Engineering bionanoreactor in bacteria for efficient hydrogen production.

Hydrogen production through water splitting is a vital strategy for renewable and sustainable clean energy. In this study, we developed an approach integrating nanomaterial engineering and synthetic biology to establish a bionanoreactor system for efficient hydrogen production. The periplasmic space (20 to 30 nm) of an electroactive bacterium, Shewanella oneidensis MR-1, was engineered to serve as a bionanoreactor to enhance the interaction between electrons and protons, catalyzed by hydrogenases for hydrogen generation. To optimize electron transfer, we used the microbially reduced graphene oxide (rGO) to coat the electrode, which improved the electron transfer from the electrode to the cells. Native MtrCAB protein complex on S. oneidensis and self-assembled iron sulfide (FeS) nanoparticles acted in tandem to facilitate electron transfer from an electrode to the periplasm. To enhance proton transport, S. oneidensis MR-1 was engineered to express Gloeobacter rhodopsin (GR) and the light-harvesting antenna canthaxanthin. This led to efficient proton pumping when exposed to light, resulting in a 35.6% increase in the rate of hydrogen production. The overexpression of native [FeFe]-hydrogenase further improved the hydrogen production rate by 56.8%. The bionanoreactor engineered in S. oneidensis MR-1 achieved a hydrogen yield of 80.4 µmol/mg protein/day with a Faraday efficiency of 80% at a potential of -0.75 V. This periplasmic bionanoreactor combines the strengths of both nanomaterial and biological components, providing an efficient approach for microbial electrosynthesis.

Implications of ammonia stress for the pathogenicity of Shewanella spp. in Oreochromis niloticus: effects on hematological, biochemical, immunological, and histopathological parameters.

Environmental stressors (such as ammonia) in aquaculture could increase the risk of pathogenicity, posing a more severe threat to farmed fish. The aim of this study was to investigate the effects of ammonia stress on the pathogenicity of Shewanella spp. in Oreochromis niloticus. First, a 96-hour static test was used to determine the median lethal concentration (LC50) of unionized ammonia to Nile tilapia. After 96 h of exposure, the Un-ionized ammonia (UIA) LC50 was estimated to be 4.26 mg/L. Second, an experiment was conducted to test the effect of unionized ammonia stress on the pathogenicity of Shewanella spp. in O. niloticus for 30 days. A study involved 180 fish divided into six groups, with the first group serving as a control. The second group (AMN1/10) and the third group (AMN1/20) were not challenged and were exposed to 1/10 (0.42 mg/L) and 1/20 (0.21 mg/L) of the 96-hour LC50 of UIA, respectively. Then 0.2 mL (0.14 × 105) of Shewanella spp. was intraperitoneally injected into the fourth (SH), fifth (SH + AMN1/10), and sixth (SH + AMN1/20) groups, which were subjected to 0, 1/10 (0.42 mg/L), and 1/20 (0.21 mg/L) of the 96-hour LC50 of UIA, respectively. The survival rate, hematological indices, immunological parameters, and antioxidant activity of the fish significantly decreased when they were exposed to ammonia and Shewanella infection separately or together. Histopathological changes were also observed in the kidney and liver. Furthermore, both individual and combined exposures significantly altered renal and hepatic function, with notable increases in glucose and cortisol levels, as well as in the expression of proinflammatory cytokine genes (TNF-α and IL-1ß). However, the detrimental effects of co-exposure to ammonia stress and Shewanella infection were greater than those of separate exposures. As a result, we may say that increased ammonia concentrations enhance the infection of Shewanella spp. These findings could contribute to a better understanding of Shewanella infection in Nile tilapia.

A large scale bacterial attraction assay: A new quantitative bacterial migration assay suitable for genetic screens.

Bacteria use various motility mechanisms to explore their environments. Chemotaxis is the ability of a motile bacterial cell to direct its movement in response to chemical gradients. A number of methods have been developed and widely used to study chemotactic responses to chemoeffectors including capillary, agar plug, microscopic slide, and microfluidic assays. While valuable, these assays are primarily designed to monitor rapid chemotactic responses to chemoeffectors on a small scale, which poses challenges in collecting large quantities of attracted bacteria. Consequently, these setups are not ideal for experiments like forward genetic screens. To overcome this limitation, we developed the Large Scale Bacterial Attraction assay (LSBA), which relies on the use of a Nalgene™ Reusable Filter Unit and other materials commonly found in laboratories. We validate the LSBA by investigating chemoeffector kinetics in the setup and by using chemoattractants to quantify the chemotactic response of wild-type, and motility impaired strains of the plant pathogenic bacterium Xanthomonas campestris pv. campestris and the environmental bacterium Shewanella oneidensis. We show that the LSBA establishes a long lasting chemoeffector gradient, that the setup can be used to quantify bacterial migration over time and that the LSBA offers the possibility to collect high numbers of attracted bacteria, making it suitable for genetic screens.

