Microbial survival and growth on non-corrodible conductive materials.

Biofilms growing aerobically on conductive substrates are often correlated with a positive, sustained shift in their redox potential. This phenomenon has a beneficial impact on microbial fuel cells by increasing their overall power output but can be detrimental when occurring on stainless steel by enhancing corrosion. The biological mechanism behind this potential shift is unresolved and a metabolic benefit to cells has not been demonstrated. Here, biofilms containing the electroautotroph 'Candidatus Tenderia electrophaga' catalysed a shift in the open circuit potential of graphite and indium tin oxide electrodes by >100 mV. Biofilms on open circuit electrodes had increased biomass and a significantly higher proportion of 'Ca. Tenderia electrophaga' compared to those on plain glass. Addition of metabolic inhibitors showed that living cells were required to maintain the more positive potential. We propose a model to describe these observations, in which 'Ca. Tenderia electrophaga' drives the shift in open circuit potential through electron uptake for oxygen reduction and CO2 fixation. We further propose that the electrode is continuously recharged by oxidation of trace redox-active molecules in the medium at the more positive potential. A similar phenomenon is possible on natural conductive substrates in the environment.

A bacterial membrane sculpting protein with BAR domain-like activity.

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here, we report a bacterial protein with BAR domain-like activity, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and influences scaffolding of OMEs into a consistent diameter and curvature. Cryo-TEM reveals that a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubules or narrow chains produced by the wild-type strain. Overexpression of BdpA promotes OME formation during planktonic growth of S. oneidensis where they are not typically observed. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli. Based on the ability of BdpA to alter membrane architecture in vivo, we propose that BdpA and its homologs comprise a newly identified class of bacterial BAR domain-like proteins.

Marinobacter atlanticus electrode biofilms differentially regulate gene expression depending on electrode potential and lifestyle.

Marinobacter spp. are opportunitrophs with a broad metabolic range including interactions with metals and electrodes. Marinobacter atlanticus strain CP1 was previously isolated from a cathode biofilm microbial community enriched from a sediment microbial fuel cell. Like other Marinobacter spp., M. atlanticus generates small amounts of electrical current when grown as a biofilm on an electrode, which is enhanced by the addition of redox mediators. However, the molecular mechanism resulting in extracellular electron transfer is unknown. Here, RNA-sequencing was used to determine changes in gene expression in electrode-attached and planktonic cells of M. atlanticus when grown at electrode potentials that enable current production (310 and 510 mV vs. SHE) compared to a potential that enables electron uptake (160 mV). Cells grown at current-producing potentials had increased expression of genes for molybdate transport, regardless of planktonic or attached lifestyle. Electrode-attached cells at current-producing potentials showed increased expression of the major export protein for the type VI secretion system. Growth at 160 mV resulted in an increase in expression of genes related to stress response and DNA repair including both RecBCD and the LexA/RecA regulatory network, as well as genes for copper homeostasis. Changes in expression of proteins with PEP C-terminal extracellular export motifs suggests that M. atlanticus is remodeling the biofilm matrix in response to electrode potential. These results improve our understanding of the physiological adaptations required for M. atlanticus growth on electrodes, and suggest a role for metal acquisition, either as a requirement for metal cofactors of redox proteins or as a possible electron shuttling mechanism.

Activation of Protein Expression in Electroactive Biofilms.

Microbes that form biofilms on electrodes and generate electrical current responses could be integrated into devices to perform sensing, conduct signals, or act as living microprocessors. A challenge in working with these species is the ability to visualize biofilm formation and protein expression in real-time while also measuring current, which is not possible with typical bio-electrochemical reactors. Here, we present a three-dimensional-printed flow cell for simultaneous electrochemistry and fluorescence imaging. Current-producing biofilms of Marinobacter atlanticus constitutively expressing green fluorescent protein were grown on the flow cell working electrode. Increasing current corresponded with increasing surface coverage and was comparable to biofilms grown in typical stirred-batch reactors. An isopropyl ß-d-1-thiogalactopyranoside (IPTG) inducible system driving yellow fluorescent protein was used to assess the spatiotemporal activation of protein expression within the biofilm at different stages of growth and induction dynamics. The response time ranged from 30 min to 5 h, depending on the conditions. These data demonstrate that the electrochemical flow cell can evaluate the performance of an electrically active environmental bacterium under conditions relevant for development as a living electronic sensor.

