Involvement of FtsE ATPase and FtsX Extracellular Loops 1 and 2 in FtsEX-PcsB Complex Function in Cell Division of Streptococcus pneumoniae D39

The FtsEX protein complex has recently been proposed to play a major role in coordinating peptidoglycan (PG) remodeling by hydrolases with the division of bacterial cells. According to this model, cytoplasmic FtsE ATPase interacts with the FtsZ divisome and FtsX integral membrane protein and powers allosteric activation of an extracellular hydrolase interacting with FtsX. In the major human respiratory pathogen Streptococcus pneumoniae (pneumococcus), a large extracellular-loop domain of FtsX (ECL1(FtsX)) is thought to interact with the coiled-coil domain of the PcsB protein, which likely functions as a PG amidase or endopeptidase required for normal cell division. This paper provides evidence for two key tenets of this model. First, we show that FtsE protein is essential, that depletion of FtsE phenocopies cell defects caused by depletion of FtsX or PcsB, and that changes of conserved amino acids in the FtsE ATPase active site are not tolerated. Second, we show that temperature-sensitive (Ts) pcsB mutations resulting in amino acid changes in the PcsB coiled-coil domain (CC(PcsB)) are suppressed by ftsX mutations resulting in amino acid changes in the distal part of ECL1(FtsX) or in a second, small extracellular-loop domain (ECL2(FtsX)). Some FtsX suppressors are allele specific for changes in CC(PcsB), and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAP(PcsB)). These results strongly support roles for both ECL1(FtsX) and ECL2(FtsX) in signal transduction to the coiled-coil domain of PcsB. Finally, we found that pcsB(CC)(Ts) mutants (Ts mutants carrying mutations in the region of pcsB corresponding to the coiled-coil domain) unexpectedly exhibit delayed stationary-phase autolysis at a permissive growth temperature.