scholarly journals Rapid Construction of a Whole-genome Transposon Insertion Collection for Shewanella oneidensis by Knockout Sudoku

2016 ◽  
Author(s):  
Michael Baym ◽  
Lev Shaket ◽  
Isao A. Anzai ◽  
Oluwakemi Adesina ◽  
Buz Barstow
Keyword(s):  
Shewanella Oneidensis ◽  
Extracellular Electron Transfer ◽  
Whole Genome ◽  
Kinetic Measurements ◽  
Predictive Algorithm ◽  
Transposon Insertion ◽  
Sequencing Library ◽  
Transposon Mutants ◽  
Technical Effort ◽  
Combinatorial Pooling

AbstractWhole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally they have needed an extraordinary technical effort to construct. Knockout Sudoku is a new method for directing the construction and purification of a curated whole-genome collection of singlegene disruption mutants generated by transposon mutagenesis. Using a simple combinatorial pooling scheme, a highly oversampled collection of transposon mutants can be condensed into a next-generation sequencing library in a single day. The identities of the mutants in the collection are then solved by a predictive algorithm based on Bayesian inference, allowing for rapid curation and validation. Starting from a progenitor collection of 39,918 transposon mutants, we compiled a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR–1 containing representatives for 3,667 genes. High-throughput kinetic measurements on this collection provide a comprehensive view of multiple extracellular electron transfer pathways operating in parallel.

scholarly journals Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Michael Baym ◽  
Lev Shaket ◽  
Isao A. Anzai ◽  
Oluwakemi Adesina ◽  
Buz Barstow
Keyword(s):  
Single Gene ◽  
Shewanella Oneidensis ◽  
Whole Genome ◽  
Sequencing Data ◽  
Kinetic Measurements ◽  
Data Set ◽  
Quinone Reduction ◽  
Transposon Insertion ◽  
Sequencing Library ◽  
Technical Effort

Abstract Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

Mind the gap: cytochrome interactions reveal electron pathways across the periplasm of Shewanella oneidensis MR-1

2012 ◽  
Vol 449 (1) ◽  
pp. 101-108 ◽  
Author(s):  
Bruno M. Fonseca ◽  
Catarina M. Paquete ◽  
Sónia E. Neto ◽  
Isabel Pacheco ◽  
Cláudio M. Soares ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Protein Interactions ◽  
Microbial Fuel Cells ◽  
Dissociation Constants ◽  
Shewanella Oneidensis ◽  
Extracellular Electron Transfer ◽  
Periplasmic Space ◽  
Protein Protein Interactions ◽  
Surface Charges ◽  
Kinetic Measurements

Extracellular electron transfer is the key metabolic trait that enables some bacteria to play a significant role in the biogeochemical cycling of metals and in bioelectrochemical devices such as microbial fuel cells. In Shewanella oneidensis MR-1, electrons generated in the cytoplasm by catabolic processes must cross the periplasmic space to reach terminal oxidoreductases found at the cell surface. Lack of knowledge on how these electrons flow across the periplasmic space is one of the unresolved issues related with extracellular electron transfer. Using NMR to probe protein–protein interactions, kinetic measurements of electron transfer and electrostatic calculations, we were able to identify protein partners and their docking sites, and determine the dissociation constants. The results showed that both STC (small tetrahaem cytochrome c) and FccA (flavocytochrome c) interact with their redox partners, CymA and MtrA, through a single haem, avoiding the establishment of stable redox complexes capable of spanning the periplasmic space. Furthermore, we verified that the most abundant periplasmic cytochromes STC, FccA and ScyA (monohaem cytochrome c5) do not interact with each other and this is likely to be the consequence of negative surface charges in these proteins. This reveals the co-existence of two non-mixing redox pathways that lead to extracellular electron transfer in S. oneidensis MR-1 established through transient protein interactions.

Enhancing Extracellular Electron Transfer of Shewanella oneidensis MR-1 through Coupling Improved Flavin Synthesis and Metal-Reducing Conduit for Pollutant Degradation

2017 ◽  
Vol 51 (9) ◽  
pp. 5082-5089 ◽  
Author(s):  
Di Min ◽  
Lei Cheng ◽  
Feng Zhang ◽  
Xue-Na Huang ◽  
Dao-Bo Li ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Shewanella Oneidensis ◽  
Extracellular Electron Transfer ◽  
Pollutant Degradation

Impact of Graphitic Structures in Pyrogenic Carbon on Microbial Reduction of Ferrihydrite

2021 ◽  
Author(s):  
wentao yu ◽  
baoliang chen
Keyword(s):  
Electron Transfer ◽  
Pyrolysis Temperature ◽  
Shewanella Oneidensis ◽  
Exchange Capacity ◽  
Microbial Reduction ◽  
Systematic Evaluation ◽  
Extracellular Electron Transfer ◽  
Pyrogenic Carbon ◽  
The Impact

<p>Pyrogenic carbon plays important roles in microbial reduction of ferrihydrite by shuttling electrons in the extracellular electron transfer (EET) processes. Despite its importance, a full assessment on the impact of graphitic structures in pyrogenic carbon on microbial reduction of ferrihydrite has not been conducted. This study is a systematic evaluation of microbial ferrihydrite reduction by Shewanella oneidensis MR-1 in the presence of pyrogenic carbon with various graphitization extents. The results showed that the rates and extents of microbial ferrihydrite reduction were significantly enhanced in the presence of pyrogenic carbon, and increased with increasing pyrolysis temperature. Combined spectroscopic and electrochemical analyses suggested that the rate of microbial ferrihydrite reduction were dependent on the electrical conductivity of pyrogenic carbon (i.e., graphitization extent), rather than the electron exchange capacity. The key role of graphitic structures in pyrogenic carbon in mediating EET was further evidenced by larger microbial electrolysis current with pyrogenic carbon prepared at higher pyrolysis temperatures. This study provides new insights into the electron transfer in the pyrogenic carbon-mediated microbial reduction of ferrihydrite.</p>

