scholarly journals Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae

2016 ◽  
Vol 5 (5) ◽  
pp. 738-752 ◽  
Author(s):  
Ruchika Bajaj ◽  
Kevin E. Bruce ◽  
Amy L. Davidson ◽  
Britta E. Rued ◽  
Cynthia V. Stauffacher ◽  
...  
Keyword(s):  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Biochemical Characterization ◽  
Peptidoglycan Hydrolysis

scholarly journals Biochemical Characterization of the CDP-D-Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F

2012 ◽  
Vol 194 (8) ◽  
pp. 1868-1874 ◽  
Author(s):  
Q. Wang ◽  
Y. Xu ◽  
A. V. Perepelov ◽  
Y. A. Knirel ◽  
P. R. Reeves ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biosynthetic Pathway ◽  
Biochemical Characterization

scholarly journals The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H)

2005 ◽  
Vol 389 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Nehmé SAKSOUK ◽  
Ludovic PELOSI ◽  
Pierre COLIN-MOREL ◽  
Manel BOUMEDIENNE ◽  
Patricia L. ABDIAN ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Biochemical Characterization ◽  
Repeat Unit ◽  
Sugar Residue ◽  
Functional Identification ◽  
Polysaccharide Biosynthesis ◽  
Sugar Addition ◽  
Galactose Residue

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one α-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([→4)-α-D-Glcp-(1→4)-α-D-Galp-(1→4)-β-D-GlcAp-(1→4)-β-D-Glcp-(1→]n). Expression of WciS–His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/ΔgalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [β-D-GlcAp-(1→4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by α-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS–His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

scholarly journals Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis

2001 ◽  
Vol 40 (1) ◽  
pp. 115-125 ◽  
Author(s):  
Andrea Feucht ◽  
Isabelle Lucet ◽  
Michael D. Yudkin ◽  
Jeffery Errington
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Biochemical Characterization ◽  
Cell Division Protein

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

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.

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

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Martin Alcorlo ◽  
Daniel Straume ◽  
Joe Lutkenhaus ◽  
Leiv Sigve Håvarstein ◽  
Juan A. Hermoso
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Structural Characterization ◽  
Bound States ◽  
Structural Information ◽  
Structural Plasticity ◽  
Membrane Complex

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.

scholarly journals Biochemical Characterization of Streptococcus pneumoniae Penicillin-Binding Protein 2b and Its Implication in β-Lactam Resistance

2004 ◽  
Vol 48 (5) ◽  
pp. 1848-1855 ◽  
Author(s):  
Estelle Pagliero ◽  
Laurent Chesnel ◽  
Julie Hopkins ◽  
Jacques Croizé ◽  
Otto Dideberg ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Biochemical Characterization ◽  
Protein A ◽  
Strain Analysis ◽  
Penicillin G ◽  
Penicillin Binding Protein ◽  
Class B ◽  
Selection Of

ABSTRACT Extensive use of β-lactam antibiotics has led to the selection of pathogenic streptococci resistant to β-lactams due to modifications of the penicillin-binding proteins (PBPs). PBP2b from Streptococcus pneumoniae is a monofunctional (class B) high-molecular-weight PBP catalyzing the transpeptidation between adjacent stem peptides of peptidoglycan. The transpeptidase domain of PBP2b isolated from seven clinical resistant (CR) strains contains 7 to 44 amino acid changes over the sequence of PBP2b from the R6 β-lactam-sensitive strain. We show that the extracellular soluble domains of recombinant PBP2b proteins (PBP2b*) originating from these CR strains have an in vitro affinity for penicillin G that is reduced by up to 99% from that of the R6 strain. The Thr446Ala mutation is always observed in CR strains and is close to the key conserved motif (S443SN). The Thr446Ala mutation in R6 PBP2b* displays a 60% reduction in penicillin G affinity in vitro compared to that for the wild-type protein. A recombinant R6 strain expressing the R6 PBP2b Thr446Ala mutation is twofold less sensitive to piperacillin than the parental S. pneumoniae strain. Analysis of the Thr446Ala mutation in the context of the PBP2b CR sequences revealed that its influence depends upon the presence of other unidentified mutations.

scholarly journals Structural characterization of the essential cell division protein FtsE and its interaction with FtsX in Streptococcus pneumoniae

2020 ◽  
Author(s):  
Martin Alcorlo ◽  
Daniel Straume ◽  
Joe Lutkenhaus ◽  
Leiv Sigve Håvarstein ◽  
Juan A. Hermoso
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Structural Characterization ◽  
Structural Information ◽  
Structural Plasticity ◽  
Central Process ◽  
Membrane Complex

ABSTRACTFtsEX 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, where FtsX is an integral membrane protein and FtsE is an ATPase, required for mechanotransmission of the 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 interact with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report the high-resolution structures of pneumococcal FtsE in complex with different nucleotides. Structural analysis reveals that FtsE contains all the conserved structural motifs associated with ATPase activity, and allowed interpretation of the in vivo dimeric arrangement in both ADP and ATP states. Interestingly, three specific FtsE regions were identified with high structural plasticity that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for FtsE contact, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology.IMPORTANCEBacterial 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, play 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 transporter, couple ATP hydrolysis by FtsE to mechanically transduce a conformational signal to activate 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 key for FtsX binding. The complementary region in FtsX has been also identified and validated in vivo. Our results provide evidences on how difference between ATP and ADP states in FtsE would dramatically alter FtsEX interaction with PG hydrolase PcsB in pneumococcal division.

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