Roles of pneumococcal DivIB in cell division

Abstract

DivIB, also known as FtsQ in gram-negative organisms, is a division protein that is conserved in most eubacteria. DivIB is localized at the division site and forms a complex with two other division proteins, FtsL and DivIC/FtsB. The precise function of these three bitopic membrane proteins, which are central to the division process, remains unknown. We report here the characterization of a divIB deletion mutant of Streptococcus pneumoniae, which is a coccus that divides with parallel planes. Unlike its homologue FtsQ in Escherichia coli, pneumococcal DivIB is not required for growth in rich medium, but the Delta divIB mutant forms chains of diplococci and a small fraction of enlarged cells with defective septa. However, the deletion mutant does not grow in a chemically defined medium. In the absence of DivIB and protein synthesis, the partner FtsL is rapidly degraded, whereas other division proteins are not affected, pointing to a role of DivIB in stabilizing FtsL. This is further supported by the finding that an additional copy of ftsL restores growth of the Delta divIB mutant in defined medium. Functional mapping of the three distinct alpha, beta, and gamma domains of the extracellular region of DivIB revealed that a complete beta domain is required to fully rescue the deletion mutant. DivIB with a truncated beta domain reverts only the chaining phenotype, indicating that DivIB has distinct roles early and late in the division process. Most importantly, the deletion of divIB increases the susceptibility to beta-lactams, more evidently in a resistant strain, suggesting a function in cell wall synthesis.

FIG. 1.

Sequence alignment of the DivIB extracytoplasmic region. The alignment was generated with ClustalW, using in addition to the sequences shown those of Yersinia pestis, Vibrio cholerae, C. crescentus, H. influenzae, Mycobacterium tuberculosis, S. coelicolor, Bacillus bacilliformis, Corynebacterium glutamicum, Rhizobium meliloti, Listeria monocytogenes, Enterococcus hirae, S. aureus, L. pneumophila, Neisseria meningitidis, Borrelia burgdorferi, Streptococcus pyogenes, and Geobacillus kaustophilus. The domains were defined by limited tryptic proteolysis of recombinant pneumococcal DivIB. Residue 336 corresponds to the C terminus of the β domain defined by the tryptic digestion of DivIB from G. stearothermophilus (43).

FIG. 2.

Phenotypes of the ΔdivIB mutant. Bacteria were grown in TH broth at 37°C under 5% CO₂ to an optical density at 600 nm of 0.45 and prepared for microscopy. (A) Chain length distribution of strains R6 (black) and B2 (gray) (165 measurements each). (B) Transmission electron micrographs of negatively stained pneumococci (images were taken at nominal magnifications of ×10,000 or ×22,000 [top and bottom, respectively]). White arrowheads indicate equatorial rings, and black arrowheads point to septa. (C) Scanning electron micrographs. (D) Transmission electron micrographs of negatively stained thin sections of B2. Arrowheads point to nascent septa. Bars, 0.5 μm.

FIG. 3.

Stability in vivo of some division proteins in the absence of DivIB. Strains R6 and B2 were grown in TH broth to an optical density at 600 nm of 0.3 prior to addition of erythromycin (40 μg/ml) to inhibit protein synthesis. Aliquots withdrawn after the time intervals indicated were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. (A to G) Immunoblots with sera against the proteins indicated on the right. (H) Corresponding Coomassie blue-stained gel of total cell lysates.

FIG. 4.

Functional characterization of the DivIB domains. The cytoplasmic N-terminal domain is in white. The transmembrane segment is hatched. The three domains of the extracellular region are in various shades of gray. Alleles encoding DivIB variants were introduced in B2 at the bgaA locus. Exponentially growing cultures were analyzed by phase-contrast microscopy. For each strain, 200 particles were measured. Growth in CD medium was monitored in 12-well plates at 37°C. +, mass doubling of about 45 min. −, absence of growth at 35 h following the inoculation.