- In the genomic age, genomes of microorganisms from various environments have been deciphered. - In this work, we attempted to use the easily accessible banknotes to search for novel microbial gene sequences.. - Results: We used high-throughput genomic sequencing technology to comprehensively characterize the diversity of microorganisms on the US dollars and Chinese Renminbis (RMBs). - In addition to finding a vast diversity of microbes, we found a significant number of novel gene sequences, including an unreported superoxide dismutase (SOD) gene, whose catalytic activity was further verified by experiments.. - Paper money or banknote, as a convenient medium of payment was first issued during the Song Dynasty of China in the eleventh century. - The concept of banknote was introduced to Europe in the thirteenth century and the first European banknotes were issued by a Swedish bank in 1661. - The widespread use of mobile de- vices and the rise of electronic payment platforms such Applepay or Alipay, as well as bitcoin in recent years. - Thus, the RMB and the dollar’s microbiological eco-system, has a certain “representative meaning”.. - 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - Full list of author information is available at the end of the article. - Our purpose of this study is to get an overview of the diversity of species on banknotes, and to explore the possibility of using paper money as economic- ally valuable microbial genetic resources.. - All raw data were uploaded to the NCBI-SRA database under the ac- cession number of SRP128023. - All scaftigs in the assem- bled results were counted as well as the distribution of scaftigs’ length in each sample. - represents the number of scaftigs and percentage. - b, SampleID indicates the name of the sample. - Total Length (bp), the overall length of the assembled scaftigs;. - Number, the total number of scaftigs assembled. - Average Length (bp), the average length of the assembled Scaftigs. - The results (Table 1) showed that for either Kit or STE extraction method, Chao1 value, Shannon and simpson indexes of the RMB samples were signifi- cantly greater than the respective index of the dollar samples. - It is noticeable that for either kit or STE extrac- tion method, the N50 and N90 of dollar samples were significantly greater than the respective N50 and N90 value of the RMB samples. - This is mainly due to the presence of more microbial species on the RMBs.. - Majority of the predicted gene sequences are between 300 and 400 bp, among which most of predicted gene sequences range from 330 bp to 360 bp (Fig. - The length of most non-redundant protein sequences is between 35 and 210 amino acids, among which the length of most non-redundant protein sequences is in the range 100–. - We performed gene function annotation for the identi- fied 207,051 unigenes using the CAZy [7], eggNOG [8]. - We found that using the KEGG database, 25% of the pathway genes are related to metabolism, 11% related to genetic information processing, 9% of annotated genes are involved in environmental information processing, and about 50% of genes are of unknown and unclassified functions (Fig. - 1 The richness and evenness of the community were considered. - 2 The probability that two randomly sampled individuals belong to different species = 1-the probability that two randomly sampled individuals belong to the same species. - 3 Chao1 algorithm is used to estimate the number of OTUs in the community. - The larger the Chao1 value, the more the total number of species. - The longitudinal axis shows the number of predicted genes (in blue) and the percentage of the predicted gene number (yellow curve). - The horizontal axis represents length of the predicted genes (in bp). - the number of genes and percentage. - the percentage of the number of genes (yellow). - The horizontal axis, the protein amino acid sequence length of the ORF.. - Using the CAZy database for function annotation, we found a large number of glyco- syl transferases and glycoside hydrolases (Fig. - Among the 350 enzymes in the Enzyme Commission EC number at Sub-subclasses level, we found a total of 225 enzyme sequences in the banknote metagenomic data.. - We also found a large number of suspected but unre- ported novel enzyme genes on the banknotes. - Since sequences were acquired by de novo sequen- cing and assembled by software, many of the identi- fied enzyme genes may not be real existence. - The horizontal axis represents different samples, and the vertical coordinate, the relative abundance of the genes of a certain function. - D, Functional annotation and abundance information of all samples based on KEGG, we selected the first 35 of the functions ranked by abundance in each sample to construct a hot map (Kegg Select the second level (Levels 2), from the functional information and the difference between the sample by two levels of clustering. - In the KEGG annotation of sequences, we found a se- quence, numbered total_314734, with only 60% nucleo- tide identity and 76% protein sequence similarity to the SOD genes using the NCBI online protein Blast program (Database version: March 2017). - We obtained the full length sequence of this gene by direct PCR using the paper money’s metagenomic DNA