| The microbial functional genes widely used in research,medical,and industrial fields come from culturable microbes.However,more than 99%of the microbes in the environment are unculturable,and their gene resources have not been effectively exploited.In this study,we developed a method to explore the functional genes of unculturable microbes by comparative metatranscriptomics and metagenomics.Furthermore,the soil microbial community in the Lanzhou industrial zone of the Yellow River was used to verify the feasibility.This study contained three parts:i)The Lanzhou Xigu industrial zone of the Yellow River is polluted by heavy metals,especially chromium?Cr?.Therefore,the development of the high-quality RNA recovery method from severely heavy-metal contaminated soil microbes was the technical support for this research;ii)During the investigation of microbial community structure and function in the Gansu industrial reach of the Yellow River,it was found that the microbial community in the Lanzhou Xigu industrial zone has a strong Cr???contamination remediation ability.Besides,the Cr???remediation genes of the microbes showed a significant response on stimulation by Cr???at the transcription level.However,the Cr???remediation ability of the microbes isolated from this area was far lower than the whole microbial community.Therefore,we speculated that the high-efficiency Cr???remediation genes were from unculturable microbes;iii)Based on the foundation of the above researches,we performed comparative metatranscriptomics and metagenomics on the microbial community of this site to explore the high-efficiency Cr???remediation genes of the unculturable microbes.Further,the application potential of the genes from unculturable microbes was evaluated in the Cr???contained industrial wastewater,and the molecular mechanisms of the novel high-efficiency Cr???remediation genes were explored.The main findings are as follows:1.An efficient high-quality microbial total RNA yield method,namely the guanidine thiocyanate-high EDTA?GTHE?for severely heavy metal contaminated soils was established and optimized.This method is the technic foundation for the molecular researches,such as q RT-PCR and metatranscriptomics.Due to the low microbial biomass in severely heavy-metal contaminated soils,we combined multiple strong denaturants and intense mechanical lysis to fully release the intracellular RNA,and thereby increase the yield of RNA.To minimize RNAase and heavy metals interference on RNA integrity,the concentrations of guanidine thiocyanate and EDTA were increased from 0.5 to 0.625 m L·g-1 soil and 10 to 100 m M,respectively.Then,the optimized GTHE method was applied to seven severely contaminated soils.The obtained RNA recovery efficiencies were 2.80-59.41?g·g-1 soil.RNA integrity and purity met the requirements of subsequent q RT-PCR and metatranscriptomic analyses.Therefore,this method served as a technical support for our follow-up research.2.Then,based on the GTHE method and Miseq sequencing,we explored the response of the soil microbial community structure and function at different levels of Cr pollution.First,we collected riparian soil samples from six sites along the Gansu industrial reach of the Yellow River based on different levels of Cr pollution.The functional phenotyping test showed that the Cr???reduction ability of the microbial community was positively correlated with the levels of Cr pollution.The results of q RT-PCR showed that the common Cr???remediation genes chr A and yie F were present in the Cr heavily contaminated samples?XG and CG?.Besides,and chr A and yie F had a transcriptional response to additional Cr???stimulation,while the copy number of them did not change.Miseq sequencing and principal coordinate analysis?PCo A?analysis showed that the six samples were divided into two categories based on the soil microbial structure,as the uncontaminated and the contaminated groups.Variation partition analysis?VPA?showed that 69.49%of the variance in the microbial structure could be explained by the detected variables,among which bioavailable Cr?SCr?,had the highest proportion among single factors?5.80%?.The above results indicated that Cr is one of the main factors altering microbial structure and function.In addition,some potential Cr contamination indicator species were also identified through spearman correlation analysis,including Geobacter,PSB-M-3,Flavobacterium,Methanosarcina.Through the analysis of microbial phenotype and bioinformatic analysis,potential high-efficiency Cr???remediation microbes and functional genes in the riparian soil collected from the Lanzhou Xigu industrial zone of the Yellow River were detected.However,the Cr???remediation ability of the isolated microbes in the lab,such as Staphylococcus aureus,Pseudomonas gessardii,Streptomyces violaceoruber,Pseudomonas monteilii,Cellulosimicrobium cellulans,etc.,was far lower than the whole microbial