scholarly journals Segregation of fourAgrobacterium tumefaciensreplicons during polar growth: PopZ and PodJ control segregation of essential replicons

2020 ◽  
Vol 117 (42) ◽  
pp. 26366-26373
Author(s):  
J. S. Robalino-Espinosa ◽  
J. R. Zupan ◽  
A. Chavez-Arroyo ◽  
P. Zambryski
Keyword(s):  
Cell Cycle ◽  
Agrobacterium Tumefaciens ◽  
Single Cell ◽  
Essential Role ◽  
Polar Growth ◽  
Cell Compartment ◽  
Circular Chromosome ◽  
Linear Chromosome ◽  
Growth Pole ◽  
Cell Compartments

Agrobacterium tumefaciensC58 contains four replicons, circular chromosome (CC), linear chromosome (LC), cryptic plasmid (pAt), and tumor-inducing plasmid (pTi), and grows by polar growth from a single growth pole (GP), while the old cell compartment and its old pole (OP) do not elongate. We monitored the replication and segregation of these four genetic elements during polar growth. The three largest replicons (CC, LC, pAt) reside in the OP compartment prior to replication; post replication one copy migrates to the GP prior to division. CC resides at a fixed location at the OP and replicates first. LC does not stay fixed at the OP once the cell cycle begins and replicates from varied locations 20 min later than CC. pAt localizes similarly to LC prior to replication, but replicates before the LC and after the CC. pTi does not have a fixed location, and post replication it segregates randomly throughout old and new cell compartments, while undergoing one to three rounds of replication during a single cell cycle. Segregation of the CC and LC is dependent on the GP and OP identity factors PopZ and PodJ, respectively. Without PopZ, replicated CC and LC do not efficiently partition, resulting in sibling cells without CC or LC. Without PodJ, the CC and LC exhibit abnormal localization to the GP at the beginning of the cell cycle and replicate from this position. These data reveal PodJ plays an essential role in CC and LC tethering to the OP during early stages of polar growth.

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 Loss of PopZAtactivity inAgrobacterium tumefaciensby Deletion or Depletion Leads to Multiple Growth Poles, Minicells, and Growth Defects

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Romain Grangeon ◽  
John Zupan ◽  
Yeonji Jeon ◽  
Patricia C. Zambryski
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Agrobacterium Tumefaciens ◽  
Caulobacter Crescentus ◽  
Polar Growth ◽  
Growth Pole ◽  
Growth Defects ◽  
Animal Pathogens ◽  
Cell Asymmetry

ABSTRACTAgrobacterium tumefaciensgrows by addition of peptidoglycan (PG) at one pole of the bacterium. During the cell cycle, the cell needs to maintain two different developmental programs, one at the growth pole and another at the inert old pole. Proteins involved in this process are not yet well characterized. To further characterize the role of pole-organizing proteinA. tumefaciensPopZ (PopZAt), we created deletions of the five PopZAtdomains and assayed their localization. In addition, we created apopZAtdeletion strain (ΔpopZAt) that exhibited growth and cell division defects with ectopic growth poles and minicells, but the strain is unstable. To overcome the genetic instability, we created an inducible PopZAtstrain by replacing the native ribosome binding site with a riboswitch. Cultivated in a medium without the inducer theophylline, the cells look like ΔpopZAtcells, with a branching and minicell phenotype. Adding theophylline restores the wild-type (WT) cell shape. Localization experiments in the depleted strain showed that the domain enriched in proline, aspartate, and glutamate likely functions in growth pole targeting. Helical domains H3 and H4 together also mediate polar localization, but only in the presence of the WT protein, suggesting that the H3 and H4 domains multimerize with WT PopZAt, to stabilize growth pole accumulation of PopZAt.IMPORTANCEAgrobacterium tumefaciensis a rod-shaped bacterium that grows by addition of PG at only one pole. The factors involved in maintaining cell asymmetry during the cell cycle with an inert old pole and a growing new pole are not well understood. Here we investigate the role of PopZAt, a homologue ofCaulobacter crescentusPopZ (PopZCc), a protein essential in many aspects of pole identity inC. crescentus. We report that the loss of PopZAtleads to the appearance of branching cells, minicells, and overall growth defects. As many plant and animal pathogens also employ polar growth, understanding this process inA. tumefaciensmay lead to the development of new strategies to prevent the proliferation of these pathogens. In addition, studies ofA. tumefacienswill provide new insights into the evolution of the genetic networks that regulate bacterial polar growth and cell division.

