Gluconic acid-producing Pseudomonas sp. prevent γ-actinorhodin biosynthesis by Streptomyces coelicolor A3(2)

Streptomyces are ubiquitous soil bacteria well known for their ability to produce a wide range of secondary metabolites including antibiotics. In their natural environments, they co-exist and interact with complex microbial communities and their natural products are assumed to play a major role in mediating these interactions. Reciprocally, their secondary metabolism can be influenced by the surrounding microbial communities. Little is known about these complex interactions and the underlying molecular mechanisms. During pairwise co-culture experiments, a fluorescent Pseudomonas, Pseudomonas fluorescens BBc6R8, was shown to prevent the production of the diffusible blue pigment antibiotic γ-actinorhodin by Streptomyces coelicolor A3(2) M145 without altering the biosynthesis of the intracellular actinorhodin. A mutant of the BBc6R8 strain defective in the production of gluconic acid from glucose and consequently unable to acidify the culture medium did not show any effect on the γ-actinorhodin biosynthesis in contrast to the wild-type strain and the mutant complemented with the wild-type allele. In addition, when glucose was substituted by mannitol in the culture medium, P. fluorescens BBc6R8 was unable to acidify the medium and to prevent the biosynthesis of the antibiotic. All together, the results show that P. fluorescens BBc6R8 impairs the biosynthesis of the lactone form of actinorhodin in S. coelicolor by acidifying the medium through the production of gluconic acid. Other fluorescent Pseudomonas and the opportunistic pathogen Pseudomonas aeruginosa PAO1 also prevented the γ-actinorhodin production in a similar way. We propose some hypotheses on the ecological significance of such interaction.     

Identification of a Pseudomonas sp. that Inhibits RHL System of Quorum Sensing

The production of many Pseudomonas aeruginosa virulence factors and secondary metabolites is regulated in concert with cell density by quorum sensing (QS). Therefore, strategies designed to inhibit QS are promising for the control of diseases. Here, we succeeded in isolating soil bacteria (56 out of 7,000 isolates) capable of inhibiting violacein production by Chromobacterium violaceum CV026. We focused on an isolate identified as a Pseudomonas sp. based on its 16S rRNA nucleotide sequence. A partially purified inhibitor factor(s) derived from culture supernatants consisted of at least three major components by HPLC analysis. A more highly purified preparation (16 μg/ml) specifically inhibited rhl-controlled pyocyanin and rhamnolipid production by wild type P. aeruginosa PAO1 (PAO1) and a QS double mutant PAO-MW1, without affecting growth. A significant inhibitory effect on elastase, protease and biofilm was also observed. These results provide compelling evidence that the inhibitor(s) interferes with the QS system. The identities of the inhibitors remain to be established.     

Discovery of Potent Broad Spectrum Antivirals Derived from Marine Actinobacteria

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds.     

Serpula lacrymans reacts with a general,unspecialized chemical response during interaction with mycoparasitic Trichoderma spp. and bacteria

Serpula lacrymans causes structural damage in timber via cellulose and hemicellulose decomposition. Serpula lacrymans shares its habitat with bacteria and fungi, interactions that often result in stress and changed behaviour of the partners. A typical response to environmental stress is the production of bioactive metabolites and pigments. The diversity and function of these metabolites in inter- and intra-kingdom interactions, is largely elusive. Using dual culture approaches and secondary metabolite fingerprinting with high performance thin layer chromatography (HPTLC) it was demonstrated that stress induces an unspecific vs. Similar metabolite patterns were identified when S. lacrymans was co-cultured with different bacteria and mycoparasitic fungi (Trichoderma spp.). By analysing the metabolites produced along the gradient from the contact zone to more remote parts of the mycelium, we show a systemic reaction of S. lacrymans and that direct contact with other microbes is not a necessity to induce secondary metabolites.     

