Transcriptional rewiring of the GcrA/CcrM bacterial epigenetic regulatory system in closely related bacteria

Abstract

Transcriptional rewiring is the regulation of different targets genes by orthologous regulators in different organisms. While this phenomenon has been observed, it has not been extensively studied, particularly in core regulatory systems. Several global cell cycle regulators are conserved in the Alphaproteobacteria, providing an excellent model to study this phenomenon. First characterized in Caulobacter crescentus, GcrA and CcrM compose a DNA methylation-based regulatory system that helps coordinate the complex life cycle of this organism. These regulators are well-conserved across Alphaproteobacteria, but the extent to which their regulatory targets are conserved is not known. In this study, the regulatory targets of GcrA and CcrM were analyzed by SMRT-seq, RNA-seq, and ChIP-seq technologies in the Alphaproteobacterium Brevundimonas subvibrioides, and then compared to those of its close relative C. crescentus that inhabits the same environment. Although the regulators themselves are highly conserved, the genes they regulate are vastly different. GcrA directly regulates 204 genes in C. crescentus, and though B. subvibrioides has orthologs to 147 of those genes, only 48 genes retained GcrA binding in their promoter regions. Additionally, only 12 of those 48 genes demonstrated significant transcriptional change in a gcrA mutant, suggesting extensive transcriptional rewiring between these organisms. Similarly, out of hundreds of genes CcrM regulates in each of these organisms, only 2 genes were found in common. When multiple Alphaproteobacterial genomes were analyzed bioinformatically for potential GcrA regulatory targets, the regulation of genes involved in DNA replication and cell division was well conserved across the Caulobacterales but not outside this order. This work suggests that significant transcriptional rewiring can occur in cell cycle regulatory systems even over short evolutionary distances.

DNA Methylation in Ensifer Species during Free-Living Growth and during Nitrogen-Fixing Symbiosis with Medicago spp.

“…Between 31 and 53 GANTC sites were repeatedly not detected as methylated on one or both strands in each of the four wild-type strains (see Data set S2 at doi.org/10.6084/m9.figshare.16556205 ), as has also been observed in other species ( 17 , 46 ). However, further work is required to determine if this hypomethylation is biologically meaningful (e.g., CcrM cannot access the site due to binding of another protein [ 46 ]) or if it reflects technical artifacts (e.g., low sequencing coverage).…”

“…Supporting this conclusion, no motif was enriched in the promoter regions of symbiosis, carbon source, or cell cycle-regulated genes, and we did not detect any motifs that were methylated specifically in bacteroids. However, we cannot rule out that one or more methylated motifs may influence specific gene expression during free-living growth, differentiation, or N 2 -fixation through extended motifs or proximity to other promoter elements, similar to the interplay between CcrM and GcrA during cell cycle regulation in C. crescentus ( 20 , 46 ).…”

“…Likely, CcrM-mediated gene regulation in the genus Ensifer is also dependent upon additional sequence elements beyond the GANTC motif. Nevertheless, as a recent study in the alphaproteobacterium Brevundimonas subvibrioides suggested ( 46 ), the CcrM and GcrA regulons as well as the GcrA-binding motif can show very little overlap between species, and therefore they will have to be defined experimentally in E. meliloti .…”

Design of Machine Learning Models for the Prediction of Transcription Factor Binding Regions in Bacterial DNA

“…Similarly, it has been found that the most significant tetramers for ML algorithms largely correspond to the motif that was found in the reference paper for the data used [2] with the MEME program, as presented in Figure 2.…”

“…Our work has been previously published as a Master's Thesis at the Universitat Oberta de Catalunya (UOC) [1]. The DNA sequences used as positive data were extracted from the article by Adhikari et al [2], obtained using the ChIP-seq technique with the GcrA TF in the bacterial organism Brevundimonas subvibrioides.…”