Roles of Two Shewanella oneidensis MR-1 Extracellular Endonucleases

Abstract

The dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 is capable of using extracellular DNA (eDNA) as the sole source of carbon, phosphorus, and nitrogen. In addition, we recently demonstrated that S. oneidensis MR-1 requires eDNA as a structural component during all stages of biofilm formation. In this study, we characterize the roles of two Shewanella extracellular endonucleases, ExeS and ExeM. While ExeS is likely secreted into the medium, ExeM is predicted to remain associated with the cell envelope. Both exeM and exeS are highly expressed under phosphate-limited conditions. Mutants lacking exeS and/or exeM exhibit decreased eDNA degradation; however, the capability of S. oneidensis MR-1 to use DNA as the sole source of phosphorus is only affected in mutants lacking exeM. Neither of the two endonucleases alleviates toxic effects of increased eDNA concentrations. The deletion of exeM and/or exeS significantly affects biofilm formation of S. oneidensis MR-1 under static conditions, and expression of exeM and exeS drastically increases during static biofilm formation. Under hydrodynamic conditions, a deletion of exeM leads to altered biofilms that consist of densely packed structures which are covered by a thick layer of eDNA. Based on these results, we hypothesize that a major role of ExeS and, in particular, ExeM of S. oneidensis MR-1, is to degrade eDNA as a matrix component during biofilm formation to improve nutrient supply and to enable detachment.

Functional Specificity of Extracellular Nucleases of Shewanella oneidensis MR-1

“…For E. coli strain WM3064, 2,6-diaminopimelic acid was added to the medium to a final concentration of 300 M. For solidification, agar was added to a final concentration of 1.5% (wt/vol). For growth assays depending on the phosphorus source, S. oneidensis strains were grown in modified M1 mineral medium as described previously (22). NaH 2 PO 4 at 0.86 mM or the indicated amount of salmon sperm DNA (Sigma, Deisenhofen, Germany) was added as the sole source of phosphorus.…”

“…Qualitative nuclease assays in medium supernatants were carried out essentially as described previously (22,48). Briefly, cells from an overnight LB preculture were incubated in fresh medium at an OD 600 of 0.05 and were grown to an OD 600 of 1.5.…”

“…Utilization of eDNA as a nutrient, in particular as a source of phosphate, requires the presence of extracellular nucleases and phosphatases, and corresponding activity was readily detected in Shewanella cultures (22,48). Recently, we characterized the role of two extracellular nucleases, ExeM and ExeS, in Shewanella oneidensis MR-1 (22).…”

“…Recently, we characterized the role of two extracellular nucleases, ExeM and ExeS, in Shewanella oneidensis MR-1 (22). ExeS is predicted to be released into the medium supernatant, while ExeM appears to remain associated with the cell envelope (9,48,54).…”

“…In contrast, the absence of both exeM and exeS had little or no effect on the use of eDNA as the sole source of phosphorus. In addition, a mutant lacking both enzymes still displayed substantial degradation of eDNA in the medium supernatant, indicating the presence of one or more additional proteins with nucleolytic function (22). That prompted us to screen for other potential extracellular nucleases in S. oneidensis MR-1.…”