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Journal of Bacteriology, July 2009, p. 4207-4217, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00057-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Janet B. Rollefson,¹ Caleb E. Levar,² and Daniel R. Bond^2,3*

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455,¹ BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108,² Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455³

Received 16 January 2009/ Accepted 14 April 2009

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.

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* Corresponding author. Mailing address: BioTechnology Institute, University of Minnesota, 140 Gortner Laboratory, 1479 Gortner Ave, St. Paul, MN 55108. Phone: (612) 624-8619. Fax: (612) 625-1700. E-mail: dbond{at}umn.edu

Published ahead of print on 24 April 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, July 2009, p. 4207-4217, Vol. 191, No. 13
0021-9193/09/$08.00+0     doi:10.1128/JB.00057-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.