Arfken Ann, Song Bongkeun, Bowman Jeff S, Piehler Michael
Department of Biological Sciences, Virginia Institute of Marine Science, Gloucester Point, Virginia, United States of America.
Integrative Oceanography Division, Scripps Institution of Oceanography, University of California, San Diego, California, United States of America.
PLoS One. 2017 Sep 21;12(9):e0185071. doi: 10.1371/journal.pone.0185071. eCollection 2017.
The eastern oyster (Crassostrea virginica) is a foundation species providing significant ecosystem services. However, the roles of oyster microbiomes have not been integrated into any of the services, particularly nitrogen removal through denitrification. We investigated the composition and denitrification potential of oyster microbiomes with an approach that combined 16S rRNA gene analysis, metabolic inference, qPCR of the nitrous oxide reductase gene (nosZ), and N2 flux measurements. Microbiomes of the oyster digestive gland, the oyster shell, and sediments adjacent to the oyster reef were examined based on next generation sequencing (NGS) of 16S rRNA gene amplicons. Denitrification potentials of the microbiomes were determined by metabolic inferences using a customized denitrification gene and genome database with the paprica (PAthway PRediction by phylogenetIC plAcement) bioinformatics pipeline. Denitrification genes examined included nitrite reductase (nirS and nirK) and nitrous oxide reductase (nosZ), which was further subdivided by genotype into clade I (nosZI) or clade II (nosZII). Continuous flow through experiments measuring N2 fluxes were conducted with the oysters, shells, and sediments to compare denitrification activities. Paprica properly classified the composition of microbiomes, showing similar classification results from Silva, Greengenes and RDP databases. Microbiomes of the oyster digestive glands and shells were quite different from each other and from the sediments. The relative abundance of denitrifying bacteria inferred by paprica was higher in oysters and shells than in sediments suggesting that oysters act as hotspots for denitrification in the marine environment. Similarly, the inferred nosZI gene abundances were also higher in the oyster and shell microbiomes than in the sediment microbiome. Gene abundances for nosZI were verified with qPCR of nosZI genes, which showed a significant positive correlation (F1,7 = 14.7, p = 6.0x10-3, R2 = 0.68). N2 flux rates were significantly higher in the oyster (364.4 ± 23.5 μmol N-N2 m-2 h-1) and oyster shell (355.3 ± 6.4 μmol N-N2 m-2 h-1) compared to the sediment (270.5 ± 20.1 μmol N-N2 m-2 h-1). Thus, bacteria carrying nosZI genes were found to be an important denitrifier, facilitating nitrogen removal in oyster reefs. In addition, this is the first study to validate the use of 16S gene based metabolic inference as a method for determining microbiome function, such as denitrification, by comparing inference results with qPCR gene quantification and rate measurements.
东方牡蛎（Crassostrea virginica）是一种基础物种，提供重要的生态系统服务。然而，牡蛎微生物群落的作用尚未被纳入任何一项服务中，尤其是通过反硝化作用去除氮。我们采用了一种结合16S rRNA基因分析、代谢推断、一氧化二氮还原酶基因（nosZ）的qPCR以及N2通量测量的方法，研究了牡蛎微生物群落的组成和反硝化潜力。基于16S rRNA基因扩增子的下一代测序（NGS），对牡蛎消化腺、牡蛎壳以及牡蛎礁附近沉积物中的微生物群落进行了检测。利用定制的反硝化基因和基因组数据库，通过paprica（基于系统发育位置的途径预测）生物信息学管道，通过代谢推断来确定微生物群落的反硝化潜力。检测的反硝化基因包括亚硝酸还原酶（nirS和nirK）和一氧化二氮还原酶（nosZ），后者根据基因型进一步细分为I类（nosZI）或II类（nosZII）。对牡蛎、贝壳和沉积物进行了连续流动实验以测量N2通量，比较反硝化活性。Paprica正确地对微生物群落的组成进行了分类，显示出与Silva、Greengenes和RDP数据库相似的分类结果。牡蛎消化腺和贝壳中的微生物群落彼此之间以及与沉积物中的微生物群落有很大不同。通过paprica推断，反硝化细菌在牡蛎和贝壳中的相对丰度高于沉积物，这表明牡蛎是海洋环境中反硝化作用的热点。同样，推断出的nosZI基因丰度在牡蛎和贝壳微生物群落中也高于沉积物微生物群落。通过nosZI基因的qPCR验证了nosZI的基因丰度，结果显示出显著的正相关（F1,7 = 14.7，p = 6.0x10-3，R2 = 0.68）。与沉积物（270.5 ± 20.1 μmol N-N2 m-2 h-1）相比，牡蛎（364.4 ± 23.5 μmol N-N2 m-2 h-1）和牡蛎壳（355.3 ± 6.4 μmol N-N2 m-2 h-1）中的N2通量率显著更高。因此，发现携带nosZI基因的细菌是重要的反硝化菌，有助于牡蛎礁中的氮去除。此外，这是第一项通过将推断结果与qPCR基因定量和速率测量结果进行比较，来验证基于16S基因的代谢推断作为确定微生物群落功能（如反硝化作用）方法的研究。
