Abstract
ABSTRACTGeobacter sulfurreducens generates electrical current by coupling intracellular oxidation of organic acids to the reduction of proteins on the cell surface that are able to interface with electrodes. This ability is attributed to the bacterium's capacity to respire other extracellular electron acceptors that require contact, such as insoluble metal oxides. To directly investigate the genetic basis of electrode-based respiration, we constructed Geobacter sulfurreducens transposon-insertion sequencing (Tn-Seq) libraries for growth, with soluble fumarate or an electrode as the electron acceptor. Libraries with >33,000 unique insertions and an average of 9 insertions/kb allowed an assessment of each gene's fitness in a single experiment. Mutations in 1,214 different genomic features impaired growth with fumarate, and the significance of 270 genes unresolved by annotation due to the presence of one or more functional homologs was determined. Tn-Seq analysis of −0.1 V versus standard hydrogen electrode (SHE) electrode-grown cells identified mutations in a subset of genes encoding cytochromes, processing systems for proline-rich proteins, sensory networks, extracellular structures, polysaccharides, and metabolic enzymes that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of select genes identified via Tn-Seq revealed a new putative porin-cytochrome conduit complex (extABCD) crucial for growth with electrodes, which was not required for Fe(III) oxide reduction. In addition, four mutants lacking components of a putative methyl-accepting chemotaxis–cyclic dinucleotide sensing network (esnABCD) were defective in electrode colonization but grew normally with Fe(III) oxides. These results suggest that G. sulfurreducens possesses distinct mechanisms for recognition, colonization, and reduction of electrodes compared to Fe(III) oxides.IMPORTANCE Since metal oxide electron acceptors are insoluble, one hypothesis is that cells sense and reduce metals using the same molecular mechanisms used to form biofilms on electrodes and produce electricity. However, by simultaneously comparing thousands of Geobacter sulfurreducens transposon mutants undergoing electrode-dependent respiration, we discovered new cytochromes and chemosensory proteins supporting growth with electrodes that are not required for metal respiration. This supports an emerging model where G. sulfurreducens recognizes surfaces and forms conductive biofilms using mechanisms distinct from those used for growth with metal oxides. These findings provide a possible explanation for studies that correlate electricity generation with syntrophic interspecies electron transfer by Geobacter and reveal many previously unrecognized targets for engineering this useful capability in other organisms.
Highlights
Geobacter sulfurreducens generates electrical current by coupling intracellular oxidation of organic acids to the reduction of proteins on the cell surface that are able to interface with electrodes
When two independent cultures were further grown with poised electrodes as the electron acceptor and separately recovered, extracted, and labeled, the number of reads mapped per gene in each independent growth experiment was reproducible, with a Pearson’s coefficient of 0.98 (Fig. 1B)
While single-gene replacement mutants lacking the omcB gene are known to grow in microbial fuel cells [52], these results indicate that no part of the ombBomaB-omcB or ombC-omaC-omcC conduit genes is required for reduction of electrodes, and two other putative trans-outer membrane conduit gene clusters are not required for electron transfer to low-potential electrodes
Summary
Geobacter sulfurreducens generates electrical current by coupling intracellular oxidation of organic acids to the reduction of proteins on the cell surface that are able to interface with electrodes. By simultaneously comparing thousands of Geobacter sulfurreducens transposon mutants undergoing electrode-dependent respiration, we discovered new cytochromes and chemosensory proteins supporting growth with electrodes that are not required for metal respiration This supports an emerging model where G. sulfurreducens recognizes surfaces and forms conductive biofilms using mechanisms distinct from those used for growth with metal oxides. As multiple overlapping electron transfer pathways exist and many electron transfer proteins have functional homologs, single mutations typically decrease rather than eliminate the growth of G. sulfurreducens [19, 20] This respiratory complexity, combined with the bottleneck of studying electron transfer in electrode biofilms, has thwarted the use of unbiased mutagenesis-based discovery tools in the study of electricity production
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