Physiological Roles of ArcA, Crp, and EtrA and Their Interactive Control on Aerobic and Anaerobic Respiration in Shewanella oneidensis

In the genome of Shewanella oneidensis, genes encoding the global regulators ArcA, Crp, and EtrA have been identified. All these proteins deviate from their counterparts in E. coli significantly in terms of functionality and regulon. It is worth investigating the involvement and relationship of these global regulators in aerobic and anaerobic respiration in S. oneidensis. In this study, the impact of the transcriptional factors ArcA, Crp, and EtrA on aerobic and anaerobic respiration in S. oneidensis were assessed. While all these proteins appeared to be functional in vivo, the importance of individual proteins in these two major biological processes differed. The ArcA transcriptional factor was critical in aerobic respiration while the Crp protein was indispensible in anaerobic respiration. Using a newly developed reporter system, it was found that expression of arcA and etrA was not influenced by growth conditions but transcription of crp was induced by removal of oxygen. An analysis of the impact of each protein on transcription of the others revealed that Crp expression was independent of the other factors whereas ArcA repressed both etrA and its own transcription while EtrA also repressed arcA transcription. Transcriptional levels of arcA in the wild type, crp, and etrA strains under either aerobic or anaerobic conditions were further validated by quantitative immunoblotting with a polyclonal antibody against ArcA. This extensive survey demonstrated that all these three global regulators are functional in S. oneidensis. In addition, the reporter system constructed in this study will facilitate in vivo transcriptional analysis of targeted promoters

Generation and Validation of a Shewanella oneidensis MR-1 Clone Set for Protein Expression and Phage Display

A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway) cloning system. A total of 3584 individual ORFs (85%) have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein expression of S. oneidensis His- or His/GST- tagged proteins in E. coli were approximately 70%. The ArcA and NarP transcription factor proteins were tested in an in vitro binding assay to demonstrate that functional proteins can be successfully produced using the clone set. Further functional validation of the clone set was obtained from phage display experiments in which a phage encoding thioredoxin was successfully isolated from a pool of 80 different clones after three rounds of biopanning using immobilized anti-thioredoxin antibody as a target. This clone set complements existing genomic (e.g., whole-genome microarray) and other proteomic tools (e.g., mass spectrometry-based proteomic analysis), and facilitates a wide variety of integrated studies, including protein expression, purification, and functional analyses of proteins both in vivo and in vitro

Oversizing the tibial component in TKAs: incidence, consequences and risk factors

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