scholarly journals PopZ and FtsZ coordinate polar growth termination and cell division in Agrobacterium tumefaciens

2018 ◽  
Author(s):  
◽  
Matthew (Matthew Lloyd) Howell
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Agrobacterium Tumefaciens ◽  
Chromosome Segregation ◽  
Morphological Changes ◽  
Genetic Material ◽  
Fluorescent Labeling ◽  
Scaffolding Proteins ◽  
Polar Growth ◽  
Cell Wall Biogenesis

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.

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 ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Streptococcus Pneumoniae ◽  
Corynebacterium Glutamicum ◽  
Chromosome Segregation ◽  
Polar Growth ◽  
Cell Wall Synthesis ◽  
Peptidoglycan Synthesis ◽  
Polarized Cell Growth

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.

scholarly journals Absence of the Polar Organizing Protein PopZ Results in Reduced and Asymmetric Cell Division in Agrobacterium tumefaciens

2017 ◽  
Vol 199 (17) ◽  
Author(s):  
Matthew Howell ◽  
Alena Aliashkevich ◽  
Anne K. Salisbury ◽  
Felipe Cava ◽  
Grant R. Bowman ◽  
...  
Keyword(s):  
Cell Division ◽  
Agrobacterium Tumefaciens ◽  
Chromosome Segregation ◽  
Caulobacter Crescentus ◽  
Length Distribution ◽  
Polar Growth ◽  
Atypical Cell ◽  
Content Type ◽  
Growth Pole ◽  
Animal Pathogens

ABSTRACT Agrobacterium tumefaciens is a rod-shaped bacterium that grows by polar insertion of new peptidoglycan during cell elongation. As the cell cycle progresses, peptidoglycan synthesis at the pole ceases prior to insertion of new peptidoglycan at midcell to enable cell division. The A. tumefaciens homolog of the Caulobacter crescentus polar organelle development protein PopZ has been identified as a growth pole marker and a candidate polar growth-promoting factor. Here, we characterize the function of PopZ in cell growth and division of A. tumefaciens. Consistent with previous observations, we observe that PopZ localizes specifically to the growth pole in wild-type cells. Despite the striking localization pattern of PopZ, we find the absence of the protein does not impair polar elongation or cause major changes in the peptidoglycan composition. Instead, we observe an atypical cell length distribution, including minicells, elongated cells, and cells with ectopic poles. Most minicells lack DNA, suggesting a defect in chromosome segregation. Furthermore, the canonical cell division proteins FtsZ and FtsA are misplaced, leading to asymmetric sites of cell constriction. Together, these data suggest that PopZ plays an important role in the regulation of chromosome segregation and cell division. IMPORTANCE A. tumefaciens is a bacterial plant pathogen and a natural genetic engineer. However, very little is known about the spatial and temporal regulation of cell wall biogenesis that leads to polar growth in this bacterium. Understanding the molecular basis of A. tumefaciens growth may allow for the development of innovations to prevent disease or to promote growth during biotechnology applications. Finally, since many closely related plant and animal pathogens exhibit polar growth, discoveries in A. tumefaciens may be broadly applicable for devising antimicrobial strategies.

scholarly journals Loss of PodJ in Agrobacterium tumefaciens Leads to Ectopic Polar Growth, Branching, and Reduced Cell Division

2016 ◽  
Vol 198 (13) ◽  
pp. 1883-1891 ◽  
Author(s):  
James C. Anderson-Furgeson ◽  
John R. Zupan ◽  
Romain Grangeon ◽  
Patricia C. Zambryski
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Agrobacterium Tumefaciens ◽  
Cell Envelope ◽  
Critical Factor ◽  
Polar Growth ◽  
Content Type ◽  
Growth Pole ◽  
Envelope Material ◽  
Z Ring

ABSTRACTAgrobacterium tumefaciensis a rod-shaped Gram-negative bacterium that elongates by unipolar addition of new cell envelope material. Approaching cell division, the growth pole transitions to a nongrowing old pole, and the division site creates new growth poles in sibling cells. TheA. tumefacienshomolog of theCaulobacter crescentuspolar organizing protein PopZ localizes specifically to growth poles. In contrast, theA. tumefacienshomolog of theC. crescentuspolar organelle development protein PodJ localizes to the old pole early in the cell cycle and accumulates at the growth pole as the cell cycle proceeds. FtsA and FtsZ also localize to the growth pole for most of the cell cycle prior to Z-ring formation. To further characterize the function of polar localizing proteins, we created a deletion ofA. tumefacienspodJ(podJAt). ΔpodJAtcells display ectopic growth poles (branching), growth poles that fail to transition to an old pole, and elongated cells that fail to divide. In ΔpodJAtcells,A. tumefaciensPopZ-green fluorescent protein (PopZAt-GFP) persists at nontransitioning growth poles postdivision and also localizes to ectopic growth poles, as expected for a growth-pole-specific factor. Even though GFP-PodJAtdoes not localize to the midcell in the wild type, deletion ofpodJAtimpacts localization, stability, and function of Z-rings as assayed by localization of FtsA-GFP and FtsZ-GFP. Z-ring defects are further evidenced by minicell production. Together, these data indicate that PodJAtis a critical factor for polar growth and that ΔpodJAtcells display a cell division phenotype, likely because the growth pole cannot transition to an old pole.IMPORTANCEHow rod-shaped prokaryotes develop and maintain shape is complicated by the fact that at least two distinct species-specific growth modes exist: uniform sidewall insertion of cell envelope material, characterized in model organisms such asEscherichia coli, and unipolar growth, which occurs in several alphaproteobacteria, includingAgrobacterium tumefaciens. Essential components for unipolar growth are largely uncharacterized, and the mechanism constraining growth to one pole of a wild-type cell is unknown. Here, we report that the deletion of a polar development gene,podJAt, results in cells exhibiting ectopic polar growth, including multiple growth poles and aberrant localization of cell division and polar growth-associated proteins. These data suggest that PodJAtis a critical factor in normal polar growth and impacts cell division inA. tumefaciens.

