Septal localization of the membrane-bound division proteins of Bacillus subtilis DivIB and DivIC is codependent only at high temperatures and requires FtsZ

Abstract

Using immunofluorescence microscopy, we have examined the dependency of localization among three Bacillus subtilis division proteins, FtsZ, DivIB, and DivIC, to the division site. DivIC is required for DivIB localization. However, DivIC localization is dependent on DivIB only at high growth temperatures, at which DivIB is essential for division. FtsZ localization is required for septal recruitment of DivIB and DivIC, but FtsZ can be recruited independently of DivIB. These localization studies suggest a more specific role for DivIB in division, involving interaction with DivIC.

FIG. 1

Western blots showing cellular levels of DivIC in BB11 (Pspac-ftsZ) (A) and in the divIB null mutant, SU321, and its wild-type parent strain, SU5 (B). (A) BB11 was grown at 37°C in IPTG-containing medium (0 min) and resuspended in either IPTG-lacking or IPTG-containing medium for 20, 40, 60, and 90 min. (B) SU321 and SU5 were shifted from 30°C at mid-exponential phase to 49°C for 1 h. Samples were collected prior to (0 min) and 20, 40, and 60 min after the shift. A₅₉₀ equivalents were loaded in each case. Standard protein positions are expressed in kilodaltons.

FIG. 2

DivIB localization in SU391 (Pspac-RRC) and SU392 (Pspac-divIC) in the presence of IPTG at 45°C. All panels show both SU391 and SU392 cells (long and short, respectively) that were mixed prior to IFM processing. (A and C) Phase-contrast images; (B and D) fluorescein isothiocyanate-immunostained images of the same field. Cells were grown at 30°C to mid-exponential phase and then shifted to 45°C for 45 min. Arrowheads indicate the long SU391 cells. Bar = 5 μm (A).

FIG. 3

DivIC and FtsZ localization in SU321 (divIB null) grown at 30°C to mid-exponential phase and then shifted to 49°C for 1 h. (B and D) Cells immunostained with fluorescein isothiocyanate; (A and C) phase-contrast image of the same field. DivIC localization is shown in panel B, while FtsZ localization is shown in panel D. Cells grown at 30°C (short) were mixed with long cells (indicated by arrowheads) that had been shifted to 49°C for 1 h. Bar = 5 μm (A).