Multifaceted regulation of siderophore synthesis by multiple regulatory systems in Shewanella oneidensis.

Siderophore-dependent iron uptake is a mechanism by which microorganisms scavenge and utilize iron for their survival, growth, and many specialized activities, such as pathogenicity. The siderophore biosynthetic system PubABC in Shewanella can synthesize a series of distinct siderophores, yet how it is regulated in response to iron availability remains largely unexplored. Here, by whole genome screening we identify TCS components histidine kinase (HK) BarA and response regulator (RR) SsoR as positive regulators of siderophore biosynthesis. While BarA partners with UvrY to mediate expression of pubABC post-transcriptionally via the Csr regulatory cascade, SsoR is an atypical orphan RR of the OmpR/PhoB subfamily that activates transcription in a phosphorylation-independent manner. By combining structural analysis and molecular dynamics simulations, we observe conformational changes in OmpR/PhoB-like RRs that illustrate the impact of phosphorylation on dynamic properties, and that SsoR is locked in the 'phosphorylated' state found in phosphorylation-dependent counterparts of the same subfamily. Furthermore, we show that iron homeostasis global regulator Fur, in addition to mediating transcription of its own regulon, acts as the sensor of iron starvation to increase SsoR production when needed. Overall, this study delineates an intricate, multi-tiered transcriptional and post-transcriptional regulatory network that governs siderophore biosynthesis.

Biological Self-Assembled Transmembrane Electron Conduits for High-Efficiency Ammonia Production in Microbial Electrosynthesis.

Usually, CymA is irreplaceable as the electron transport hub in Shewanella oneidensis MR-1 bidirectional electron transfer. In this work, biologically self-assembled FeS nanoparticles construct an artificial electron transfer route and implement electron transfer from extracellular into periplasmic space without CymA involvement, which present similar properties to type IV pili. Bacteria are wired up into a network, and more electron transfer conduits are activated by self-assembled transmembrane FeS nanoparticles (electron conduits), thereby substantially enhancing the ammonia production. In this study, we achieved an average NH4+-N production rate of 391.8 µg·h-1·L reactor-1 with the selectivity of 98.0% and cathode efficiency of 65.4%. Additionally, the amide group in the protein-like substances located in the outer membrane was first found to be able to transfer electrons from extracellular into intracellular with c-type cytochromes. Our work provides a new viewpoint that contributes to a better understanding of the interconnections between semiconductor materials and bacteria and inspires the exploration of new electron transfer chain components.

Insights into the biosynthesis of palladium nanoparticles for oxygen reduction reaction by genetically engineered bacteria of Shewanella oneidensis MR-1.

Owing to the increasing need for green synthesis and environmental protection, the utilization of biological organism-derived carbons as supports for noble-metal electrocatalysts has garnered public interest. Nevertheless, the mechanism by which microorganisms generate nanometals has not been fully understood yet. In the present study, we used genetically engineered bacteria of Shewanella oneidensis MR-1 (∆SO4317, ∆SO4320, ∆SO0618 and ∆SO3745) to explore the effect of surface substances including biofilm-associated protein (bpfA), protein secreted by type I secretion systems (TISS) and type II secretion systems (T2SS), and lipopolysaccharide in microbial synthesis of metal nanoparticles. Results showed Pd/∆SO4317 (the catalyst prepared with the mutant ∆SO4317) shows better performance than other biocatalysts and commercial Pd/C, where the mass activity (MA) and specific activity (SA) of Pd/∆SO4317 are 3.1 and 2.1 times higher than those of commercial Pd/C, reaching 257.49 A g-1 and 6.85 A m-2 respectively. It has been found that the exceptional performance is attributed to the smallest particle size and the presence of abundant functional groups. Additionally, the absence of biofilms has been identified as a crucial factor in the formation of high-quality bio-Pd. Because the absence of biofilm can minimize metal agglomeration, resulting in uniform particle size dispersion. These findings provide valuable mechanical insights into the generation of biogenic metal nanoparticles and show potential industrial and environmental applications, especially in accelerating oxygen reduction reactions.

