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

Abstract

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 (podJ At ). ⌬podJ At cells display ectopic growth poles (branching), growth poles that fail to transition to an old pole, and elongated cells that fail to divide. In ⌬podJ At cells, A. tumefaciens PopZgreen fluorescent protein (PopZ At -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-PodJ At does not localize to the midcell in the wild type, deletion of podJ At 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 PodJ At is a critical factor for polar growth and that ⌬podJ At cells 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 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, podJ At , 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 PodJ At is a critical factor in normal polar growth and impacts cell division in A. tumefaciens.

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

“…Remarkably, the phenotype described here for the ΔpopZ mutant is strikingly similar to the phenotype of a ΔpodJ mutant (11). Loss of either PopZ or PodJ causes ectopic pole formation, mislocalization of cell division proteins, and asymmetric cell division, suggesting that both proteins contribute to the regulation of divisome assembly.…”

“…Due to the predicted molecular scaffolding properties of PopZ, as well as its localization to the growth pole, PopZ has been hypothesized to function in unipolar growth of A. tumefaciens (5,11). While our tracking of flagellar basal bodies suggests that polar insertion of peptidoglycan continues in the ΔpopZ cells, we used fluorescent D-amino acids (FDAAs) to probe the growth pattern in the presence and absence of PopZ.…”

“…A. tumefaciens PopZ (PopZ At ) does not exhibit bipolar localization but rather is found strictly at the new pole (5) and accumulates in ectopic poles formed in a mutant with cell division defects (11). Based largely on its strict localization to growing poles, the polar organizing protein, PopZ, has been identified as a candidate protein for promoting polar growth in A. tumefaciens (5,11).…”

“…PopZ Cc also localizes to the old pole, where it does not tether the chromosome but rather functions in the localization of polar regulatory proteins, including histidine kinases which function in cell cycle control (8,10). A. tumefaciens PopZ (PopZ At ) does not exhibit bipolar localization but rather is found strictly at the new pole (5) and accumulates in ectopic poles formed in a mutant with cell division defects (11). Based largely on its strict localization to growing poles, the polar organizing protein, PopZ, has been identified as a candidate protein for promoting polar growth in A. tumefaciens (5,11).…”

Modularity and determinants of a (bi-)polarization control system from free-living and obligate intracellular bacteria

“…For example, PopZ is also required for stalk biogenesis in C. crescentus (Bowman et al, 2010), a structure that requires specialized peptidoglycan (PG) biosynthesis enzymes (Kühn et al, 2010). As several Rhizobiales members also sequester PopZ to the site of PG synthesis to promote unipolar growth (Anderson-Furgeson et al, 2016; Brown et al, 2012; Curtis and Brun, 2014; Grangeon et al, 2015), the current challenge is to determine which proteins interact with PopZ in the different α-proteobacterial branches, particularly in the Rhizobiales. PopZ is unipolar in this branch (Deghelt et al, 2014; Grangeon et al, 2015) and no ZitP orthologs are encoded, suggesting that unknown PopZ-control mechanisms exist (Figure 6H,Figure 1—figure supplement 1A).…”