# Identification and comparative genomic analysis of prophage sequences and CRISPR‒Cas immunity in Methylococcus genomes: insights into industrial methane bioconversion

## TL;DR

This study explores phage threats to Methylococcus bacteria used in methane-based industrial processes and identifies genetic elements that could help prevent phage infections.

## Contribution

The study provides a comprehensive analysis of prophage diversity and CRISPR-Cas systems in Methylococcus genomes for industrial risk mitigation.

## Key findings

- Eleven prophage sequences were identified in nine Methylococcus genomes, with nine potentially functional.
- Phylogenetic analysis showed prophages cluster with γ-proteobacteria phages, indicating diverse origins.
- CRISPR-Cas systems were annotated, offering insights into phage-bacteria interactions and immunity mechanisms.

## Abstract

Methylococcus species utilize methane as the sole carbon and energy source, converting it into biomass and other metabolic end products. Owing to this metabolic capacity, they hold particular promise in industrial C1 biotechnology, especially for the production of protein-rich feed. However, the industrial cultivation of Methylococcus-based consortia on methane is inherently nonsterile, exposing the process to potential biological risks that may compromise the stability, duration and productivity of cultivation. One of the most critical threats is bacteriophage infection, whose triggers for rapid phage-mediated lysis and resulting economic losses remain incompletely understood. Elucidating these processes is paramount for devising strategies to mitigate or prevent detrimental outcomes.

In this investigation, nine publicly accessible genomes of Methylococcus species were examined, culminating in the identification of eleven prophage sequences distributed variably among the genomes. Sequence annotations revealed that nine prophages are potentially functional and intact, whereas the rest carry incomplete gene sets indicative of nonviability. Phylogenetic analyses corroborated the substantial diversity of prophages, which formed distinct clusters related to γ-proteobacteria phages. Furthermore, comparative genomic analyses demonstrated a high degree of structural conservation despite the presence of rearrangements. The annotation of the CRISPR‒Cas systems provided insights into additional dimensions of phage‒bacteria interactions. Examination of prophage integration sites did not reveal any disruption of metabolic gene structures, thus suggesting minimal risk of deleterious phenotypic outcomes.

These findings considerably advance the current understanding of the genetic diversity and biological properties of prophages infecting Methylococcus species, underscoring the importance of holistic approaches for the detection and analysis of these elements. Our findings underscore the need for routine prophage monitoring in industrial methanotrophic consortia, with the pipeline established here serving as a foundational framework for future refinement and industrial adaptation.

The online version contains supplementary material available at 10.1186/s13068-026-02738-6.