Screening Of Cadmium-Tolerant Bacteria From Rice Rhizosphere Soil And Its Cadmium-Tolerance Mechanism

Cadmium is a kind of heavy metal elements with severe toxicity, it can be easily assimilated by cropsand then transported into the food chain, which causing harmful effects on human's health. How toremediate the soil polluted by cadmium pollutant has drawn widely concern by local goverment.Microorganism, on account of its potential application perspective, has become one of the most importantresources for remediating the environment polluted by heavy metals. To screen out some microorganismswith strong cadmium resistance and adsorption capacity, it is meaningful to elucidating the mechanism ofhow heavy metal cadmium absorbed and accumulated in the microorganisms, together with reducingcadmium pollution and ensuring a guarantee to food security.In this paper, plate method, adding cadmiumas culture medium, was used to screen cadmium tolerant bacterial strains from rice rhizosphere soils. Weevaluated its physiological and and biochemical characteristics and its absorption characteristics ofcadmium and its cadmium-resistant ability as well, in addition, we discusses differentially expressedproteins under cadmium stress condition. The main results are as follows.(1) Three strains of cadmium-tolerant bacteria were screened out from rhizosphere soil of ricePI312777. The three strains can growth normally under the treatment of cadmium concentration at300mg L^-1, and they also can grow at a higher cadmium concentration at700mg L^-1.(2) The consequence analysis of cadmium-resistant ability on three strains showed that highercadmium-ability was found in strain A,giving a stronger cadmium resistance at the concentration of900mg L^-1.(3) The observation of microbial cell morphology by electronic microscope and the determinationof strains growth curve indicated that there was no significant inhibition effect on the growth rates of thethree strains under the treatment of cadmium concentration at^-100mg L^-1, while some adaptivemorphological changes were found. Under the treatment of cadmium concentration higher than300mg L^-1, the growth rates of the strains were obviously inhibited. Nevertheless, the three strains couldtolerant a higher cadmium concentration at700mg L^-1, with longer time up to stationary phase and lowergrowth rate after logarithmic growing phase.(4) Based on the physiological and biochemical characteristics identification, as well as by^-16S rDNAsequence ananlysis, the three strains were classified as Pseudomonas sp. Furthermore, chemical analysisshowed that cadmium contents in the bacterial strains were9.04mg g^-1,4.96mg g^-1and28.58mg g^-1by dry weight, and the bioconcentration factors were90.4,49.6and285.8, respectively, under the treatmentof cadmium concentration at^-100mg L-1. It suggests that all the three strains performed stronger toleranceand absorption abilities of cadmium, which will provided important potential microbial resources for thepaddy soil cadmium contamination remediation.(5) In order to analyze the carbon sources metabolism functional diversity of three cadmium-tolerantBacteria(A, B and C), thirty-one carbon sources utilization characteristics of the strains were studied byusing BIOLOG ECO plate method. The results showed the average well color development(AWCD),which expressing as the abilities of the carbon sources utilization efficiency, performing in strain A washigher than strain B and strain C. Higher utilization abilities of following substrate were detected, such asN-Acetyl-D-glucosamine, L-Arginine, L-Asparagine, Itaconic acid, Putrescicine,4-Hydroxy benzoic acid,D-Mannitol, Pyruvic acid methyl ester, Tween40and Tween80.(6) There were57differential expressed proteins were found in strain A under the stress of cadmiumconcentration at300mg L^-1treatments by proteomics analysis. Among the57identified proteins,42proteins belong to intracellular proteins,the rest^-15proteins are extracellular proteins. As classified by thefunctions of those proteins we have identified,28proteins are involved in protein, amino acidandnucleotide metabolism;7proteins are related to glucose metabolism;8proteins are about stress response;4proteins that related with immune response;2proteins are refer to ethanol metabolic;2proteins arebelongs to signal transduction;2proteins are considered as material transport protein;3proteins areparticipate in electron transport; and the rest proteins touched upon other metabolic pathway.(7) Accoding to the expression mode, among the forty-two intracellular proteins, the expression of17proteins were up-regulated, including Putative ClpA/B protease ATP binding subunit, pyruvatedehydrogenase subunit E1, acetyl-CoA synthetase, probable isocitrate lyase, NAD+dependentacetaldehyde dehydrogenase, citrate synthase, fumarylacetoacetase, glutaryl-CoAdehydrogenase(twospots), GTPcycolhydrolaseII/3,4-dihydroxy-2-buanone-4-phosphate synthase,4-hydroxyphenylpyruvate,OprF, branch-chain amino acid aminotransferase,3-hydroxybutyrate dehydrogenase, DsbA, peroxidase,nucleoside diphosphate kinase Ndk. The expression of25proteins were down-regulated, there arephosphatase(two spots),30S ribosomal protein S1, Serine/Threonine Protein Kinase, argininedeiminase(two spots), elongation factor Tu, Anaerobically-induced outer membrane porin OprE precursor,branched-chain amino acid ABC transporter, Putative isovaleryl-CoA dehydrogenase, OprF, alcoholdehydrogenase, carbamate kinase, ABC transporter permease protein, amino acid (lysine/arginine/ornithine/histidine/octopine) ABC transporter periplasmic binding protein, succinatedehydrogenase subunit B, probable iron-sulfur protein, Chain X, Crystal Structure Of The OuterMembrane Protein Oprg From Pseudomonas Aeruginosa, peroxidase, Transcription elongation factorGreA, nucleoside diphosphate kinase, dna-binding stress protein, ssDNA binding protein(two spots), PilA.(8) Among the15identified extracellular proteins, the expression of12proteins were up-regulated,they are polynucleotide phosphorylase, chaperonin GroEL, Outer membrane protein OprF, ABCtransporter periplasmic protein, TonB-dependent receptor, dehydrogenase, peroxidase(three spots),lipid-binding START domain-containing protein, Chain D, Azurin(two spots), while the expression of3proteins down-regulated, they are molecular chaperone DnaK,30S ribosomal protein S1and flagellin.