Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity

Aim: To screen for novel antagonistic Pseudomonas strains producing both phenazines and biosurfactants that are as effective as Pseudomonas aeruginosa PNA1 in the biocontrol of cocoyam root rot caused by Pythium myriotylum. Material and Results: Forty pseudomonads were isolated from the rhizosphere of healthy white and red cocoyam plants appearing in natural, heavily infested fields in Cameroon. In vitro tests demonstrated that Py. myriotylum antagonists could be retrieved from the red cocoyam rhizosphere. Except for one isolate, all antagonistic isolates produced phenazines. Results from whole-cell protein profiling showed that the antagonistic isolates are different from other isolated pseudomonads, while BOX-PCR revealed high genomic similarity among them. 16S rDNA sequencing of two representative strains within this group of antagonists confirmed their relatively low similarity with validly described Pseudomonas species. These antagonists are thus provisionally labelled as unidentified Pseudomonas strains. Among the antagonists, Pseudomonas CMR5c and CMR12a were selected because of their combined production of phenazines and biosurfactants. For strain CMR5c also, production of pyrrolnitrin and pyoluteorin was demonstrated. Both CMR5c and CMR12a showed excellent in vivo biocontrol activity against Py. myriotylum to a similar level as Ps. aeruginosa PNA1. Conclusion: Pseudomonas CMR5c and CMR12a were identified as novel and promising biocontrol agents of Py. myriotylum on cocoyam, producing an arsenal of antagonistic metabolites. Significance and Impact of the Study: Present study reports the identification of two newly isolated fluorescent Pseudomonas strains that can replace the opportunistic human pathogen Ps. aeruginosa PNA1 in the biocontrol of cocoyam root rot and could be taken into account for the suppression of many plant pathogens

A bacterial symbiont is converted from an inedible producer of beneficial molecules into food by a single mutation in the gacA gene

Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria\u27s host is a farmer clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer\u27s spore production and depresses a nonfarmer\u27s spore production. Genome sequence and phylogenetic analyses identify a derived point mutation in the food strain that generates a premature stop codon in a global activator (gacA), encoding the response regulator of a two-component regulatory system. Generation of a knockout mutant of this regulatory gene in the nonfood bacterial strain altered its secondary metabolite profile to match that of the food strain, and also, independently, converted it into a food source. These results suggest that a single mutation in an inedible ancestral strain that served a protective role converted it to a domesticated food source

Burkholderia from fungus gardens of fungus-growing ants produce antifungals that inhibit the specialized parasite Escovopsis.

Within animal-associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for microbial symbiosis, to determine the potential defensive roles of key bacterial taxa present in the ants’ fungus gardens. Fungus gardens serve as an external digestive system for the ants, with mutualistic fungi in the genus Leucoagaricus converting the plant substrate into energy for the ants. The fungus garden is host to specialized parasitic fungi in the genus Escovopsis. Here, we examine the potential role of Burkholderia spp. that occur within ant fungus gardens in inhibiting Escovopsis. We isolated members of the bacterial genera Burkholderia and Paraburkholderia from 50% of the 52 colonies sampled, indicating that members of the family Burkholderiaceae are common inhabitants in the fungus gardens of a diverse range of fungus-growing ant genera. Using antimicrobial inhibition bioassays, we found that 28 out of 32 isolates inhibited at least one Escovopsis strain with a zone of inhibition greater than 1cm. Genomic assessment of fungus garden-associated Burkholderiaceae indicated that isolates with strong inhibition all belonged to the genus Burkholderia and contained biosynthetic gene clusters that encoded the production of two antifungals: burkholdine1213 and pyrrolnitrin. Organic extracts of cultured isolates confirmed that these compounds are responsible for antifungal activities that inhibit Escovopsis but, at equivalent concentrations, not Leucoagaricus spp. Overall, these new findings, combined with previous evidence, suggest that members of the fungus garden microbiome play an important role in maintaining the health and function of fungus-growing ant colonies.National Institutes of Health/[U19 TW009872]/NIH/Estados UnidosNational Institutes of Health/[U19 AI142720]/NIH/Estados UnidosNational Institutes of Health/[T32 AI055397]/NIH/Estados UnidosNational Science Foundation/[DEB-1927155]/NSF/Estados UnidosSão Paulo Research Foundation/[2013/50954-0]/FAPESP/BrasilUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Estructuras Microscópicas (CIEMIC)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

