Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen

Abstract

SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity.

Fig. 1.

Asymmetric cell division and the role of SpoIIE. (A) In the pre-divisional cell (upper) and the mother cell (MC) following cell division (lower), SpoIIAA (AA) is phosphorylated and σ^F is in a complex with SpoIIAB (AB₂). In the forespore (FS), σ^F is free and AA and AB are in complex. SpoIIE (E) accumulates at the asymmetric septum, a double membrane structure. (B) The putative three domain structure of SpoIIE. The limits of the putative FtsZ-binding domain are uncertain.

Fig. 2.

Screening of the truncation library. The fluorescence signals from a small section of the colony array are shown. The array was probed for the presence of biotin with a steptavidin-Alexa fluor, which gives a green fluorescent signal as an indicator of solubility of the clone (left) and for hexa-histidine with conjugated anti-His₆ antibody, which gives a red fluorescent signal (right). Comparison of the two signals permits screening out of degraded or truncated constructs.

Fig. 3.

Mapping soluble SpoIIE constructs onto the primary structure. The sequenced fragments of SpoIIE are represented as black bars. The numbers in bold script represent the residue numbers of full-length SpoIIE. The fragment identifier and position of the first residue are indicated. Dotted boxes indicate clusters of similarly sized fragments, representing possible windows of solubility. The approximate domain organisation of SpoIIE inferred from earlier work is indicated.

Fig. 4.

Gel electrophoretic analysis of samples of the purified SpoIIE fragments. Coomassie blue staining of gels following resolution of the samples by electrophoresis. (A) 12.5% SDS-polyacrylamide gel. Lanes 1 and 5, low molecular markers (Bio-rad) with masses indicated; lane 2, the B1′ fragment SpoIIE(590–827); lane 3, the H1 fragment SpoIIE(412–827) and lane 4, the B2–B1 fragment SpoIIE(375–595) and (B) 7.5% non-denaturing polyacrylamide gel lane 1, Fragment B1′; lane 2, Fragment H1 and lane 3, the B2–B1 fragment.

Fig. 5.

Assays of SpoIIE fragments. (A) SpoIIAA-phosphate dephosphorylation by the H1 and B1′ fragments monitored by native gel electrophoresis. Lane 1, SpoIIAA∼P (5 μg); lane 2, SpoIIAA (5 μg); lanes 3 and 4, SpoIIAA∼P (5 μg) incubated in the presence of the H1 fragment at 100:1 and 400:1 molar ratios, respectively; lanes 5 and 6, SpoIIAA∼P (5 μg) incubated in the presence of the B1′ fragment at 100:1 and 400:1 molar ratios, respectively. The conversion of SpoIIAA∼P to the lower mobility SpoIIAA species upon incubation with the SpoIIE fragments is evident. (B) Gel mobility shift assay of FtsZ binding by the H1 and B1′ fragments. Lane 1, FtsZ (7 μg); lane 2, FtsZ (7 μg) + 1 mM GTP; lane 3, SpoIIE H1 fragment (7 μg); lane 4, FtsZ (7 μg) + SpoIIE H1 (7 μg) + 1 mM GTP; lane 5, SpoIIE B1′ fragment (7 μg); lane 6, FtsZ (7 μg) + SpoIIE B1′ (7 μg) + 1 mM GTP; lane 7, FtsZ (7 μg) + 1mM GTP. There is no mobility shift evident from these gels other than the additional staining of material at the top of lane 4. (C) SEC-MALLS traces of the molecular mass and differential refractive index (dRI) versus time, of the eluate from a Superdex S200 column. The bold lines give molecular mass of the eluting species calculated from measurements of the refractive index and the multi-angle laser light scattering. Three traces for the (i) H1 (red), (ii) B1′ (green) and (iii) B2–B1 (blue) fragments are overlaid.