scholarly journals DivIVA Is Required for Polar Growth in the MreB-Lacking Rod-Shaped Actinomycete Corynebacterium glutamicum

2008 ◽  
Vol 190 (9) ◽  
pp. 3283-3292 ◽  
Author(s):  
Michal Letek ◽  
Efrén Ordóñez ◽  
José Vaquera ◽  
William Margolin ◽  
Klas Flärdh ◽  
...  

ABSTRACT The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVACg) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVACg-depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVACg localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.

2020 ◽  
Vol 203 (2) ◽  
pp. e00463-20
Author(s):  
Amit Bhambhani ◽  
Isabella Iadicicco ◽  
Jules Lee ◽  
Syed Ahmed ◽  
Max Belfatto ◽  
...  

ABSTRACTPrevious work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.


2020 ◽  
Author(s):  
Amit Bhambhani ◽  
Isabella Iadicicco ◽  
Jules Lee ◽  
Syed Ahmed ◽  
Max Belfatto ◽  
...  

ABSTRACTPrevious work identified gp56, encoded by the lytic bacteriophage SP01, as responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here we show that expression of the predicted 9.3-kDa gene product 56 (gp56) of SP01 inhibits latter stages of B. subtilis cell division without altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. While its localization permits recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analysis suggest that gp56 localization and activity depends on its interaction with mid-recruited proteins DivIC and/or FtsL. Together these data support a model where gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCEResearch over the past decades has uncovered bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. Phage factors that cause cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanism of several identified phage factors that inhibit cytokinesis remain unexplored, including gp56 of bacteriophage SP01 of Bacillus subtilis. Here, we show that unlike related published examples of phage inhibition of cyotkinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and block recruitment of proteins needed for the septal cell wall synthesis.


eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Saman Hussain ◽  
Carl N Wivagg ◽  
Piotr Szwedziak ◽  
Felix Wong ◽  
Kaitlin Schaefer ◽  
...  

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.


2021 ◽  
Vol 12 ◽  
Author(s):  
Nicholas S. Briggs ◽  
Kevin E. Bruce ◽  
Souvik Naskar ◽  
Malcolm E. Winkler ◽  
David I. Roper

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.


2016 ◽  
Author(s):  
Alexandre W. Bisson Filho ◽  
Yen-Pang Hsu ◽  
Georgia R. Squyres ◽  
Erkin Kuru ◽  
Fabai Wu ◽  
...  

AbstractHow bacteria produce a septum to divide in two is not well understood. This process is mediated by periplasmic cell-wall producing enzymes that are positioned by filaments of the cytoplasmic membrane-associated actin FtsA and the tubulin FtsZ (FtsAZ). To understand how these components act in concert to divide cells, we visualized their movements relative to the dynamics of cell wall synthesis during cytokinesis. We find that the division septum is built at discrete sites that move around the division plane. Furthermore, FtsAZ filaments treadmill in circumferential paths around the division ring, pulling along the associated cell-wall-synthesizing enzymes. We show that the rate of FtsZ treadmilling controls both the rate of cell wall synthesis and cell division. The coupling of both the position and activity of the cell wall synthases to FtsAZ treadmilling guides the progressive insertion of new cell wall, synthesizing increasingly small concentric rings to divide the cell.One-sentence summaryBacterial cytokinesis is controlled by circumferential treadmilling of FtsAZ filaments that drives the insertion of new cell wall.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kevin D. Whitley ◽  
Calum Jukes ◽  
Nicholas Tregidgo ◽  
Eleni Karinou ◽  
Pedro Almada ◽  
...  

AbstractDespite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.


