Levar, C. E. | bioRxiv Science

bioRxiv2022

Single amino acid residues control potential-dependent inactivation of an inner membrane bc-cytochrome

During extracellular electron transfer, Geobacter sulfurreducens constitutively expresses the bc-cytochrome CbcL, yet cells containing only this menaquinone oxidase fail to respire above -0.1 V vs. SHE. By identifying mutations within cbcL that permit growth at higher potentials, we provide evidence that this cytochrome is regulated by redox potential. Strains expressing CbcLV205A, CbcLV205G, and CbcLF525Y were capable of growth with high potential electron acceptors including Fe(III) citrate, Mn(IV) oxides, and electrodes poised at +0.1 V vs. SHE. Electrochemical characterization of wild type CbcL revealed oxidative inactivation of electron transfer above -0.1 V, while CbcLV205A, CbcLV205G, and CbcLF525Y remained active. Growth yields of CbcLV205A, CbcLV205G, and CbcLF525Y were only 50% of WT, consistent with CbcL-dependent electron transfer conserving less energy. These data support the hypothesis that CbcL has evolved to rapidly shut off in response to redox potential to divert electrons to higher yield oxidases that coexist in the Geobacter membrane. TOC image and captionO_ST_ABSTunnel diode behaviorC_ST_ABSElectron flux from cells utilizing the menaquinone oxidase CbcL is attenuated by increased redox potential, preventing use of this low-efficiency pathway when driving forces are high enough to conserve energy via other oxidases. Single amino acid substitutions eliminate this switch-off effect and allow function at all potentials. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/506072v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@11c9b5corg.highwire.dtl.DTLVardef@11be4c6org.highwire.dtl.DTLVardef@4883bborg.highwire.dtl.DTLVardef@c09223_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2016

Genome scale mutational analysis of Geobacter sulfurreducens reveals distinct molecular mechanisms for respiration of poised electrodes vs. Fe(III) oxides

Geobacter sulfurreducens generates electricity by coupling intracellular oxidation of organic acids with electron transfer to the cell exterior, while maintaining a conductive connection to electrode surfaces. This unique ability has been attributed to the bacteriums capacity to also respire extracellular terminal electron acceptors that require contact, such as insoluble metal oxides. To expand the molecular understanding of electricity generation mechanisms, we constructed Geobacter sulfurreducens transposon mutant (Tn-Seq) libraries for growth with soluble fumarate or an electrode surface as the electron acceptor. Mutant libraries with over 33,000 unique transposon insertions and an average of 9 transposon insertions per kb allowed identification of 1,214 genomic features essential for growth with fumarate, including over 270 genes with one or more functional homologs that could not be resolved by previous annotation or in silico modeling. Tn-Seq analysis of electrode-grown cells identified mutations in over 50 genes encoding cytochromes, processing systems for proline-rich proteins, sensory systems, extracellular structures, polysaccharides, metabolic enzymes and hypothetical proteins that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of genes identified via Tn-Seq revealed a new putative c-type cytochrome conduit complex (extABCD) essential for growth with electrodes, which was not required for Fe(III)-oxide reduction. In addition, mutants lacking components of a putative methyl-accepting chemotaxis/cyclic dinucleotide sensing network (esnABCD) were defective in electrode growth, 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 other environmental electron acceptors.

microbiology↗