Efficient nitrate and Cr(VI) removal by denitrifier: The mechanism of S. oneidensis MR-1 promoting electron production, transportation and consumption.

When Cr(VI) and nitrate coexist, the efficiency of both bio-denitrification and Cr(VI) bio-reduction is poor because chromate hinders bacterial normal functions (i.e., electron production, transportation and consumption). Moreover, under anaerobic condition, the method about efficient nitrate and Cr(VI) removal remained unclear. In this paper, the addition of Shewanella oneidensis MR-1 to promote the electron production, transportation and consumption of denitrifier and cause an increase in the removal of nitrate and Cr(VI). The efficiency of nitrate and Cr(VI) removal accomplished by P. denitrificans as a used model denitrifier increased respectively from 51.3% to 96.1% and 34.3% to 99.8% after S. oneidensis MR-1 addition. The mechanism investigations revealed that P. denitrificans provided S. oneidensis MR-1 with lactate, which was utilized to secreted riboflavin and phenazine by S. oneidensis MR-1. The riboflavin served as coenzymes of cellular reductants (i.e., thioredoxin and glutathione) in P. denitrificans, which created favorable intracellular microenvironment conditions for electron generation. Meanwhile, phenazine promoted biofilm formation, which increased the adsorption of Cr(VI) on the cell surface and accelerated the Cr(VI) reduction by membrane bound chromate reductases thereby reducing damage to other enzymes respectively. Overall, this strategy reduced the negative effect of chromate, thus improved the generation, transportation, and consumption of electrons. SYNOPSIS: The presence of S. oneidensis MR-1 facilitated nitrate and Cr(VI) removal by P. denitrificans through decreasing the negative effect of chromate due to the metabolites' secretion.

Shewanella oneidensis MR-1 dissimilatory reduction of ferrihydrite to highly enhance mineral transformation and reactive oxygen species production in redox-fluctuating environments.

Diverse paths generated by reactive oxygen species (ROS) can mediate contaminant transformation and fate in the soil/aquatic environments. However, the pathways for ROS production upon the oxygenation of redox-active ferrous iron minerals are underappreciated. Ferrihydrite (Fh) can be reduced to produce Fe(II) by Shewanella oneidensis MR-1, a representative strain of dissimilatory iron-reducing bacteria (DIRB). The microbial reaction formed a spent Fh product named mr-Fh that contained Fe(II). Material properties of mr-Fh were characterized with X-ray diffraction (XRD), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). Magnetite could be observed in all mr-Fh samples produced over 1-day incubation, which might greatly favor the Fe(II) oxygenation process to produce hydroxyl radical (•OH). The maximum amount of dissolved Fe(II) can reach 1.1 mM derived from added 1 g/L Fh together with glucose as a carbon source, much higher than the 0.5 mM generated in the case of the Luria-Bertani carbon source. This may confirm that MR-1 can effectively reduce Fh and produce biogenetic Fe(II). Furthermore, the oxygenation of Fe(II) on the mr-Fh surface can produce abundant ROS, wherein the maximum cumulative •OH content is raised to about 120 µM within 48 h at pH 5, but it is decreased to about 100 µM at pH 7 for the case of MR-1/Fh system after a 7-day incubation. Thus, MR-1-mediated Fh reduction is a critical link to enhance ROS production, and the •OH species is among them the predominant form. XPS analysis proves that a conservable amount of Fe(II) species is subject to adsorption onto mr-Fh. Here, MR-1-mediated ROS production is highly dependent on the redox activity of the form Fe(II), which should be the counterpart presented as the adsorbed Fe(II) on surfaces. Hence, our study provides new insights into understanding the mechanisms that can significantly govern ROS generation in the redox-oscillation environment.

Mechanisms of oxidative response during biodegradation of malathion by S. oneidensis MR-1.

