Mathes, D. J. | bioRxiv Science

bioRxiv2026

Growth rate dependent DNA methylation patterns along bacterial chromosomes

DNA methylation plays critical roles in gene regulation in bacteria, from regulating essential processes like the cell cycle to phenotypes of practical interest like pathogenicity and motility. Synthetic manipulation of global methylation levels has broad impacts on cellular physiology, changing expression patterns of hundreds of genes. However, whether or how environmental variation in natural settings similarly impacts DNA methylation patterns has been unclear. In this work, using the alphaproteobacteria Methylobacterium extorquens and Caulobacter crescentus as model systems, we discover the methylome is highly fluid in response to environmental variation, with different environments leading to distinct patterns of increased or decreased methylation levels along the chromosome. Despite a heterogeneous effect of different environments on methylation patterns, we find a general principle where the dependence of methylation states on position in the genome decreases in proportion to growth rate. A simple model that considers the methylation state through different phases of the cell cycle as a function of distance from an origin provides a framework to interpret the effects of different stressors upon the observed environmental responsiveness of the methylation patterns. Our work highlights how sequencing data alone can shed light on important aspects of microbial physiology. Significance StatementDNA methylation is known to profoundly impact gene regulation in prokaryotes, with both distinct methylation states at specific loci and global levels of DNA methylation modulating critical cellular phenotypes. Yet whether or how DNA methylation patterns depend on environmental variation remains unclear. Using Methylobacterium and Caulobacter as model systems, we combine experiments and theory to uncover general principles of how global patterns of DNA methylation are shaped by the environment. In particular, we discover a positive relationship between growth rate and the magnitude of the genomic position-dependent methylation level, highlighting how sequencing data can provide a culture-independent approach to estimating microbial traits like growth rate in natural settings. Our results resolve an open question, highlighting that DNA methylation patterns in bacteria can rapidly change in response to environmental shifts and revealing rules by which methylation patterns can help understand cellular phenotypes.

microbiology↗

bioRxiv2026

Evolutionary Integration of a Foreign Aromatic Catabolic Pathway Drives Metabolic Trade-offs in Methylobacterium extorquens PA1

Bacteria often acquire novel metabolic functions through horizontal gene transfer, allowing them to utilize new carbon sources. However, to benefit from these new pathways, they must be integrated with the hosts native metabolism. In nature, this process is fine-tuned via selection, enabling bacteria to exploit new niches. Alternate routes for pathway integration might yield distinct patterns of trade-offs, leading to differentiation within the adapting population. Understanding the metabolic mechanisms underlying these trade-offs provides insight into what maintains metabolic diversity in the environment. Lignin, a complex aromatic biopolymer, serves as an ideal substrate for exploring these questions, as monomers require complex metabolic pathways to break aromatic rings and be fed directly into central metabolism. In this study, we used the phyllosphere bacterium Methylobacterium extorquens PA1 to examine how a previously engineered catabolic gene cluster enabling lignin monomer utilization integrates with central metabolism. To this end, we experimentally evolved strains on lignin monomers vanillate (VA) and protocatechuate (PCA). Whole-genome sequencing revealed substrate-specific mutations that collectively reprogram stress responses and carbon storage regulation. These mutations resulted in myriad metabolic trade-offs: VA adapted strains showed diminished growth on PCA, PCA adapted strains largely cross-adapted to VA, and both VA and PCA evolution decreased growth rate on non-aromatic native substrates like succinate. These findings illuminate how evolution optimizes catabolic flux and resource allocation to efficiently integrate a foreign pathway following horizontal gene transfer, and the pleiotropic effects of such optimization. ImportanceMicrobial use of aromatics derived from lignin, whether in natural ecosystems or in biomass conversion, has been a major research focus. While aromatic pathways are well-known to have broad substrate specificity, pulling in aromatic molecules at various levels of conversion, it is unclear how much evolutionary constraint there is upon adaptation to each of these steps. By experimentally evolving Methylobacterium extorquens carrying a foreign lignin-derived aromatic catabolic pathway, we demonstrate that adaptation involves not just increased pathway flux but also regulatory rewiring and reallocation of resources, especially through PHB metabolism and the TCA cycle. The results reveal substrate-specific trade-offs and cross-adaptation, illustrating how new functions reshape fitness landscapes. These insights on metabolic innovation, pleiotropy, and diversification following horizontal gene transfer have broad implications for microbial evolution and metabolic engineering.

microbiology↗