community.Therefore,we speculated that the high-efficiency Cr???remediation genes in this sample are from unculturable microbes.3.Based on the above researches,we proposed exploring highly efficient Cr???remediation genes from uncultured microbes in the Lanzhou Xigu industrial zone of the Yellow River by comparative metatranscriptomics and metagenomics.According to the standard of high-quality RNA yield and largest fold changes in gene expression of Cr???remediation genes,the most suitable Cr???treatment condition was determined to be 1 m M Cr???for 30 min.The gene ontology?GO?analysis showed that the differentially expressed genes were evenly distributed in 21 functional groups,of which at least seven groups responded to Cr???stimulation.This indicated that the Cr???remediation genes were actively expressed under the pressure of 1 m M Cr???for 30 min.The average sequence length of genes in the metagenomic library was longer than that in the metagenomic library,indicating that it is feasible to obtain the full-length sequence of the differentially expressed genes in the metatranscriptomic library from the metagenomic library.Cluster of orthologous groups?COG?results showed that the‘unknown genes'category accounted for a much higher proportion of the 25 functional categories than others,indicating that a large number of genes are function-unknown in this microbial community.With the screen standard[false discovery rate?FDR??0.05 and|log 2 FC|>1],241 differentially expressed genes were finally obtained in the comparative metatranscriptomic library.The unannotated genes?77?in the GO and kyoto encyclopedia of genes and genomes?KEGG?databases were defined as novel genes in this study.Finally,six genes?crr1,crr2,crr3,crr4,gsr,mcr?having a complete open reading frame?ORF?in the metagenomics library and the corresponding amino acid sequence length exceeding150 were selected for subsequent assays.The PCR results showed that six novel genes can be amplified with the g DNA extracted from fresh soil,but not from the conventional medium-cultured microbes.In addition,the bioinformatic analysis of genes showed that these six genes have never been studied.Phylogenetic comparisons of the corresponding novel protein analysis showed that each of the novel proteins was separated into an independent clade.These results indicated that by comparative metatranscriptomics and metagenomics,diverse novel genes with the same function can be explored.4.To compare the Cr???remediation ability of the above six novel genes and a known high-efficient Cr???remediation gene?yie F?in industrial wastewater treatment,they were constructed into E.coli to form different engineered strains.Cr???remediation ability test showed that the six novel genes all improved the Cr???resistance/reduction ability of E.coli.Their corresponding proteins were all identified by SDS-PAGE and western blotting?WB?.In the 200-600?M Cr???-contained industrial wastewater treatment,E-mcr and E-gsr presented the highest Cr???treatment efficiency without a decrease in efficiency over 17 days.In the treatment of200?M Cr???-contained industrial wastewater,the Cr???removal efficiencies of E-mcr,E-gsr,E-crr2,and E-crr3 were at least?7%higher than E-yie F.Under 600?M Cr???pressure,E-mcr and E-gsr still maintained Cr???removal efficiency of48.7%and 62.2%,respectively,while the removal efficiencies of E-P28 and E-yie F were 36.8%and 45.6%,respectively.In addition,the stress resistance assay of engineered strains under different extreme conditions showed that when Na Cl concentration was 15-30 g·L-1,the growth rate of E-mcr was higher than that of E-yie F.E-gsr presented high stress resistance ability under different adverse conditions?p H,temperature,osmotic pressure?.The above results showed that gsr and mcr have a great potential for practical application in Cr???-contaminated industrial wastewater treatment.5.Finally,we explored the mechanisms of the two most efficient Cr???remediation genes gsr and mcr,which also did not have any functional information reference.The resting cell assay showed that E-mcr and E-yie F had similar Cr???remediation abilities.However,in the Cr valence distribution assay,E-mcr presented?40?M higher extracellular Cr?III?compared to E-yie F.Transmission electron microscope?TEM?and energy dispersive X-ray spectrometry?EDX?analysis further demonstrated a large number of black agglomerates on the surface of E-mcr cells.Thus,mcr was identified as a membrane chromate reductase gene and it is likely to reduce Cr???through the respiratory pathway.The resting cell and enzyme activity assays showed that gsr did not have Cr???reduction ability.However,based on the fact that under different extreme conditions?p H,osmotic pressure,temperature,Cr???concentrations?,gsr increased the growth rate of E.coli by at least 30%in Chapter 5,gsr was determined as a general stress-response gene. |
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