Quorum-sensing signaling is required for production of the antibiotic pyrrolnitrin in a rhizospheric biocontrol strain of Serratia plymuthica

One mechanism that bacteria have adopted to regulate the production of antimicrobial compounds is population-density-dependent LuxRI-type quorum sensing (QS), exploiting the production of N-acyl homoserine lactone (AHL) autoinducer signals. In biocontrol bacteria, most known cases involve the AHL control of phenazine antibiotics production by rhizospheric pseudomonads. This work is the first to demonstrate that phenazines are not the only group of biocontrol-related antibiotics whose production is regulated by QS systems. Strain HRO-C48 of Serratia plymuthica isolated from the rhizosphere of oilseed rape and described as a chitinolytic bacterium, which protects crops against Verticillium wilt, was also shown to produce wide-range antibiotic pyrrolnitrin and several AHLs, including N-butanoyl-HSL, N-hexanoyl-HSL and N-3-oxo-hexanoyl-HSL (OHHL). The genes splI and splR, which are analogues of luxI and luxR genes from other Gram-negative bacteria, were cloned and sequenced. The mutant AHL-4 (splI::miniTn5) was simultaneously deficient in the production of AHLs and pyrrolnitrin, as well as in its ability to suppress the growth of several fungal plant pathogens in vitro. However, pyrrolnitrin production could be restored in this mutant by introduction of the splIR genes cloned into a plasmid or by addition of the conditioned medium from strain C48 or OHHL standard to the growth mediu

The bacterium Pseudomonas protegens antagonizes the microalga Chlamydomonas reinhardtii using a blend of toxins

The unicellular alga Chlamydomonas reinhardtii and the bacterium Pseudomonas protegens serve as a model to study the interactions between photosynthetic and heterotrophic microorganisms. P . protegens secretes the cyclic lipopeptide orfamide A that interferes with cytosolic Ca 2+ homeostasis in C . reinhardtii resulting in deflagellation of the algal cells. Here, we studied the roles of additional secondary metabolites secreted by P . protegens using individual compounds and co‐cultivation of algae with bacterial mutants. Rhizoxin S2, pyrrolnitrin, pyoluteorin, 2,4‐diacetylphloroglucinol (DAPG) and orfamide A all induce changes in cell morphology and inhibit the growth of C . reinhardtii . Rhizoxin S2 exerts the strongest growth inhibition, and its action depends on the spatial structure of the environment (agar versus liquid culture). Algal motility is unaffected by rhizoxin S2 and is most potently inhibited by orfamide A (IC 50 = 4.1 μM). Pyrrolnitrin and pyoluteorin both interfere with algal cytosolic Ca 2+ homeostasis and motility whereas high concentrations of DAPG immobilize C . reinhardtii without deflagellation or disturbance of Ca 2+ homeostasis. Co‐cultivation with a regulatory mutant of bacterial secondary metabolism (Δ gacA ) promotes algal growth under spatially structured conditions. Our results reveal how a single soil bacterium uses an arsenal of secreted antialgal compounds with complementary and partially overlapping activities

Molecular Characterisation of the Interaction of Microbes with the Insulin Pathway

