Movement Dynamics of Divisome and Penicillin-Binding Proteins (PBPs) in Cells of Streptococcus pneumoniae

ABSTRACTBacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae (Spn). Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase binding partner relative to FtsZ treadmilling in Spn cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in Spn.SignificanceThis study answers two long-standing questions about FtsZ dynamics and its relationship to septal PG synthesis in Spn for the first time. In previous models, FtsZ concertedly moves from midcell septa to MapZ rings that have reached the equators of daughter cells. Instead, the results presented here show that FtsZ, FtsA, and EzrA filaments/bundles move continuously out from early septa as part of MapZ rings. In addition, this study establishes that the movement of bPBP2x:FtsW complexes in septal PG synthesis depends on and likely mirrors new PG synthesis and is not correlated with the treadmilling of FtsZ filaments/bundles. These findings are consistent with a mechanism where septal FtsZ rings organize directional movement of bPBP2x:FtsW complexes dependent on PG substrate availability.

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Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816018116 ◽

2019 ◽

Vol 116 (8) ◽

pp. 3211-3220 ◽

Cited By ~ 36

Author(s):

Amilcar J. Perez ◽

Yann Cesbron ◽

Sidney L. Shaw ◽

Jesus Bazan Villicana ◽

Ho-Ching T. Tsui ◽

...

Keyword(s):

Cell Division ◽

Streptococcus Pneumoniae ◽

Single Molecule ◽

Protein Complexes ◽

Bacterial Cell Division ◽

Binding Partner ◽

Dynamic Movement ◽

Movement Dynamics ◽

Tightly Coupled ◽

Filament Bundles

Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae. Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.

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Benzamide Derivatives Targeting the Cell Division Protein FtsZ: Modifications of the Linker and the Benzodioxane Scaffold and Their Effects on Antimicrobial Activity

Antibiotics ◽

10.3390/antibiotics9040160 ◽

2020 ◽

Vol 9 (4) ◽

pp. 160 ◽

Cited By ~ 2

Author(s):

Valentina Straniero ◽

Lorenzo Suigo ◽

Andrea Casiraghi ◽

Victor Sebastián-Pérez ◽

Martina Hrast ◽

...

Keyword(s):

Antimicrobial Activity ◽

Cell Division ◽

Single Molecule ◽

Efflux Pump ◽

Computational Study ◽

Structural Features ◽

Temperature Sensitive ◽

Bacterial Cell Division ◽

Gram Positive ◽

Prokaryotic Protein

Filamentous temperature-sensitive Z (FtsZ) is a prokaryotic protein with an essential role in the bacterial cell division process. It is widely conserved and expressed in both Gram-positive and Gram-negative strains. In the last decade, several research groups have pointed out molecules able to target FtsZ in Staphylococcus aureus, Bacillus subtilis and other Gram-positive strains, with sub-micromolar Minimum Inhibitory Concentrations (MICs). Conversely, no promising derivatives active on Gram-negatives have been found up to now. Here, we report our results on a class of benzamide compounds, which showed comparable inhibitory activities on both S. aureus and Escherichia coli FtsZ, even though they proved to be substrates of E. coli efflux pump AcrAB, thus affecting the antimicrobial activity. These surprising results confirmed how a single molecule can target both species while maintaining potent antimicrobial activity. A further computational study helped us decipher the structural features necessary for broad spectrum activity and assess the drug-like profile and the on-target activity of this family of compounds.

