Cargando…

New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing

Gene encoding the large subunit of As(III) oxidase (AioA), an important component of the microbial As(III) oxidation system, is a widely used biomarker to characterize As(III)-oxidizing communities in the environment. However, many studies were restricted to a few sequences generated by clone librar...

Descripción completa

Detalles Bibliográficos
Autores principales: Hu, Min, Li, Fangbai, Qiao, Jiangtao, Yuan, Chaolei, Yu, Huanyun, Zhuang, Li
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8527091/
https://www.ncbi.nlm.nih.gov/pubmed/34690945
http://dx.doi.org/10.3389/fmicb.2021.691913
_version_ 1784586009568083968
author Hu, Min
Li, Fangbai
Qiao, Jiangtao
Yuan, Chaolei
Yu, Huanyun
Zhuang, Li
author_facet Hu, Min
Li, Fangbai
Qiao, Jiangtao
Yuan, Chaolei
Yu, Huanyun
Zhuang, Li
author_sort Hu, Min
collection PubMed
description Gene encoding the large subunit of As(III) oxidase (AioA), an important component of the microbial As(III) oxidation system, is a widely used biomarker to characterize As(III)-oxidizing communities in the environment. However, many studies were restricted to a few sequences generated by clone libraries and Sanger sequencing, which may have underestimated the diversity of As(III)-oxidizers in natural environments. In this study, we designed a primer pair, 1109F (5′-ATC TGG GGB AAY RAC AAY TA−3′) and 1548R (5′-TTC ATB GAS GTS AGR TTC AT−3′), targeting gene sequence encoding for the conserved molybdopterin center of the AioA protein, yielding amplicons approximately 450 bp in size that are feasible for highly parallel amplicon sequencing. By utilizing in silico analyses and the experimental construction of clone libraries using Sanger sequencing, the specificity and resolution of 1109F/1548R are approximated with two other previously published and commonly used primers, i.e., M1-2F/M3-2R and deg1F/deg1R. With the use of the 1109F/1548R primer pair, the taxonomic composition of the aioA genes was similar both according to the Sanger and next-generation sequencing (NGS) platforms. Furthermore, high-throughput amplicon sequencing using the primer pair, 1109F/1548R, successfully identified the well-known As(III)-oxidizers in paddy soils and sediments, and they also revealed the differences in the community structure and composition of As(III)-oxidizers in above two biotopes. The random forest analysis showed that the dissolved As(III) had the highest relative influence on the Chao1 index of the aioA genes. These observations demonstrate that the newly designed PCR primers enhanced the ability to detect the diversity of aioA-encoding microorganisms in environments using highly parallel short amplicon sequencing.
format Online
Article
Text
id pubmed-8527091
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-85270912021-10-21 New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing Hu, Min Li, Fangbai Qiao, Jiangtao Yuan, Chaolei Yu, Huanyun Zhuang, Li Front Microbiol Microbiology Gene encoding the large subunit of As(III) oxidase (AioA), an important component of the microbial As(III) oxidation system, is a widely used biomarker to characterize As(III)-oxidizing communities in the environment. However, many studies were restricted to a few sequences generated by clone libraries and Sanger sequencing, which may have underestimated the diversity of As(III)-oxidizers in natural environments. In this study, we designed a primer pair, 1109F (5′-ATC TGG GGB AAY RAC AAY TA−3′) and 1548R (5′-TTC ATB GAS GTS AGR TTC AT−3′), targeting gene sequence encoding for the conserved molybdopterin center of the AioA protein, yielding amplicons approximately 450 bp in size that are feasible for highly parallel amplicon sequencing. By utilizing in silico analyses and the experimental construction of clone libraries using Sanger sequencing, the specificity and resolution of 1109F/1548R are approximated with two other previously published and commonly used primers, i.e., M1-2F/M3-2R and deg1F/deg1R. With the use of the 1109F/1548R primer pair, the taxonomic composition of the aioA genes was similar both according to the Sanger and next-generation sequencing (NGS) platforms. Furthermore, high-throughput amplicon sequencing using the primer pair, 1109F/1548R, successfully identified the well-known As(III)-oxidizers in paddy soils and sediments, and they also revealed the differences in the community structure and composition of As(III)-oxidizers in above two biotopes. The random forest analysis showed that the dissolved As(III) had the highest relative influence on the Chao1 index of the aioA genes. These observations demonstrate that the newly designed PCR primers enhanced the ability to detect the diversity of aioA-encoding microorganisms in environments using highly parallel short amplicon sequencing. Frontiers Media S.A. 2021-10-06 /pmc/articles/PMC8527091/ /pubmed/34690945 http://dx.doi.org/10.3389/fmicb.2021.691913 Text en Copyright © 2021 Hu, Li, Qiao, Yuan, Yu and Zhuang. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Hu, Min
Li, Fangbai
Qiao, Jiangtao
Yuan, Chaolei
Yu, Huanyun
Zhuang, Li
New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing
title New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing
title_full New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing
title_fullStr New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing
title_full_unstemmed New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing
title_short New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing
title_sort new arsenite oxidase gene (aioa) pcr primers for assessing arsenite-oxidizer diversity in the environment using high-throughput sequencing
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8527091/
https://www.ncbi.nlm.nih.gov/pubmed/34690945
http://dx.doi.org/10.3389/fmicb.2021.691913
work_keys_str_mv AT humin newarseniteoxidasegeneaioapcrprimersforassessingarseniteoxidizerdiversityintheenvironmentusinghighthroughputsequencing
AT lifangbai newarseniteoxidasegeneaioapcrprimersforassessingarseniteoxidizerdiversityintheenvironmentusinghighthroughputsequencing
AT qiaojiangtao newarseniteoxidasegeneaioapcrprimersforassessingarseniteoxidizerdiversityintheenvironmentusinghighthroughputsequencing
AT yuanchaolei newarseniteoxidasegeneaioapcrprimersforassessingarseniteoxidizerdiversityintheenvironmentusinghighthroughputsequencing
AT yuhuanyun newarseniteoxidasegeneaioapcrprimersforassessingarseniteoxidizerdiversityintheenvironmentusinghighthroughputsequencing
AT zhuangli newarseniteoxidasegeneaioapcrprimersforassessingarseniteoxidizerdiversityintheenvironmentusinghighthroughputsequencing