Malathion, an extensively used organophosphorus pesticide, poses a high potential risk of toxicity to humans and the environment. Shewanella (S.) oneidensis MR-1 has been proposed as a strain with excellent bioremediation capabilities, capable of efficiently removing a wide range of hard-to-degrade pollutants. However, the physiological and biochemical response of S. oneidensis MR-1 to malathion is unknown. Therefore, this study aimed to examine how S. oneidensis MR-1 responds physiologically and biochemically to malathion while also investigating the biodegradation properties of the pesticide. The results showed that the 7-day degradation rates of S. oneidensis MR-1 were 84.1, 91.6, and 94.0% at malathion concentrations of 10, 20, and 30 mg/L, respectively. As the concentration of malathion increased, superoxide dismutase and catalase activities were inhibited, leading to a significant rise in malondialdehyde content. This outcome can be attributed to the excessive production of reactive oxygen species (ROS) triggered by malathion stress. In addition, ROS production stimulates the secretion of soluble polysaccharides, which alleviates oxidative stress caused by malathion. Malathion-induced oxidative damage further exacerbated the changes in the cellular properties of S. oneidensis MR-1. During the initial stages of degradation, the cell density and total intracellular protein increased significantly with increasing malathion exposure. This can be attributed to the remarkable resistance of S. oneidensis MR-1 to malathion. Based on scanning electron microscopy observations, continuous exposure to contaminants led to a reduction in biomass and protein content, resulting in reduced cell activity and ultimately leading to cell rupture. In addition, this was accompanied by a decrease in Na+/K+- ATPase and Ca2+/Mg2+-ATPase levels, suggesting that malathion-mediated oxidative stress interfered with energy metabolism in S. oneidensis MR-1. The findings of this study provide new insights into the environmental risks associated with organophosphorus pesticides, specifically malathion, and their potential for bioremediation.

Colony-Based Electrochemistry Reveals Electron Conduction Mechanisms Mediated by Cytochromes and Flavins in Shewanella oneidensis.

Bacteria utilize electron conduction in their communities to drive their metabolism, which has led to the development of various environmental technologies, such as electrochemical microbial systems and anaerobic digestion. It is challenging to measure the conductivity among bacterial cells when they hardly form stable biofilms on electrodes. This makes it difficult to identify the biomolecules involved in electron conduction. In the present study, we aimed to identify c-type cytochromes involved in electron conduction in Shewanella oneidensis MR-1 and examine the molecular mechanisms. We established a colony-based bioelectronic system that quantifies bacterial electrical conductivity, without the need for biofilm formation on electrodes. This system enabled the quantification of the conductivity of gene deletion mutants that scarcely form biofilms on electrodes, demonstrating that c-type cytochromes, MtrC and OmcA, are involved in electron conduction. Furthermore, the use of colonies of gene deletion mutants demonstrated that flavins participate in electron conduction by binding to OmcA, providing insight into the electron conduction pathways at the molecular level. Furthermore, phenazine-based electron transfer in Pseudomonas aeruginosa PAO1 and flavin-based electron transfer in Bacillus subtilis 3610 were confirmed, indicating that this colony-based system can be used for various bacteria, including weak electricigens.

Application of dissimilatory iron-reducing bacteria for the remediation of soil and water polluted with chlorinated organic compounds: Progress, mechanisms, and directions.

Chlorinated organic compounds are widely used as solvents, but they are pollutants that can have adverse effects on the environment and human health. Dissimilatory iron-reducing bacteria (DIRB) such as Shewanella and Geobacter have been applied to treat a wide range of halogenated organic compounds due to their specific biological properties. Until now, there has been no systematic review on the mechanisms of direct or indirect degradation of halogenated organic compounds by DIRB. This work summarizes the discussion of DIRB's ability to enhance the dechlorination of reaction systems through different pathways, both biological and biochemical. For biological dechlorination, some DIRB have self-dechlorination capabilities that directly dechlorinate by hydrolysis. Adjustment of dechlorination genes through genetic engineering can improve the dechlorination capabilities of DIRB. DIRB can also adjust the capacity for the microbial community to dechlorinate and provide nutrients to enhance the expression of dechlorination genes in other bacteria. In biochemical dechlorination, DIRB bioconverts Fe(III) to Fe(II), which is capable of dichlorination. On this basis, the DIRB-driven Fenton reaction can efficiently degrade chlorinated organics by continuously maintaining anoxic conditions to generate Fe(II) and oxic conditions to generate H2O2. DIRB can drive microbial fuel cells due to their electroactivity and have a good dechlorination capacity at low levels of energy consumption. The contribution of DIRB to the removal of pesticides, antibiotics and POPs is summarized. Then the DIRB electron transfer mechanism is discussed, which is core to their ability to dechlorinate. Finally, the prospect of future work on the removal of chlorine-containing organic pollutants by DIRB is presented, and the main challenges and further research directions are suggested.

