Metabolites produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. enable a Gram-positive bacterium to achieve extracellular electron transfer

Previous studies revealed the abundance of Pseudomonas sp. in the microbial community of a microbial fuel cell (MFC). These bacteria can transfer electrons to the electrode via self-produced phenazine-based mediators. A MFC fed with acetate where several Pseudomonas sp. were present was found to be rich in a Gram-positive bacterium, identified as Brevibacillus sp. PTH1. Remarkably, MFCs operated with only the Brevibacillus strain in their anodes had poor electricity generation. Upon replacement of the anodic aqueous part of Brevibacillus containing MFCs with the cell-free anodic supernatants of MFCs operated with Pseudomonas sp. CMR12a, a strain producing considerable amounts of phenazine-1-carboxamide (PCN) and biosurfactants, the electricity generation was improved significantly. Supernatants of Pseudomonas sp. CMR12a_Reg, a regulatory mutant lacking the ability to produce PCN, had no similar improvement effect. Purified PCN, together with rhamnolipids as biosurfactants (1 mg L⁻¹), could clearly improve electricity generation by Brevibacillus sp. PTH1, as well as enable this bacterium to oxidize acetate with concomitant reduction of ferric iron, supplied as goethite (FeOOH). When added alone, PCN had no observable effects on Brevibacillus’ electron transfer. This work demonstrates that metabolites produced by Pseudomonas sp. enable Gram-positive bacteria to achieve extracellular electron transfer. Possibly, this bacterial interaction is a key process in the anodic electron transfer of a MFC, enabling Brevibacillus sp. PTH1 to achieve its dominance. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lines of Evidence for Horizontal Gene Transfer of a Phenazine Producing Operon into Multiple Bacterial Species

Phenazines are secondary metabolites with broad-spectrum antibiotic activity against bacteria, fungi, and eukaryotes. In pseudomonad species, a conserved seven-gene phenazine operon (phzABCDEFG) is required for the conversion of chorismic acid to the broad-spectrum antibiotic phenazine-1-carboxylate. Previous analyses of genes involved in phenazine production from nonpseudomonad species uncovered a high degree of sequence similarity to pseudomonad homologues. The analyses undertaken in this study wished to eluciadate the evolutionary history of genes involved in the production of phenazines. Furthermore, I wanted to determine if the phenazine operon has been transferred through horizontal gene transfer. Analyses of GC content, codon usage patterns, frequency of 3:1 dinucleotides, sequence similarities, and phylogenetic reconstructions were undertaken to map the evolutionary history of phenazine genes from multiple bacterial species. Patchy phyletic distribution, high sequence similarities, and phylogenetic evidence infer that pseudomonad, Streptomyces cinnamonensis, Pantoea agglomerans, Burkholderia cepacia, Pectobacterium atrosepticum, Brevibacterium linens, and Mycobacterium abscessus species all contain a phenazine operon which has most likely been transferred among these species through horizontal gene transfer. The acquisition of an antibiotic-associated operon is significant, as it may increase the relative fitness of the recipient species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of platforms for functional characterization and production of phenazines using a multi-chassis approach via CRAGE

Phenazines (Phzs), a family of chemicals with a phenazine backbone, are secondary metabolites with diverse properties such as antibacterial, anti-fungal, or anticancer activity. The core derivatives of phenazine, phenazine-1-carboxylic acid (PCA) and phenazine-1,6-dicarboxylic acid (PDC), are themselves precursors for various other derivatives. Recent advances in genome mining tools have enabled researchers to identify many biosynthetic gene clusters (BGCs) that might produce novel Phzs. To characterize the function of these BGCs efficiently, we performed modular construct assembly and subsequent multi-chassis heterologous expression using chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE allowed rapid integration of a PCA BGC into 23 diverse γ-proteobacteria species and allowed us to identify top PCA producers. We then used the top five chassis hosts to express four partially refactored PDC BGCs. A few of these platforms produced high levels of PDC. Specifically, Xenorhabdus doucetiae and Pseudomonas simiae produced PDC at a titer of 293 mg/L and 373 mg/L, respectively, in minimal media. These titers are significantly higher than those previously reported. Furthermore, selectivity toward PDC production over PCA production was improved by up to 9-fold. The results show that these strains are promising chassis for production of PCA, PDC, and their derivatives, as well as for function characterization of Phz BGCs identified via bioinformatics mining.     

