Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes.

Shewanella oneidensis is known for its ability to respire on extracellular electron acceptors. The spectrum of these acceptors also includes anode surfaces. Based on this activity, a versatile S. oneidensis based biosensor strain was constructed in which electricity production can be modulated. Construction started with the identification of a usable rate-limiting step of electron transfer to an anode. Thereafter, the sensor strain was genetically engineered to produce a protein complex consisting of the three proteins MtrA, MtrB and MtrF. This complex is associated to the outer membrane and most probably enables membrane spanning electron transfer. MtrF is an outer membrane cytochrome that catalyzes electron transfer reactions on the cell surface. Under anoxic conditions, wild type cells do not express MtrF but rather MtrC as electron transferring outer membrane cytochrome. Still, our analysis revealed that MtrF compared to MtrC overexpression is less toxic to the cell which gives MtrF a superior position for biosensor based applications. Transcription of mtrA, mtrB and mtrF was linked up to an inducible promoter system, which positively reacts to rising l-arabinose concentrations. Anode reduction mediated by this strain was linearly dependent on the arabinose content of the medium. This linear dependency was detectable over a wide range of arabinose concentrations. The l-arabinose biosensor presented in this study proves the principle of an outer membrane complex based sensing method which could be easily modified to different specificities by a simple change of the regulatory elements.

Outer-membrane cytochrome-independent reduction of extracellular electron acceptors in Shewanella oneidensis.

Dissimilatory metal reduction under pH-neutral conditions is dependent on an extended respiratory chain to the cell surface. The final reduction is catalysed by outer-membrane cytochromes that transfer respiratory electrons either directly to mineral surfaces and metal ions bound in larger organic complexes such as Fe(III) citrate, or indirectly using endogenous or exogenous electron shuttles such as humic acids and flavins. Consequently, a Shewanella oneidensis deletion mutant devoid of outer-membrane cytochromes is unable to reduce Fe(III) citrate or manganese oxide minerals and reduces humic acids at lower rates. Surprisingly, the phenotype of this quintuple deletion mutant can be rescued by a suppressor mutation, which enables metal or humic acid reduction without any outer-membrane cytochrome. Furthermore, the type II secretion system, essential for metal reduction in wild-type S. oneidensis, is not necessary for the suppressor strain. Using genome sequencing we identified two point mutations in key genes for metal reduction: mtrA and mtrB. These mutations are necessary and sufficient to account for the observed phenotype. This study is the first evidence for a catabolic, outer-membrane cytochrome-independent electron transport chain to ferric iron, manganese oxides and humic acid analogues operating in a mesophilic organism. Available bioinformatic data allow the hypothesis that outer-membrane cytochrome-independent electron transfer might resemble an evolutionary intermediate between ferrous iron-oxidizing and ferric iron-reducing micro-organisms.

Involvement of the Shewanella oneidensis decaheme cytochrome MtrA in the periplasmic stability of the beta-barrel protein MtrB.

The Shewanella oneidensis outer membrane ß-barrel protein MtrB is part of a membrane-spanning protein complex (MtrABC) which is necessary for dissimilatory iron reduction. Quantitative PCR, heterologous gene expression, and mutant studies indicated that MtrA is required for periplasmic stability of MtrB. DegP depletion compensated for this MtrA dependence.

A simple and fast method to analyze the orientation of c-type cytochromes in the outer membrane of Gram-negative bacteria.

Outer membrane cytochromes catalyze the final reduction step of respiratory chains to electron acceptors that cannot diffuse through the outer membrane of Gram-negative bacteria. We developed an in vivo method to detect the orientation of outer membrane cytochromes via analysis of electron transfer reactions between these enzymes and riboflavin.

Involvement and specificity of Shewanella oneidensis outer membrane cytochromes in the reduction of soluble and solid-phase terminal electron acceptors.

The formation of outer membrane (OM) cytochromes seems to be a key step in the evolution of dissimilatory iron-reducing bacteria. They are believed to be the endpoints of an extended respiratory chain to the surface of the cell that establishes the connection to insoluble electron acceptors such as iron or manganese oxides. The gammaproteobacterium Shewanella oneidensis MR-1 contains the genetic information for five putative OM cytochromes. In this study, the role and specificity of these proteins were investigated. All experiments were conducted using a markerless deletion mutant in all five OM cytochromes that was complemented via the expression of single, plasmid-encoded genes. MtrC and MtrF were shown to be potent reductases of chelated ferric iron, birnessite, and a carbon anode in a microbial fuel cell. OmcA-producing cells were unable to catalyze iron and electrode reduction, although the protein was correctly produced and oriented. However, OmcA production resulted in a higher birnessite reduction rate compared with the mutant. The presence of the decaheme cytochrome SO_2931 as well as the diheme cytochrome SO_1659 did not rescue the phenotype of the deletion mutant.