Silk fibroin as an additive for cell-free protein synthesis.

Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions. Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing, point-of-need manufacturing, and responsive materials. Meanwhile, silk fibroin from the silk worm, Bombyx mori, has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes. In this work, we explore the effects of silk fibroin as an additive in cell-free protein synthesis (CFPS) reactions. Impacts of silk fibroin on CFPS activity and stability after drying, as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed. We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%, which we attribute to macromolecular crowding effects. However, we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes. Further, the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture. Crosslinking attempts did not impact CFPS activity, but did yield localized protein aggregates rather than a hydrogel. We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.

Lactobacillus acidophilus Membrane Vesicles as a Vehicle of Bacteriocin Delivery.

Recent reports have shown that Gram-positive bacteria actively secrete spherical nanometer-sized proteoliposome membrane vesicles (MVs) into their surroundings. Though MVs are implicated in a broad range of biological functions, few studies have been conducted to examine their potential as delivery vehicles of antimicrobials. Here, we investigate the natural ability of Lactobacillus acidophilus MVs to carry and deliver bacteriocin peptides to the opportunistic pathogen, Lactobacillus delbrueckii. We demonstrate that upon treatment with lactacin B-inducing peptide, the proteome of the secreted MVs is enriched in putative bacteriocins encoded by the lab operon. Further, we show that purified MVs inhibit growth and compromise membrane integrity in L. delbrueckii, which is confirmed by confocal microscopy imaging and spectrophotometry. These results show that L. acidophilus MVs serve as conduits for antimicrobials to competing cells in the environment, suggesting a potential role for MVs in complex communities such as the gut microbiome. With the potential for controlling their payload through microbial engineering, MVs produced by L. acidophilus may be an interesting platform for effecting change in complex microbial communities or aiding in the development of new biomedical therapeutics.

Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application.

Melanin is a pigment produced by organisms throughout all domains of life. Due to its unique physicochemical properties, biocompatibility, and biostability, there has been an increasing interest in the use of melanin for broad applications. In the vast majority of studies, melanin has been either chemically synthesized or isolated from animals, which has restricted its use to small-scale applications. Using bacteria as biocatalysts is a promising and economical alternative for the large-scale production of biomaterials. In this study, we engineered the marine bacterium Vibrio natriegens, one of the fastest-growing organisms, to synthesize melanin by expressing a heterologous tyrosinase gene and demonstrated that melanin production was much faster than in previously reported heterologous systems. The melanin of V. natriegens was characterized as a polymer derived from dihydroxyindole-2-carboxylic acid (DHICA) and, similarly to synthetic melanin, exhibited several characteristic and useful features. Electron microscopy analysis demonstrated that melanin produced from V. natriegens formed nanoparticles that were assembled as "melanin ghost" structures, and the photoprotective properties of these particles were validated by their protection of cells from UV irradiation. Using a novel electrochemical reverse engineering method, we observed that melanization conferred redox activity to V. natriegens Moreover, melanized bacteria were able to quickly adsorb the organic compound trinitrotoluene (TNT). Overall, the genetic tractability, rapid division time, and ease of culture provide a set of attractive properties that compare favorably to current E. coli production strains and warrant the further development of this chassis as a microbial factory for natural product biosynthesis.IMPORTANCE Melanins are macromolecules that are ubiquitous in nature and impart a large variety of biological functions, including structure, coloration, radiation resistance, free radical scavenging, and thermoregulation. Currently, in the majority of investigations, melanins are either chemically synthesized or extracted from animals, which presents significant challenges for large-scale production. Bacteria have been used as biocatalysts to synthesize a variety of biomaterials due to their fast growth and amenability to genetic engineering using synthetic biology tools. In this study, we engineered the extremely fast-growing bacterium V. natriegens to synthesize melanin nanoparticles by expressing a heterologous tyrosinase gene with inducible promoters. Characterization of the melanin produced from V. natriegens-produced tyrosinase revealed that it exhibited physical and chemical properties similar to those of natural and chemically synthesized melanins, including nanoparticle structure, protection against UV damage, and adsorption of toxic compounds. We anticipate that producing and controlling melanin structures at the nanoscale in this bacterial system with synthetic biology tools will enable the design and rapid production of novel biomaterials for multiple applications.