Fabrication of bio-abiotic hybrid living hydrogel for bifunctional electrochemical conversion.

Design and intelligent use renewable natural bioenergy is an important challenge. Electric microorganism-based materials are being serve as an important part of bioenergy devices for energy release and collection, calling for suitable skeleton materials to anchor live microbes. Herein we verified the feasibility of constructing bio-abiotic hybrid living materials based on the combination of gelatin, Li-ions and exoelectrogenic bacteria Shewanella oneidensis manganese-reducing-1 (MR-1). The gelatin-based mesh contains abundant pores, allowing microbes to dock and small molecules to diffuse. The hybrid materials hold plentiful electronegative groups, which effectively anchor Li-ions and facilitate their transition. Moreover, the electrochemical characteristics of the materials can be modulated through changing the ratios of gelatin, bacteria and Li-ions. Based on the gelatin-Li-ion-microorganism hybrid materials, a bifunctional device was fabricated, which could play dual roles alternatively, generation of electricity as a microbial fuel cell and energy storage as a pseudocapacitor. The capacitance and the maximum voltage output of the device reaches 68 F g-1 and 0.67 V, respectively. This system is a new platform and fresh start to fabricate bio-abiotic living materials for microbial electron storage and transfer. We expect the setup will extend to other living systems and devices for synthetic biological energy conversion.

Evolution of paralogous multicomponent systems for site-specific O-sialylation of flagellin in Gram-negative and Gram-positive bacteria.

Many bacteria glycosylate flagellin on serine or threonine residues using pseudaminic acid (Pse) or other sialic acid-like donor sugars. Successful reconstitution of Pse-dependent sialylation by the conserved Maf-type flagellin glycosyltransferase (fGT) may require (a) missing component(s). Here, we characterize both Maf paralogs in the Gram-negative bacterium Shewanella oneidensis MR-1 and reconstitute Pse-dependent glycosylation in heterologous hosts. Remarkably, we uncovered distinct acceptor determinants and target specificities for each Maf. Whereas Maf-1 uses its C-terminal tetratricopeptide repeat (TPR) domain to confer flagellin acceptor and O-glycosylation specificity, Maf-2 requires the newly identified conserved specificity factor, glycosylation factor for Maf (GlfM), to form a ternary complex with flagellin. GlfM orthologs are co-encoded with Maf-2 in Gram-negative and Gram-positive bacteria and require an invariant aspartate in their four-helix bundle to function with Maf-2. Thus, convergent fGT evolution underlies distinct flagellin-binding modes in tripartite versus bipartite systems and, consequently, distinct O-glycosylation preferences of acceptor serine residues with Pse.

Elucidation of high-pressure processing toward microbial inhibition, physicochemical properties, collagen fiber and muscle structure of blood clam edible portion.

Impact of high-pressure processing (HP-P) on microbial inactivation, protein oxidation, collagen fiber, and muscle structure of the edible portion (EP) of blood clams (BC) was investigated. Aerobic plate count, Vibrio parahaemolyticus, V. vulnificus, other Vibrio spp. and Shewanella algae counts were not detectable when HP-P pressure of ≥300 MPa was applied. Carbonyl, disulphide bond content, and surface hydrophobicity upsurged as HP-P with augmenting pressure was employed. Protein with ∼53 kDa appeared when HP-P at 100 and 200 MPa was implemented. Increased pressure enhanced gap formation and abnormal muscle cell structure arrangements. HP-P also affected connective tissue, causing size reduction and disruption of the collagen filament fibers. However, firmness and toughness of BC-EP with HP-P ≤ 300 MPa were comparable to those of the control. HP-P at 300 MPa was therefore appropriate for treatment of BC with maintained textural properties, while less protein oxidation, collagen fiber and muscle structure disruption occurred.

Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

Chlorophyll a acts as a natural photosensitizer to drive nitrate reduction in nonphotosynthetic microorganisms.

