Data_Sheet_1_Construction of a Geobacter Strain With Exceptional Growth on Cathodes.DOCX

<p>Insoluble extracellular electron donors are important sources of energy for anaerobic respiration in biogeochemical cycling and in diverse practical applications. The previous lack of a genetically tractable model microorganism that could be grown to high densities under anaerobic conditions in pure culture with an insoluble extracellular electron donor has stymied efforts to better understand this form of respiration. We report here on the design of a strain of Geobacter sulfurreducens, designated strain ACL, which grows as thick (ca. 35 μm) confluent biofilms on graphite cathodes poised at -500 mV (versus Ag/AgCl) with fumarate as the electron acceptor. Sustained maximum current consumption rates were >0.8 A/m², which is >10-fold higher than the current consumption of the wild-type strain. The improved function on the cathode was achieved by introducing genes for an ATP-dependent citrate lyase, completing the complement of enzymes needed for a reverse TCA cycle for the synthesis of biosynthetic precursors from carbon dioxide. Strain ACL provides an important model organism for elucidating the mechanisms for effective anaerobic growth with an insoluble extracellular electron donor and may offer unique possibilities as a chassis for the introduction of synthetic metabolic pathways for the production of commodities with electrons derived from electrodes.</p

Confocal scanning laser microscopy images of <i>Geobacter sulfurreducens</i> grown with different electron acceptors.

<p>Confocal scanning laser microscopy images of current harvesting and fumarate control biofilms of wild type <i>G. sulfurreducens.</i> Metabolically active (green) and inactive (red) cells where differentiated with a LIVE/DEAD kit based on the permeability of the cell membrane. A. 3-D projection, top view, fumarate control biofilm; B. slices through biofilm parallel to electrode large panel and perpendicular to electrode top and side panel, fumarate control biofilm; C. 3-D projection, top view, current harvesting biofilm; D. slices through biofilm parallel to electrode large panel and perpendicular to electrode top and side panel, current harvesting biofilm.</p

OmcZ sequence and identification.

<p>A. Amino acid sequence of OmcZ. The predicted cleavage site for mature OmcZ indicated by arrow. Confirmed heme-binding domains (CXXCH) are enclosed in boxes. OmcZ has 473 amino acid residues and 7 heme binding domains. B. Cytochrome content of loosely bound outer membrane protein-enriched fractions from fumarate and current-harvesting biofilms. Proteins (10 µg/lane) were separated by 12% Tris-Tricine denaturing polyacryamide gel electrophoresis and stained for heme. C. Peptides detected from two different sizes of OmcZs, fragments detected in the 50 KDa OmcZ are indicated in panel A in red; fragments detected in the 30 KDa OmcZ are indicated in panel A in green and fragments detected in both size OmcZ are indicated in blue.</p

Primers used for mutant construction, complementation and RT-PCR analyses.

<p>Primers used for mutant construction, complementation and RT-PCR analyses.</p

Maximal current production of fuel cells inoculated with wild-type cells of <i>G. sulfurreducens</i> or strains with the designated genes deleted or the deleted genes complemented via expression of the designated gene on a plasmid.

<p>Values are the average±the standard deviation of triplicates.</p

Current production of <i>Geobacter sulfurreducens</i>.

<p>A. Current production time courses of wild type <i>G. sulfurreducens</i> grown entirely as current harvesting (arrow indicates the switch from original feed of 10 mM acetate to continual feed) and current production time course of fully grown fumarate control biofilms switched to current harvesting of wild type <i>G. sulfurreducens</i>. These data are representative time courses for multiple replicates of each treatment. B–C. Confocal scanning laser microscopy images of fumarate control swapped to current harvesting biofilms of <i>G. sulfurreducens</i> . Metabolically active (green) and inactive (red) cells where differentiated with a LIVE/DEAD kit based on the permeability of the cell membrane. B. 3-D projection, top view; C. slices through biofilm parallel to electrode large panel and perpendicular to electrode top and side panel.</p

Gene transcripts whose expression (arithmetic) was significantly increased when <i>G. sulfurreducens</i> was grown within a current-harvesting 10 mA potentiostat biofilm versus a fumarate control biofilm.

<p>Limma Analysis with a maximum p value of 0.001 was used for statistical analysis.</p

Gene transcripts whose expression (arithmetic) was significantly decreased when <i>G. sulfurreducens</i> was grown within a current-harvesting 10 mA potentiostat biofilm versus a fumarate control biofilm.

<p>Limma Analysis with a maximum p value of 0.001 was used for statistical analysis. Genes included has log2 ratios of greater than 1.</p

RT-PCR of genes up-regulated in microarray analyses.

<p>Fold change of <i>pilA</i>, <i>omcB</i>, <i>omcE</i>, <i>omcS</i>, <i>omcZ</i> and GSU1497 at different amounts of current produced compared to a soluble Fe(III) control as determined by quantitative RT-PCR.</p