Use of Pseudomonas species producing phenazine-based metabolites in the anodes of microbial fuel cells to improve electricity generation

The rate of anodic electron transfer is one of the factors limiting the performance of microbial fuel cells (MFCs). It is known that phenazine-based metabolites produced by Pseudomonas species can function as electron shuttles for Pseudomonas themselves and also, in a syntrophic association, for Gram-positive bacteria. In this study, we have investigated whether phenazine-based metabolites and their producers could be used to improve the electricity generation of a MFC operated with a mixed culture. Both anodic supernatants obtained from MFCs operated with a Pseudomonas strain (P-PCA) producing phenazine-1-carboxylic acid (PCA) and those from MFCs operated with a strain (P-PCN) producing phenazine-1-carboxamide (PCN) exerted similarly positive effects on the electricity generation of a mixed culture. Replacing supernatants of MFCs operated with a mixed culture with supernatants of MFCs operated with P-PCN could double the currents generated. Purified PCA and purified PCN had similar effects. If the supernatant of an engineered strain overproducing PCN was used, the effect could be maintained over longer time courses, resulting in a 1.5-fold increase in the production of charge. Bioaugmentation of the mixed culture MFCs using slow release tubes containing P-PCN not only doubled the currents but also maintained the effect over longer periods. The results demonstrated the electron-shuttling effect of phenazine-based compounds produced by Pseudomonas species and their capacity to improve the performance of MFCs operated with mixed cultures. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Spectroscopic and viscometric determination of DNA-binding modes of some bioactive dibenzodioxins and phenazines

Push-pull dibenzodioxins and phenazines having ‘anthracene-like’ planar structures and good charge transfer character had been previously synthesised in our laboratory. The dibenzodioxins had earlier proven their anti-proliferative nature against HeLa tumor cell lines. Since phenazines are structural analogues of the former, these molecules were evaluated in course of the current study for their cytotoxic action against HeLa cell lines and they exhibited strong anti-tumor activity. This behavior could be related to their good DNA binding property. The DNA binding modes of molecules 1–4 (Fig. 1) were evaluated using various experimental techniques and they interacted with DNA in a non-covalently by both intercalative as well as groove binding mechanisms. Molecule 1 follows predominantly intercalative binding mode whereas molecules 2 and 3 have nearly equal and opposite preferences for both groove binding and intercalative modes. For molecule 4, groove binding is preferred mode of binding to DNA. A rationale for such differential binding behaviour is provided based on the subtle structural differences in our synthesised dibenzodioxins and phenazines. Elucidation of the mode of a molecule-DNA-binding event is relevant for understanding the mechanism of action of these molecules and will help promote further research into designing better DNA targeting small molecules.     

A phenazine-inspired framework for identifying biological functions of microbial redox-active metabolites

While the list of small molecules known to be secreted by environmental microbes continues to grow, our understanding of their in situ biological functions remains minimal. The time has come to develop a framework to parse the meaning of these “secondary metabolites,” which are ecologically ubiquitous and have direct applications in medicine and biotechnology. Here, we focus on a particular subset of molecules, redox active metabolites (RAMs), and review the well-studied phenazines as archetypes of this class. We argue that efforts to characterize the chemical, physical and biological makeup of the microenvironments, wherein these molecules are produced, coupled with measurements of the molecules' basic chemical properties, will enable significant progress in understanding the precise roles of novel RAMs.     

Metabolic engineering of E. coli for pyocyanin production

Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P. aeruginosa strains, limiting its practical applicability. In this study, the pyocyanin biosynthetic pathway was engineered for the first time for high level production of this compound in a heterologous host. Escherichia coli cells harboring the nine-gene pathway divided into two plasmids were able to produce and secrete pyocyanin at higher levels than some Pseudomonas aeruginosa strains. The influence of culture and induction parameters were evaluated, and the optimized conditions led to an increase of 3.5-fold on pyocyanin accumulation. Pathway balancing was achieved by testing a set of plasmids with different copy numbers to optimize the expression levels of pyocyanin biosynthetic genes, resulting in a fourfold difference in product titer among the engineered strains. Further improvements were achieved by co-expression of Vitreoscilla hemoglobin Vhb, which relieved oxygen limitations and led to a final titer of 18.8 mg/L pyocyanin. These results show promise to use E. coli for phenazines production, and the engineered strain developed here has the potential to be used in electro-fermentation systems where pyocyanin plays a role as electron-shuttle.