The Chemotaxis Mechanism Towards Electron Acceptor And Gene Regulation In Shewanella Species

Dissimilatory Metal Reducing Bacteria(DMRB)can couple the reduction of metal oxide and electrodes to cellular metabolism and growth,and thus have gained wide attention due to their great roles in the biogeochemical cycling of metals,environmental remediation,and energy generation from wastes.The genus Shewanella are one of the model DMRB which have been extensively explored.Chemotaxis is the responsive of bacteria movement toward a chemical stimulus.As one of the basic physiological response of bacteria,chemotaxis is important for bacteria to search for electron donors or acceptors,and escape from poor living conditions.Exploring the chemotaxis mechanisms toward several electron acceptors including environmental pollutants in Shewanella species not only deepen the understanding of the bacterial chemotaxis,but also provides a new strategy for pollution remediation.Moreover,developing the new gene expression and editing systems in Shewanella species will facilitate the constructions of engineered strains and broaden their applications in ecological remediation and energy recovery.This dissertation aims to investigate the chemotaxis mechanism towards different electron acceptors and establish the emerging gene expression and editing tools in Shewanella species.The main contents and results of this dissertation are listed below:1.The chemotaxis mechanism toward toxic arsenate in Shewanella putrefaciens CN-32.The chemotactic behavior toward As(?)in S.putrefaciens CN-32 was firstly confirmed,and the molecular mechanism of chemotaxis toward As(?)was further elucidated.First,a chemotaxis response toward As(?)and diverse electron acceptors in S.putrefaciens CN-32 was observed using three different chemotaxis assays.Next,some constructed strains and their chemotactic phenotypes showed that functional arsenate respiratory reductase but not metal-reducing-like respiratory pathway was required for the chemotaxis toward As(?)in S.putrefaciens CN-32.The chemotactic behavior toward As(?)was mediated by some methyl-accepting chemotaxis proteins(MCPs),but not a specific MCP,and governed by energy taxis.The chemotactic signal transduction pathway containing histidine kinase and flagella-mediated motility were essential for the chemotaxis toward As(V).This study reverses the conventional view on arsenate as a chemotactic inhibitor to microbes and provides the new ideas for arsenic remediation with DMRB.2.Decreased biofilm formation and electroactivity mediated by insertion sequence(IS)elements in Shewanella oneidensis MR-1.The spontaneous mutant of S.oneidensis MR-1 with non-motile phenotype(NMM)was discovered by chance at our lab,and the underlying molecular basis of phenotypic changes was further investigated.By combining PCR amplification,quantitative reverse transcription-PCR,and sequencing results,an ISSod2 element was found in flrA encoding the master regulator for flagella synthesis,which caused the non-motile and biofilm-deficient phenotypes in NMM.Moreover,there were no significant differences in colonial morphologies and growth rates between NMM and the wild-type strain,and the two strains were easily confused.The deteriorated electroactivity of the variant NMM was observed in both Cr(?)immobilization and in bioelectrochemical systems.Given the IS elements are widely distributed in DMRB,their negative influences on the electroactivity or biofilm-forming capability of DMRB should not be ignored.It is also necessary to prevent IS-mediated spontaneous mutations by adopting appropriate preservation and cultivation conditions.3.The mechanism of motility recovery mediated by IS excision in S.oneidensis MR-1.Based on the motility recovery of the strain NMM under aerobic and static culture in LB medium,the underlying molecular mechanism of IS excision was further explored.By comparing the phenotypic changes of several constructed mutants under different culture conditions,oxygen and aerotaxis were confirmed to play important roles in motility recovery of the non-motile strain NMM.Different from the known mechanisms of IS excision,the specific excision of ISSod2 inserted into flrA didn't require the transposase encoded by itself.Meanwhile,the excision mechanism did not match the model of replicational slippage.According to the phenotypic and genotypic changes of the knock-in Shewanella or Escherichia coli strains under specific conditions,a similar mechanism of ISSod2 excision could be found in other IS element belonging to IS3 family.This work can broaden our understanding about how IS elements affect the bacterial genome plasticity.4.Engineering the rhamnose-inducible system for precise control of gene expression in Shewanella species.First,the rhaBAD promoter of E.coli was introduced into multiple Shewanella species.This rhaBAD promoter was functional in Shewanella species,with a linear response to inducer concentration over a wide range and no basal expression in the absence of inducer.Moreover,the inducer is not metabolized by nor toxic to Shewanella species.Finally,the rhamnose-inducible system was applied to control the extracellular electron transfer ability of Shewanella species for pollutant transformation.Such a highly efficient rhamnose-inducible system presented here has enriched the transcriptional regulation tools available in Shewanella species and will facilitate more efficient utilization of the Shewanella species for practical applications.5.Developing a base editor for enhanced pollutant degradation in S.oneidensis MR-1.A highly efficient base-editing tool pCBEso was firstly constructed by fusing a Cas9 nickase with the cytidine deaminase.Based on systematic evaluation of such a base-editing tool,the target C within the editable window was confirmed to be readily and efficiently converted to T without requiring repair templates or double-strand break.Furthermore,double-locus simultaneous editing could be easily achieved with high efficiency.With the base editor,the essential genes associated with glucose or N-acetylglucosamine(GlcNAc)metabolism in S.oneidensis MR-1 were quickly identified.An engineered strain with expanded carbon source utilization spectra was rapidly constructed.The constructed strain exhibited a higher degradation and transformation capability of several organic pollutants when using glucose or GlcNAc as the sole carbon source.Such a tool could be easily introduced into other DMRB and greatly facilitate their applications in ecological remediation.