Stress-Induced Outer Membrane Vesicle Production by Pseudomonas aeruginosa

As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt and survive changes and stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved defense mechanisms, including the production of an exopolysaccharide capsule and the secretion of a myriad of degradative proteases and lipases. The production of outer membrane-derived vesicles (OMVs) serves as a secretion mechanism for virulence factors as well as a general bacterial response to envelope-acting stressors. This study investigated the effect of sublethal physiological stressors on OMV production by P. aeruginosa and whether the Pseudomonas quinolone signal (PQS) and the MucD periplasmic protease are critical mechanistic factors in this response. Exposure to some environmental stressors was determined to increase the level of OMV production as well as the activity of AlgU, the sigma factor that controls MucD expression. Overexpression of AlgU was shown to be sufficient to induce OMV production; however, stress-induced OMV production was not dependent on activation of AlgU, since stress caused increased vesiculation in strains lacking algU. We further determined that MucD levels were not an indicator of OMV production under acute stress, and PQS was not required for OMV production under stress or unstressed conditions. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. Together, these results demonstrate that distinct mechanisms exist for stress-induced OMV production in P. aeruginosa.     

Interspecific Small Molecule Interactions between Clinical Isolates of Pseudomonas aeruginosa and Staphylococcus aureus from Adult Cystic Fibrosis Patients

Pseudomonas aeruginosa and Staphylococcus aureus are the most prevalent pathogens in airway infections of cystic fibrosis (CF) patients. We studied how these pathogens coexist and interact with each other. Clinical isolates of both species were retrieved from adult CF patients. Culture supernatants from 63 P. aeruginosa isolates triggered a wide range of biofilm-stimulatory activities when added to the culture of a control S. aureus strain. The extent of biofilm formation by S. aureus was positively correlated to the levels of the 2-alkyl-4-(1H)-quinolones (AQs) Pseudomonas Quinolone Signal (PQS) and 2-heptyl-4-hydroxy quinoline N-oxide (HQNO) produced by the P. aeruginosa isolates. Supernatants from P. aeruginosa isogenic mutants deficient in PQS and HQNO production stimulated significantly less biofilm formation by S. aureus than that seen with the parental strain PA14. When studying co-isolated pairs of P. aeruginosa and S. aureus retrieved from patients showing both pathogens, P. aeruginosa supernatants stimulated less biofilm production by the S. aureus counterparts compared to that observed using the control S. aureus strain. Accordingly, some P. aeruginosa isolates produced low levels of exoproducts and also some of the clinical S. aureus isolates were not stimulated by their co-isolates or by PA14 despite adequate production of HQNO. This suggests that colonization of the CF lungs promotes some type of strain selection, or that co-existence requires specific adaptations by either or both pathogens. Results provide insights on bacterial interactions in CF.     

Differential Habitat Use and Niche Partitioning by <Emphasis Type="Italic">Pseudomonas</Emphasis> Species in Human Homes

Many species of Pseudomonas have the ability to use a variety of resources and habitats, and as a result Pseudomonas are often characterized as having broad fundamental niches. We questioned whether actual habitat use by Pseudomonas species is equally broad. To do this, we sampled extensively to describe the biogeography of Pseudomonas within the human home, which presents a wide variety of habitats for microbes that live in close proximity to humans but are not part of the human flora, and for microbes that are opportunistic pathogens, such as Pseudomonas aeruginosa. From 960 samples taken in 20 homes, we obtained 163 Pseudomonas isolates. The most prevalent based on identification using the SepsiTest BLAST analysis of 16S rRNA () were Pseudomonas monteilii (42 isolates), Pseudomonas plecoglossicida, Pseudomonas fulva, and P. aeruginosa (approximately 25 each). Of these, all but P. fulva differed in recovery rates among evaluated habitat types (drains, soils, water, internal vertebrate sites, vertebrate skin, inanimate surfaces, and garbage/compost) and all four species also differed in recovery rates among subcategories of habitat types (e.g., types of soils or drains). We also found that at both levels of habitat resolution, each of these six most common species (the four above plus Pseudomonas putida and Pseudomonas oryzihabitans) were over- or under-represented in some habitats relative to their contributions to the total Pseudomonas collected across all habitats. This pattern is consistent with niche partitioning. These results suggest that, whereas Pseudomonas are often characterized as generalists with broad fundamental niches, these species in fact have more restricted realized niches. Furthermore, niche partitioning driven by competition among Pseudomonas species may be contributing to the observed variability in habitat use by Pseudomonas in this system.     

