Characterization of ferredoxins involved in electron transfer pathways for nitrogen fixation implicates differences in electronic structure in tuning 2[4Fe4S] Fd activity.

Ferredoxins (Fds) are small proteins which shuttle electrons to pathways like biological nitrogen fixation. Physical properties tune the reactivity of Fds with different pathways, but knowledge on how these properties can be manipulated to engineer new electron transfer pathways is lacking. Recently, we showed that an evolved strain of Rhodopseudomonas palustris uses a new electron transfer pathway for nitrogen fixation. This pathway involves a variant of the primary Fd of nitrogen fixation in R. palustris, Fer1, in which threonine at position 11 is substituted for isoleucine (Fer1T11I). To understand why this substitution in Fer1 enables more efficient electron transfer, we used in vivo and in vitro methods to characterize Fer1 and Fer1T11I. Electrochemical characterization revealed both Fer1 and Fer1T11I have similar redox transitions (-480 mV and - 550 mV), indicating the reduction potential was unaffected despite the proximity of T11 to an iron­sulfur (FeS) cluster of Fer1. Additionally, disruption of hydrogen bonding around an FeS cluster in Fer1 by substituting threonine with alanine (T11A) or valine (T11V) did not increase nitrogenase activity, indicating that disruption of hydrogen bonding does not explain the difference in activity observed for Fer1T11I. Electron paramagnetic resonance spectroscopy studies revealed key differences in the electronic structure of Fer1 and Fer1T11I, which indicate changes to the high spin states and/or spin-spin coupling between the FeS clusters of Fer1. Our data implicates these electronic structure differences in facilitating electron flow and sets a foundation for further investigations to understand the connection between these properties and intermolecular electron transfer.

Evolving a New Electron Transfer Pathway for Nitrogen Fixation Uncovers an Electron Bifurcating-Like Enzyme Involved in Anaerobic Aromatic Compound Degradation.

Nitrogenase is the key enzyme involved in nitrogen fixation and uses low potential electrons delivered by ferredoxin (Fd) or flavodoxin (Fld) to reduce dinitrogen gas (N2) to produce ammonia, generating hydrogen gas (H2) as an obligate product of this activity. Although the phototrophic alphaproteobacterium Rhodopseudomonas palustris encodes multiple proteins that can reduce Fd, the FixABCX complex is the only one shown to support nitrogen fixation, and R. palustris Fix- mutants grow poorly under nitrogen-fixing conditions. To investigate how native electron transfer chains (ETCs) can be redirected toward nitrogen fixation, we leveraged the strong selective pressure of nitrogen limitation to isolate a suppressor of an R. palustris ΔfixC strain that grows under nitrogen-fixing conditions. We found two mutations were required to restore growth under nitrogen-fixing conditions in the absence of functional FixABCX. One mutation was in the gene encoding the primary Fd involved in nitrogen fixation, fer1, and the other mutation was in aadN, which encodes a homolog of NAD+-dependent Fd:NADPH oxidoreductase (Nfn). We present evidence that AadN plays a role in electron transfer to benzoyl coenzyme A reductase, the key enzyme involved in anaerobic aromatic compound degradation. Our data support a model where the ETC for anaerobic aromatic compound degradation was repurposed to support nitrogen fixation in the ΔfixC suppressor strain. IMPORTANCE There is increasing evidence that protein electron carriers like Fd evolved to form specific partnerships with select electron donors and acceptors to keep native electron transfer pathways insulated from one another. This makes it challenging to integrate a Fd-dependent pathway such as biological nitrogen fixation into non-nitrogen-fixing organisms and provide the high-energy reducing power needed to fix nitrogen. Here, we show that amino acid substitutions in an electron donor for anaerobic aromatic compound degradation and an Fd involved in nitrogen fixation enabled electron transfer to nitrogenase. This study provides a model system to understand electron transfer chain specificity and how new electron transfer pathways can be evolved for biotechnologically valuable pathways like nitrogen fixation.

CsrA-Controlled Proteins Reveal New Dimensions of Acinetobacter baumannii Desiccation Tolerance.

Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that it is a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 40% RH, we found the global posttranscriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. Proteomics experiments identified proteins that were differentially present in wild-type and csrA mutant cells. Subsequent analysis of mutants in genes encoding some of these proteins revealed six genes that were required for wild-type levels of desiccation tolerance. These include genes for catalase, a universal stress protein, a hypothetical protein, and a biofilm-associated protein. Two genes of unknown function had very strong desiccation phenotypes, with one of the two genes predicting an intrinsically disordered protein (IDP) that binds to DNA. Intrinsically disordered proteins are widespread in eukaryotes but less so in prokaryotes. Our results suggest there are new mechanisms underlying desiccation tolerance in bacteria and identify several key functions involved. IMPORTANCE Acinetobacter baumannii is found in terrestrial environments but can cause nosocomial infections in very sick patients. A factor that contributes to the prevalence of A. baumannii in hospital settings is that it is intrinsically resistant to dry conditions. Here, we established the virulent strain A. baumannii AB5075 as a model for studies of desiccation tolerance at very low relative humidity. Our results show that this trait depends on two proteins of unknown function, one of which is predicted to be an intrinsically disordered protein. This category of protein is critical for the small animals named tardigrades to survive desiccation. Our results suggest that A. baumannii may have novel strategies to survive desiccation that have not previously been seen in bacteria.