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The role of CsrA in controls the extracellular electron transfers in Geobacter sulfurreducens

ABSTRACT: CsrA is a post-transcriptional regulator that controls biofilm formation, virulence, carbon metabolism, and motility, among other phenotypes in bacteria. Although CsrA has been extensively studied in γ-proteobacteria and firmicute, it is unknown which cellular processes are targeted for regulation in δ-proteobacteria. In this work, we constructed and characterized the ΔcsrA mutant strain in Geobacter sulfurreducens to determine the involvement of the CsrA protein in the regulation of biofilm and extracellular electron transfer. The ΔcsrA mutant strain grows on acetate-fumarate and reduces soluble Fe(III) similarly to the wild type strain, but has higher rates of insoluble Fe(III) reduction than the wild type. Quantification and characterization of biofilms by violet staining and confocal laser scanning microscopy showed that the ΔcsrA strain produces up to twice as much biofilm as the wild type strain, containing more than 95% viable cells. Transcriptome analysis by RNA-seq shows that in ΔcsrA biofilms developed on an inert support, 244 (103 upregulated and 141 downregulated) genes changed their expression, including those related to extracellular electron transfer, exopolysaccharide synthesis, c-di-GMP synthesis and degradation. To validate the transcriptome data, RT-qPCR confirmed the differential expression of several selected genes in the ΔcsrA strain. Current production in microbial fuel cells was tested and the ΔcsrA strain was found to produce 45-50% more current than the wild type. To verify the genes that changed expression in the graphite electrodes in the ΔcsrA strain, a transcriptome analysis was performed. 181 genes changed their expression in the ΔcsrA biofilms of which 113 genes showed differentially expression only in MFC and 68 genes changed their expression as well as transcriptome from biofilms grown on inert support. In silico analysis of the 5'-UTR regions revealed that 76 genes that changed expression in the RNA-seq analysis have a consensus sequence for CsrA binding. This is the first report describing the involvement of CsrA in the regulation of extracellular electron transfer and biofilm in a member of the γ-proteobacteria.

ORGANISM(S): Geobacter sulfurreducens

PROVIDER: GSE282747 | GEO | 2025/02/28

REPOSITORIES: GEO

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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:The conductive pili of Geobacter sulfurreducens are essential for optimal extracellular electron transfer to Fe(III) and long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene for PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, slow rates of Fe(III) reduction were detected after extended (> 30 days) incubation in the presence of Fe(III) oxide. After seven consecutive transfers the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, proteomic, and gene deletion studies indicated that this adaptation was associated with greater production of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every three days, the wild-type strain out-competed the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron-shuttling producing Fe(III) reducers in most soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current; consistent with the concept that long-range electron transport through G. sulfurreducens biofilms cannot be achieved without PilA-pili. An eight-chip study using total RNA recovered from four separate cultures of Geobacter sulfurreducens JS-1 (experimental condition) or Geobacter sulfurreducens KN400 (control condition) grown with acetate (10mM)-Fe(III) oxide (100 mmol l-1) exponential growth. Each chip measures the expression level of 3,328 genes from Geobacter sulfurreducens KN400 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

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The role of CsrA in controls the extracellular electron transfers in Geobacter sulfurreducens

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