With the death and decomposition of widely distributed photosynthetic organisms, free natural pigments are often detected in surface water, sediment and soil. Whether free pigments can act as photosensitizers to drive biophotoelectrochemical metabolism in nonphotosynthetic microorganisms has not been reported. In this work, we provide direct evidence for the photoelectrophic relationship between extracellular chlorophyll a (Chl a) and nonphotosynthetic microorganisms. The results show that 10 µg of Chl a can produce significant photoelectrons (∼0.34 A/cm2) upon irradiation to drive nitrate reduction in Shewanella oneidensis. Chl a undergoes structural changes during the photoelectric process, thus the ability of Chl a to generate a photocurrent decreases gradually with increasing illumination time. These changes are greater in the presence of microorganisms than in the absence of microorganisms. Photoelectron transport from Chl a to S. oneidensis occurs through a direct pathway involving the cytochromes MtrA, MtrB, MtrC and CymA but not through an indirect pathway involving riboflavin. These findings reveal a novel photoelectrotrophic linkage between natural photosynthetic pigments and nonphototrophic microorganisms, which has important implications for the biogeochemical cycle of nitrogen in various natural environments where Chl a is distributed.

Enhancing extracellular membrane vesicle productivity of Shewanella vesiculosa HM13, a prospective host for vesiculation-mediated protein secretion, by weakening outer membrane-peptidoglycan linkage.

Shewanella vesiculosa HM13, a psychrotrophic gram-negative bacterium isolated from the intestinal contents of horse mackerel, produces abundant extracellular membrane vesicles (EMVs) by budding the outer membrane. The EMVs of this bacterium carry a single major cargo protein, P49, of unknown function, which may be useful as a carrier for the secretory production of heterologous proteins as cargoes of EMVs. In this study, to increase the utility of S. vesiculosa HM13 as a host for EMV-mediated protein production, we improved its EMV productivity by weakening the linkage between the outer membrane and underlying peptidoglycan layer. In gram-negative bacteria, the outer membrane is connected to peptidoglycans predominantly through Braun's lipoprotein (Lpp), and the formation of this linkage is catalyzed by an l,d-transpeptidase (Ldt). We constructed gene-disrupted mutants of Lpp and Ldt and assessed their EMV productivity. The EMVs of the lpp- and ldt-disrupted mutants grown at 18 °C were evaluated using nanoparticle tracking analysis, and their morphologies were observed using transmission electron microscopy. As a result, an approximately 2.5-fold increase in EMV production was achieved, whereas the morphology of the EMVs of these mutants remained almost identical to that of the parent strain. In accordance with the increase in EMV production, the mutants secreted approximately 2-fold higher amounts of P49 than the parent strain into the culture broth as the EMV cargo. These findings will contribute to the development of an EMV-based secretory production system for heterologous proteins using S. vesiculosa HM13 as a host.

Morphological, Microstructural, and In Situ Chemical Characteristics of Siderite Produced by Iron-Reducing Bacteria.

Dissimilatory iron-reducing bacteria (DIRB) oxidize organic matter or hydrogen and reduce ferric iron to form Fe(II)-bearing minerals, such as magnetite and siderite. However, compared with magnetite, which was extensively studied, the mineralization process and mechanisms of siderite remain unclear. Here, with the combination of advanced electron microscopy and synchrotron-based scanning transmission X-ray microscopy (STXM) approaches, we studied in detail the morphological, structural, and chemical features of biogenic siderite via a growth experiment with Shewanella oneidensis MR-4. Results showed that along with the growth of cells, Fe(II) ions were increasingly released into solution and reacted with CO32- to form micrometer-sized siderite minerals with spindle, rod, peanut, dumbbell, and sphere shapes. They are composed of many single-crystal siderite plates that are fanned out from the center of the particles. Additionally, STXM revealed Fh and organic molecules inside siderite. This suggests that the siderite crystals might assemble around a Fh-organic molecule core and then continue to grow radially. This study illustrates the biomineralization and assembly of siderite by a successive multistep growth process induced by DIRB, also provides evidences that the distinctive shapes and the presence of organic molecules inside might be morphological and chemical features for biogenic siderite.

Dual-mode harvest solar energy for photothermal Cu2-xSe biomineralization and seawater desalination by biotic-abiotic hybrid.

