Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation

Abstract

In limiting oxygen as an electron acceptor, the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 rapidly forms nanowires, extensions of its outer membrane containing the cytochromes MtrC and OmcA needed for extracellular electron transfer. RNA sequencing (RNA-Seq) analysis was employed to determine differential gene expression over time from triplicate chemostat cultures that were limited for oxygen. We identified 465 genes with decreased expression and 677 genes with increased expression. The coordinated increased expression of heme biosynthesis, cytochrome maturation, and transport pathways indicates that S. oneidensis MR-1 increases cytochrome production, including the transcription of genes encoding MtrA, MtrC, and OmcA, and transports these decaheme cytochromes across the cytoplasmic membrane during electron acceptor limitation and nanowire formation. In contrast, the expression of the mtrA and mtrC homologs mtrF and mtrD either remains unaffected or decreases under these conditions. The ompW gene, encoding a small outer membrane porin, has 40-fold higher expression during oxygen limitation, and it is proposed that OmpW plays a role in cation transport to maintain electrical neutrality during electron transfer. The genes encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP) and the extracytoplasmic function sigma factor RpoE are among the transcription factor genes with increased expression. RpoE might function by signaling the initial response to oxygen limitation. Our results show that RpoE activates transcription from promoters upstream of mtrC and omcA. The transcriptome and mutant analyses of S. oneidensis MR-1 nanowire production are consistent with independent regulatory mechanisms for extending the outer membrane into tubular structures and for ensuring the electron transfer function of the nanowires. IMPORTANCEShewanella oneidensis MR-1 has the capacity to transfer electrons to its external surface using extensions of the outer membrane called bacterial nanowires. These bacterial nanowires link the cell's respiratory chain to external surfaces, including oxidized metals important in bioremediation, and explain why S. oneidensis can be utilized as a component of microbial fuel cells, a form of renewable energy. In this work, we use differential gene expression analysis to focus on which genes function to produce the nanowires and promote extracellular electron transfer during oxygen limitation. Among the genes that are expressed at high levels are those encoding cytochrome proteins necessary for electron transfer. Shewanella coordinates the increased expression of regulators, metabolic pathways, and transport pathways to ensure that cytochromes efficiently transfer electrons along the nanowires.

Extracellular reduction of solid electron acceptors byShewanella oneidensis

“…Targets for RpoE are genes involved in outer membrane lipoprotein transport and folding, lipopolysaccharide production and periplasmic proteases (Dai et al, 2015). Moreover, RpoE binding sites were detected upstream of the genes for the outer membrane cytochromes OmcA and MtrC as well as genes involved in c-type cytochrome maturation and heme biosynthesis (Barchinger et al, 2016). Expression of the rpoE gene itself is upregulated twofold in the absence of oxygen (Barchinger et al, 2016) and the presence of metals and thiosulfate (Beliaev et al, 2005).…”

Distinct Electron Conductance Regimes in Bacterial Decaheme Cytochromes

“…Within the resolution of our calculations,the rate-limiting steps in MtrC and MtrF are comparable,w ith differences of less than one order of magnitude (Supporting Information, Tables S7 and S8). This result suggests that EET is not the main factor determining higher expression of MtrABC relative to MtrDEF in anoxic environments; [16] however, more precise calculations are necessary to confidently establish whether the two proteins differ in their EET efficiency.…”

“…[11,12] Different mechanisms of EET (direct versus mediated) are associated with distinct voltammetry signatures observed in vivo. [16] TheX -ray structures of both outer membrane cytochromes,M trC and MtrF,a re now available. Forexample, and in contrast to MtrABC,n oi ncrease in MtrDEF expression was observed under O 2 -limited conditions.…”

“…Forexample, and in contrast to MtrABC,n oi ncrease in MtrDEF expression was observed under O 2 -limited conditions. [16] TheX -ray structures of both outer membrane cytochromes,M trC and MtrF,a re now available. [7,17] Figure 1 highlights the intriguing staggered-cross arrangement of the heme-c cofactors.Itishypothesized that heme 10 is the redox center that receives electrons from the periplasmic side of the membrane,while heme 5, the farthest exposed cofactor, is the exit heme that serves as an electron donor for solid acceptors outside the cell.…”

Electron transfer and transport through multi-heme proteins: recent progress and future directions

“…1 When cytoplasmatic O 2 becomes scarce it starts to grow µm-long electrically conducting cellular appendages, which allows the bacterium to export electrons from the cytoplasm to extracellular space for reduction of extracellular electron acceptors in place of O 2 (see Figure 1A). [2][3][4] Until not too long ago it was speculated that these conducting appendages are bacterial pili, believed to be relevant for extracellular electron transport in other organisms, e.g. G. sulfurreducens.…”