Identification of an Extracellular Polysaccharide Network Essential for Cytochrome Anchoring and Biofilm Formation in Geobacter sulfurreducens

Abstract

Transposon insertions in Geobacter sulfurreducens GSU1501, part of an ATP-dependent exporter within an operon of polysaccharide biosynthesis genes, were previously shown to eliminate insoluble Fe(III) reduction and use of an electrode as an electron acceptor. Replacement of GSU1501 with a kanamycin resistance cassette produced a similarly defective mutant, which could be partially complemented by expression of GSU1500 to GSU1505 in trans. The Δ1501 mutant demonstrated limited cell-cell agglutination, enhanced attachment to negatively charged surfaces, and poor attachment to positively charged poly-d-lysine- or Fe(III)-coated surfaces. Wild-type and mutant cells attached to graphite electrodes, but when electrodes were poised at an oxidizing potential inducing a positive surface charge (+0.24 V versus the standard hydrogen electrode [SHE]), Δ1501 mutant cells detached. Scanning electron microscopy revealed fibrils surrounding wild-type G. sulfurreducens which were absent from the Δ1501 mutant. Similar amounts of type IV pili and pilus-associated cytochromes were detected on both cell types, but shearing released a stable matrix of c-type cytochromes and other proteins bound to polysaccharides. The matrix from the mutant contained 60% less sugar and was nearly devoid of c-type cytochromes such as OmcZ. The addition of wild-type extracellular matrix to Δ1501 cultures restored agglutination and Fe(III) reduction. The polysaccharide binding dye Congo red preferentially bound wild-type cells and extracellular matrix material over mutant cells, and Congo red inhibited agglutination and Fe(III) reduction by wild-type cells. These results demonstrate a crucial role for the xap (extracellular anchoring polysaccharide) locus in metal oxide attachment, cell-cell agglutination, and localization of essential cytochromes beyond the Geobacter outer membrane.

Extracellular reduction of uranium via Geobacter conductive pili as a protective cellular mechanism

“…Some of the most abundant metalloproteins on the outer membrane of G. sulfurreducens also are loosely bound to and easily detach from the membrane (40)(41)(42), providing a natural mechanism for releasing the U deposits. Some of these cytochromes also may be anchored to a recently identified exopolysaccharide matrix (43), which may promote the extracellular reduction of U. Furthermore, although some areas of the outer membrane were voided of U reductase activity, U(VI) was effectively prevented from permeating into the periplasm.…”

Microbial interspecies electron transfer via electric currents through conductive minerals

“…4B, nanoparticles were seen at the surface of bacterial cells, and, in some parts, these two bacteria were connected via these particles. Extracellular appendages (24) and polysaccharide networks (25,26) were not observed, even though our samplepreparation procedure was the same as that used in a previous study (25), in which polysaccharide networks were observed. In the hematite-supplemented culture, nanoparticles also adhered to surfaces of bacterial cells (Fig.…”

The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer

“…4, Table S1). The e-pilin gene is found within a cluster of genes including pilB and pilT (both ATPase proteins involved in pilin assembly and retraction), pilC (platform protein), pilS and pilR (two-component regulator proteins involved in pilin expression) and genes involved in synthesis and export of surface polysaccharides and biofilm formation (xap genes) (Rollefson et al, 2011). A gene coding for shikimate dehydrogenase (aroE) is also frequently found in this gene cluster.…”