Full version unavailable due to 3rd party copyright restrictions.Exposure to microorganisms is considered an environmental factor which can contribute to diabetes mellitus via cytotoxicity or autoimmune responses against pancreatic cells. Firstly, the effects on rat insulinoma pancreatic β-cell line of secondary metabolites pyrrolnitrin (Burkholderia spp), phenazine compounds (Pseudomonas aeruginosa and Burkholderia spp) were investigated. Both compounds separately showed significant cytotoxicity after 24 h and at concentrations of 10 & 100 ng/ml potentiated insulin gene transcription, Ca2+ content and glucose-stimulated insulin secretion (GSIS). Furthermore, the outward membrane current was inhibited by phenazine (100 ng/ml) or pyrrolnitrin (10 or 100 ng/ml). Secondly, the capacity of 45 microbial species to bind insulin was screened in order to assess how common insulin binding was amongst microorganisms Burkholderia multivorans, B. cenocepacia and Aeromonas salmonicida bound insulin. A genomic library of B. multivorans was constructed in λ Zap Express and screened successfully for insulin binding recombinants. Recombinant phagemids p1 & p2 were excised, p1, encoded an insulin binding protein (IBP1 30 kDa) with homology to the iron complex siderophore receptor. For p2, two IBPs were detected at 20 & 30 kDa (IBP2 & IBP3), representing an intracellular and outer membrane peptide transporter. Comparison of IBP1 and human insulin receptor (HIR) produced 6 linear epitopes, and for IBP2 & IBP3 produced 3 epitopes. Thirdly, glutamic acid decarboxylase GAD65 is a major pancreatic autoantigen contributing to autoimmune diabetes. To assess the likelihood that microorganisms possess epitopes that mimic regions on GAD, 45 microbial species were tested for homology. This was facilitated by purifying recombinant GAD protein which was used to produce GAD antiserum. Four E. coli cross-reacting proteins were identified using mass spectrometry, outer-membrane protein A, formate dehydrogenase, superoxide dismutase and DNA starvation protein. Epitopes occurred at the C-terminal region of GAD65 (residues 419–565), a region previously reported to be targeted by autoantibodies. This study suggests that pyrrolnitrin and phenazine are cytotoxic to pancreatic β-cells and B. multivorans IBPs linear epitopes may be diabetogenic, particularly in patients with cystic fibrosis related diabetes (CFRD) who suffer a long term infections with Pseudomonas and Burkholderia species. Furthermore, microbial GAD epitopes could potentially induce an autoimmune response leading to diabetes

Burkholderia bacteria produce multiple potentially novel molecules that inhibit carbapenem-resistant gram-negative bacterial pathogens

Antimicrobial resistance in Gram-negative pathogens represents a global threat to human health. This study determines the antimicrobial potential of a taxonomically and geographically diverse collection of 263 Burkholderia (sensu lato) isolates and applies natural product dereplication strategies to identify potentially novel molecules. Antimicrobial activity is almost exclusively present in Burkholderia sensu stricto bacteria and rarely observed in the novel genera Paraburkholderia, Caballeronia, Robbsia, Trinickia, and Mycetohabitans. Fourteen isolates show a unique spectrum of antimicrobial activity and inhibited carbapenem-resistant Gram-negative bacterial pathogens. Dereplication of the molecules present in crude spent agar extracts identifies 42 specialized metabolites, 19 of which represented potentially novel molecules. The known identified Burkholderia metabolites include toxoflavin, reumycin, pyrrolnitrin, enacyloxin, bactobolin, cepacidin, ditropolonyl sulfide, and antibiotics BN-227-F and SF 2420B, as well as the siderophores ornibactin, pyochelin, and cepabactin. Following semipreparative fractionation and activity testing, a total of five potentially novel molecules are detected in active fractions. Given the molecular formula and UV spectrum, two of those putative novel molecules are likely related to bactobolins, and another is likely related to enacyloxins. The results from this study confirm and extend the observation that Burkholderia bacteria present exciting opportunities for the discovery of potentially novel bioactive molecules

Antagonistic Compounds Producing Plant Growth Promoting Rhizobacteria: A Tool for Management of Plant Disease

Agriculture is facing struggle to meet the various confront of reducing plant diseases for an increasing world population food security. Great quantities of synthetic fertilizers and pesticides are required for high productivity which can damage ecosystem structures and functions, including the soil microbial community which plays an important role in agriculture sustainability. Soil is an excellent niche of growth of much plant growth promoting rhizobacteria. PGPR are naturally occurring soil bacteria that aggressively colonize in plant roots and play a vital role in crop protection, growth promotion and in the improvement of soil health. Scientific researchers involve multidisciplinary approaches to understand adaptation of PGPR, effects on plant physiology and growth induced systemic resistance, biocontrol of plant pathogens and biofertilization. The primary mechanism of biocontrol by PGPR involves the production of antibiotics such as carboxylic acid, 2,4-diacetyl phloroglucinoloomycin,pyoluteorin,pyrrolnitrin,kanosamine,zwittemycin-A and pantocin. A cascade of endogenous signals such as sensor kinases, N-acyl homoserine lactones and sigma factors regulates the synthesis of antibiotics. Some of these antibiotics have broad spectrum against many plant pathogens like fungi, viruses and bacteria, affecting crop plants. These antibiotics also serve as determinants in triggering induced systemic resistance (ISR) in the plant system