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Synthetic biology has been traditionally associated with electronics through the application of circuit design concepts towards the genetic engineering of microbes. Due to recent advances in the understanding of extracellular electron transfer in the bacterium Shewanella oneidensis (Shewanella), synthetic biology advances now have the potential of being used towards electronics applications. Towards this end, there is a need for tools that enable the systematic optimisation of genetic circuits in Shewanella. With the introduction of an RK2 origin of transfer cassette, we show that a modular plasmid system constructed prior for synthetic biology efforts in the bacterium Escherichia coli (E. coli) can be ported to Shewanella. In the process, it is also shown that different replication origins can be maintained in Shewanella and that multiple-plasmid strains can be realised in the bacterium. The results suggest that parts accumulated from E. coli synthetic biology efforts over the past decade and a half may be able to be ported to Shewanella, enabling the future engineering of systems where microbes interface with electronics (e.g. biosensors). [ABSTRACT FROM AUTHOR]

Abstract Background EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood. Results The expression of the nap genes, nrfA, cymA and hcp was significantly reduced in etrA deletion mutant EtrA7-1; however, limited anaerobic growth and nitrate reduction occurred, suggesting that multiple regulators control nitrate reduction in this strain. Dimethyl sulfoxide (DMSO) and fumarate reductase gene expression was down-regulated at least 2-fold in the mutant, which, showed lower or no reduction of these electron acceptors when compared to the wild type, suggesting both respiratory pathways are under EtrA control. Transcript analysis further suggested a role of EtrA in prophage activation and down-regulation of genes implicated in aerobic metabolism. Conclusion In contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and, in conjunction with other regulators, fine-tunes the expression of genes involved in anaerobic metabolism in S. oneidensis strain MR-1. Transcriptomic and sequence analyses of the genes differentially expressed showed that those mostly affected by the mutation belonged to the "Energy metabolism" category, while stress-related genes were indirectly regulated in the mutant possibly as a result of a secondary perturbation (e.g. oxidative stress, starvation). We also conclude based on sequence, physiological and expression analyses that this regulator is more appropriately termed Fnr and recommend this descriptor be used in future publications.

Extracellular electron transfer (EET) is the physiological process that enables the reduction or oxidation of molecules and minerals beyond the surface of a microbial cell. The first bacteria characterized with this capability were Shewanella and Geobacter , both reported to couple their growth to the reduction of iron or manganese oxide minerals located extracellularly. A key difference between EET and nearly every other respiratory activity on Earth is the need to transfer electrons beyond the cell membrane. The past decade has resolved how well-conserved strategies conduct electrons from the inner membrane to the outer surface. However, recent data suggest a much wider and less well understood collection of mechanisms enabling electron transfer to distant acceptors. This review reflects the current state of knowledge from Shewanella and Geobacter , specifically focusing on transfer across the outer membrane and beyond-an activity that enables reduction of highly variable minerals, electrodes, and even other organisms.

Amide peptide backbone methylation is a characteristic post-translational modification found in a family of ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) called borosins. Previously, we bioinformatically identified >1500 putative borosin pathways in bacteria; however, none of the pathways were associated with a known secondary metabolite. Through in-depth characterization of a borosin pathway in Shewanella oneidensis MR-1, we have now identified a bacterially derived borosin natural product named Shewanellamide A. Borosin identification was facilitated by the creation and analysis of a series of precursor variants and crystallographic interrogation of variant precursor and methyltransferase complexes. Along with assaying two proteases from S. oneidensis , probable boundaries for proteolytic maturation of the metabolite were projected and confirmed via comparison of S. oneidensis knockout and overexpression strains. All in all, the S. oneidensis natural product was found to be a 16-mer linear peptide featuring two backbone methylations, establishing Shewanellamide A as one of the few borosin metabolites yet identified, and the first from bacteria., Competing Interests: M. F. F. is an inventor on patent WO2017EP58327, US20190112583A1, WO2021168399, and US-20230090771-A1. All other authors declare no competing interests., (This journal is © The Royal Society of Chemistry.)