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Acyldepsipeptide antibiotics – current state of knowledge

Polish Journal of Microbiology ◽

10.33073/pjm-2015-013 ◽

2015 ◽

Vol 64 (2) ◽

pp. 85-92

Author(s):

MICHAŁ T. PSTRĄGOWSKI ◽

MAGDALENA BUJALSKA-ZADROŻNY

Keyword(s):

Staphylococcus Aureus ◽

Bacillus Subtilis ◽

Cell Division ◽

Streptococcus Pneumoniae ◽

Animal Studies ◽

Potential Mechanism ◽

Bacterial Cell Division ◽

Resistance Development ◽

Bacillus Subtilis 168 ◽

Current State

The objective of this paper is to review and summarize the antimicrobial efficacy of the acyldepsipeptides and to indicate the prospects of the therapeutic values of these compounds. This work is enriched by the description of the mutations within the clpP1clpP2 and c1pP3clpP4 operons of Streptomyces lividans, which are considered to be the potential mechanism of the acyldepsipeptide (ADEP)-resistance development. The researchers' conclusions demonstrated a significant impact on microorganisms including the destabilization of bacterial cell division in Bacillus subtilis 168, Staphylococcus aureus HG001 and Streptococcus pneumoniae G9A strains. The results of animal studies show higher bactericidal effectiveness of the acyldepsipeptides ADEP-2 and ADEP-4 compared to linezolid. ADEPs may be considered as a very important mechanism of defense against the increasing resistance of microorganisms . They also might prevent or reduce the risk of many epidemiological events.

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LocZ Is a New Cell Division Protein Involved in Proper Septum Placement in Streptococcus pneumoniae

mBio ◽

10.1128/mbio.01700-14 ◽

2014 ◽

Vol 6 (1) ◽

Cited By ~ 36

Author(s):

Nela Holečková ◽

Linda Doubravová ◽

Orietta Massidda ◽

Virginie Molle ◽

Karolína Buriánková ◽

...

Keyword(s):

Cell Division ◽

Streptococcus Pneumoniae ◽

Protein Kinase ◽

Bacterial Cell Division ◽

Content Type ◽

Daughter Cells ◽

Division Site ◽

Z Ring ◽

Specific Shape ◽

Cell Division Protein

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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Mechanical stress compromises multicomponent efflux complexes in bacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909562116 ◽

2019 ◽

Vol 116 (51) ◽

pp. 25462-25467 ◽

Cited By ~ 1

Author(s):

Lauren A. Genova ◽

Melanie F. Roberts ◽

Yu-Chern Wong ◽

Christine E. Harper ◽

Ace George Santiago ◽

...

Keyword(s):

Cell Division ◽

Mechanical Stress ◽

Single Molecule ◽

Protein Complexes ◽

Hydrostatic Stress ◽

Cell Envelope ◽

Mechanical Forces ◽

Bacterial Survival ◽

Physical Forces ◽

Membrane Components

Physical forces have a profound effect on growth, morphology, locomotion, and survival of organisms. At the level of individual cells, the role of mechanical forces is well recognized in eukaryotic physiology, but much less is known about prokaryotic organisms. Recent findings suggest an effect of physical forces on bacterial shape, cell division, motility, virulence, and biofilm initiation, but it remains unclear how mechanical forces applied to a bacterium are translated at the molecular level. In Gram-negative bacteria, multicomponent protein complexes can form rigid links across the cell envelope and are therefore subject to physical forces experienced by the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that octahedral shear (but not hydrostatic) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used byEscherichia colito resist copper and silver toxicity. By promoting disassembly of this protein complex, mechanical forces within the cell envelope make the bacteria more susceptible to metal toxicity. These findings demonstrate that mechanical forces can inhibit the function of cell envelope protein assemblies in bacteria and suggest the possibility that other multicomponent, transenvelope efflux complexes may be sensitive to mechanical forces including complexes involved in antibiotic resistance, cell division, and translocation of outer membrane components. By modulating the function of proteins within the cell envelope, mechanical stress has the potential to regulate multiple processes required for bacterial survival and growth.

