Study On The Responses And Mechanisms Of Pseudomonas Putida MnB1 Biofilm To Environmental Factors Mn???and H₂O₂

Background:Environmental stress,including environmental toxin,depletion of nutrient,extreme temperature,high salt,ultraviolet,ionizing radiation,and contaminants etc.leads to overproduction and accumulation of reactive oxygen species?ROS?in bacterial,resulting in oxidative stress.The maintenance of intracellular oxidative/anti-oxidative homeostasis is one of the protective mechanisms against environmental stress.Another newly reported mechanism is the formation of biofilm in microorganisms.Relevant reports mainly focus on pathogenic microorganisms of human and animal,remaining the environmental micro-organisms not deeply investigated.Microorganisms participate in the chemical cycle of elements in earth.Geological microbes contribute to the elements circulation on earth and promote the change of environment.Accordingly,the physiological and biochemical processes,as well as the functions of microorganisms are changed.In turn the complex geological environment drives the adaption and evolution of the microorganisms.Pseudomonas putida?Pseudomonas pudida,P.putida?variety of polluted environments,containing some highly conserved housekeeping genes which encode catabolic pathways and enzymes.Moreover,the effective defense mechanisms to environmental stress function well in P.putida.P.putida MnB1 is a well-studied bacteria model of manganese oxidation.Acquisition and oxidation of exogenous manganese can beapplied as energy for growth and proliferation Furthermore,manganese is reported to be involved in ROS scavengence,biofilm formation and other biological processes.In the present study,the effect of environmental factors,such as divalent manganese and H₂O₂ on the growth,nutrient substrate acquisition,oxidative/anti-oxidative homeostasis and biofilm formation in P.putida MnBI was foucsed.We hope to recover the adaptive and protective mechanism of environmental microbes against the environmental stress.Aim:Investigate the effects of MnCl₂ and H₂O₂ on the biofilm formation of P.putida MnB1 and associated mechanism,as well as recovering the adaptive and protective mechanism of environmental microbes against the environmental stress.Methods:MnCl₂?40-5000 ?M?and H₂O₂?40-1000 ?M?were added in the pure cultivation of P.putida MnB1.The effects of MnCl₂ and H₂O₂ on bacterial growth were assayed with Turbidimetric method.The effects of MnCl₂ and H₂O₂ on the biofilm formation were investigated by crystal violet staining,scanning electron microscopy and laser confocal microscopy.Effect of GTP on the biofilm formation was conducted with crystal violet staining.Based on the whole genome sequence of P.putida GB-1,6 target genes,encoding multicopper oxidase?Mco?.manganses ABC transporter,?MntABC??superoxide dismutase?Sod?.glutathione reductase?Gr??phosphodiesterase?Pde?were amplified and sequenced.The sequence homology alignment of nucleic acid and protein were conducted.The expression differences of Sod,Gr and Pde gene in suspended and colonized cells were studied by real-time quantitative Q-PCR.The effect of MnCl₂ on the expression of Mco,MntABC,Sod and Pde gene was assayed by real-time quantitative Q-PCR.The effect of H₂O₂ on the expression of Sod?Gr?Pde gene was assayed by real-time quantitative Q-PCR.Results:40-5000 ?M MnCl₂ promoted the growth of P.putida MnBl and suppresses the biofilm formation.The expression level of Mco gene did not differ at significance between planktonic and colonized cells in biofilm,while did not respond to the addition of exogenous MnCl₂.At the same time,exogenous MnCl₂ promoted the expression of MntABC gene.Exogenous manganese reversed the expression trends of Sod gene during the procedure of biofilm formation,and the Sod gene expression was significantly decreased under MnCl₂.Increases of Pde gene expression were detected during the intermediate phase of biofilm formation under exogenous MnCl₂ treatment.40-1000 ?M H₂O₂ had no obvious cytotoxicity on cell viability.1000 ?M group retarded the proliferation before 24h.H₂O₂ at different initial concentrations can be completely degraded by P.putida MnB1 after 6 h.Crystal violet staining,scanning electron microscope?SEM?and laser scanning confocal microscope?CLSM?revealed that H₂O₂ promoted the formation of biofilm at early stage.H₂O₂ suppressed the expression levels of Gr at the stable stage.H₂O₂ did not restore the significant activation of Sod gene expression during the biofilm formation.During the whole process of biofilm formation,Pde gene expression significantly altered.The expression of Pde gene was suppressed at significance in the early stage of biofilm formation under H₂O₂Conclusion:The manganese-rich condition promoted the growth of P.putida MnB1 and manganese acquisition.Decrease in intracellular oxidative stress was accompanied by the inhibition of biofilm formation,suggesting that the favorable environmental factors may negatively modulate the biofilm formation.H₂O₂ promoted the biofilm formation of P.putida MnB1,accompanying with increase in intracellular oxidative stress.Therefore,the biofilm formation is important in the protective effect of P.putida MnB1 against unfavorable environmental factors,in which Pde gene might play a crucial role.At the same time,biofilm is the primary mechanism of response to environmental stress,since it is more sensitive than the oxidative/anti-oxidative homeostasis in P.putida MnBl.