How the xap Locus Put Electrical “Zap” in Geobacter sulfurreducens Biofilms

Investigation of microbial mineral respiration remains an experimental challenge. In this issue of Journal of Bacteriology, Rollefson et al. (11) present a foundational study on the functionality of the biofilm matrix in Geobacter sulfurreducens, a model dissimilatory metal respiring bacterium (DMRB). In this study, the investigators identify an extracellular polysaccharide scaffold or network that entraps redox-active proteins, thus positioning these proteins for optimal electron transfer from the membrane-bound respiratory supercomplexes to a mineral phase electron acceptor. The distinguishing feature of this study is the perspective, in that the team examined specifically exopolysaccharide formation and how it enables entrapment and tethering of redox proteins in the vicinity of the cell. Previous studies on Geobacter (10) and Shewanella (4) have focused primarily on the presence and functionality of conductive pili and nanowires, proteinaceous structures that also enable and enhance extracellular electron transfer. Rollefson et al. remind investigators in this field that many microbial systems have redundancy in essential functions, and in the case of DMRB, it is clearly critical that more than one mechanism exists to ensure vectoral electron transport to mineral phase electron acceptors. The major findings of Rollefson et al. (11) were (i) identification of a biofilm locus in G. sulfurreducens that harbors exopolysaccharide synthesis and export genes; (ii) detection of c-type cytochromes in exopolysaccharide materials; (iii) genetic mutation of a gene (xapD [GSU1501], an ATP-dependent ABC transporter) in the locus which results in reduced functionality of the biofilm matrix, i.e., reduced agglutination and attachment, and less matrix-bound cytochrome; and (iv) microscopic confirmation of exopolysaccharide materials in the biofilm matrix. The research employed a combination of traditional microbiological techniques, genetic manipulation and mutation, and fluorescence and electron microscopy to obtain an integrated view of biofilm matrix composition and functionality in G. sulfurreducens. The elegance of the study is in its relative simplicity. For example, while agglutination has been examined in bacteria for decades, basic assessment of this phenomenon has not been made in many DMRB models, thus showing that classical microbiology still can reveal a great deal about DMRB biofilms. For example, Fig. 2 and 3 of the Rollefson et al. paper clearly reveal both macroscopic and microscopic differences in culture and biofilm morphology. Differential extraction procedures purify pili and exopolysaccharide and demonstrate binding of redox proteins and positioning of an extracellular biomolecular conductive network, and although one can argue the efficacy and resolution of these methods, the results seem to suggest that protein-exopolysaccharide complexes can be successfully isolated from biofilm cultures, and these approaches can be reliably used on other model DMRB systems. Unique experiments using polysaccharide binding stains coupled with scanning electron microscopy (SEM) reveal that exopolysaccharide is a key component for attachment and colonization of mineral and electrode surfaces. Indeed, the exopolysaccharide appears as a network of strands wiring cells together and to the substrate. Blocking techniques with safranin O work to strengthen the interpretation of the SEM images by better visualization of the extracellular matrix material.