Global transcriptional analysis of Geobacter sulfurreducens gsu1771-mutant in response to biofilm development in different supports. Global transcriptional analysis of Geobacter sulfurreducens gsu1771-mutant in response to biofilm development in different supports

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. Overall design: Comparative gene expression profiling analysis of RNA-seq data for Geobacter sulfurreducens biofilm from wild type and gsu1771-mutant strains grown on a glass support and graphite electrode. From each sample, experimental duplicates were used.

电活性生物膜的形成是生物发电与生物修复的核心过程。硫还原地杆菌（G. sulfurreducens）是一种异化金属还原微生物，可将有机物的氧化过程与胞外电子传递偶联，以不同不溶性电子受体作为末端受体。该菌能够在不溶性金属氧化物表面形成生物膜，并可在生物电化学系统的电极表面形成导电生物膜。针对该菌的电活性生物膜形成过程，学界已从生理学、遗传学、物理学及电化学等多个维度开展研究。
在硫还原地杆菌中，我们发现转录调控因子GSU1771参与调控电子传递与生物膜形成相关的关键基因的表达。GSU1771基因缺失菌株的Fe(III)还原能力增强，且可产生更多的c型细胞色素与胞外多糖。此外，该缺失菌株形成的生物膜较野生型菌株更厚，且电活性更强。
本研究通过RNA测序（RNA-seq）技术，对比分析了在非导电载体（玻璃）与呼吸型石墨电极表面生长的Δgsu1771突变株生物膜的全局基因表达谱。对玻璃载体表面生长的Δgsu1771突变株生物膜进行RNA测序分析后发现，其与野生型生物膜相比，共存在467个差异表达基因，其中167个基因上调、300个基因下调。与此同时，在呼吸型石墨电极表面形成的Δgsu1771突变株生物膜中，我们共检测到119个与野生型生物膜存在差异表达的基因，其中79个上调、40个下调。此外，在玻璃载体与石墨电极表面形成的Δgsu1771突变株生物膜中，共有67个基因存在共同的转录表达变化，其中56个基因的调控方向一致，11个基因的调控方向相反。
我们在Δgsu1771突变株生物膜中鉴定出了参与胞外多糖合成的潜在靶标基因（gsu1961-63、gsu1959、gsu1972-73、gsu1976-77），这些基因均呈现上调表达。我们通过实时定量PCR（RT-qPCR）验证了gsu1979、gsu0972、gsu0783、pgcA、omcM、aroG、panC、gnfK、gsu2507等基因的上调表达，以及asnA、ato-1、gsu0810、pilA、csrA、ppcD、gsu3356等基因的下调表达。DNA-蛋白质结合实验证实，GSU1771调控因子可直接结合pgcA、pulF、relA及gsu3356的启动子区域。此外，血红素染色与蛋白质免疫印迹（Western blotting）实验结果显示，Δgsu1771突变株生物膜中的c型细胞色素（如OmcS、OmcZ）含量显著升高。
综上，本研究数据表明，GSU1771是一种全局性调控因子，参与调控胞外电子传递与胞外多糖合成过程，而这两类过程正是导电生物膜形成所必需的。
实验设计概述：对在玻璃载体与石墨电极表面生长的野生型及gsu1771突变株硫还原地杆菌生物膜的RNA测序数据进行比较基因表达谱分析。每个样本设置2次生物学重复。