Biotic-abiotic hybrid photocatalytic system is an innovative strategy to capture solar energy. Diversifying solar energy conversion products and balancing photoelectron generation and transduction are critical to unravel the potential of hybrid photocatalysis. Here, we harvest solar energy in a dual mode for Cu2-xSe nanoparticles biomineralization and seawater desalination by integrating the merits of Shewanella oneidensis MR-1 and biogenic nanoparticles. Photoelectrons generated by extracellular Se0 nanoparticles power Cu2-xSe synthesis through two pathways that either cross the outer membrane to activate periplasmic Cu(II) reduction or are directly delivered into the extracellular space for Cu(I) evolution. Meanwhile, photoelectrons drive periplasmic Cu(II) reduction by reversing MtrABC complexes in S. oneidensis. Moreover, the unique photothermal feature of the as-prepared Cu2-xSe nanoparticles, the natural hydrophilicity, and the linking properties of bacterium offer a convenient way to tailor photothermal membranes for solar water production. This study provides a paradigm for balancing the source and sink of photoelectrons and diversifying solar energy conversion products in biotic-abiotic hybrid platforms.

A biocompatible electrode/exoelectrogens interface augments bidirectional electron transfer and bioelectrochemical reactions.

Bidirectional electron transfer is about that exoelectrogens produce bioelectricity via extracellular electron transfer at anode and drive cytoplasmic biochemical reactions via extracellular electron uptake at cathode. The key factor to determine above bioelectrochemical performances is the electron transfer efficiency under biocompatible abiotic/biotic interface. Here, a graphene/polyaniline (GO/PANI) nanocomposite electrode specially interfacing exoelectrogens (Shewanella loihica) and augmenting bidirectional electron transfer was conducted by in-situ electrochemical modification on carbon paper (CP). Impressively, the GO/PANI@CP electrode tremendously improved the performance of exoelectrogens at anode for wastewater treatment and bioelectricity generation (about 54 folds increase of power density compared to blank CP electrode). The bacteria on electrode surface not only showed fast electron release but also exhibited high electricity density of extracellular electron uptake through the proposed direct electron transfer pathway. Thus, the cathode applications of microbial electrosynthesis and bio-denitrification were developed via GO/PANI@CP electrode, which assisted the close contact between microbial outer-membrane cytochromes and nanocomposite electrode for efficient nitrate removal (0.333 mM/h). Overall, nanocomposite modified electrode with biocompatible interfaces has great potential to enhance bioelectrochemical reactions with exoelectrogens.

Total Synthesis of the Conjugation-Ready Tetrasaccharide Repeating Unit of Shewanella japonica Type Strain KMM 3299T.

Here we report the first total synthesis of the conjugation-ready tetrasaccharide repeating unit of Shewanella japonica type strain KMM 3299T. The presence of rare deoxyamino sugars and installation of three consecutive 1,2-cis glycosidic linkages makes the synthesis formidable. The challenging late-stage oxidation was overcome by using a galacturonate donor. The total synthesis was completed via a longest linear sequence of 22 steps in an overall yield of 3.5% starting from d-mannose.

Structural study of a polysaccharide component of nfnB mutant of Shewanella vesiculosa HM13.

Shewanella vesiculosa HM13 is a Gram-negative bacterium able to produce a large amount of extracellular membrane vesicles. These nanoparticles carry a major protein P49, the loading of which seems to be influenced by the glycans decorating the membrane. Here we report the structural characterization, using chemical analyses and NMR spectroscopy, of the capsular polysaccharides isolated from the nfnB-mutant strain of S. vesiculosa HM13, which is unable to load P49 on the membrane vesicles. In addition to the polysaccharide corona isolated and characterized from the parental strain, the nfnB-mutant strain released another polysaccharide composed of disaccharide repeating units having the following structure. →4)-ß-D-Glc-(1 â†’ 3)-ß-D-GlcNAc-(1→.

Shewanella phaeophyticola sp. nov. and Vibrio algarum sp. nov., isolated from marine brown algae.

