Sir2-domain associated short prokaryotic Argonautes provide defence against invading mobile genetic elements through NAD+ depletion

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID, and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). Intriguingly, the majority (~60%) of prokaryotic Agos (pAgos) identified bioinformatically are shorter (comprised of only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here, we show that short pAgos from Geobacter sulfurreducens, Caballeronia cordobensis and Paraburkholderia graminis, together with the NAD+-bound Sir2-proteins form a stable heterodimeric Sir2/Ago complex that recognizes invading plasmid or phage DNA through the pAgos subunit and activates Sir2 subunit triggering the endogenous NAD+ depletion and cell death thus preventing the propagation of invading DNA. This is the first demonstration that short Sir2-associated pAgos provide defence against phages and plasmids and underscores the diversity of mechanisms of prokaryotic Agos.

Sir2-domain associated short prokaryotic Argonautes provide defence against invading mobile genetic elements through NAD+ depletion

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID, and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). Intriguingly, the majority (~60%) of prokaryotic Agos (pAgos) identified bioinformatically are shorter (comprised of only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here, we show that short pAgos from Geobacter sulfurreducens, Caballeronia cordobensis and Paraburkholderia graminis, together with the NAD+-bound Sir2-proteins form a stable heterodimeric Sir2/Ago complex that recognizes invading plasmid or phage DNA through the pAgos subunit and activates Sir2 subunit triggering the endogenous NAD+ depletion and cell death thus preventing the propagation of invading DNA. This is the first demonstration that short Sir2-associated pAgos provide defence against phages and plasmids and underscores the diversity of mechanisms of prokaryotic Agos.

Making protons tag along with electrons

Every living cell needs to get rid of leftover electrons when metabolism extracts energy through the oxidation of nutrients. Common soil microbes such as Geobacter sulfurreducens live in harsh environments that do not afford the luxury of soluble, ingestible electron acceptors like oxygen. Instead of resorting to fermentation, which requires the export of reduced compounds (e.g. ethanol or lactate derived from pyruvate) from the cell, these organisms have evolved a means to anaerobically respire by using nanowires to export electrons to extracellular acceptors in a process called extracellular electron transfer (EET) [ 1]. Since 2005, these nanowires were thought to be pili filaments [ 2]. But recent studies have revealed that nanowires are composed of multiheme cytochromes OmcS [ 3, 4] and OmcZ [ 5] whereas pili remain inside the cell during EET and are required for the secretion of nanowires [ 6]. However, how electrons are passed to these nanowires remains a mystery ( Figure 1A). Periplasmic cytochromes (Ppc) called PpcA-E could be doing the job, but only two of them (PpcA and PpcD) can couple electron/proton transfer — a necessary condition for energy generation. In a recent study, Salgueiro and co-workers selectively replaced an aromatic with an aliphatic residue to couple electron/proton transfer in PpcB and PpcE (Biochem. J. 2021, 478 (14): 2871–2887). This significant in vitro success of their protein engineering strategy may enable the optimization of bioenergetic machinery for bioenergy, biofuels, and bioelectronics applications.

Proteomic Analysis of a Syntrophic Coculture of Syntrophobacter fumaroxidans MPOBT and Geobacter sulfurreducens PCAT

We established a syntrophic coculture of Syntrophobacter fumaroxidans MPOBT (SF) and Geobacter sulfurreducens PCAT (GS) growing on propionate and Fe(III). Neither of the bacteria was capable of growth on propionate and Fe(III) in pure culture. Propionate degradation by SF provides acetate, hydrogen, and/or formate that can be used as electron donors by GS with Fe(III) citrate as electron acceptor. Proteomic analyses of the SF-GS coculture revealed propionate conversion via the methylmalonyl-CoA (MMC) pathway by SF. The possibility of interspecies electron transfer (IET) via direct (DIET) and/or hydrogen/formate transfer (HFIT) was investigated by comparing the differential abundance of associated proteins in SF-GS coculture against (i) SF coculture with Methanospirillum hungatei (SF-MH), which relies on HFIT, (ii) GS pure culture growing on acetate, formate, hydrogen as propionate products, and Fe(III). We noted some evidence for DIET in the SF-GS coculture, i.e., GS in the coculture showed significantly lower abundance of uptake hydrogenase (43-fold) and formate dehydrogenase (45-fold) and significantly higher abundance of proteins related to acetate metabolism (i.e., GltA; 62-fold) compared to GS pure culture. Moreover, SF in the SF-GS coculture showed significantly lower abundance of IET-related formate dehydrogenases, Fdh3 (51-fold) and Fdh5 (29-fold), and the rate of propionate conversion in SF-GS was 8-fold lower than in the SF-MH coculture. In contrast, compared to GS pure culture, we found lower abundance of pilus-associated cytochrome OmcS (2-fold) and piliA (5-fold) in the SF-GS coculture that is suggested to be necessary for DIET. Furthermore, neither visible aggregates formed in the SF-GS coculture, nor the pili-E of SF (suggested as e-pili) were detected. These findings suggest that the IET mechanism is complex in the SF-GS coculture and can be mediated by several mechanisms rather than one discrete pathway. Our study can be further useful in understanding syntrophic propionate degradation in bioelectrochemical and anaerobic digestion systems.