Iron is an essential element for nearly all life on Earth, primarily for its value as a redox active cofactor. Iron exists predominantly in two biologically relevant redox states: ferric iron, the oxidized state (Fe 3+ ), and ferrous iron, the reduced state (Fe 2+ ). Fe 2+ is well known to facilitate electron transfer reactions that can lead to the generation of reactive oxygen species. Less is known about why iron is toxic to cells in the absence of oxygen, yet this phenomenon is critically important for our understanding of life on early Earth and in iron-rich ecosystems today. In this brief review, we will highlight our current understanding of anaerobic Fe 2+ toxicity, focusing on molecular mechanistic studies in several model systems., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Shewanella oneidensis bacteria use an abiotic reaction to help shuttle electrons outside of the cell., Competing Interests: BC, JG No competing interests declared, (© 2019, Conley and Gralnick.)

A stoichiometric model describing the central metabolism of Shewanella oneidensis MR-1 wild-type and derivative strains was developed and used in elementary mode analysis (EMA). Shewanella oneidensis MR-1 can anaerobically respire a diverse pool of electron acceptors, and may be applied in several biotechnology settings, including bioremediation of toxic metals, electricity generation in microbial fuel cells, and whole-cell biocatalysis. The metabolic model presented here was adapted and verified by comparing the growth phenotypes of 13 single- and 1 double-knockout strains, while considering respiration via aerobic, anaerobic fumarate, and anaerobic metal reduction (Mtr) pathways, and utilizing acetate, n-acetylglucosamine (NAG), or lactate as carbon sources. The gene ppc, which encodes phosphoenolpyruvate carboxylase (Ppc), was determined to be necessary for aerobic growth on NAG and lactate, while not essential for growth on acetate. This suggests that Ppc is the only active anaplerotic enzyme when cultivated on lactate and NAG. The application of regulatory and substrate limitations to EMA has enabled creation of metabolic models that better reflect biological conditions, and significantly reduce the solution space for each condition, facilitating rapid strain optimization. This wild-type model can be easily adapted to include utilization of different carbon sources or secretion of different metabolic products, and allows the prediction of single- and multiple-knockout strains that are expected to operate under defined conditions with increased efficiency when compared to wild type cells., (© 2011. Published by Elsevier Ireland Ltd.)

The deleterious effect of superoxide radicals on cell growth and survival is predominantly caused by rapid oxidation of labile [Fe-S] clusters in proteins. Oxidation of these clusters releases Fe(II) ions, which participate in Fenton chemistry that damages DNA. Here it is shown that elevated levels of the YggX protein increase the resistance of Salmonella enterica to superoxide stress, reverse enzymatic defects attributed to oxidized [Fe-S] clusters, and decrease the spontaneous mutation frequency. The data are consistent with a model in which YggX protects protein [Fe-S] clusters from oxidation.

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella enterica serovar Typhimurium and other bacteria. In addition to genes encoding enzymes in the biosynthetic pathway, mutations in other metabolic loci have been shown to prevent thiamine synthesis. The latter loci identify the integration of the thiamine biosynthetic pathway with other metabolic processes and can be uncovered when thiamine biosynthesis is challenged. Mutations in gshA, encoding gamma-L-glutamyl-L-cysteine synthetase, prevent the synthesis of glutathione, the major free thiol in the cell, and are shown here to result in a thiamine auxotrophy in some of the strains tested, including S. enterica LT2. Phenotypic characterization of the gshA mutants indicated they were similar enough to apbC and apbE mutants to warrant the definition of a class of mutants unified by (i) a requirement for both the hydroxymethyl pyrimidine (HMP) and thiazole (THZ) moiety of thiamine, (ii) the ability of L-tryosine to satisfy the THZ requirement, (iii) suppression of the thiamine requirement by anaerobic growth, and (iv) suppression by a second-site mutation at a single locus. Genetic data indicated that a defective ThiH generates the THZ requirement in these strains, and we suggest this defect is due to a reduced ability to repair a critical [Fe-S] cluster.