Two Gram-stain-negative, rod-shaped bacteria, designated as strains KJ10-1T and KJ40-1T, were isolated from marine brown algae. Both strains were catalase-positive, oxidase-positive, and facultative aerobic. Strain KJ10-1T exhibited optimal growth at 25 °C, pH 7.0, and 3 % NaCl, whereas strain KJ40-1T showed optimal growth at 25 °C, pH 7.0, and 2 % NaCl. The respiratory quinones of strain KJ10-1T were ubiquinone-8, ubiquinone-7, menaquinone-7, and methylated menaquinone-7, while the respiratory quinone of strain KJ40-1T was only ubiquinone-8. As major fatty acids, strain KJ10-1T contained C16 : 0, C17 : 1 ω8c, iso-C15 : 0, and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and strain KJ40-1T contained C16 : 0 and summed features 3 and 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The major polar lipids in strain KJ10-1T were phosphatidylethanolamine, phosphatidylglycerol, and an unidentified aminolipid, whereas those in strain KJ40-1T were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G+C contents of strains KJ10-1T and KJ40-1T were 42.1 and 40.8 mol%, respectively. Based on 16S rRNA gene sequences, strains KJ10-1T and KJ40-1T exhibited the closest relatedness to Shewanella saliphila MMS16-UL250T (98.6 %) and Vibrio rumoiensis S-1T (95.4 %), respectively. Phylogenetic analyses, based on both 16S rRNA and 92 housekeeping genes, showed that the strains formed distinct phylogenic lineages within the genera Shewanella and Vibrio. Digital DNA-DNA hybridization and orthologous average nucleotide identity values between strain KJ10-1T and other Shewanella species, as well as between strain KJ40-1T and other Vibrio species, were below the thresholds commonly accepted for prokaryotic species delineation. Based on the phenotypic, chemotaxonomic, and phylogenetic data, strains KJ10-1T and KJ40-1T represent novel species of the genera Shewanella and Vibrio, respectively, for which the names Shewanella phaeophyticola sp. nov. and Vibrio algarum sp. nov. are proposed, respectively. The type strains of S. phaeophyticola and V. algarum are KJ10-1T (=KACC 22589T=JCM 35409T) and KJ40-1T (=KACC 22588T=JCM 35410T), respectively.

Enhanced biotoxicity by co-exposure of aged polystyrene and ciprofloxacin: the adsorption and its influence factors.

Microplastics (MPs), as emerging contaminants, usually experience aging processes in natural environments and further affect their interactions with coexisted contaminants, resulting in unpredictable ecological risks. Herein, the effect of MPs aging on their adsorption for coexisting antibiotics and their joint biotoxicity have been investigated. Results showed that the adsorption capacity of aged polystyrene (PS, 100 d and 50 d) for ciprofloxacin (CIP) was 1.10-4.09 times higher than virgin PS due to the larger BET surface area and increased oxygen-containing functional groups of aged PS. Following the increased adsorption capacity of aged PS, the joint toxicity of aged PS and CIP to Shewanella Oneidensis MR-1 (MR-1) was 1.03-1.34 times higher than virgin PS and CIP. Combined with the adsorption process, CIP posed higher toxicity to MR-1 compared to aged PS due to the rapid adsorption of aged PS for CIP in the first 12 h. After that, the adsorption process tended to be gentle and hence the joint toxicity to MR-1 was gradually dominated by aged PS. A similar transformation between the adsorption rate and the joint toxicity of PS and CIP was observed under different conditions. This study supplied a novel perception of the synergistic effects of PS aging and CIP on ecological health.

Efficient Enhancement of Extracellular Electron Transfer in Shewanella oneidensis MR-1 via CRISPR-Mediated Transposase Technology.

Electroactive bacteria, exemplified by Shewanella oneidensis MR-1, have garnered significant attention due to their unique extracellular electron-transfer (EET) capabilities, which are crucial for energy recovery and pollutant conversion. However, the practical application of MR-1 is constrained by its EET efficiency, a key limiting factor, due to the complexity of research methodologies and the challenges associated with the practical use of gene editing tools. To address this challenge, a novel gene integration system, INTEGRATE, was developed, utilizing CRISPR-mediated transposase technologies for precise genomic insertion within the S. oneidensis MR-1 genome. This system facilitated the insertion of extensive gene segments at different sites of the Shewanella genome with an efficiency approaching 100%. The inserted cargo genes could be kept stable on the genome after continuous cultivation. The enhancement of the organism's EET efficiency was realized through two primary strategies: the integration of the phenazine-1-carboxylic acid synthesis gene cluster to augment EET efficiency and the targeted disruption of the SO3350 gene to promote anodic biofilm development. Collectively, our findings highlight the potential of utilizing the INTEGRATE system for strategic genomic alterations, presenting a synergistic approach to augment the functionality of electroactive bacteria within bioelectrochemical systems.

