title: 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.
authors.affiliation: BioTechnology Institute, University of Minnesota, St. Paul, MN 55108, USA.
authors.forename: Janet B
authors.initials: JB
authors.lastname: Rollefson
authors.forename: Camille S
authors.initials: CS
authors.lastname: Stephen
authors.forename: Ming
authors.initials: M
authors.lastname: Tien
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Bond
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development*; D003580:Cytochromes / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D045302:Geobacter / Q000502:physiology* / Q000648:ultrastructure; D005810:Multigene Family; D009154:Mutation; D011135:Polysaccharides, Bacterial / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D003580:Cytochromes; D011135:Polysaccharides, Bacterial