CRISPRi–sRNA: Transcriptional–Translational Regulation of Extracellular Electron Transfer in Shewanella oneidensis

2017 ◽  
Vol 6 (9) ◽  
pp. 1679-1690 ◽  
Author(s):  
Yingxiu Cao ◽  
Xiaofei Li ◽  
Feng Li ◽  
Hao Song
Keyword(s):  
Electron Transfer ◽  
Translational Regulation ◽  
Shewanella Oneidensis ◽  
Extracellular Electron Transfer

scholarly journals Spin-Dependent Electron Transport through Bacterial Cell Surface Multiheme Electron Conduits

2019 ◽  
Author(s):  
Suryakant Mishra ◽  
Sahand Pirbadian ◽  
Amit Kumar Mondal ◽  
Moh El-Naggar ◽  
Ron Naaman
Keyword(s):  
Electron Transport ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Shewanella Oneidensis ◽  
Extracellular Electron Transfer ◽  
Long Distance ◽  
Force Microscopy ◽  
Bacterial Cell Surface ◽  
Redox Active ◽  
Gain Energy

Multiheme cytochromes, located on the bacterial cell surface, function as long-distance (> 10 nm) electron conduits linking intracellular reactions to external surfaces. This extracellular electron transfer process, which allows microorganisms to gain energy by respiring solid redox-active minerals, also facilitates the wiring of cells to electrodes. While recent studies suggested that a chiral induced spin selectivity effect is linked to efficient electron transmission through biomolecules, this phenomenon has not been investigated in the extracellular electron conduits. Using magnetic conductive probe atomic force microscopy, Hall voltage measurements, and spin-dependent electrochemistry of the decaheme cytochromes MtrF and OmcA from the metal-reducing bacterium <i>Shewanella oneidensis</i> MR-1, we show that electron transport through these extracellular conduits is spin-selective. Our study has implications for understanding how spin-dependent interactions and magnetic fields may control electron transport across biotic-abiotic interfaces in both natural and biotechnological systems.

Identifying the potential extracellular electron transfer pathways from a c-type cytochrome network

2014 ◽  
Vol 10 (12) ◽  
pp. 3138-3146 ◽  
Author(s):  
De-Wu Ding ◽  
Jun Xu ◽  
Ling Li ◽  
Jian-Ming Xie ◽  
Xiao Sun
Keyword(s):  
Electron Transfer ◽  
Shewanella Oneidensis ◽  
Extracellular Electron Transfer ◽  
Genome Wide ◽  
A Genome ◽  
Electron Transfer Pathways

A genome-wide c-type cytochrome network was constructed to explore the extracellular electron transfer pathways in Shewanella oneidensis MR-1.

scholarly journals Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI)

Science ◽  
2017 ◽  
Vol 356 (6334) ◽  
pp. 189-194 ◽  
Author(s):  
Chongyi Chen ◽  
Dong Xing ◽  
Longzhi Tan ◽  
Heng Li ◽  
Guangyu Zhou ◽  
...  
Keyword(s):  
Single Cell ◽  
Whole Genome ◽  
Linear Amplification ◽  
Transposon Insertion ◽  
Genome Analyses

scholarly journals A whole-genome screen identifies Salmonella enterica serovar Typhi genes involved in fluoroquinolone susceptibility

2020 ◽  
Vol 75 (9) ◽  
pp. 2516-2525
Author(s):  
A Keith Turner ◽  
Sabine E Eckert ◽  
Daniel J Turner ◽  
Muhammud Yasir ◽  
Mark A Webber ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Insertion Site ◽  
Whole Genome ◽  
Genome Screen ◽  
Polysaccharide Biosynthesis ◽  
Salmonella Enterica Serovar Typhi ◽  
Transposon Insertion ◽  
Associated Functions ◽  
The Impact ◽  
Insertion Mutations

Abstract Objectives A whole-genome screen at sub-gene resolution was performed to identify candidate loci that contribute to enhanced or diminished ciprofloxacin susceptibility in Salmonella enterica serovar Typhi. Methods A pool of over 1 million transposon insertion mutants of an S. Typhi Ty2 derivative were grown in a sub-MIC concentration of ciprofloxacin, or without ciprofloxacin. Transposon-directed insertion site sequencing (TraDIS) identified relative differences between the mutants that grew following the ciprofloxacin treatment compared with the untreated mutant pool, thereby indicating which mutations contribute to gain or loss of ciprofloxacin susceptibility. Results Approximately 88% of the S. Typhi strain’s 4895 annotated genes were assayed, and at least 116 were identified as contributing to gain or loss of ciprofloxacin susceptibility. Many of the identified genes are known to influence susceptibility to ciprofloxacin, thereby providing method validation. Genes were identified that were not known previously to be involved in susceptibility, and some of these had no previously known phenotype. Susceptibility to ciprofloxacin was enhanced by insertion mutations in genes coding for efflux, other surface-associated functions, DNA repair and expression regulation, including phoP, barA and marA. Insertion mutations that diminished susceptibility were predominantly in genes coding for surface polysaccharide biosynthesis and regulatory genes, including slyA, emrR, envZ and cpxR. Conclusions A genomics approach has identified novel contributors to gain or loss of ciprofloxacin susceptibility in S. Typhi, expanding our understanding of the impact of fluoroquinolones on bacteria and of mechanisms that may contribute to resistance. The data also demonstrate the power of the TraDIS technology for antibacterial research.

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