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

Abstract

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, twocomponent 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.

Voltammetry and Growth Physiology of Geobacter sulfurreducens Biofilms as a Function of Growth Stage and Imposed Electrode Potential

“…These values can be compared to estimates based on standard protein:cell ratios (0.3 Â 10 À6 mg protein:cell [37], and an assumption that a cell occupies ca. 1 mm 2 ); which produces a similar monolayer estimate at 30 mg/cm [23,35,36].…”

“…To determine when biofilms were transitioning beyond the monolayer stage, levels of attached protein were correlated with confocal microscopy images of identical electrodes, (representative images are available in two previous works, [35,36], and are also similar to [23]). According to these measurements, monolayer coverage of G. sulfurreducens occurred near levels of attached protein of ca.…”

Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens

“…Many genes in the pilA-C-adjacent extracellular polysaccharide (xap) gene cluster (GSU1498-GSU1508), which has been shown to be involved in biofilm formation (Rollefson et al, 2009), were significantly upregulated in Fe(III) oxide-grown cells (Table S2); however, only the putative methyltransferase gene xapI (GSU1506) also had increased transcript abundance during growth on Mn(IV) oxide (Table S4). The xapD gene (GSU1501) coding for the ATPase subunit of an inner membrane transporter complex, which had significantly more transcripts in Fe(III) oxide-grown cells (Table 1), is required for the reduction of anodes and Fe(III) oxide, is likely to be involved in attachment of G. sulfurreducens to metal oxides and in cell-cell agglutination and possibly facilitates the localization of essential cytochromes beyond the Geobacter outer membrane (Rollefson et al, 2011).…”

Widespread Head-to-Head Hydrocarbon Biosynthesis in Bacteria and Role of OleA

“…All organisms, including recombinant strains, were grown aerobically in 50-ml culture flasks on a rotary shaker at 225 rpm, except for Geobacter strains, which were grown in a 100-ml anaerobic culture flask flushed for 30 min with a nitrogen/carbon dioxide gas mix prior to culture inoculation (50). All organisms were grown at 30°C (7,11,35,37,50) except for Shewanella amazonensis (35°C) (69), Shewanella frigidimarina (22°C) (70), Opitutaceae bacterium TAV2 (22°C) (57), Brevibacterium fuscum (22°C) (35), Colwellia psychrerythraea (4°C) (75), Chloroflexus aurantiacus (55°C) (48), and all Escherichia coli strains (37°C) (52). The organisms were allowed to achieve stationary phase prior to hydrocarbon extraction and analysis.…”