scholarly journals PopZ identifies the new pole, and PodJ identifies the old pole during polar growth in Agrobacterium tumefaciens

2015 ◽  
Vol 112 (37) ◽  
pp. 11666-11671 ◽  
Author(s):  
Romain Grangeon ◽  
John R. Zupan ◽  
James Anderson-Furgeson ◽  
Patricia C. Zambryski
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Agrobacterium Tumefaciens ◽  
Time Lapse ◽  
Polar Growth ◽  
Superresolution Microscopy ◽  
Growth Pole ◽  
Pole Asymmetry ◽  
Specific Localization ◽  
Polar Organelle

Agrobacterium tumefaciens elongates by addition of peptidoglycan (PG) only at the pole created by cell division, the growth pole, whereas the opposite pole, the old pole, is inactive for PG synthesis. How Agrobacterium assigns and maintains pole asymmetry is not understood. Here, we investigated whether polar growth is correlated with novel pole-specific localization of proteins implicated in a variety of growth and cell division pathways. The cell cycle of A. tumefaciens was monitored by time-lapse and superresolution microscopy to image the localization of A. tumefaciens homologs of proteins involved in cell division, PG synthesis and pole identity. FtsZ and FtsA accumulate at the growth pole during elongation, and improved imaging reveals FtsZ disappears from the growth pole and accumulates at the midcell before FtsA. The L,D-transpeptidase Atu0845 was detected mainly at the growth pole. A. tumefaciens specific pole-organizing protein (Pop) PopZAt and polar organelle development (Pod) protein PodJAt exhibited dynamic yet distinct behavior. PopZAt was found exclusively at the growing pole and quickly switches to the new growth poles of both siblings immediately after septation. PodJAt is initially at the old pole but then also accumulates at the growth pole as the cell cycle progresses suggesting that PodJAt may mediate the transition of the growth pole to an old pole. Thus, PopZAt is a marker for growth pole identity, whereas PodJAt identifies the old pole.

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 Draft Genome Sequence of Agrobacterium tumefaciens Strain 1D1526

2019 ◽  
Vol 8 (45) ◽  
Author(s):  
Naxin Huo ◽  
Yong Gu ◽  
Kent F. McCue ◽  
Diaa Alabed ◽  
James G. Thomson
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genome Sequence ◽  
Draft Genome ◽  
Draft Genome Sequence ◽  
Circular Chromosome ◽  
Linear Chromosome ◽  
Content Type

This work reports the draft genome sequence of Agrobacterium tumefaciens strain 1D1526. The assembled genome is composed of a 2,881,823-bp circular chromosome, a 2,235,711-bp linear chromosome, and a 44,582-bp unassembled contig.

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.

scholarly journals Replicon-Specific Regulation of Small Heat Shock Genes in Agrobacterium tumefaciens

2004 ◽  
Vol 186 (20) ◽  
pp. 6824-6829 ◽  
Author(s):  
Sylvia Balsiger ◽  
Curdin Ragaz ◽  
Christian Baron ◽  
Franz Narberhaus
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Heat Shock ◽  
Elevated Temperatures ◽  
Heat Shock Gene ◽  
Circular Chromosome ◽  
Linear Chromosome ◽  
Shsp Gene ◽  
Shine Dalgarno Sequence ◽  
Specific Regulation ◽  
Shock Proteins

ABSTRACT Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner. While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (σ32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5′ untranslated region. Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence. The hspC gene was barely expressed (if at all) and not temperature responsive.

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 Combined Genetic and Physical Map of the Complex Genome of Agrobacterium tumefaciens

1999 ◽  
Vol 181 (17) ◽  
pp. 5160-5166 ◽  
Author(s):  
Brad W. Goodner ◽  
Brian P. Markelz ◽  
M. Casey Flanagan ◽  
Chris B. Crowell ◽  
Jodi L. Racette ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physical Mapping ◽  
Physical Map ◽  
Genomic Structure ◽  
Genetic Maps ◽  
Mapping Data ◽  
Circular Chromosome ◽  
Linear Chromosome ◽  
Single Chromosome ◽  
Complex Genome

ABSTRACT A combined genetic and physical map of the Agrobacterium tumefaciens A348 (derivative of C58) genome was constructed to address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two nonhomologous chromosomes. The combined map confirms the two-chromosome genomic structure and the correspondence of the initial genetic maps to the circular chromosome. The linear chromosome is almost devoid of auxotrophic markers, which probably explains why it was missed by genetic mapping studies.

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