Studies on the Regulation of Acetyl-CoA Carboxylase and its Influence on Bacillus subtilis Differentiation

In starving Bacillus subtilis cells, the accDA operon encoding two subunits of the essential enzyme acetyl-CoA carboxylase (ACC) is a direct target of the master regulator of sporulation, Spo0A. Binding of Spo0A to a cis-element (0A box) in the promoter region of the operon has recently been identified as a strict requirement for biofilm formation and sporulation, two widely-studied cell differentiation processes which occur after the exponential growth phase. Here, we present evidence to conclude ongoing debates in B. subtilis AccDA research and propose new mechanisms that shed light on the complex regulation of the accDA operon. We provide experimental evidence for the location of the disputed accD start codon, show that the transition state regulator AbrB upregulates accDA expression during growth and the stationary phase, and identify the site at which AbrB binds to the accDA promoter. We also determine that two 0A boxes, one previously known (0A-1) and another newly discovered (0A-2), in the accDA promoter region are positively and negatively regulated by Spo0A~P, respectively. Finally, we demonstrate that the ACC enzyme localizes on or near the cell membrane by directly observing a functional GFP fusion to the AccA subunit. Collectively, we propose a revised model in which the accDA operon is regulated by a triple-input AND gate composed of σA-RNA polymerase, AbrB, and Spo0A~P that fine-tunes accDA expression from growth to starvation to support proper biofilm formation and sporulation.Biology and Biochemistry, Department o