as template. - All primers used in this article was shown in the Supple- mental Table S1. - In this specific case, we demonstrated that the metagen- ome of banknotes could be a potentially important. - In addition, we performed phylogenetic analysis of amino acid sequences of this enzyme. - The sequence of total_314734 was submitted to Genbank under the accession number of MK681865.. - The number of non-redundant genes per GB base of raw sequence we found on banknotes was more than that of the intestinal [17] and soil [18]. - Of note is that the amount of raw sequence data in this study is much lower than that of previous studies and the number of samples was far less (Table 6). - This may indicate that our findings could be only a very small fraction of the whole microbiota on banknotes.. - From metabolism analysis of the KEGG annotation (Fig. - Table 3 Proportion of gene functions annotated using the eggNOG database for the 4 samples. - E: Amino acid transport and metabolism . - I: Lipid transport and metabolism . - F: Nucleotide transport and metabolism . - G: Carbohydrate transport and metabolism . - H: Coenzyme transport and metabolism . - Table 2 Proportion of gene functions annotated using the KEGG database for the samples. - This suggests that the microbes on the banknotes might form a certain social network to adapt to the special environment on banknotes. - However, the abundance of enzyme genes found in this study was still unexpected, considering that the data were derived from only 24 banknotes. - Many of the putative enzyme sequences have a low identity value with previously identified sequences in the public databases, as exempli- fied by our discovery of a novel SOD enzyme gene vari- ant, which was successfully expressed and shown to have activity. - Table 5 Overview of seven important enzymes found in this study. - Name Ec number The number of. - of the superoxide (O 2. - It is useful in the food and cosmetic industry.. - polysaccharides, useful in the food industry.. - Penicillin amidase Used in the production of beta lactam antibiotic. - Beta-D-Glucodidase Catalyzes the hydrolysis of the glycosidic bonds. - Table 4 Proportion of gene functions for the 4 samples using the CAZy database. - We collected RMB in China and US dollars in the United States, one in the eastern hemisphere and the other in the western hemisphere. - The dollar samples and the RMB samples are treated separately, to avoid cross contamination. - In this study, we collected 12 one Yuan bills of RMB in China, and 12 one dollar bills in the United States. - The surface of each bill was washed with sterile water, and the liquid was filtered through a 0.22 μm filter to collect the microbes. - In order to obtain the most complete informa- tion on the metagenomic DNA, We used two genomic DNA extraction methods (Supplemental Methods S1), the classic STE buffer (sodium chloride, Tris-HCl, EDTA) and Mobio kit, to isolate bacterial genomic DNA from banknotes. - But the harsh cell grinding and disrupting procedure in this method may damage the genomic DNA of some fragile microbes. - In this study, four DNA samples of the meta- genome were studied, which were labeled as follow:. - steD: metagenomic DNA from dollars extracted using STE method. - KitD: metagenomic DNA from dollars using Mobio Kit. - metagenomic DNA from RMB using STE method. - KitR: metagenomic DNA from RMB using Mobio Kit. - PCR amplification was performed on the ligated products using an adaptor spe- cific primer pair. - An Illumina Hiseq2500 sequencer was used for high-throughput sequencing of the four DNA samples and paired-end reads were generated. - The bio- informatics analysis method for NGS data of this study was shown in the Supplemental Methods S2.. - The Alpha diversity index analysis is based on the re- sults of assembly for species annotation analysis, for which the scaftigs data was used. - The numbers above the branch points denote the confidence levels of the relationship of the clustered sequences determined by boot strap statistical analysis [15]. - The evolutionary history was inferred by using the Maximum Likelihood method based on the Poisson correction model (Zuckerkandl and Pauling 1965).. - The bootstrap consensus tree inferred from 70 repli- cates is taken to represent the evolutionary history of the taxa analyzed. - The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (70 replicates) are shown next to the branches (Felsenstein 1985). - The bioinformatics analysis method for NGS data of this study.. - Primer sequences used in this paper.. - This work was financially supported by National Natural Science Foundation of China The Critical Patented Project of The Science &. - All raw data was uploaded to the NCBI-SRA database under the accession number of SRP128023. - 576.7Gb mapped to the 319,812 target genes. - this study. - Toward a metagenomic understanding on the bacterial composition and Resistome in Hong Kong banknotes. - KEGG for linking genomes to life and the environment. - Superoxide dismutases-a review of the metal-associated mechanistic variations. - Mierendorf RC, Morris BB, Hammer B, Novy RE: Expression and Purification of Recombinant Proteins Using the pET System
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