Microbial fermentation of polyethylene terephthalate (PET) plastic waste for the production of chemicals and electricity

Ideonella sakaiensis (I. sakaiensis) can grow on polyethylene terephthalate (PET) as the sole carbon and energy source. Previous work has shown that conversion of the hydrolysis products terephthalic acid (TPA) and ethylene glycol (EG) under aerobic conditions released carbon dioxide and water while yielding adenosine triphosphate (ATP) through oxidative phosphorylation. This study demonstrates that under anaerobic conditions I. sakaiensis ferments PET to the feedstock chemicals acetate and ethanol while co-producing ATP by substrate-level phosphorylation. In addition to PET, maltose, EG, and ethanol can also serve as fermenting substrates. Co-culturing of I. sakaiensis with electrogenic Geobacter sulfurreducens produced electricity from PET or EG. This newly identified plastic fermentation process by I. sakaiensis provides a novel biosynthetic route to produce high-value chemicals and electricity from plastic waste streams.

Algae-Assisted Microbial Desalination Cell: Analysis of Cathode Performance and Desalination Efficiency Assessment

Algae-assisted microbial desalination cells represent a sustainable technology for low-energy fresh water production in which microalgae culture is integrated into the system to enhance oxygen reduction reaction in the cathode chamber. However, the water production (desalination rate) is low compared to conventional technologies (i.e., reverse osmosis and/or electrodialysis), as biocathodes provide low current generation to sustain the desalination process. In this sense, more research efforts on this topic are necessary to address this bottleneck. Thus, this study provides analysis, from the electrochemical point of view, on the cathode performance of an algae-assisted microbial desalination cell (MDC) using Chlorella vulgaris. Firstly, the system was run with a pure culture of Chlorella vulgaris suspension in the cathode under conditions of an abiotic anode to assess the cathodic behavior (i.e., cathode polarization curves in light-dark conditions and oxygen depletion). Secondly, Geobacter sulfurreducens was inoculated in the anode compartment of the MDC, and the desalination cycle was carried out. The results showed that microalgae could generate an average of 9–11.5 mg/L of dissolved oxygen during the light phase, providing enough dissolved oxygen to drive the migration of ions (i.e., desalination) in the MDC system. Moreover, during the dark phase, a residual concentration of oxygen (ca. 5.5–8 mg/L) was measured, indicating that oxygen was not wholly depleted under our experimental conditions. Interestingly, the oxygen concentration was restored (after complete depletion of dissolved oxygen by flushing with N2) as soon as microalgae were exposed to the light phase again. After a 31 h desalination cycle, the cell generated a current density of 0.12 mA/cm2 at an efficiency of 60.15%, 77.37% salt was removed at a nominal desalination rate of 0.63 L/m2/h, coulombic efficiency was 9%, and 0.11 kWh/m3 of electric power was generated. The microalgae-assisted biocathode has an advantage over the air diffusion and bubbling as it can self-sustain a steady and higher concentration of oxygen, cost-effectively regenerate or recover from loss and sustainably retain the system’s performance under naturally occurring conditions. Thus, our study provides insights into implementing the algae-assisted cathode for sustainable desalination using MDC technology and subsequent optimization.

Microbial fuel cell power overshoot studied with microfluidics: from quantification to elimination

Power overshoot can hinder determination of maximum power densities in microbial fuel cells (MFCs). In this work, a microfluidic approach was used to study overshoot in an MFC containing a pure culture of electroactive biofilms (EAB) containing Geobacter sulfurreducens. After 1-month operation under constant flow of an ideal nutrient medium, the MFC health began to degrade, marked by voltage loss and the appearance of anomalies in the power density curves. One such anomaly was a chronic power overshoot, accompanying a loss of both measured power and current density on the high-current side of the power density curve. The degree of power overshoot was quantified while certain flow-based interventions were applied, notably the shear erosion of the EAB outer layer. Next, two approaches to acclimation were demonstrated to treat the remaining overshoot. The standard approach, which acclimates the MFC to high currents before a standard polarization test, eliminated the remaining overshoot and returned maximum power densities to initial levels, but maximum current density remained lower than the initial level. A microfluidic-assisted “long-hold polarization test” enabled efficient in situ acclimation of each external resistor during the measurement. Despite the health-compromised MFC, this method provided long-term stability during the polarization test, resulting in power and current density measurements that exceeded those made on the healthy MFC using the standard polarization test. We conclude that slower electron transfer kinetics in unhealthy MFCs can provoke overshoot by prolonging the time to reach steady state during the polarization test, but a properly designed measurement overcomes this problem.