Phosphorylation of the cell division protein GpsB regulates PrkC kinase activity through a negative feedback loop in Bacillus subtilis

Abstract

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.

Splitsville: structural and functional insights into the dynamic bacterial Z ring

“…Curiously, the ZipA requirement for preseptal peptidoglycan synthesis can be bypassed by FtsA*, suggesting that assembly state of FtsZ may ultimately govern this activity. In low-GC Gram-positive species such as B. subtilis , EzrA plays an indirect role in recruitment of PBPs 22,99 , which is tied to post-translational regulation via the serine-threonine protein kinase family member PrkC 100–102 .…”

Subunit Arrangement in GpsB, a Regulator of Cell Wall Biosynthesis

“…Perhaps the gpsB null phenotype of phosphomimetic mutations reflects the introduction of a net negative charge in this region affecting the ability of GpsB to interact with PrkC or another divisome component. A molecular rationale for the gpsB null phenotype associated with the phosphomimetic mutation reported earlier 19 and herein remains to be determined.…”

“…19 The phosphomimetic mutations T75D and T75E appeared to have the same salt-sensitive phenotype as a gpsB deletion. 19 Likewise, we found that the introduction of the analogous T88D exchange into Lm GpsB almost completely inactivated the protein. However, the phosphoablative T88A mutation was without effect on complementation activity of Lm GpsB (Fig.…”

“…The position of F91 and L94 close to or at the interface between subunits is particularly interesting in view of the recent identification that, in B. subtilis , the nearby T75 (equivalent to T88 in L. monocytogenes ) is phosphorylated by the kinase PrkC 19 and that phosphomimetic mutations at this position produce a gpsB null-like phenotype both in B. subtilis and in L. monocytogenes . Similarly, phosphoablative mutations have no demonstrable effect in either species.…”

“…First, GpsB has been reported to interact with EzrA in two hybrid assays, although only unidirectionally, 5 potentially providing an indirect link between FtsZ and PBPs, given that numerous in vitro and in vivo studies have shown that EzrA interacts with FtsZ ( e.g ., 6–9). Second, it has been reported that GpsB is phosphorylated in B. subtilis by PrkC, 19 a eukaryotic-like membrane-embedded Ser/Thr kinase with an extracellular PASTA domain 20 that binds peptidoglycan fragments. 21 The relationship between GpsB and PrkC suggests that GpsB could be involved in transducing alterations in cell wall structure on the outside of the cell to the interior.…”