Secondary metabolites from plant-associated Pseudomonas are overproduced in biofilm

Plant rhizosphere soil houses complex microbial communities in which microorganisms are often involved in intraspecies as well as interspecies and inter-kingdom signalling networks. Some members of these networks can improve plant health thanks to an important diversity of bioactive secondary metabolites. In this competitive environment, the ability to form biofilms may provide major advantages to microorganisms. With the aim of highlighting the impact of bacterial lifestyle on secondary metabolites production, we performed a metabolomic analysis on four fluorescent Pseudomonas strains cultivated in planktonic and biofilm colony conditions. The untargeted metabolomic analysis led to the detection of hundreds of secondary metabolites in culture extracts. Comparison between biofilm and planktonic conditions showed that bacterial lifestyle is a key factor influencing Pseudomonas metabolome. More than 50% of the detected metabolites were differentially produced according to planktonic or biofilm lifestyles, with the four Pseudomonas strains overproducing several secondary metabolites in biofilm conditions. In parallel, metabolomic analysis associated with genomic prediction and a molecular networking approach enabled us to evaluate the impact of bacterial lifestyle on chemically identified secondary metabolites, more precisely involved in microbial interactions and plant-growth promotion. Notably, this work highlights the major effect of biofilm lifestyle on acyl-homoserine lactone and phenazine production in P. chlororaphis strains.     

Improved PCR for identification of Pseudomonas aeruginosa

The aim of the present study was to develop a noble and specific marker for a quantitative polymerase chain reaction (PCR) assay for the species-specific detection of Pseudomonas aeruginosa based on the O-antigen acetylase gene. It is an important challenge to characterize populations of the bacterium P. aeruginosa, an opportunist by virtue of its physiological and genetic adaptability. However, molecular and serological methods currently available for sensitive and specific detection of P. aeruginosa are by no means satisfactory because there have been critical defects in the diagnosis and identification of P. aeruginosa strains in that these assays also detect other Pseudomonas species, or do not obtain amplified products from P. aeruginosa strains. Therefore, a primer set was designed based on the O-antigen acetylase gene of P. aeruginosa PA01 because it has been known that this gene is structurally diverse among species. The specificity of the primer set was evaluated using genomic DNA from six isolates of P. aeruginosa, 18 different species of Pseudomonas, and 23 other reference pathogenic bacteria. The primer set used in the PCR assay amplified a 232-bp amplicon for only six P. aeruginosa strains. The assay was also able to detect at least 1.41?×?10³?copies/μl of cloned amplified target DNA using purified DNA, or 2.7?×?10² colony-forming unit per reaction when using calibrated cell suspension. In conclusion, this assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of water with a low level or latent infection of P. aeruginosa.     

Breath gas metabolites and bacterial metagenomes from cystic fibrosis airways indicate active pH neutral 2,3-butanedione fermentation

