Determination of the transcription unit landscape and associated regulatory elements in 5.

Methanotrophic bacteria show significant promise for methane bioconversion. Despite their ecological and biotechnological importance, the understanding of their transcriptional regulation and genetic regulatory elements remains limited. Here, we applied high-throughput sequencing to elucidate the transcriptional regulatory landscape of 5, a type II methanotroph. With its genome sequence completion, we identified 1,983 transcription start sites (TSSs) and 1,483 transcript 3'-ends (TEPs), which collectively defined 1,431 transcription units (TUs). This comprehensive analysis revealed diverse regulatory elements, including promoters, untranslated regions (UTRs), terminators, and regulatory RNAs in 5. A consensus promoter motif comprizing conserved -10 (TATAHT) and -35 (TYGMSV) elements, recognized by the housekeeping sigma factor RpoD, was predominant, particularly upstream of genes involved in methane and central carbon metabolism. We also uncovered diverse -regulatory motifs associated with nitrogen metabolism and cell division, including binding sites for RpoN (σ) and the transcription regulator CtrA. TEP analysis identified three classes of transcript ends with distinct sequence features and termination strengths. I-shaped intrinsic terminators were most prevalent, while L-shaped terminators were enriched in highly expressed genes, ensuring efficient transcription termination. Integration of TSSs and TEPs revealed functionally related genes within polycistronic TUs and identified previously unannotated small RNAs, including EcpR1 and αr45. This study provides the first genome-scale map of transcriptional regulation in a methanotroph, offering foundational insights into regulatory architecture and enabling future strain engineering for enhanced methane bioconversion.IMPORTANCEMethanotrophic bacteria offer a sustainable solution for converting methane into valuable products. However, the molecular mechanisms governing gene expression regulation in methanotrophs remain poorly understood. In this study, we applied high-throughput sequencing approaches to elucidate gene organization and transcriptional regulation in 5 during growth on methane. By identifying key regulatory features including promoter sequences, diverse -regulatory elements, and transcript boundaries, we revealed the coordinated gene expression mechanisms in this organism. This represents the first genome-wide transcriptome architecture in a methanotrophic bacterium. The regulatory elements provide a valuable resource for future genetic engineering of 5 and related methanotrophs. Our findings significantly advance the understanding of gene regulation in methanotrophs and support their development as microbial platforms for methane-based biomanufacturing.