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

UNLABELLED: Agrobacterium tumefaciens is 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. The A. tumefaciens homolog of the Caulobacter crescentus polar organizing protein PopZ localizes specifically to growth poles. In contrast, the A. tumefaciens homolog of the C. crescentus polar 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 of A. tumefaciens podJ (podJAt). ΔpodJAt cells display ectopic growth poles (branching), growth poles that fail to transition to an old pole, and elongated cells that fail to divide. In ΔpodJAt cells, A. tumefaciens PopZ-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-PodJAt does not localize to the midcell in the wild type, deletion of podJAt impacts 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 PodJAt is a critical factor for polar growth and that ΔpodJAt cells display a cell division phenotype, likely because the growth pole cannot transition to an old pole. IMPORTANCE: How 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 as Escherichia coli, and unipolar growth, which occurs in several alphaproteobacteria, including Agrobacterium 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 PodJAt is a critical factor in normal polar growth and impacts cell division in A. tumefaciens.

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

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.

The essential features and modes of bacterial polar growth.

Polar growth represents a surprising departure from the canonical dispersed cell growth model. However, we know relatively little of the underlying mechanisms governing polar growth or the requisite suite of factors that direct polar growth. Underscoring how classic doctrine can be turned on its head, the peptidoglycan layer of polar-growing bacteria features unusual crosslinks and in some species the quintessential cell division proteins FtsA and FtsZ are recruited to the growing poles. Remarkably, numerous medically important pathogens utilize polar growth, accentuating the need for intensive research in this area. Here we review models of polar growth in bacteria based on recent research in the Actinomycetales and Rhizobiales, with emphasis on Mycobacterium and Agrobacterium species.

Peptidoglycan synthesis machinery in Agrobacterium tumefaciens during unipolar growth and cell division.

UNLABELLED: 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.

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens.

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes.

Peptide linkage mapping of the Agrobacterium tumefaciens vir-encoded type IV secretion system reveals protein subassemblies.

Numerous bacterial pathogens use type IV secretion systems (T4SS) to deliver virulence factors directly to the cytoplasm of plant, animal, and human host cells. Here, evidence for interactions among components of the Agrobacterium tumefaciens vir-encoded T4SS is presented. The results derive from a high-resolution yeast two-hybrid assay, in which a library of small peptide domains of T4SS components was screened for interactions. The use of small peptides overcomes problems associated with assaying for interactions involving membrane-associated proteins. We established interactions between VirB11 (an inner membrane pore-forming protein), VirB9 (a periplasmic protein), and VirB7 (an outer membrane-associated lipoprotein and putative pilus component). We provide evidence for an interaction pathway, among conserved members of a T4SS, spanning the A. tumefaciens envelope and including a potential pore protein. In addition, we have determined interactions between VirB1 (a lytic transglycosylase likely involved in the local remodeling of the peptidoglycan) and primarily VirB8, but also VirB4, VirB10, and VirB11 (proteins likely to assemble the core structure of the T4SS). VirB4 interacts with VirB8, VirB10, and VirB11, also establishing a connection to the core components. The identification of these interactions suggests a model for assembly of the T4SS.