Interface Visualization of Bio-material Interaction Via Cryo-AEM Using the Biosynthesis of Iron-Based Nanoparticles as a Model.

Although interaction between organisms and nonorganisms is vital in environmental processes, it is difficult to characterize at nanoscale resolution. Biosynthesis incorporates intracellular and extracellular processes involving crucial interfacial functions and electron and substance transfer processes, especially on the inorganic-organic interface. This work chooses the biosynthesis of iron-based nanoparticles (nFe) as a model for biomaterial interaction and employs Cryo-AEM (i.e., S/TEM, EELS, and EDS analysis based on sample preparation with cryo-transfer holder system), combined with CV, Raman, XPS, and FTIR to reveal the inorganic-organic interface process. The inorganic-organic interactions in the biosynthesis of iron-based nanoparticles by Shewanella oneidensis MR-1 (M-nFe) were characterized by changes in electron cloud density, and the corresponding chemical shifts of Fe and C EELS edges confirm that M-nFe acquires electrons from MR-1 on the interface. Capturing intact filamentous-like, slightly curved, and bundled structure provides solid evidence of a "circuit channel" for electron transfer between organic and inorganic interface. CV results also confirm that adding M-nFe can enhance electron transfer from MR-1 to ferric ions. A mechanism for the synthesis of M-nFe with MR-1 based on intracellular and extracellular conditions under facultative anaerobic was visualized, providing a protocol for investigating the organic-inorganic interface.

Red-Light-Induced Genetic System for Control of Extracellular Electron Transfer.

Optogenetics is a powerful tool for spatiotemporal control of gene expression. Several light-inducible gene regulators have been developed to function in bacteria, and these regulatory circuits have been ported to new host strains. Here, we developed and adapted a red-light-inducible transcription factor for Shewanella oneidensis. This regulatory circuit is based on the iLight optogenetic system, which controls gene expression using red light. A thermodynamic model and promoter engineering were used to adapt this system to achieve differential gene expression in light and dark conditions within a S. oneidensis host strain. We further improved the iLight optogenetic system by adding a repressor to invert the genetic circuit and activate gene expression under red light illumination. The inverted iLight genetic circuit was used to control extracellular electron transfer within S. oneidensis. The ability to use both red- and blue-light-induced optogenetic circuits simultaneously was also demonstrated. Our work expands the synthetic biology capabilities in S. oneidensis, which could facilitate future advances in applications with electrogenic bacteria.

Identifying components of the Shewanella phage LambdaSo lysis system.

Phage-induced lysis of Gram-negative bacterial hosts usually requires a set of phage lysis proteins, a holin, an endopeptidase, and a spanin system, to disrupt each of the three cell envelope layers. Genome annotations and previous studies identified a gene region in the Shewanella oneidensis prophage LambdaSo, which comprises potential holin- and endolysin-encoding genes but lacks an obvious spanin system. By a combination of candidate approaches, mutant screening, characterization, and microscopy, we found that LambdaSo uses a pinholin/signal-anchor-release (SAR) endolysin system to induce proton leakage and degradation of the cell wall. Between the corresponding genes, we found that two extensively nested open-reading frames encode a two-component spanin module Rz/Rz1. Unexpectedly, we identified another factor strictly required for LambdaSo-induced cell lysis, the phage protein Lcc6. Lcc6 is a transmembrane protein of 65 amino acid residues with hitherto unknown function, which acts at the level of holin in the cytoplasmic membrane to allow endolysin release. Thus, LambdaSo-mediated cell lysis requires at least four protein factors (pinholin, SAR endolysin, spanin, and Lcc6). The findings further extend the known repertoire of phage proteins involved in host lysis and phage egress. IMPORTANCE: Lysis of bacteria can have multiple consequences, such as the release of host DNA to foster robust biofilm. Phage-induced lysis of Gram-negative cells requires the disruption of three layers, the outer and inner membranes and the cell wall. In most cases, the lysis systems of phages infecting Gram-negative cells comprise holins to disrupt or depolarize the membrane, thereby releasing or activating endolysins, which then degrade the cell wall. This, in turn, allows the spanins to become active and fuse outer and inner membranes, completing cell envelope disruption and allowing phage egress. Here, we show that the presence of these three components may not be sufficient to allow cell lysis, implicating that also in known phages, further factors may be required.