Project description:The formation of electroactive biofilms is a crucial process for the generation of bioelectricity and bioremediation. G. sulfurreducens is a dissimilatory metal-reducing microorganism that can couple oxidation of organic matter with extracellular electron transfer to different insoluble electron acceptors. It has the capability to form biofilms in insoluble metal oxides and electroconductive biofilms in electrodes in bioelectrochemical systems. The formation of electroactive biofilms in this microorganism is a process that has been studied from a physiological, genetic, physical, and electrochemical approach. In G. sulfurreducens, we found that the transcriptional regulator GSU1771 participates in the gene expression of essential genes involved in electron transfer and biofilm formation. Strains deficient in GSU1771 increases Fe(III) reduction, produces more c-type cytochromes and exopolysaccharides. Furthermore, the biofilms produced are thicker and more electroactive than wild-type. In this work, we investigate the global gene expression profile performing RNA-seq comparing Δgsu1771 mutant biofilm grown in non-conductive support (glass) and respiring-graphite electrode. RNA-seq analysis of Δgsu1771 biofilm grown in glass support revealed a total of 467 (167 upregulated and 300 downregulated) differentially expressed genes versus the wild-type biofilm. Meanwhile, in Δgsu1771 biofilm developed in respiring-electrode graphite, we detect 119 (79 upregulated and 40 downregulated) differentially expressed genes with respect to wild-type biofilm. Moreover, transcriptional changes of 67 (56 with the same regulation and in 11 counterregulation) genes were shared in Δgsu1771 biofilm developed in glass and graphite electrodes. We locate upregulated in Δgsu1771 biofilms potential target genes, involved in exopolysaccharide synthesis (gsu1961-63, gsu1959, gsu1972-73, gsu1976-77). We confirmed the upregulation of gsu1979, gsu0972, gsu0783, pgcA, omcM, aroG, panC gnfK, gsu2507, and the downregulation of asnA, ato-1, gsu0810, pilA, csrA, ppcD, and gsu3356 genes by RT-qPCR. DNA-protein binding assay shows direct binding of the GSU1771 regulator to the promoter region of pgcA, pulF, relA, and gsu3356. Also, heme-staining and western blotting revealed an increase of c-type cytochromes in Δgsu1771 biofilms such as OmcS and OmcZ. In general, our data shows that GSU1771 is a global regulator involved in controlling the extracellular electron transfer and exopolysaccharide synthesis, processes required for electroconductive biofilm development.

Project description:Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms, as well as responding leukocytes, may impede wound healing. In this study, we used oxygen microsensors to measure oxygen transects through in vitro-cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse model, and ex vivo human chronic wound specimens. The results show that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17-72 mmHg on live mice and 6.4-1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. No oxygen gradients were observed for heat-killed mouse scabs, suggesting that active metabolism by the viable bacteria and host cells contributed to the reduced oxygen partial pressure of the scabs. To characterize the metabolic activities of the bacteria in the mouse scabs, we performed transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds using Affymetrix microarrays. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results indicated that the bacteria within the wounds also experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results support the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions. Transcriptional profiling of two independent biological replicates of Pseudomonas aeruginosa biofilms, as grown to 72 hours and used as inocula applied to the murine wounds, was performed. A principle components analysis (PCA) was used to provide an overview of the transcriptome data from the 28-day mouse wound scab, comparing the data to the biofilm inoculum, and to published reports of P. aeruginosa biofilm and planktonic samples. The analysis shows that the transcriptome of the mouse wound scab was distinct from the biofilm inoculum that was applied to the wound, demonstrating a shift in biofilm gene expression following 28 days of infection. We sought to characterize P. aeruginosa activity within biofilms in the mouse wound model by isolating and identifying mRNA from the biofilms used as inocula and from the wound scabs 28 days post infection.

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Bioreactor metagenome

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