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.

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.

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.

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.

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.