Central carbon metabolism acts as a regulator of bacterial intracellular activity based on environmental conditions. Understanding the processes regulated by metabolism will provide insight into how bacteria grow and develop for applications in biotechnology and infectious disease. Acetyl-Coenzyme A (acetyl-CoA), a molecular intermediate of carbon metabolism, is essential for various cellular processes including energy production, cell development, and protein acetylation. The acetate pathway acts as a relief mechanism for excess carbon metabolism by converting acetyl-CoA into acetate, which is secreted by the cell. Inactivation of the acetate pathway causes a buildup of acetyl-CoA accompanied by delayed growth and cell death in Bacillus subtilis, a Gram-positive soil dwelling bacterium commonly used as a model for molecular microbiology research. However, the mechanism behind these acetyl-CoA-mediated growth defects are unknown. The acetate pathway was inactivated in B. subtilis by knocking out the ackA gene (ΔackA). ΔackA suppressor mutants were isolated based on recovered growth, and morphology studies and whole genome sequencing were performed to investigate underlying pathways. ΔackA cells exhibited altered morphology, including elongated cells and blebbing, which indicated defects in cell division and cell wall biosynthesis. The suppressors, however, exhibited significantly recovered growth and morphology resembling wild type cells. Whole genome sequencing revealed that several suppressors had mutations in pyruvate dehydrogenase complex genes pdhA and pdhC, likely inhibiting production of excess acetyl-CoA. An additional mutation was observed in dapH, a gene involved in the acetylation of a cell wall precursor. These results suggest that acetyl-CoA levels act as a signaling mechanism to connect metabolic status to cell wall biosynthesis and that the cell maintains a balance of acetyl metabolites for proper growth. This study deepens our understanding of metabolism as a regulatory mechanism for cellular processes.

Bacillus subtilis biofilms are effective models for studying prokaryotic multicellular systems. These biofilms, multicellular bacterial communities encased in a protective polysaccharide and protein matrix, are associated with several bacterial and chronic infections but can also serve beneficial purposes in agriculture and wastewater treatment. Recent investigation into post‐translational protein modification in bacteria has elucidated important regulatory mechanisms for growth and biofilm formation. Protein acetylation has been observed to regulate bacterial multicellularity, and it is closely linked to cellular metabolism by using acetyl metabolites as donors of acetyl groups. The goal of this study is to examine the role of protein lysine acetylation as a regulatory mechanism for metabolism B. subtilis. We hypothesize that bacteria use acetylation as a buffering mechanism for acetyl metabolites to balance levels and prevent metabolite toxicity by increasing acetylation during times of high acetyl metabolite levels and deacetylating proteins to restore metabolites during times of low levels. Wild type bacteria as well as knockout mutants affecting protein acetylation and the carbon overflow pathway are grown in biofilm‐inducing minimal media, and cell and supernatant samples are collected at various ODs. Acetyl‐CoA, acetyl‐phosphate, and acetate levels over the course of growth are measured using fluorometric and colorimetric metabolite test level kits. Dynamics of global lysine acetylation levels are also measured by an anti‐acetyl lysine Western blot. Work so far has focused primarily on optimizing and troubleshooting metabolite assay kits by modifying growth and sample preparation conditions. Acetate dynamics have been measured for the wild type bacteria, showing a continuous secretion of acetate into the supernatant throughout exponential growth. This result is expected, as rapid growth should coincide with continuous utilization of the carbon overflow pathway. Initial Western blots show increased acetylation in the ΔackAΔacuCΔsrtN triple knockout mutant, which is expected due to a buildup of acetyl phosphate and inhibition of deacetylase activity. Future research will also focus on analyzing mutated genes in Δpta suppressor mutants to uncover genes related to acetyl‐CoA‐mediated cell death. Findings from this study will promote the understanding of the regulatory mechanisms behind biofilm formation and potentially uncover novel information about bacterial survival strategies and programmed cell death.Support or Funding InformationThis project was funded by grants from the National Science Foundation, the Northeastern University Honors Program, and the Northeastern University Office of Undergraduate Research and Fellowships.