scholarly journals Agrobacterium tumefaciens Growth Pole Ring Protein: C Terminus and Internal Apolipoprotein Homologous Domains Are Essential for Function and Subcellular Localization

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
John Zupan ◽  
Zisheng Guo ◽  
Trevor Biddle ◽  
Patricia Zambryski
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Agrobacterium Tumefaciens ◽  
Polar Growth ◽  
Content Type ◽  
Growth Pole ◽  
Human Apolipoprotein ◽  
Apolipoprotein A ◽  
C Terminus ◽  
Organizing Center

ABSTRACT The Agrobacterium growth pole ring (GPR) protein forms a hexameric ring at the growth pole (GP) that is essential for polar growth. GPR is large (2,115 amino acids) and contains 1,700 amino acids of continuous α-helices. To dissect potential GPR functional domains, we created deletions of regions with similarity to human apolipoprotein A-IV (396 amino acids), itself composed of α-helical domains. We also tested deletions of the GPR C terminus. Deletions were inducibly expressed as green fluorescent protein (GFP) fusion proteins and tested for merodiploid interference with wild-type (WT) GPR function, for partial function in cells lacking GPR, and for formation of paired fluorescent foci (indicative of hexameric rings) at the GP. Deletion of domains similar to human apolipoprotein A-IV in GPR caused defects in cell morphology when expressed in trans to WT GPR and provided only partial complementation to cells lacking GPR. Agrobacterium-specific domains A-IV-1 and A-IV-4 contain predicted coiled coil (CC) regions of 21 amino acids; deletion of CC regions produced severe defects in cell morphology in the interference assay. Mutants that produced the most severe effects on cell shape also failed to form paired polar foci. Modeling of A-IV-1 and A-IV-4 reveals significant similarity to the solved structure of human apolipoprotein A-IV. GPR C-terminal deletions profoundly blocked complementation. Finally, peptidoglycan (PG) synthesis is abnormally localized circumferentially in cells lacking GPR. The results support the hypothesis that GPR plays essential roles as an organizing center for membrane and PG synthesis during polar growth. IMPORTANCE Bacterial growth and division are extensively studied in model systems (Escherichia coli, Bacillus subtilis, and Caulobacter crescentus) that grow by dispersed insertion of new cell wall material along the length of the cell. An alternative growth mode—polar growth—is used by some Actinomycetales and Proteobacteria species. The latter phylum includes the family Rhizobiaceae, in which many species, including Agrobacterium tumefaciens, exhibit polar growth. Current research aims to identify growth pole (GP) factors. The Agrobacterium growth pole ring (GPR) protein is essential for polar growth and forms a striking hexameric ring structure at the GP. GPR is long (2,115 amino acids), and little is known about regions essential for structure or function. Genetic analyses demonstrate that the C terminus of GPR, and two internal regions with homology to human apolipoproteins (that sequester lipids), are essential for GPR function and localization to the GP. We hypothesize that GPR is an organizing center for membrane and cell wall synthesis during polar growth.

scholarly journals An ABC transporter Wzm–Wzt catalyzes translocation of lipid-linked galactan across the plasma membrane in mycobacteria

2021 ◽  
Vol 118 (17) ◽  
pp. e2023663118
Author(s):  
Karin Savková ◽  
Stanislav Huszár ◽  
Peter Baráth ◽  
Zuzana Pakanová ◽  
Stanislav Kozmon ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Abc Transporter ◽  
Morphological Changes ◽  
Transport Systems ◽  
Host Immune System ◽  
Mycolic Acids ◽  
Cell Wall Biogenesis ◽  
Covalently Linked ◽  
High Level

