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Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae.

ABSTRACT: FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily. The component FtsX is an integral membrane protein, whereas FtsE is an ATPase and is required for the transmission of a conformational signal from the cytosol through the membrane to regulate the activity of cell wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium Streptococcus pneumoniae, and FtsX interacts with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report high-resolution structures of pneumococcal FtsE bound to different nucleotides. Structural analysis revealed that FtsE contains all the conserved structural motifs associated with ATPase activity and afforded interpretation of the in vivo dimeric arrangement in both the ADP and ATP states. Interestingly, three specific FtsE regions with high structural plasticity were identified that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for contacting FtsE, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology.IMPORTANCE Bacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, plays a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the type VII family of the ABC-superfamily, but instead of being a transporter, it couples the ATP hydrolysis catalyzed by FtsE to mechanically transduce a conformational signal that provokes the activation of peptidoglycan (PG) hydrolases. So far, no structural information is available for FtsE. Here, we provide the structural characterization of FtsE, confirming its ATPase nature and revealing regions with high structural plasticity which are key for FtsE binding to FtsX. The complementary binding region in FtsX has also been identified and validated in vivo Our results provide evidence on how the difference between the ATP/ADP-bound states in FtsE would dramatically alter the interaction of FtsEX with the PG hydrolase PcsB in pneumococcal division.

SUBMITTER: Alcorlo M

PROVIDER: S-EPMC7468199 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae.

Alcorlo Martin M Straume Daniel D Lutkenhaus Joe J Håvarstein Leiv Sigve LS Hermoso Juan A JA

mBio 20200901 5

FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily. The component FtsX is an integral membrane protein, whereas FtsE is an ATPase and is required for the transmission of a conformational signal from the cytosol through the membrane to regulate the ...[more]

PMID: 32873757

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Project description: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 (ECL1FtsX) 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 (CCPcsB) are suppressed by ftsX mutations resulting in amino acid changes in the distal part of ECL1FtsX or in a second, small extracellular-loop domain (ECL2FtsX). Some FtsX suppressors are allele specific for changes in CCPcsB, and no FtsX suppressors were found for amino acid changes in the catalytic PcsB CHAP domain (CHAPPcsB). These results strongly support roles for both ECL1FtsX and ECL2FtsX in signal transduction to the coiled-coil domain of PcsB. Finally, we found that pcsBCC(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. Little is known about how FtsX interacts with cognate PG hydrolases in any bacterium, besides that ECL1FtsX domains somehow interact with coiled-coil domains. This work used powerful genetic approaches to implicate a specific region of pneumococcal ECL1FtsX and the small ECL2FtsX in the interaction with CCPcsB. These findings identify amino acids important for in vivo signal transduction between FtsX and PcsB for the first time. This paper also supports the central hypothesis that signal transduction between pneumococcal FtsX and PcsB is linked to ATP hydrolysis by essential FtsE, which couples PG hydrolysis to cell division. The classical genetic approaches used here can be applied to dissect interactions of other integral membrane proteins involved in PG biosynthesis. Finally, delayed autolysis of the pcsBCC(Ts) mutants suggests that the FtsEX-PcsB PG hydrolase may generate a signal in the PG necessary for activation of the major LytA autolysin as pneumococcal cells enter stationary phase.

Project description:Streptococcus pneumoniae is an ovoid-shaped Gram-positive bacterium that grows by carrying out peripheral and septal peptidoglycan (PG) synthesis, analogous to model bacilli, such as Escherichia coli and Bacillus subtilis In the model bacilli, FtsZ and FtsA proteins assemble into a ring at midcell and are dedicated to septal PG synthesis but not peripheral PG synthesis; hence, inactivation of FtsZ or FtsA results in long filamentous cells unable to divide. Here, we demonstrate that FtsA and FtsZ colocalize at midcell in S. pneumoniae and that partial depletion of FtsA perturbs septum synthesis, resulting in elongated cells with multiple FtsZ rings that fail to complete septation. Unexpectedly, complete depletion of FtsA resulted in the delocalization of FtsZ rings and ultimately cell ballooning and lysis. In contrast, depletion or deletion of gpsB and sepF, which in B. subtilis are synthetically lethal with ftsA, resulted in enlarged and elongated cells with multiple FtsZ rings, with deletion of sepF mimicking partial depletion of FtsA. Notably, cell ballooning was not observed, consistent with later recruitment of these proteins to midcell after Z-ring assembly. The overproduction of FtsA stimulates septation and suppresses the cell division defects caused by the deletion of sepF and gpsB under some conditions, supporting the notion that FtsA shares overlapping functions with GpsB and SepF at later steps in the division process. Our results indicate that, in S. pneumoniae, both GpsB and SepF are involved in septal PG synthesis, whereas FtsA and FtsZ coordinate both peripheral and septal PG synthesis and are codependent for localization at midcell.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is a clinically important human pathogen for which more therapies against unexploited essential targets, like cell growth and division proteins, are needed. Pneumococcus is an ovoid-shaped Gram-positive bacterium with cell growth and division properties that have important distinctions from those of rod-shaped bacteria. Gaining insights into these processes can thus provide valuable information to develop novel antimicrobials. Whereas rods use distinctly localized protein machines at different cellular locations to synthesize peripheral and septal peptidoglycans, we present evidence that S. pneumoniae organizes these two machines at a single location in the middle of dividing cells. Here, we focus on the properties of the actin-like protein FtsA as an essential orchestrator of peripheral and septal growth in this bacterium.

Project description:UnlabelledHow bacteria control proper septum placement at midcell, to guarantee the generation of identical daughter cells, is still largely unknown. Although different systems involved in the selection of the division site have been described in selected species, these do not appear to be widely conserved. Here, we report that LocZ (Spr0334), a newly identified cell division protein, is involved in proper septum placement in Streptococcus pneumoniae. We show that locZ is not essential but that its deletion results in cell division defects and shape deformation, causing cells to divide asymmetrically and generate unequally sized, occasionally anucleated, daughter cells. LocZ has a unique localization profile. It arrives early at midcell, before FtsZ and FtsA, and leaves the septum early, apparently moving along with the equatorial rings that mark the future division sites. Consistently, cells lacking LocZ also show misplacement of the Z-ring, suggesting that it could act as a positive regulator to determine septum placement. LocZ was identified as a substrate of the Ser/Thr protein kinase StkP, which regulates cell division in S. pneumoniae. Interestingly, homologues of LocZ are found only in streptococci, lactococci, and enterococci, indicating that this close phylogenetically related group of bacteria evolved a specific solution to spatially regulate cell division.ImportanceBacterial cell division is a highly ordered process regulated in time and space. Recently, we reported that the Ser/Thr protein kinase StkP regulates cell division in Streptococcus pneumoniae, through phosphorylation of several key proteins. Here, we characterized one of the StkP substrates, Spr0334, which we named LocZ. We show that LocZ is a new cell division protein important for proper septum placement and likely functions as a marker of the cell division site. Consistently, LocZ supports proper Z-ring positioning at midcell. LocZ is conserved only among streptococci, lactococci, and enterococci, which lack homologues of the Min and nucleoid occlusion effectors, indicating that these bacteria adapted a unique mechanism to find their middle, reflecting their specific shape and symmetry.

Dataset Information

Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae.

Publications

Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae.

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