GlnR was reported to repress the transcription of the glnRA, tnrA and ureABC operons under conditions of excess nitrogen. As GlnR and TnrA regulators share the same DNA binding motifs, a genome-wide mapping of in vivo GlnR-binding sites was still needed to clearly define the set of GlnR/TnrA motifs directly bound by GlnR. METHODS: We used chromatin immunoprecipitation coupled with hybridization to DNA tiling arrays (ChIP-on-chip) to identify the GlnR DNA-binding sites, in vivo, at the genome scale. RESULTS: We provide evidence that GlnR binds reproducibly to 61 regions on the chromosome. Among those, 20 regions overlap the previously defined in vivo TnrA-binding sites. In combination with real-time in vivo transcriptional profiling using firefly luciferase, we identified the alsT gene as a new member of the GlnR regulon. Additionally, we characterized the GlnR secondary regulon, which is composed of promoter regions harboring a GlnR/TnrA box and bound by GlnR in vivo. However, the growth conditions revealing a GlnR-dependent regulation for this second category of genes are still unknown. CONCLUSIONS: Our findings show an extended overlap between the GlnR and TnrA in vivo binding sites. This could allow efficient and fine tuning of gene expression in response to nitrogen availability. GlnR appears to be part of complex transcriptional regulatory networks, which involves interactions between different regulatory proteins.

Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis.

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho-null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks.

Genetic engineering of the branched fatty acid metabolic pathway of Bacillus subtilis for the overproduction of surfactin C14 isoform.

Surfactin, a lipopeptide produced by Bacillus subtilis, is one of the most powerful biosurfactants known. This molecule consists of a cyclic heptapeptide linked to a ß-hydroxy fatty acid chain. The isomery and the length of the fatty acid (FA) chain are responsible for the surfactin's activities. In this study, the gene codY, which encode for the global transcriptional regulator and the gene lpdV, located in the bkd operon (lpdV, bkdAA, bkdAB and bkdB genes), which is responsible for the last step of the branched chain amino acid (BCAA) degradation in acyl-CoA were deleted. The influence of these deletions on the quantitative and qualitative surfactin production was analysed. The surfactin production was quantified by RP-HPLC and the surfactin isoforms were characterized using LC-MS-MS and GC-MS analysis. The results obtained in the mutants showed an enhancement of surfactin specific production by a factor of 5.8 for the codY mutant and 1.4 for lpdV mutant. Moreover qualitative analysis of the lpdV mutant reveals that it mainly produced surfactin C14 isoform (2 fold more than the wild type) with linear FA chain. Complete analysis of the extracellular metabolites using 1 H quantitative NMR reveals a reduced production of acetoin in this mutant. This work demonstrates for the first time an original approach to overproduce specifically surfactin with C14 FA chain.

The MarR-like protein PchR (YvmB) regulates expression of genes involved in pulcherriminic acid biosynthesis and in the initiation of sporulation in Bacillus subtilis.

BACKGROUND: Cyclodipeptides and their derivatives constitute a large class of peptide natural products with noteworthy biological activities. In some yeasts and bacterial species, pulcherriminic acid derived from cyclo-L-leucyl-L-leucyl is excreted and chelates free ferric ions to form the pulcherrimin. In Bacillus subtilis, the enzymes YvmC and CypX are known to be involved in pulcherriminic acid biosynthesis. However, the mechanisms controlling the transcription of the yvmC-cypX operon are still unknown. RESULTS: In this work, we demonstrated that the B. subtilis YvmB MarR-like regulator is the major transcription factor controlling yvmC-cypX expression. A comprehensive quantitative proteomic analysis revealed a wide and prominent effect of yvmB deletion on proteins involved in cellular processes depending on iron availability. In addition, expression of yvmB depends on iron availability. Further analysis with real-time in vivo transcriptional profiling allowed us to define the YvmB regulon. We identified yvmBA, yvmC-cypX and yvnB for negative regulation and yisI for positive regulation. In combination with genetic approaches, gel mobility shift assays indicated that a 14-bp palindromic motif constitutes the YvmB binding site. It was unexpected that YvmB controls expression of yisI, whose encoding protein plays a negative role in the regulation of the sporulation initiation pathway. YvmB appears as an additional regulatory element into the cell's decision to grow or sporulate. CONCLUSION: Our findings reveal a possible role of the B. subtilis YvmB regulator in the regulatory networks connected to iron metabolism and to the control of proper timing of sporulation. YvmB was renamed as PchR controlling the pulcherriminic acid biosynthetic pathway of B. subtilis.