Mycobacterium tuberculosis, one of the deadliest pathogens in human history, is distinguished by a unique, multilayered cell wall, which offers the bacterium a high level of protection from the attacks of the host immune system. The primary structure of the cell wall core, composed of covalently linked peptidoglycan, branched heteropolysaccharide arabinogalactan, and mycolic acids, is well known, and numerous enzymes involved in the biosynthesis of its components are characterized. The cell wall biogenesis takes place at both cytoplasmic and periplasmic faces of the plasma membrane, and only recently some of the specific transport systems translocating the metabolic intermediates between these two compartments have been characterized [M. Jackson, C. M. Stevens, L. Zhang, H. I. Zgurskaya, M. Niederweis, Chem. Rev., 10.1021/acs.chemrev.0c00869 (2020)]. In this work, we use CRISPR interference methodology in Mycobacterium smegmatis to functionally characterize an ATP-binding cassette (ABC) transporter involved in the translocation of galactan precursors across the plasma membrane. We show that genetic knockdown of the transmembrane subunit of the transporter results in severe morphological changes and the accumulation of an aberrantly long galactan precursor. Based on similarities with structures and functions of specific O-antigen ABC transporters of gram-negative bacteria [C. Whitfield, D. M. Williams, S. D. Kelly, J. Biol. Chem. 295, 10593-10609 (2020)], we propose a model for coupled synthesis and export of the galactan polymer precursor in mycobacteria.

scholarly journals Tol-Pal System and Rgs Proteins Interact to Promote Unipolar Growth and Cell Division in Sinorhizobium meliloti

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Elizaveta Krol ◽  
Hamish C. L. Yau ◽  
Marcus Lechner ◽  
Simon Schäper ◽  
Gert Bange ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Cell Growth ◽  
Cell Elongation ◽  
Sinorhizobium Meliloti ◽  
Rgs Proteins ◽  
Polar Growth ◽  
Content Type ◽  
Cell Pole ◽  
Growing Cell

ABSTRACT Sinorhizobium meliloti is an alphaproteobacterium belonging to the Rhizobiales. Bacteria from this order elongate their cell wall at the new cell pole, generated by cell division. Screening for protein interaction partners of the previously characterized polar growth factors RgsP and RgsM, we identified the inner membrane components of the Tol-Pal system (TolQ and TolR) and novel Rgs (rhizobial growth and septation) proteins with unknown functions. TolQ, Pal, and all Rgs proteins, except for RgsE, were indispensable for S. meliloti cell growth. Six of the Rgs proteins, TolQ, and Pal localized to the growing cell pole in the cell elongation phase and to the septum in predivisional cells, and three Rgs proteins localized to the growing cell pole only. The putative FtsN-like protein RgsS contains a conserved SPOR domain and is indispensable at the early stages of cell division. The components of the Tol-Pal system were required at the late stages of cell division. RgsE, a homolog of the Agrobacterium tumefaciens growth pole ring protein GPR, has an important role in maintaining the normal growth rate and rod cell shape. RgsD is a periplasmic protein with the ability to bind peptidoglycan. Analysis of the phylogenetic distribution of the Rgs proteins showed that they are conserved in Rhizobiales and mostly absent from other alphaproteobacterial orders, suggesting a conserved role of these proteins in polar growth. IMPORTANCE Bacterial cell proliferation involves cell growth and septum formation followed by cell division. For cell growth, bacteria have evolved different complex mechanisms. The most prevalent growth mode of rod-shaped bacteria is cell elongation by incorporating new peptidoglycans in a dispersed manner along the sidewall. A small share of rod-shaped bacteria, including the alphaproteobacterial Rhizobiales, grow unipolarly. Here, we identified and initially characterized a set of Rgs (rhizobial growth and septation) proteins, which are involved in cell division and unipolar growth of Sinorhizobium meliloti and highly conserved in Rhizobiales. Our data expand the knowledge of components of the polarly localized machinery driving cell wall growth and suggest a complex of Rgs proteins with components of the divisome, differing in composition between the polar cell elongation zone and the septum.

scholarly journals Peptidoglycan Synthesis Machinery in Agrobacterium tumefaciens During Unipolar Growth and Cell Division

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Todd A. Cameron ◽  
James Anderson-Furgeson ◽  
John R. Zupan ◽  
Justin J. Zik ◽  
Patricia C. Zambryski
Keyword(s):  
Cell Division ◽  
Agrobacterium Tumefaciens ◽  
Fluorescent Protein ◽  
Polar Growth ◽  
Growth Strategies ◽  
Cell Width ◽  
Content Type ◽  
E Coli ◽  
Cell Compartments ◽  
New Material

ABSTRACT The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli. IMPORTANCE Many rod-shaped bacteria, including pathogens such as Brucella and Mycobacteriu, grow by adding new material to their cell poles, and yet the proteins and mechanisms contributing to this process are not yet well defined. The polarly growing plant pathogen Agrobacterium tumefaciens was used as a model bacterium to explore these polar growth mechanisms. The results obtained indicate that polar growth in this organism is facilitated by repurposed cell division components and an otherwise obscure class of alternative peptidoglycan transpeptidases (l,d-transpeptidases). This growth results in dynamically changing cell widths as the poles expand to maturity and contrasts with the tightly regulated cell widths characteristic of canonical rod-shaped growth. Furthermore, the abundance and/or activity of l,d-transpeptidases appears to associate with polar growth strategies, suggesting that these enzymes may serve as attractive targets for specifically inhibiting growth of Rhizobiales, Actinomycetales, and other polarly growing bacterial pathogens.

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