Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria

Abstract

Anaerobic oxidation of methane (AOM) coupled to nitrite reduction (AOM-NIR) is ecologically significant for mitigating the methane-induced greenhouse effect. The microbes responsible for this reaction, NC10 bacteria, have been widely detected in diverse ecosystems. However, some defects were discovered in the commonly used NC10-specific primers, 202F and qP1F. In the present work, the primers were redesigned and improved to overcome the defects found in the previous primers. A new nested PCR method was developed using the improved primers to amplify 16S ribosomal RNA (rRNA) genes from NC10 bacteria. In the new nested PCR method, the qP1mF/1492R and 1051F/qP2R primer sets were used in the first and second rounds, respectively. The PCR products were sequenced, and more operational taxonomic units (OTUs) of the NC10 phylum were obtained using the new primers compared to the previous primers. The sensitivity of the new nested PCR was tested by the serial dilution method, and the limit of detection was approximately 10(3) copies g(-1) dry sed. for the environmental samples compared to approximately 10(5) copies g(-1) dry sed. by the previous method. Finally, the improved primer, qP1mF, was used in quantitative PCR (qPCR) to determine the abundance of NC10 bacteria, and the results agreed well with the activity of AOM-NIR measured by isotope tracer experiments. The improved primers are able to amplify NC10 16S rRNA genes more efficiently than the previous primers and useful to explore the microbial community of the NC10 phylum in different systems.

Denitrifying Anaerobic Methane Oxidation: A Previously Overlooked Methane Sink in Intertidal Zone

“…The abundance of ANME-2d archaea and NC10 bacteria was determined based on the quantification of their 16S rRNA genes. , The results showed that the NC10 bacteria and ANME-2d archaea were present in all samples. The 16S rRNA gene copy number of ANME-2d archaea was 4.3 × 10 4 ∼ 1.2 × 10 5 copies g –1 (dry sediment), and the copy number of NC10 bacteria was 4.9 × 10 4 ∼ 3.3 × 10 5 copies g –1 (dry sediment) (Figure a).…”

“…The specific information for the primers used in the study were shown in SI Table S4. The PCR reaction systems for quantitative PCR of ANME-2d archaea and NC10 bacteria were as previously described. , The standard curve was constructed with a series of 10-fold dilutions of plasmid DNA with a known copy number (amplification efficiency εC > 0.91).…”

Nitrate decreases methane production also by increasing methane oxidation through stimulating NC10 population in ruminal culture

“…To date, NC10 bacteria are the only known bacteria that can anaerobically oxidize methane; all other anaerobic methanotrophs are archaea (He et al 2016). ANME-2d and ‘ M. oxyfera ’ are two anaerobic methanotrophs and exist ubiquitously in the anaerobic environment abundant in methane, such as freshwater sludge, landfill and wastewater (Wu et al 2012).…”

Microbial Diversity: The Gap between the Estimated and the Known

“…Because microscopy-based identification is time consuming and laborious, researchers commonly choose to proceed with the molecular identification, even without collecting morphological data on the species. The molecular identification of microorganisms is performed based on the amplification of the 16S rRNA gene for bacteria, cyanobacteria, and archaea (e.g., [90][91][92]), and on the 18S and/or 28S rRNA gene for fungi (e.g., [93,94]). The sequences of these genes are used in microbial taxonomic studies for several reasons: the 16S rRNA gene is found in almost all bacteria, cyanobacteria, and archaea, often as a multigene family or within operons; its function has not changed over time, suggesting that random changes in sequences are a more accurate measure of time (evolution); and the size of this gene, approximately 1500 bp, is large enough for computing purposes [95].…”