The localization of key Bacillus subtilis penicillin binding proteins during cell growth is determined by substrate availability

Abstract

The shape of bacteria is maintained by the cell wall. The main component of the cell wall is peptidoglycan (PG) that is synthesized by penicillin binding proteins (PBPs). The correct positioning of PBPs is essential for the maintenance of cell shape. In the literature, two different models for localization of PBPs have been proposed - localization through interaction with a cytoskeletal structure or localization through the presence of substrate. Here, we show that the localization of PBPs critical for the rod shape of Bacillus subtilis is altered when the substrate LipidII is delocalized by treatment of the cells with nisin. Alteration of this localization is only seen in a LipidII-dependent manner and is not influenced by dissipation of the membrane potential, a secondary effect of nisin treatment. Our results strongly suggest that the localization of PG synthesis at the periphery of the cell is substrate-driven, even in bacteria that contain actin-like MreB cytoskeletal structures.

In Vivo Cluster Formation of Nisin and Lipid II Is Correlated with Membrane Depolarization

“…Control cells show a large amount of lipid II in the septum and additional lipid II on the cell edges, whereas nisin and PP-nisin induce the formation of spotty clusters with the loss of defined fluorescent cell edges ( Fig. 1A and B), as reported previously (12,13,19,20), although nisin was more potent at lower concentrations. ⌬⌬-Nisin was less potent in cluster formation, with only 27% of cells showing clusters at 30 g/ml and a minimum concentration at which cluster formation was observed of 20 g/ml.…”

“…PP-nisin was expected not to affect the membrane potential of live cells (7); however, we found that PP-nisin induced membrane potential loss (13). This compromised the localization of many membraneassociated proteins, including MreB (14).…”

“…Recently, we used PP-nisin as a tool to cluster lipid II into domains to determine the effect of delocalized lipid II on the localization of proteins involved in PG synthesis (13). PP-nisin was expected not to affect the membrane potential of live cells (7); however, we found that PP-nisin induced membrane potential loss (13).…”

“…This suggests that lantibiotics have a stronger pore-forming activity on live cell membranes, which could be caused by either differences in lipid composition, the presence of protein in the membranes, or the presence of a membrane potential. Neither lipid II binding (by nisin 1-22 or mersacidin) nor membrane depolarization (e.g., by CCCP [13]) alone is sufficient to form lipid II clusters. This strongly suggests that nisin-lipid II cluster formation results in depolarization, although we cannot formally exclude that depolarization results in clustering.…”

Strategies for Circumventing Bacterial Resistance Mechanisms

“…This force is, of course, necessary for bacterial vitality: it contributes directly, for example, to the correct localization of an essential highmolecular-mass penicillin-binding protein in B. subtilis as shown by the collapse of this force following exposure of the bacterium to the lantibiotic, nisin (Lages et al 2013). And while the collapse of this force is intimately connected to the cellular damage arising from oxidative stress (Ezraty et al 2013), the enzymes that sustain the proton-motive force represent independent targets for antibacterial discovery.…”