Using the housekeeping gene cpn60 as a marker for microbial community profiling and viability assessment

Abstract

Abstract High-throughput sequencing has revolutionized microbiome research, yet distinguishing viable from nonviable microbes in complex communities remains challenging. Microbial viability underlines microbiome function, which is mostly driven by living, active members. Transcriptional activity of genes has been suggested as a viability marker but not systematically explored in complex communities. Here, we find that both community-wide transcription and cpn60 gene activity can effectively quantify community composition and identify viable members. For targeted cpn60 amplicons, protein-coding RNA activity better captured the differential structure of the active community than 16S rRNA amplicons. To enable scalable application, we constructed an expanded database integrating diverse cpn60 protein and nucleotide sequences, substantially extending the existing cpnDB database. In Human Microbiome Project II data, cpn60 protein-based taxonomy strongly agreed with shotgun metagenomic profiles. C pn60 transcript abundance correlated positively with bacterial growth rates estimated by the bPTR method, indicating its potential as a marker of microbial transcriptional activity and viability. It also distinguished resident gut microbes from potentially transient (e.g., oral-typical) members. Together, these results demonstrate that integrating DNA and RNA sequences at the single cpn60 locus enables accurate profiling of microbiome composition and activity, offering a cost-effective framework extendable to other rationally selected genes across diverse ecosystems.