Broad range shuttle vector construction and promoter evaluation for the use of Lactobacillus plantarum WCFS1 as a microbial engineering platform.

As the field of synthetic biology grows, efforts to deploy complex genetic circuits in nonlaboratory strains of bacteria will continue to be a focus of research laboratories. Members of the Lactobacillus genus are good targets for synthetic biology research as several species are already used in many foods and as probiotics. Additionally, Lactobacilli offer a relatively safe vehicle for microbiological treatment of various health issues considering these commensals are often minor constituents of the gut microbial community and maintain allochthonous behavior. In order to generate a foundation for engineering, we developed a shuttle vector for subcloning in Escherichia coli and used it to characterize the transcriptional and translational activities of a number of promoters native to Lactobacillus plantarum WCFS1. Additionally, we demonstrated the use of this vector system in multiple Lactobacillus species, and provided examples of non-native promoter recognition by both L. plantarum and E. coli strains that might allow a shortcut assessment of circuit outputs. A variety of promoter activities were observed covering a range of protein expression levels peaking at various times throughout growth, and subsequent directed mutations were demonstrated and suggested to further increase the degree of output tuning. We believe these data show the potential for L. plantarum WCFS1 to be used as a nontraditional synthetic biology chassis and provide evidence that our system can be transitioned to other probiotic Lactobacillus species as well.

Development of a Genetic System for Marinobacter atlanticus CP1 (sp. nov.), a Wax Ester Producing Strain Isolated From an Autotrophic Biocathode.

Here, we report on the development of a genetic system for Marinobacter sp. strain CP1, previously isolated from the Biocathode MCL community and shown to oxidize iron and grow as a cathodic biofilm. Sequence analysis of the small and large subunits of the 16S rRNA gene of CP1, as well as comparison of select conserved proteins, indicate that it is most closely related to Marinobacter adhaerens HP15 and Marinobacter sp. ES.042. In silico DNA-DNA hybridization using the genome-to-genome distance calculator (GGDC) predicts CP1 to be a new species of Marinobacter described here as Marinobacter atlanticus. CP1 is competent for transformation with plasmid DNA using conjugation with Escherichia coli donor strain WM3064 and constitutive expression of green fluorescent protein (GFP) is stable in the absence of antibiotic selection. Targeted double deletion mutagenesis of homologs for the M. aquaeoli fatty acyl-CoA reductase (acrB) and fatty aldehyde reductase (farA) genes resulted in a loss of production of wax esters; however, single deletion mutants for either gene resulted in an increase in total wax esters recovered. Genetic tools presented here for CP1 will enable further exploration of wax ester synthesis for biotechnological applications, as well as furthering our efforts to understand the role of CP1 within the Biocathode MCL community.

MR imaging of inflammation during myelin-specific T cell-mediated autoimmune attack in the EAE mouse spinal cord.

PURPOSE: The purpose of this study is to detect myelin-specific T cells, key pathological mediators in early multiple sclerosis, and the corresponding animal model, experimental autoimmune encephalomyelitis (EAE), in the mouse spinal cord. PROCEDURES: T cells were labeled with the iron-based, magnetic resonance (MR) contrast reagent, Feridex, and the transfection reagent, protamine sulfate, resulting in approximately 100% iron-labeling efficiency. Feridex-labeling did not alter the induction of EAE by T cells, and recipients were imaged by a 12-T MR instrument. RESULTS: Focal hypointense lesions were resolvable to gray or white matter of the lumbar spinal cord in T(2)-weighted images of the recipients of Feridex-labeled T cells. Lesions corresponded to histological evidence of inflammatory lesions and iron-labeled cells in eight-of-eight mice. In contrast, hypointense lesions were not observed eight-of-eight recipients of unlabeled T cells. CONCLUSIONS: These results demonstrate and provide methodologies for labeling, detecting, and extracting MRI-detectable foci of iron-labeled cells.