Aaptation Mechanism Of Deep-sea Bacterium Shewanella Piezotolerans WP3-anaerobic Respiratory System Response To Different Environmental And Regulatory Factors

In this study, we used deep-sea isolated Shewanella piezotolerans WP3as a model tostudy its adaptation to deep-sea environment, and focused on understanding therelationship between its anaerobic respiratory system and the deep-sea environmentalfactors (such as hydrostatic pressure, as well as changes in the redox state and the ironconcentration). Meanwhile, the impacts and the mechanisms of the global regulators (suchas iron homeostasis regulator_Fur protein and small RNA chaperone_Hfq protein) on thedeep-sea environmental adaptation were also under in-depth analysis.Firstly, a series of mutants related to metal reduction (mtr-omc cluster) wereconstructed. Each mutant was tested for its insoluble iron reduction ability at atmosphericpressure and high hydrostatic pressure, respectively. As a result, the high hydrostaticpressure had some influence on the insoluble iron reduction system. We found that theMtrABC system had certain enhancements under high hydrostatic pressure condition,while three omcA-like proteins and the MtrDEF system were not subject to highhydrostatic pressure. By comparing the transcriptomic changes of WP3using oxygen (thehighest redox potential) and fumarate (a lower redox potential) as electron acceptors, wefound that the anaerobic electron transfer system, the energy generating system, theABC-transporters of cofactors related to anaerobic respiration and chemotaxis wereregulated to help WP3handle the dramatic changes of the redox state in deep-seaenvironment.Secondly, a fur deletion mutant of WP3was constructed, and physiological studiesrevealed that severe growth defects were observed under anaerobic conditions when different electron acceptors were provided, especially under DMSO-respiring condition.Comparative transcriptomic analysis demonstrated that Fur expanded its existingregulatory networks by controlling anaerobic electron transport system, the hemebiosynthesis system, and the cytochrome c maturation system for the direct or indirectregulation of anaerobic respiration. Bioinformatic analysis and the electrophoreticmobility shift assays revealed a primary regulation network for Fur in WP3: Fur proteincan positively regulate genes involved in anaerobic electron transport system by directlybinding to Fur Box in the promoter region, such as omcA and napD, or indirectly regulateanaerobic respiration system by other important regulatory factors, such as Crp-likeprotein, ArcA/B two-component protein and small RNA_RyhB. These results suggestedthat Fur might act as a sensor for anoxic conditions to trigger and influence the anaerobicrespiratory system. As Fur protein influenced the anaerobic respiratory system includingiron-respiring process, we compared iron reduction and subsequent mineralizationprocesses in WP3wild type and the fur deletion mutant (Δfur). The two processesdecreased in the Δfur, but the final mineralization products appeared non-affected,especially regarding to the mineralogy, concentration, crystallinity and grain size based onthe magnetic hysteresis and the X-ray diffraction spectrum of the products.Thirdly, we constructed an hfq deletion mutant of WP3and physiological studiesrevealed that severe growth defects were observed under anaerobic conditions whendifferent electron acceptors were provided. Transcriptomic and proteomic analysis showeda complex network regulated by Hfq protein in WP3, which is different from otherbacteria. Hfq protein activated the substrate-level phosphorylation and glyoxylate cycleunder anaerobic conditions, and positively regulated the expressions of Crp regulator, thefumarate and nitrite reductases as well as molybdenum cofactor biosysthesis system.Furthermore, its impacts on the redundant Nap and DMSO anaerobic respiratory systemshad different regulatory strategies. Meanwhile, Hfq protein can function as a pleiotropicregulatory factor on WP3deep environmental adaptability by controlling cell motility, amino acid metabolism, regulation of iron homeostasis, signal transduction pathways andsigma factor expression.At last, a low-temperature inducible protein expression vector (pSW2) based on afilamentous phage (SW1) of the deep-sea bacterium WP3was constructed. This vectorreplicated stably in Escherichia coli and Shewanella species, and its copy numberincreased at low temperature. Promoters of low-temperature inducible genes in WP3were fused into the vector to construct a series of vectors for enhancing low-temperatureprotein expression. Together with its accessory vectors containing promoters oflow-temperature inducible genes, pSW2can be utilized for protein expression as acomplementary, overexpression and low-temperature protein expression vector in WP3and potentially used in other Shewanella species.