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Biochemical characterization of an E. coli cell division factor FtsE shows ATPase cycles similar to the NBDs of ABC-transporters.

ABSTRACT: The peptidoglycan (PG) layer is an intricate and dynamic component of the bacterial cell wall, which requires a constant balance between its synthesis and hydrolysis. FtsEX complex present on the inner membrane is shown to transduce signals to induce PG hydrolysis. FtsE has sequence similarity with the nucleotide-binding domains (NBDs) of ABC transporters. The NBDs in most of the ABC transporters couple ATP hydrolysis to transport molecules inside or outside the cell. Also, this reaction cycle is driven by the dimerization of NBDs. Though extensive studies have been carried out on the Escherchia coli FtsEX complex, it remains elusive regarding how FtsEX complex helps in signal transduction or transportation of molecules. Also, very little is known about the biochemical properties and ATPase activities of FtsE. Because of its strong interaction with the membrane-bound protein FtsX, FtsE stays insoluble upon overexpression in E. coli, and thus, most studies on E. coli FtsE (FtsEEc) in the past have used refolded FtsE. Here in the present paper, for the first time, we report the soluble expression, purification, and biochemical characterization of FtsE from E. coli. The purified soluble FtsE exhibits high thermal stability, exhibits ATPase activity and has more than one ATP-binding site. We have also demonstrated a direct interaction between FtsE and the cytoplasmic loop of FtsX. Together, our findings suggest that during bacterial division, the ATPase cycle of FtsE and its interaction with the FtsX cytoplasmic loop may help to regulate the PG hydrolysis at the mid cell.

SUBMITTER: Mallick S

PROVIDER: S-EPMC7791547 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Biochemical characterization of an E. coli cell division factor FtsE shows ATPase cycles similar to the NBDs of ABC-transporters.

Mallick Sunanda S Kumar Ashish A Dodia Hiren H Alexander Cyrus C Vasudevan Dileep D Beuria Tushar Kant TK

Bioscience reports 20210101 1

The peptidoglycan (PG) layer is an intricate and dynamic component of the bacterial cell wall, which requires a constant balance between its synthesis and hydrolysis. FtsEX complex present on the inner membrane is shown to transduce signals to induce PG hydrolysis. FtsE has sequence similarity with the nucleotide-binding domains (NBDs) of ABC transporters. The NBDs in most of the ABC transporters couple ATP hydrolysis to transport molecules inside or outside the cell. Also, this reaction cycle i ...[more]

PMID: 33320186

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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.

Dataset Information

Biochemical characterization of an E. coli cell division factor FtsE shows ATPase cycles similar to the NBDs of ABC-transporters.

Publications

Biochemical characterization of an E. coli cell division factor FtsE shows ATPase cycles similar to the NBDs of ABC-transporters.

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