Extracellular organic disulfide reduction by Shewanella oneidensis MR-1.

Microbial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c-type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling.IMPORTANCEOrganic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c-type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems.

DIET-like and MIET-like mutualism of S. oneidensis MR-1 and metal-reducing function microflora boosts Cr(VI) reduction.

Microbial treatment of Cr(VI) is an environmentally friendly and low-cost approach. However, the mechanism of mutualism and the role of interspecies electron transfer in Cr(VI) reducing microflora are unclear. Herein, we constructed an intersymbiotic microbial association flora to augment interspecies electron transfer via functionalizing electroactive Shewanella oneidensis MR-1 with metal-reducing microflora, and thus the efficiency of Cr(VI) reduction. The findings suggest that the metal-reducing active microflora could converts glucose into lactic acid and riboflavin for S. oneidensis MR-1 to act as a carbon source and electron mediator. Thus, when adding initial 25 mg/L Cr (VI), this microflora exhibited an outstanding Cr (VI) removal efficiency (100%) at 12 h and elevated Cr (III) immobilization efficiency (80%) at 60 h with the assistance of 25 mg/L Cu(II). A series of electrochemical experiments proved this remarkable removal efficiency were ascribed to the improved interspecies electron transfer efficiency through direct interspecies electron transfer and riboflavin through mediated interspecies electron transfer. Furthermore, the metagenomic analysis revealed the expression level of the electron transport pathway was promoted. Intriguing high abundance of genes participating in the bio-reduction and biotransformation of Cr(VI) was also observed in functional microflora. These outcomes give a novel strategy for enhancing the reduction and fixation of harmful heavy metals by coculturing function microflora with electrogenic microorganisms.

Acetylation of xenogeneic silencer H-NS regulates biofilm development through the nitrogen homeostasis regulator in Shewanella.

Adjusting intracellular metabolic pathways and adopting suitable live state such as biofilms, are crucial for bacteria to survive environmental changes. Although substantial progress has been made in understanding how the histone-like nucleoid-structuring (H-NS) protein modulates the expression of the genes involved in biofilm formation, the precise modification that the H-NS protein undergoes to alter its DNA binding activity is still largely uncharacterized. This study revealed that acetylation of H-NS at Lys19 inhibits biofilm development in Shewanella oneidensis MR-1 by downregulating the expression of glutamine synthetase, a critical enzyme in glutamine synthesis. We further found that nitrogen starvation, a likely condition in biofilm development, induces deacetylation of H-NS and the trimerization of nitrogen assimilation regulator GlnB. The acetylated H-NS strain exhibits significantly lower cellular glutamine concentration, emphasizing the requirement of H-NS deacetylation in Shewanella biofilm development. Moreover, we discovered in vivo that the activation of glutamine biosynthesis pathway and the concurrent suppression of the arginine synthesis pathway during both pellicle and attached biofilms development, further suggesting the importance of fine tune nitrogen assimilation by H-NS acetylation in Shewanella. In summary, posttranslational modification of H-NS endows Shewanella with the ability to respond to environmental needs by adjusting the intracellular metabolism pathways.

Co-metabolic degradation and metabolite detection of hexabromocyclododecane by Shewanella oneidensis MR-1.

