Evaluation of Protein Coding Marker Genes Reveals rpoB as a Robust Complement to 16S rRNA for Detecting Total and Transcriptionally Active Bacterial Communities in Environmental Samples.
The small-subunit ribosomal gene (16S rRNA) has transformed microbial ecology by providing unprecedented access to microbiome composition. However, inherent limitations of the 16S rRNA gene such as multiple genomic copies, intragenomic sequence heterogeneity, and the lack of correlation between rRNA abundance and taxa activity have led to growing interest in complementary protein coding markers genes. Although several protein coding genes and associated PCR primers have been proposed as alternatives or complements to 16S rRNA, their performance has not been comprehensively evaluated and compared. In this study, we designed de-novo PCR primers targeting more 44 protein coding marker genes and systematically compared the performance of both newly designed and previously published primer sets. From these, we selected the best performing primers, targeting the rpoB and gyrB genes for PCR testing. We demonstrate that one newly designed rpoB primer pair (1528F/2041R) outperforms existing primers when amplifying environmental DNA and cDNA. Additionally, we carried out an in silico assessment of commonly used 16S rRNA primer sets and identified the best performing pair (515F/926R), which we used as a benchmark against the new rpoB primers for quantifying (Q-PCR) and sequencing (Illumina NextSeq) environmental soil and sediment DNA and cDNA. Using both the 515F/926R 16S rRNA and the improved rpoB primers proposed here alongside an expanded rpoB nucleotide database comprising over 20,000 more curated full length or near full length open reading frames compared to the best available database (GTDB), we observed strong agreement in the detection of dominant bacterial phyla across environments for the two marker genes. However, each marker gene uniquely detected several low abundance taxa, highlighting their complementary value. Combined rpoB and 16S rRNA DNA and cDNA quantification and sequencing revealed similar dominant transcriptionally active taxa, but only rpoB showed a positive and significant correlation between absolute cDNA abundance and cDNA:DNA ratios, highlighting its potential to identify active taxa in the environment.