2019 ◽  
Author(s):  
◽  
Michelle A. Williams

All bacteria elongate and divide to faithfully reproduce their cell shape. Understanding the mechanisms that drive bacterial morphology requires an intimate knowledge of how the cell wall is synthesized. During cell division, most bacteria synthesize new cell wall at mid-cell and the mechanism underlying this process is highly conserved. In contrast, there is a high degree of diversity in bacterial growth patterning during elongation. Bacteria in the Rhizobiales exhibit an atypical form of unipolar elongation, and the molecular mechanisms of how new cell wall is synthesized during growth and division currently remains unexplored. Using microfluidics and fluorescent cell wall probes we first investigated whether polar growth is conserved in a morphologically complex bacterium, Prosthecomicrobium hirschii. We showed that P. hirschii has a dimorphic lifestyle and can switch between a long-stalked, non-motile form and a short-stalked, motile form. Furthermore, we found that all morphotypes of P. hirschii elongate using polar growth, suggesting the polar elongation is a widespread feature of bacteria in this order. Next, we used the rod-shaped bacterium Agrobacterium tumefaciens as a model to investigate the precise mechanisms that drive polar elongation. We characterized a comprehensive set of cell wall synthesis enzymes in A. tumefaciens and identified penicillin-binding protein 3a (PBP3a) and PBP3b as a synthetic lethal pair that function during cell division, and PBP1a as an essential enzyme required for polar growth and maintenance of rod shape. Compositional analysis of the PBP1a depletion, suggested that LD-transpeptidase (LDT) enzymes may play an important role in polar growth. We identified three LDTs that likely function in polar growth. We also observed subpolar localization of LDTs, suggesting bacteria in the Rhizobiales may insert or remodel cell wall material in a subpolar zone during growth. Finally, we used RNA-seq to explore changes in gene expression during PBP1a depletion, revealing that that loss of PBP1a induces a lifestyle switch which mimics the switch from a free-living bacterium into a plant-associated state. The change in lifestyle is characterized by increased exopolysaccharide production and Type VI Secretion System activity and a decrease in flagella-mediated motility. This finding indicates that bacteria have a mechanism to sense changes in cell wall composition or integrity due to the loss of PBP1a and respond through changes in gene expression that impact physiology and behavior. This finding opens the door to future studies on the link between changes in cell wall composition and complex bacterial behaviors and lifestyles. Overall, this research provides mechanistic insights about the roles of cell wall synthesis during cell growth and division in the A. tumefaciens, which are conserved in other Rhizobiales, including agriculturally and medically species such as Sinorhizobium and Brucella.


2018 ◽  
Author(s):  
Gro Anita Stamsås ◽  
Ine Storaker Myrbråten ◽  
Daniel Straume ◽  
Zhian Salehian ◽  
Jan-Willem Veening ◽  
...  

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.


Author(s):  
Kevin D. Whitley ◽  
Calum Jukes ◽  
Nicholas Tregidgo ◽  
Eleni Karinou ◽  
Pedro Almada ◽  
...  

ABSTRACTDespite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments1,2. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear2–5. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a novel combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.


2018 ◽  
Author(s):  
◽  
Matthew (Matthew Lloyd) Howell

Understanding how bacterial cells expand their cell walls is an important question with relevance to development of antibiotics. While many studies have focused on the regulation of bacterial elongation utilizing lateral cell wall biogenesis, polar growth in bacteria is less well understood. Yet, polar growth has been observed across taxonomically diverse bacteria like Actinobacteria and the alphaproteobacterial clade Rhizobiales (Howell and Brown, 2016). Interestingly, polar-growing bacteria within Rhizobiales lack canonical scaffolding proteins for spatial and temporal regulation of peptidoglycan synthesis during elongation. Here, we dissect the role of two candidate scaffolding proteins in directing cell wall synthesis in the bacterial plant pathogen, Agrobacterium tumefaciens. Since cell wall (peptidoglycan) biosynthesis during elongation and cell division is vital for bacterial survival, we expected many key proteins involved in these processes to be essential for cell survival. Thus, we developed a depletion system for A. tumefaciens (Figureroa-Cuilan et al. 2016). We further optimized a suite of target-specific fluorescent labeling techniques which allow us to visualize morphological changes during essential cell processes (Howell, Daniel, and Brown, 2017). We use these techniques to dissect the contributions of PopZ and FtsZ to polar growth and cell division. Although PopZ is not required for polar growth, it is required for proper coordination of polar growth, chromosome segregation, and cell division. This PopZ-mediated coordination ensures that daughter cells are the proper size and contain a complete complement of genetic material (Howell et al 2017). Next, we find that FtsZ is required for both termination of polar growth and cell division. This finding suggests that FtsZ has at least two important functions in regulation of cell wall biogenesis. First, FtsZ enables cell wall biogenesis machinery to be released or inactivated from the growth pole. Second, FtsZ must recruit additional proteins to mid cell to assemble the divisome, enabling activation of cell wall biogenesis to promote septum formation and cell separation. While further research is needed to understand how growth is targeted to the pole during elongation, our work provides mechanistic insights about the coordination of polar growth termination, chromosome segregation, and cell division. We hypothesize that our findings will be applicable to other closely related polar growing Rhizobiales, including plant, animal, and human pathogens.


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