Subunit Arrangement in GpsB, a Regulator of Cell Wall Biosynthesis

Abstract

GpsB, a key regulator of cell division in Gram-positive bacteria, interacts with a key peptidoglycan synthase at the cell division septum, the penicillin binding protein PBP1 (a.k.a. PonA). Bacillus subtilis GpsB has been reported to interact with other components of the cell division machinery, including EzrA, MreC, and PrkC. In this study, we report an analysis of the arrangement of subunits in Listeria monocytogenes GpsB by small-angle X-ray scattering. The resulting model has an elongated shape with residues critical for interaction with PBP1 and the cell membrane clustered at one end of the molecule. Mutations that destabilize the hexameric assembly of the wild-type protein have a gpsB null phenotype, indicating that oligomerization is critical for the correct function of GpsB. We suggest a model in which a single GpsB hexamer can interact with multiple PBP1 molecules and can therefore influence the arrangement of PBP1 molecules within the cell division machinery, a dynamic multiprotein complex called the divisome, consistent with a role for GpsB in modulating the synthesis of the cell wall.

The GpsB files: the truth is out there

“…Recent results in Listeria monocytogenes are in general support of these findings – here GpsB was found to switch between septal and side‐wall sites of active PG synthesis (Rismondo et al ., ) and the direct interaction between GpsB and PBPA1, the listerial orthologue of B. subtilis PBP1, was confirmed in vitro as well as in vivo (Cleverley et al ., ; Rismondo et al ., ). Structural and biochemical studies determined that a negatively‐charged surface depression in GpsB interacted with the positively‐charged cytoplasmic domain of PBPA1 (Cleverley et al ., ; Rismondo et al ., ). Phosphomimetic mutations at Thr88 (the equivalent to Thr75 in B. subtilis ) had a gpsB null‐like phenotype, but phosphoablative mutations had no effect on growth of L. monocytogenes and neither class of substitution had any significant impact on GpsB folding, assembly or stability in vitro (Cleverley et al ., ).…”

“…Structural and biochemical studies determined that a negatively‐charged surface depression in GpsB interacted with the positively‐charged cytoplasmic domain of PBPA1 (Cleverley et al ., ; Rismondo et al ., ). Phosphomimetic mutations at Thr88 (the equivalent to Thr75 in B. subtilis ) had a gpsB null‐like phenotype, but phosphoablative mutations had no effect on growth of L. monocytogenes and neither class of substitution had any significant impact on GpsB folding, assembly or stability in vitro (Cleverley et al ., ). It would thus seem that GpsB functions in rod‐shaped bacteria to shuttle PBP1 away from the cell pole to the side‐wall for elongation and that the Z‐ring regulator EzrA returns PBP1/GpsB back to the septum for division, but the role of phosphorylation is yet to be clarified.…”

“…The EzrA dimer (PDBid 4BR8) is coloured wheat and sand; a dimer of FtsA (4A2A) is shown in pale and lime green; an FtsZ dimer (1W5A) is light and sky blue; the dimeric extracellular domain of MreC (2J5U) is blue‐white; the glycosyltransferase (GT) and transpeptidase (TP) domains of PBP2a (3ZG7) are coloured pale cyan and cyan, respectively; the pedestal and TP domains of PBP2b (2WAD) are light orange and orange, respectively; the pedestal, PASTA and TP domains of PBP2x (1QME) are coloured light pink, violet and purple, respectively, and the extracellular, PASTA and intracellular, catalytic domains of StkP (3PY9 and 1O6Y) are both grey. The N‐terminus of the GpsB hexamer from SAXS analysis is pale yellow and the C‐terminal domain is lemon (Cleverley et al ., ). Trans‐membrane helices are black and the N‐terminal, cytoplasmic domains of the three PBPs are coloured salmon.…”

“…GpsB is phosphorylated on Thr75 by the kinase PrkC (Pompeo et al ., ), but this does not seem to regulate cell division in B. subtilis , instead GpsB appears to be required for PrkC function and the phosphorylation of GpsB may actually be part of a negative feedback loop (Pompeo et al ., ). Recent results in Listeria monocytogenes are in general support of these findings – here GpsB was found to switch between septal and side‐wall sites of active PG synthesis (Rismondo et al ., ) and the direct interaction between GpsB and PBPA1, the listerial orthologue of B. subtilis PBP1, was confirmed in vitro as well as in vivo (Cleverley et al ., ; Rismondo et al ., ). Structural and biochemical studies determined that a negatively‐charged surface depression in GpsB interacted with the positively‐charged cytoplasmic domain of PBPA1 (Cleverley et al ., ; Rismondo et al ., ).…”

PynA is a pyrimidine 5′‐nucleotidase that functions as an antimutator protein inStreptococcus pneumoniae

“…However, in light of our recent findings, we hypothesize that downregulation of spr1057 (pynA) and other neighbouring genes involved in DNA repair, including spr1054 (mutX) and spr1055 (ung) [9], may enable cells to acquire suppressor mutations that allows for the slowgrowth phenotype to be bypassed in the strain lacking stkP. GpsB is an adaptor protein that mediates interactions between peptidoglycan synthases and other cell division proteins and coordinates cell wall synthesis with the bacterial cell cycle [43,44]. GpsB is an adaptor protein that mediates interactions between peptidoglycan synthases and other cell division proteins and coordinates cell wall synthesis with the bacterial cell cycle [43,44].…”