The airways of cystic fibrosis (CF) patients are chronically colonized by patient-specific polymicrobial communities. The conditions and nutrients available in CF lungs affect the physiology and composition of the colonizing microbes. Recent work in bioreactors has shown that the fermentation product 2,3-butanediol mediates cross-feeding between some fermenting bacteria and Pseudomonas aeruginosa, and that this mechanism increases bacterial current production. To examine bacterial fermentation in the respiratory tract, breath gas metabolites were measured and several metagenomes were sequenced from CF and non-CF volunteers. 2,3-butanedione was produced in nearly all respiratory tracts. Elevated levels in one patient decreased during antibiotic treatment, and breath concentrations varied between CF patients at the same time point. Some patients had high enough levels of 2,3-butanedione to irreversibly damage lung tissue. Antibiotic therapy likely dictates the activities of 2,3-butanedione-producing microbes, which suggests a need for further study with larger sample size. Sputum microbiomes were dominated by P. aeruginosa, Streptococcus spp. and Rothia mucilaginosa, and revealed the potential for 2,3-butanedione biosynthesis. Genes encoding 2,3-butanedione biosynthesis were disproportionately abundant in Streptococcus spp, whereas genes for consumption of butanedione pathway products were encoded by P. aeruginosa and R. mucilaginosa. We propose a model where low oxygen conditions in CF lung lead to fermentation and a decrease in pH, triggering 2,3-butanedione fermentation to avoid lethal acidification. We hypothesize that this may also increase phenazine production by P. aeruginosa, increasing reactive oxygen species and providing additional electron acceptors to CF microbes.     

Variation of secondary metabolite levels in maize seedling roots induced by inoculation with Azospirillum, Pseudomonas and Glomus consortium under field conditions

Background and aims

Many plant-beneficial microorganisms can influence secondary plant metabolism, but whether these effects add up when plants are co-inoculated is unclear. This issue was assessed, under field conditions, by comparing the early impacts of seed inoculation on secondary metabolite profiles of maize at current or reduced mineral fertilization levels.

Methods

Maize seeds were inoculated singly with selected strains from bacterial genera Pseudomonas and Azospirillum or mycorrhizal genus Glomus, or with these strains combined two by two or all three together. At 16?days, maize root methanolic extracts were analyzed by RP-HPLC and secondary metabolites (phenolics, flavonoids, xanthones, benzoxazionoids, etc.) identified by LC/MS.

Results

Inoculation did not impact on plant biomass but resulted in enhanced total root surface, total root volume and/or root number in certain inoculated treatments, at reduced fertilization. Inoculation led to qualitative and quantitative modifications of root secondary metabolites, particularly benzoxazinoids and diethylphthalate. These modifications depended on fertilization level and microorganism(s) inoculated. The three selected strains gave distinct results when used alone, but unexpectedly all microbial consortia gave somewhat similar results.

Conclusions

The early effects on maize secondary metabolism were not additive, as combining strains gave effects similar to those of Glomus alone. This is the first study demonstrating and analyzing inoculation effects on crop secondary metabolites in the field.     

Interspecific allelopathy in cyanobacteria: Cylindrospermopsin and Cylindrospermopsis raciborskii effect on the growth and metabolism of Microcystis aeruginosa

The biological role of cyanobacteria secondary metabolites is relatively unknown although several possible hypotheses have been discussed. In the following study the effect of cylindrospermopsin (CYN) and metabolites of non-CYN producing Cylindrospermopsis raciborskii strain on growth, alkaline phosphatase (ALP) activity and microcystin-LR (MC-LR) production in Microcystis aeruginosa was evaluated. Higher concentrations of CYN (10 and 50 μg L⁻¹) induced toxicity effects demonstrated by significant growth inhibition and M. aeruginosa cell necrosis. Lower concentrations of CYN (1 and 5 μg L⁻¹) slightly decreased growth rates but significantly up-regulated ALP activity. Moreover, under all studied CYN concentrations MC-LR production strongly decreased. Spent C. raciborskii medium mimicked the CYN action by inducing strong inhibition of M. aeruginosa growth and MC-LR production and through up-regulation of ALP activity. On the other hand, spent M. aeruginosa medium did not affect C. raciborskii growth and no alterations in ALP activity were observed. Co-culturing of these two species resulted in an increase of C. raciborskii contribution at the expense of M. aeruginosa. From the results we conclude that CYN can be involved in interspecific competition in cyanobacteria and that non-CYN producing C. raciborskii strains may produce a hitherto unknown bioactive compound(s) which can mimic CYN action.     

