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Identification of potential regulatory domains within the MreC and MreD components of the cell elongation machinery.

ABSTRACT: The bacterial peptidoglycan (PG) cell wall maintains cell shape and prevents osmotic lysis. During growth of rod-shaped cells, PG is incorporated along the cell cylinder by the RodA-PBP2 synthase of the multi-protein Rod system (elongasome). Filaments of the actin-like MreB protein orient synthesis of the new PG material. They are connected to the RodA-PBP2 synthase in part through the RodZ component. MreC and MreD are other conserved components of the system, but their function is not well understood. Amino acid changes in RodA-PBP2 were recently identified that bypass a requirement for MreC and MreD function, suggesting the Mre proteins act as activators of the synthase. To further investigate their function, we developed a genetic strategy to identify dominant-negative alleles of mreC and mreD in Escherichia coli Residues essential for Rod system function were identified at the junction of two subdomains within MreC and in a predicted ligand-binding pocket of MreD. Additionally, we found that although the proline-rich C-terminal domain of MreC is non-essential, substitutions within this region disrupt its function. Based on these results, we propose that the C-terminus of MreC and the putative ligand-binding domain of MreD play regulatory roles in controlling Rod system activity.IMPORTANCE: Cell shape in bacteria is largely determined by the cell wall structure that surrounds them. The multi-protein machine called the Rod system (elongasome) has long been implicated in rod-shape determination in bacilli. However, the functions of many of its conserved components remain unclear. Here, we describe a new genetic system to dissect the function of these proteins and how we used it to identify potential regulatory domains within them that may modulate the function of the shape-determining machinery.

SUBMITTER: Rohs PDA

PROVIDER: S-EPMC8092158 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Genome wide association studies (GWAS) are a population-scale approach to the identification of segments of the genome in which genetic variations may contribute to disease risk. Current methods focus on the discovery of single nucleotide polymorphisms (SNPs) associated with disease traits. As there are many SNPs within identified risk loci, and the majority of these are situated within non-coding regions, a key challenge is to identify and prioritize variants affecting regulatory sequences that are likely to contribute to the phenotype assessed.We focused investigation on SNPs within lung and breast cancer GWAS loci that reached genome-wide significance for potential roles in gene regulation with a specific focus on SNPs likely to disrupt transcription factor binding sites. Within risk loci, the regulatory potential of sub-regions was classified using relevant open chromatin and epigenetic high throughput sequencing data sets from the ENCODE project in available cancer and normal cell lines. Furthermore, transcription factor affinity altering variants were predicted by comparison of position weight matrix scores between disease and reference alleles. Lastly, ChIP-seq data of transcription associated factors and topological domains were included as binding evidence and potential gene target inference.The sets of SNPs, including both the disease-associated markers and those in high linkage disequilibrium with them, were significantly over-represented in regulatory sequences of cancer and/or normal cells; however, over-representation was generally not restricted to disease-relevant tissue specific regions. The calculated regulatory potential, allelic binding affinity scores and ChIP-seq binding evidence were the three criteria used to prioritize candidates. Fitting all three criteria, we highlighted breast cancer susceptibility SNPs and a borderline lung cancer relevant SNP located in cancer-specific enhancers overlapping multiple distinct transcription associated factor ChIP-seq binding sites.Incorporating high throughput sequencing epigenetic and transcription factor data sets from both cancer and normal cells into cancer genetic studies reveals potential functional SNPs and informs subsequent characterization efforts.

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Identification of potential regulatory domains within the MreC and MreD components of the cell elongation machinery.

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