Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens

Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe Geobacter sulfurreducens requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in Geobacter spp. and identify novel genes involved in this process. Escherichia coli mating strains and plasmids were used to develop a conjugation protocol and deliver mini-Himar transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in Geobacter spp.Geobacter sulfurreducens is a member of the metal-reducing Geobacteraceae family and was originally isolated based on its ability to transfer electrons from internal oxidative reactions to extracellular electron acceptors such as insoluble Fe(III) or Mn(IV) oxides (5). G. sulfurreducens is also able to use an electrode as its sole electron acceptor for respiration, a phenotype which has many possible biotechnological applications (28, 29), and serves as a useful tool for direct measurement of electron transfer rates (2, 31). As G. sulfurreducens was the first Geobacteraceae genome sequence available (34) and the only member of this family with a robust genetic system (7), it serves as a model organism for extracellular electron transfer studies.The proteins facilitating electron transfer to insoluble Fe(III) oxides by individual Geobacter cells and how these cells interact in multicellular biofilms are not fully understood. Many genes implicated in Fe(III) and electrode reduction were identified based on proteomic and microarray analysis of cultures grown with fumarate versus Fe(III) citrate as a terminal electron acceptor (9, 15, 35). More recently, similar expression data from Fe(III) oxide and electrode-grown cultures have also become available (8, 12, 16). In most extracellular electron transfer studies, outer membrane proteins (such as c-type cytochromes) have been the focus (4, 23, 27, 32), leading to targeted knockout studies of at least 14 cytochromes to date.To reduce an insoluble electron acceptor, Geobacter spp. must achieve direct contact with the substrate (36). While contact with small Fe(III) oxide particles may be transient, growth on Fe(III)-coated surfaces or electron-accepting electrodes requires biofilm formation (31, 39). For example, when G. sulfurreducens produces an exponentially increasing rate of electron transfer at an electrode, this demonstrates that all newly divided cells remain embedded in the growing, conductive biofilm (2, 31). Thus, in addition to the need for an array of outer membrane cytochromes, there is also a need for control of both cell-cell contact and cell-surface contact.While a genetic system for G. sulfurreducens has been developed, conjugal transfer of a plasmid or a transposon has not been reported (7). The broad-host-range cloning vector pBBR1MCS-2 has previously been electroporated into G. sulfurreducens, but its mobilization capabilities were not utilized (7). Similarly, a number of suicide vectors have been identified for G. sulfurreducens, but none have been used to deliver transposons for mutagenesis. mariner-based transposon mutagenesis systems have been successful in a variety of Bacteria and Archaea, producing random insertions (20, 25, 40, 41, 43, 46, 48, 49). For example, genes involved in Shewanella oneidensis cytochrome maturation were discovered using the modified transposon mini-Himar RB1 (3).In this work, we describe a system for the conjugal transfer of the pBBR1MCS family of plasmids from Escherichia coli to G. sulfurreducens, which allowed transposon mutagenesis based on pMiniHimar RB1. Under strictly anaerobic conditions, a library of insertion mutants was constructed and screened to identify genes putatively involved in attachment and Fe(III) citrate reduction. Approximately 8,000 insertion mutants were isolated, with insertions distributed throughout the G. sulfurreducens chromosome. Subsequent characterization revealed mutants defective in metal reduction but unaffected in all aspects of electrode reduction, as well as mutants able to reduce metals but incapable of electrode reduction. These observations greatly expand the list of Geobacter mutants with defects in respiration or biofilm formation, and this library serves as a resource for further screening of extracellular electron transfer phenotypes.