Metabolism of Tryptophans by Pseudomonas aureofaciens: VI. Production of Pyrrolnitrin by Selected Pseudomonas Species

Twenty-nine strains of Pseudomonas, classified as P. fluorescens biotype D or E or as P. multivorans, were examined for the production of pyrrolnitrin, an antifungal agent synthesized in P. aureofaciens. Eight strains were shown to produce pyrrolnitrin in shake-flask fermentation. Four cultures were from the multivorans taxon, and the remaining four were members of the fluorescens group. The antifungal agent produced in these strains was isolated and shown to be pyrrolnitrin by comparison with an authentic sample. The strains differed markedly with respect to the amount of pyrrolnitrin produced and in their utilization of exogenous tryptophan. Secondary metabolites, not related to pyrrolnitrin, were also examined and compared with those synthesized in P. aureofaciens. Marked differences were noted in both phenazine pigments and phenolic metabolites. The results of the study suggest that the production of pyrrolnitrin may be widespread in selected taxonomic groups of Pseudomonas.     

S-thanatin in vitro prevents colistin resistance and improves its efficacy in an animal model of Pseudomonas aeruginosa sepsis

An experimental study was performed to evaluate the interaction between s-thanatin and colistin both in vitro and in vivo, using two Pseudomonas aeruginosa strains with different patterns of susceptibilities. We evaluated whether selecting for colistin-resistant P. aeruginosa could be prevented in vitro by combining colistin with s-thanatin. The strains were serially exposed in broth to twofold stepwise increasing concentrations of colistin alone or in combination with a fixed concentration [0.25× minimum inhibitory concentration (MIC)] of s-thanatin. We also performed an in vitro synergy study. For in vivo studies, a mouse model of Pseudomonas sepsis has been used. Main outcome measures were lethality and quantitative blood cultures. Exposure to colistin alone gradually selected for Pseudomonas strains with an increased MIC. In vitro studies, s-thanatin showed a positive interaction with colistin, and was able to prevent its resistance. In vivo studies, s-thanatin combined with colistin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of this combination and provide a future therapeutic alternative in severe Pseudomonas infections.     

Characterization of rhamnolipids produced by wild-type and engineered Burkholderia kururiensis

Biosurfactants are a class of functional molecules produced and secreted by microorganisms, which play important roles in cell physiology such as flagellum-dependent or -independent bacterial spreading, cell signaling, and biofilm formation. They are amphipathic compounds and comprise a variety of chemical structures, including rhamnolipids, typically produced by Pseudomonas spp. and also reported within other bacterial genera. The present study is focused on Burkholderia kururiensis KP23^T, a trichloroethylene (TCE)-degrading, N-fixing, and plant growth-promoting bacterium. Herein, we describe the production of rhamnolipids by B. kururiensis, and its characterization by LTQ-Orbitrap Hybrid Mass Spectrometry, a powerful tool that allowed efficient identification of molecular subpopulations, due to its high selectivity, mass accuracy, and resolving power. The population of rhamnolipids produced by B. kururiensis revealed molecular species commonly observed in Pseudomonas spp. and/or Burkholderia spp. In addition, this strain was used as a platform for expression of two Pseudomonas aeruginosa biosynthetic enzymes: RhlA, which directly utilizes β-hydroxydecanoyl-ACP intermediates in fatty acid synthesis to generate the HAA, and RhlB, the rhamnosyltransferase 1, which catalyzes the transfer of dTDP-L-rhamnose to β-hydroxy fatty acids in the biosynthesis of rhamnolipids. We show that rhamnolipid production by the engineered B. kururiensis was increased over 600 % when compared to the wild type. Structural analyses demonstrated a molecular population composed mainly of monorhamnolipids, as opposed to wild-type B. kururiensis and P. aeruginosa in which dirhamnolipids are predominant. We conclude that B. kururiensis is a promising biosurfactant-producing organism, with great potential for environmental and biotechnological applications due to its non-pathogenic characteristics and efficiency as a platform for metabolic engineering and production of tailor-made biosurfactants.