Hexabromocyclododecane (HBCD) is a widely used brominated flame retardant; however, it is a persistent organic pollutant as well as affects the human thyroid hormones and causes cancer. However, the degradation of HBCD has received little attention from researchers. Due to its bioaccumulative and hazardous properties, an appropriate strategy for its remediation is required. In this study, we investigated the biodegradation of HBCD using Shewanella oneidensis MR-1 under optimized conditions. The Box-Behnken design (BBD) was implemented for the optimization of the physical degradation parameters of HBCD. S. oneidensis MR-1 showed the best degradation performance at a temperature of 30 °C, pH 7, and agitation speed of 115 rpm, with an HBCD concentration of 1125 µg/L in mineral salt medium (MSM). The strain tolerated up to 2000 µg/L HBCD. Gas chromatography-mass spectrometry analysis identified three intermediates, including 2-bromo dodecane, 2,7,10-trimethyldodecane, and 4-methyl-1-decene. The results provide an insightful understanding of the biodegradation of HBCD by S. oneidensis MR-1 under optimized conditions and could pave the way for further eco-friendly applications. KEY POINTS: • HBCD biodegradation by Shewanella oneidensis • Optimization of HBCD biodegradation by the Box-Behnken analysis • Identification of useful metabolites from HBCD degradation.

The electron transport chain of Shewanella oneidensis MR-1 can operate bidirectionally to enable microbial electrosynthesis.

Extracellular electron transfer is a process by which bacterial cells can exchange electrons with a redox-active material located outside of the cell. In Shewanella oneidensis, this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive the microbial electrosynthesis (MES) of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (BDH). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via BDH. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and oxygen (O2) reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical system (BES), and the translocation of protons across the membrane during O2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death.IMPORTANCEMicrobial electrosynthesis (MES) is increasingly employed for the generation of specialty chemicals, such as biofuels, bioplastics, and cancer therapeutics. For these systems to be viable for industrial scale-up, it is important to understand the energetic requirements of the bacteria to mitigate unnecessary costs. This work demonstrates sustained production of an industrially relevant chemical driven by a cathode. Additionally, it optimizes a previously published system by removing any requirement for phototrophic energy, thereby removing the additional cost of providing a light source. We also demonstrate the severe impact of oxygen intrusion into bioelectrochemical systems, offering insight to future researchers aiming to work in an anaerobic environment. These studies provide insight into both the thermodynamics of electrosynthesis and the importance of the bioelectrochemical systems' design.

Anaerobic biodegradation of azo dye reactive black 5 by a novel strain Shewanella sp. SR1: Pathway and mechanisms.

The efficiency of microbial populations in degrading refractory pollutants and the impact of adverse environmental factors often presents challenges for the biological treatment of azo dyes. In this study, the genome analysis and azo dye Reactive Black 5 (RB5) degrading capability of a newly isolated strain, Shewanella sp. SR1, were investigated. By analyzing the genome, functional genes involved in dye degradation and mechanisms for adaptation to low-temperature and high-salinity conditions were identified in SR1. The addition of co-substrates, such as glucose and yeast extract, significantly enhanced RB5 decolorization efficiency, reaching up to 87.6%. Notably, SR1 demonstrated remarkable robustness towards a wide range of NaCl concentrations (1-30 g/L) and temperatures (10-30 °C), maintaining efficient decolorization and high biomass concentration. The metabolic pathways of RB5 degradation were deduced based on the metabolites and genes detected in the genome, in which the azo bond was first cleaved by FMN-dependent NADH-azoreductase and NAD(P)H-flavin reductase, followed by deamination, desulfonation, and hydroxylation mediated by various oxidoreductases. Importantly, the degradation metabolites exhibited reduced toxicity, as revealed by toxicity analysis. These findings highlighted the great potential of Shewanella sp. SR1 for bioremediation of wastewaters contaminated with azo dyes.

Metabolic regulation of Shewanella oneidensis for microbial electrosynthesis: From extracellular to intracellular.

