SummaryAlthough many membrane Ser/Thr-kinases with PASTA motifs have been shown to control bacterial cell division and morphogenesis, inactivation of the Ser/Thr-kinase PrkC does not impact Bacillus subtilis cell division. In this study, we show that PrkC localizes at the division septum. In addition, three proteins involved in cell division/elongation, GpsB, DivIVA and EzrA are required for stimulating PrkC activity in vivo. We show that GpsB interacts with the catalytic subunit of PrkC that, in turn, phosphorylates GpsB. These observations are not made with DivIVA and EzrA. Consistent with the phosphorylated residue previously detected for GpsB in a high-throughput phosphoproteomic analysis of B. subtilis, we show that threonine 75 is the single PrkC-mediated phosphorylation site in GpsB. Importantly, the substitution of this threonine by a phospho-mimetic residue induces a loss of PrkC kinase activity in vivo and a reduced growth under high salt conditions as observed for gpsB and prkC null mutants. Conversely, substitution of threonine 75 by a phospho-ablative residue does not induce such growth and PrkC kinase activity defects. Altogether, these data show that proteins of the divisome control PrkC activity and thereby phosphorylation of PrkC substrates through a negative feedback loop in B. subtilis.

During cell division, Gram-negative bacteria must coordinate inner membrane (IM) 4 invagination, peptidoglycan (PG) synthesis and cleavage and outer membrane (OM) 5 constriction. The OM constriction remains largely enigmatic and the nature of this 6 process, passive or active, is under debate. The proton-motive force (PMF) 7 dependent Tol-Pal system performs a network of interactions within these three 8 compartments. Here we confirm that the trans-envelope Tol-Pal complex 9 accumulates at constriction site in Escherichia coli. We show that the IM complex 10 composed of TolA, TolQ and TolR recruits the OM complex TolB-Pal to the septum, 11 in an energy-dependent process. Pal recruitment then allows its binding to PG and 12 subsequently, OM constriction. Our results provide evidence that the constriction of 13 the OM is an energized process. 14