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The Pneumococcal Divisome: Dynamic Control of Streptococcus pneumoniae Cell Division

Frontiers in Microbiology ◽

10.3389/fmicb.2021.737396 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nicholas S. Briggs ◽

Kevin E. Bruce ◽

Souvik Naskar ◽

Malcolm E. Winkler ◽

David I. Roper

Keyword(s):

Cell Wall ◽

Cell Division ◽

Streptococcus Pneumoniae ◽

Virulence Factors ◽

Protein Complexes ◽

Dynamic Control ◽

Capsular Polysaccharides ◽

Control Mechanisms ◽

Cell Wall Synthesis ◽

Molecular Events

Cell division in Streptococcus pneumoniae (pneumococcus) is performed and regulated by a protein complex consisting of at least 14 different protein elements; known as the divisome. Recent findings have advanced our understanding of the molecular events surrounding this process and have provided new understanding of the mechanisms that occur during the division of pneumococcus. This review will provide an overview of the key protein complexes and how they are involved in cell division. We will discuss the interaction of proteins in the divisome complex that underpin the control mechanisms for cell division and cell wall synthesis and remodelling that are required in S. pneumoniae, including the involvement of virulence factors and capsular polysaccharides.

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Membrane Topology of the Streptococcus pneumoniae FtsW Division Protein

Journal of Bacteriology ◽

10.1128/jb.184.7.1925-1931.2002 ◽

2002 ◽

Vol 184 (7) ◽

pp. 1925-1931 ◽

Cited By ~ 33

Author(s):

Philippe Gérard ◽

Thierry Vernet ◽

André Zapun

Keyword(s):

Alkaline Phosphatase ◽

Cell Division ◽

Streptococcus Pneumoniae ◽

Membrane Protein ◽

Theoretical Prediction ◽

Membrane Topology ◽

Bacterial Cell Division ◽

Terminal Part ◽

Membrane Spanning ◽

Hydropathy Analysis

ABSTRACT The topology of FtsW from Streptococcus pneumoniae, an essential membrane protein involved in bacterial cell division, was predicted by computational methods and probed by the alkaline phosphatase fusion and cysteine accessibility techniques. Consistent results were obtained for the seven N-terminal membrane-spanning segments. However, the results from alkaline phosphatase fusions did not confirm the hydropathy analysis of the C-terminal part of FtsW, whereas the accessibility of introduced cysteine residues was in agreement with the theoretical prediction. Based on the combined results, we propose the first topological model of FtsW, featuring 10 membrane-spanning segments, a large extracytoplasmic loop, and both N and C termini located in the cytoplasm.

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Mechanical stress compromises multicomponent efflux complexes in bacteria

10.1101/571398 ◽

2019 ◽

Author(s):

Lauren A. Genova ◽

Melanie F. Roberts ◽

Yu-Chern Wong ◽

Christine E. Harper ◽

Ace George Santiago ◽

...

Keyword(s):

Cell Division ◽

Mechanical Stress ◽

Single Molecule ◽

Protein Complexes ◽

Cell Envelope ◽

Mechanical Forces ◽

Growth And Survival ◽

Physical Forces ◽

Bacterial Growth And Survival ◽

Membrane Components

Physical forces have long been recognized for their effects on the growth, morphology, locomotion, and survival of eukaryotic organisms1. Recently, mechanical forces have been shown to regulate processes in bacteria, including cell division2, motility3, virulence4, biofilm initiation5,6, and cell shape7,8, although it remains unclear how mechanical forces in the cell envelope lead to changes in molecular processes. In Gram-negative bacteria, multicomponent protein complexes that form rigid links across the cell envelope directly experience physical forces and mechanical stresses applied to the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that shear (but not tensile) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used by E. coli to resist copper and silver toxicity, thereby making bacteria more susceptible to metal toxicity. These findings provide the first demonstration that mechanical forces, such as those generated during colony overcrowding or bacterial motility through submicron pores, can inhibit the contact and function of multicomponent complexes in bacteria. As multicomponent, trans-envelope efflux complexes in bacteria are involved in many processes including antibiotic resistance9, cell division10, and translocation of outer membrane components11, our findings suggest that the mechanical environment may regulate multiple processes required for bacterial growth and survival.

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