Shewanella oneidensis MR-1 (S. oneidensis MR-1) has been shown to benefit from microbial electrosynthesis (MES) due to its exceptional electron transfer efficiency. In this study, genes involved in both extracellular electron uptake (EEU) and intracellular CO2 conversion processes were examined and regulated to enhance MES performance. The key genes identified for MES in the EEU process were mtrB, mtrC, mtrD, mtrE, omcA and cctA. Overexpression of these genes resulted in 1.5-2.1 times higher formate productivity than that of the wild-type strains (0.63 mmol/(L·µg protein)), as 0.94-1.61 mmol/(L·µg protein). In the intracellular CO2 conversion process, overexpression of the nadE, nadD, nadR, nadV, pncC and petC genes increased formate productivity 1.3-fold-3.4-fold. Moreover, overexpression of the formate dehydrogenase genes fdhA1, fdhB1 and fdhX1 in modified strains led to a 2.3-fold-3.1-fold increase in formate productivity compared to wild-type strains. The co-overexpression of cctA, fdhA1 and nadV in the mutant strain resulted in 5.59 times (3.50 mmol/(L·µg protein)) higher formate productivity than that of the wild-type strains. These findings revealed that electrons of MES derived from the electrode were utilized in the energy module for synthesizing ATP and NADH, followed by the synthesis of formate in formate dehydrogenase by the combinatorial effects of ATP, NADH, electrons and CO2. The results provide new insights into the mechanism of MES in S. oneidensis MR-1 and pave the way for genetic improvements that could facilitate the further application of MES.

Enforcing energy consumption promotes microbial extracellular respiration for xenobiotic bioconversion.

Extracellular electron transfer (EET) empowers electrogens to catalyse the bioconversion of a wide range of xenobiotics in the environment. Synthetic bioengineering has proven effective in promoting EET output. However, conventional strategies mainly focus on modifications of EET-related genes or pathways, which leads to a bottleneck due to the intricate nature of electrogenic metabolic properties and intricate pathway regulation that remain unelucidated. Herein, we propose a novel EET pathway-independent approach, from an energy manipulation perspective, to enhance microbial EET output. The Controlled Hydrolyzation of ATP to Enhance Extracellular Respiration (CHEER) strategy promotes energy utilization and persistently reduces the intracellular ATP level in Shewanella oneidensis, a representative electrogenic microbe. This approach leads to the accelerated consumption of carbon substrate, increased biomass accumulation and an expanded intracellular NADH pool. Both microbial electrolysis cell and microbial fuel cell tests exhibit that the CHEER strain substantially enhances EET capability. Analysis of transcriptome profiles reveals that the CHEER strain considerably bolsters biomass synthesis and metabolic activity. When applied to the bioconversion of model xenobiotics including methyl orange, Cr(VI) and U(VI), the CHEER strain consistently exhibits enhanced removal efficiencies. This work provides a new perspective and a feasible strategy to enhance microbial EET for efficient xenobiotic conversion.

Electron Transfer Mechanism at the Interface of Multi-Heme Cytochromes and Metal Oxide.

Electroactive microbial cells have evolved unique extracellular electron transfer to conduct the reactions via redox outer-membrane (OM) proteins. However, the electron transfer mechanism at the interface of OM proteins and nanomaterial remains unclear. In this study, the mechanism for the electron transfer at biological/inorganic interface is investigated by integrating molecular modeling with electrochemical and spectroscopic measurements. For this purpose, a model system composed of OmcA, a typical OM protein, and the hexagonal tungsten trioxide (h-WO3 ) with good biocompatibility is selected. The interfacial electron transfer is dependent mainly on the special molecular configuration of OmcA and the microenvironment of the solvent exposed active center. Also, the apparent electron transfer rate can be tuned by site-directed mutagenesis at the axial ligand of the active center. Furthermore, the equilibrium state of the OmcA/h-WO3 systems suggests that their attachment is attributed to the limited number of residues. The electrochemical analysis of OmcA and its variants reveals that the wild type exhibits the fastest electron transfer rate, and the transient absorption spectroscopy further shows that the axial histidine plays an important role in the interfacial electron transfer process. This study provides a useful approach to promote the site-directed mutagenesis and nanomaterial design for bioelectrocatalytic applications.

