The Deep-sea Microorganism Bacillus Cereus 98 Tolerates Heavy Metals And Pseudomonas Stutzeri 273 Inhibits Biofilm Mechanism

Adverse environments are ubiquity for microbes,and they evolve various tolerance mechanisms against environmental stresses.Heavy metal stress is one of the most representative abiotic environmental stresses.Cadmium(Cd),a common toxic heavy metal,which causes series of detrimental effects in the environment,and urges bacteria to evolve different strategies coping with high concentrations of cadmium stresses.In addition,microorganisms usually compete with other microorganisms for space and resource in the process of growth and reproduction.Among them,biofilms are one of the main weapons for the competitiveness of microorganisms,which also prompt other microorganisms to evolve various mechanisms to deal with the formation of biofilms.In this study,on the one hand,Bacillus cereus 98,a strain with high tolerance to heavy metal cadmium,was used to explore the mechanism of calcium(Ca)enhancing its response to cadmium stress.On the other hand,Pseudomonas stutzeri 273,which produce exopolysaccharide EPS273,was used to explore the antibiofilm mechanism against Pseudomonas aeruginosa.Seven bacterial strains with high tolerance to cadmium were screened from more than300 bacterial strains isolated from deep-sea cold spring sediments,and it was found that addition of calcium could enhance the tolerance of these seven strains to cadmium,and the tolerance of B.cereus 98 to cadmium was increased most obviously.In order to explore its underlying mechanism,and to explore the expression of enzymes and related metabolic pathways at the protein level,proteomics was analyzed.The results showed that the expression of nitric oxide synthase(NOS)in B.cereus 98 was significantly up-regulated,and the expression of nitric oxide dioxygenase(NOD),that converts nitrogen(NO)to nitrate,was significantly down-regulated after cadmium was added,while the protein expression levels related to NO production were completely opposite after calcium was added.Further experiments proved that cadmium could induce the synthesis of NO,and addition of calcium could significantly reduce the intracellular NO content of B.cereus 98.Iron acquisition also contributed to the reduction of calcium-mediated toxicity of cadmium.In addition,proteomics analysis also showed that the expressions of nitrate reductase(Nar)and nitrite reductase(Nir)were significantly increased when calcium was added under the condition of cadmium,and the determination of the corresponding ions indicated that the concentrations of nitrate ion,nitrite ion and ammonium ion in group with calcium addition were significantly higher than those in the group without calcium addition,indicating that theaddition of calcium could promote the transformation of NO into corresponding ions.Therefore,combined with previous reports,we preliminarily speculated that under the stress of heavy metal Cd,B.cereus 98 can produce a large amount of NO,and NO will produce nitrogen oxidation stress,which ultimately leads to cell damage.The addition of Ca can induce the activity of related enzymes,and finally convert NO into ammonium ions to participate in the nitrogen cycle,thereby eliminating the nitrogen oxidation stress produced by NO and avoiding the occurrence of cell damage.In addition,the addition of calcium could also enhance the tolerance of B.cereus 98 to heavy metals copper,chromium,manganese,nickel,and furthermore calcium-mediated reduction of cadmium toxicity was also applicable to zebrafish.In addition,in order to explore the antibiofilm mechanism of P.stutzeri 273 against P.aeruginosa PAO1,transcriptomics analysis was performed to investigate the regulated genes by exopolysaccharide EPS273,which was produced by P.stutzeri 273,and functions of related genes.The results showed that after P.aeruginosa PAO1 was treated with exopolysaccharide EPS273,the expression levels of the two-component regulatory system and quorum sensing system,which are closely related to biofilm formation,were significantly reduced,and the expression levels of genes related to the formation of biofilm extracellular matrix,flagella and cell movement as well as the acquisition of iron also changed significantly.In addition,gene phoP and phoQ related to the two-component regulatory system and gene eddA related to eDNA degradation were disrupted,and the formation of biofilm and detection of the mobility of corresponding mutants further verified the results of transcriptome analysis.Therefore,it is preliminarily speculated that EPS273 may regulate the expression of genes of two-component regulatory system PhoP/PhoQ of P.aeruginosa PAO1,which then regulates the expression of genes of the quorum sensing system LasI / LasR,RhlI / RhlR,and the quorum sensing system further regulates the expression of genes involved in biofilm formation.In conclusion,on the one hand,we explored the mechanism of calcium enhancing tolerance of B.cereus 98 to cadmium stress.On the other hand,we investigated the antibiofilm mechanism of EPS273,which was produced by P.stutzeri 273,against Pseudomonas aeruginosa,thus revealing the mechanism of environmental adaptation of deep-sea microorganisms from two different aspects in this study.