Physiological Consequences of Overexpression of a Twin-Arginine Translocase in Bacillus subtilis Revealed by ¹⁴ N/ ¹⁵ N Labeling

Bacillus subtilis is a Gram-positive bacterium widely used in biotechnology due to its efficient secretion systems. Among these, the twin-arginine (Tat) pathway facilitates the export of fully folded cofactor-containing proteins across the cytoplasmic membrane. The TatAyCy translocase, which is expressed constitutively, is key to this process. Previous studies showed that this translocase not only consists of the TatAy and TatCy subunits, but that it also recruits the LiaH protein upon overexpression. Presumably, the recruitment of LiaH represents an intrinsic protective mechanism against the potentially detrimental effects of facilitating the membrane passage of large, fully folded proteins. However, to date the full spectrum of physiological consequences of protein translocation via TatAyCy has remained elusive. In this study, we employed a 14N/15N metabolic labeling approach combined with subcellular fractionation to quantitatively analyze proteomic changes in the cytoplasm, membrane, and extracellular milieu upon TatAyCy overexpression. Our findings show that high-level TatAyCy expression leads to a prolonged vegetative state and disrupts key cellular processes, including genetic competence, motility, chemotaxis, and biofilm formation. Notably, arginine metabolism emerges as a central factor in the cellular adaptation to TatAyCy-induced stress.