A common inducer molecule enhances sugar utilization by Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is an electroactive bacterium that is a promising host for bioelectrochemical technologies, which makes it a common target for genetic engineering, including gene deletions and expression of heterologous pathways. Expression of heterologous genes and gene knockdown via CRISPRi in S. oneidensis are both frequently induced by ß-D-1-thiogalactopyranoside (IPTG), a commonly used inducer molecule across many model organisms. Here, we report and characterize an unexpected phenotype; IPTG enhances the growth of wild-type S. oneidensis MR-1 on the sugar substrate N-acetylglucosamine (NAG). IPTG improves the carrying capacity of S. oneidensis growing on NAG while the growth rate remains similar to cultures without the inducer. Extracellular acetate accumulates faster and to a higher concentration in cultures without IPTG than those with it. IPTG appears to improve acetate metabolism, which combats the negative effect that acetate accumulation has on the growth of S. oneidensis with NAG. We recommend using extensive experimental controls and careful data interpretation when using both NAG and IPTG in S. oneidensis cultures.

Bacterial TANGO2 homologs are heme-trafficking proteins that facilitate biosynthesis of cytochromes c.

Heme, an essential molecule for virtually all living organisms, acts primarily as a cofactor in a large number of proteins. However, how heme is mobilized from the site of synthesis to the locations where hemoproteins are assembled remains largely unknown in cells, especially bacterial ones. In this study, with Shewanella oneidensis as the model, we identified HtpA (SO0126) as a heme-trafficking protein and homolog of TANGO2 proteins found in eukaryotes. We showed that HtpA homologs are widely distributed in all domains of living organisms and have undergone parallel evolution. In its absence, the cytochrome (cyt) c content and catalase activity decreased significantly. We further showed that both HtpA and representative TANGO2 proteins bind heme with 1:1 stoichiometry and a relatively low dissociation constant. Protein interaction analyses substantiated that HtpA directly interacts with the cytochrome c maturation system. Our findings shed light on cross-membrane transport of heme in bacteria and extend the understanding of TANGO2 proteins. IMPORTANCE The intracellular trafficking of heme, an essential cofactor for hemoproteins, remains underexplored even in eukaryotes, let alone bacteria. Here we developed a high-throughput method by which HtpA, a homolog of eukaryotic TANGO2 proteins, was identified to be a heme-binding protein that enhances cytochrome c biosynthesis and catalase activity in Shewanella oneidensis. HtpA interacts with the cytochrome c biosynthesis system directly, supporting that this protein, like TANGO2, functions in intracellular heme trafficking. HtpA homologs are widely distributed, but a large majority of them were found to be non-exchangeable, likely a result of parallel evolution. By substantiating the heme-trafficking nature of HtpA and its eukaryotic homologs, our findings provide general insight into the heme-trafficking process and highlight the functional conservation along evolution in all living organisms.

Extracellular polymeric substances sustain photoreduction of Cr(VI) by Shewanella oneidensis-CdS biohybrid system.

Photosensitized biohybrid system (PBS) enables bacteria to exploit light energy harvested by semiconductors for rapid pollutants transformation, possessing a promising future for water reclamation. Maintaining a biocompatible environment under photocatalytic conditions is the key to developing PBS-based treatment technologies. Natural microbial cells are surrounded by extracellular polymeric substances (EPS) that either be tightly bound to the cell wall (i.e., tightly bound EPS, tbEPS) or loosely associated with cell surface (i.e., loosely bound EPS, lbEPS), which provide protection from unfavorable environment. We hypothesized that providing EPS fractions can enhance bacterial viability under adverse environment created by photocatalytic reactions. We constructed a model PBS consisting of Shewanella oneidensis and CdS using Cr(VI) as the target pollutant. Results showed complete removal of 25 mg/L Cr(VI) within 90 min without an electron donor, which may mainly rely on the synergistic effect of CdS and bacteria on photoelectron transfer. Long-term cycling experiment of pristine PBS and PBS with extra EPS fractions (including lbEPS and tbEPS) for Cr(VI) treatment showed that PBS with extra lbEPS achieved efficient Cr(VI) removal within five consecutive batch treatment cycles, compared to the three cycles both in pristine PBS and PBS with tbEPS. After addition of lbEPS, the accumulation of reactive oxygen species (ROS) was greatly reduced via the EPS-capping effect and quenching effect, and the toxic metal internalization potential was lowered by complexation with Cd and Cr, resulting in enhanced bacterial viability during photocatalysis. This facile and efficient cytoprotective method helps the rational design of PBS for environmental remediation.