Essential PcsB putative peptidoglycan hydrolase interacts with the essential FtsXSpn cell division protein in Streptococcus pneumoniae D39

ABSTRACTHow 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 inStreptococcus pneumoniae. We show thatlocZis 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.

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Structure of the large extracellular loop of FtsX and its interaction with the essential peptidoglycan hydrolase PcsB inStreptococcus pneumoniae

10.1101/432344 ◽

2018 ◽

Author(s):

Britta E. Rued ◽

Martín Alcorlo ◽

Katherine A. Edmonds ◽

Siseth Martínez-Caballero ◽

Daniel Straume ◽

...

Keyword(s):

Cell Division ◽

Streptococcus Pneumoniae ◽

Membrane Protein ◽

Cell Viability ◽

Integral Membrane Protein ◽

Peptidoglycan Hydrolase ◽

Physical Interaction ◽

Extracellular Loop ◽

Large Extracellular Loop

ABSTRACTStreptococcus pneumoniaeis a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically targeting essential proteins for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises an integral membrane protein (FtsX) and cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present NMR solution structural and crystallographic models of the large extracellular domain of FtsX, denoted ECL1. The structure of ECL1 reveals an upper extended β-hairpin and a lower α-helical lobe, each extending from a mixed α-β core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB, via the coiled-coil domain of PcsB (PcsB-CC). Characterization ofS. pneumoniaeD39 derived strains harboring mutations in the α-helical lobe shows that this subdomain is essential for cell viability and required for proper cell division ofS. pneumoniae.IMPORTANCEFtsX is a ubiquitous bacterial integral membrane protein involved in cell division that regulates the activity of peptidoglycan (PG) hydrolases. FtsX is representative of a large group of ABC3 superfamily proteins that function as “mechanotransmitters,” proteins that relay signals from inside to the outside of the cell. Here we present a structural characterization of the large extracellular loop (ECL1) of FtsX from the human opportunistic pathogenStreptococcus pneumoniae. We show a direct interaction between the peptidoglycan hydrolase PcsB and FtsX, and demonstrate that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.

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1H, 13C, 15N resonance assignments of the extracellular loop 1 domain (ECL1) of Streptococcus pneumoniae D39 FtsX, an essential cell division protein

Biomolecular NMR Assignments ◽

10.1007/s12104-015-9644-9 ◽

2015 ◽

Vol 10 (1) ◽

pp. 89-92 ◽

Cited By ~ 3

Author(s):

Yue Fu ◽

Kevin E. Bruce ◽

Britta Rued ◽

Malcolm E. Winkler ◽

David P. Giedroc

Keyword(s):

Cell Division ◽

Streptococcus Pneumoniae ◽

Resonance Assignments ◽

Extracellular Loop ◽

Cell Division Protein

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CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus

10.1101/256560 ◽

2018 ◽

Author(s):

Gro Anita Stamsås ◽

Ine Storaker Myrbråten ◽

Daniel Straume ◽

Zhian Salehian ◽

Jan-Willem Veening ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Cell Division ◽

Streptococcus Pneumoniae ◽

Normal Cell ◽

Double Mutant ◽

Spherical Shape ◽

Cell Wall Synthesis ◽

Morphological Defects ◽

Cell Division Protein

SummaryStaphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozESpn of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the ΔcozEaΔcozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the ΔcozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with the cell division protein EzrA, and that EzrA-GFP mislocalizes in the absence of CozEa and CozEb. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozESpn-depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA.

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