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 (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.

FIG 1

podJ genomic contexts in A. tumefaciens, S. meliloti, and C. crescentus. Atu numbers refer to A. tumefaciens strain C58 locus tags, Sm11_chr numbers refer to S. meliloti strain SM11 locus tags, and CC_ numbers refer to C. crescentus CB15N locus tags. Open reading frames that are similarly shaded or patterned are reciprocal best BLAST hits. Open reading frames that are white are not homologous to any other open reading frame shown. Breaks in S. meliloti and C. crescentus open reading frames reflect open reading frames that extend beyond the area shown. The ΔpodJ_At strain was created using allelic exchange to remove both podJ_At and the small overlapping reading frame Atu0500 in the region between the arrows.

FIG 2

ΔpodJ_At cells are elongated, branched, multiply constricted, and swollen and show ectopic polar growth. (A and B) FM4-64-labeled ΔpodJ_At cells. Cells with morphological deviations from rod shape, such as cells with multiple constrictions, branched cells, swollen cells, and bent cells, are indicated with circles or ovals. (C and D) SEM images of ΔpodJ_At cells displaying deviations from rod-shape morphology, including elongated cells, branched cells, and cells with multiple constrictions. Arrowheads denote cell constrictions (variations in cell width). Plus signs indicate branch tips. (E) Time-lapse images of ΔpodJ_At cells undergoing polar growth (white arrow) and a division event (black arrow). White arrowheads denote cell constrictions. Bars, 3 μm.

FIG 3

Frequency of morphological abnormalities in ΔpodJ_At cells. Percentages of WT cells (n = 283), ΔpodJ_At cells (n = 262), ΔpodJ_At cells expressing GFP-PodJ_At (n = 340), and ΔpodJ_At cells expressing PodJ_At (n = 247) displaying different morphologies are shown below representative images. Bar, 3 μm.

FIG 4

Complementation of ΔpodJ_At with GFP-PodJ_At restores cell morphology and dynamic polar localization of GFP-PodJ_At. In the early stages of the cell cycle (0 to 20 min), GFP-PodJ_At localizes as a large focus at the old pole (arrow). Arrowheads indicate the growth pole postdivision at 0 min and future growth poles at 100 min. A small GFP-PodJ_At focus develops at the postdivision growth pole at 40 min. Postdivision, GFP-PodJ_At localizes as large foci at old poles (100 min, arrows). For simplicity, arrows and arrowheads are shown only in the first and last panels. Bar, 3 μm.

FIG 5

PopZ_At-GFP localizes to ectopic growth poles in ΔpodJ_At cells. (A) The ΔpodJ_At cell on the top displays a PopZ_At-GFP focus (red) at a growing pole (white arrow) that continues to grow postdivision. The black arrow indicates a pole produced by division (60 min) that should normally be a growth pole, but it does not grow and does not label with PopZ_At-GFP. In contrast, the lower sibling cell grows from the site of division, as in the WT (arrowhead), and this new growth pole does label with PopZ_At-GFP. (B) ΔpodJ_At cell displaying splitting of the growth pole and producing two PopZ_At-GFP foci (arrows). (C) ΔpodJ_At cell with PopZ_At-GFP focus at a growth pole emerging from a convex point along the sidewall of the cell (arrow). Bars, 3 μm.

FIG 6

FtsA-GFP localizes in polar foci, lateral foci, and rings in elongated ΔpodJ_At cells. ΔpodJ_At cells expressing FtsA-GFP stained with FM4-64 display FtsA-GFP patterns seen in WT cells: unipolar (A), unipolar and midcell (B), and midcell (C) localizations. ΔpodJ_At cells also display localization patterns of FtsA-GFP not observed in WT cells: bipolar and midcell (D), multiple rings (E), bipolar (F), polar and lateral foci (G), and polar clusters (H). The percentages of ΔpodJ_At cells (out of 234 cells total) showing these localization types are shown under representative images, and the percentages of WT cells showing these localization types are shown in parentheses and are from reference . Bar, 3 μm. (I) Gaussian kernel density estimates of the distribution of cell lengths of ΔpodJ_At cells with different FtsA-GFP localization patterns. Density estimates were weighted by the proportion of cells displaying the different localization types so that the height of the peaks corresponds to the proportion of cells displaying each localization pattern. Colors of density curves correspond to color outlines of representative images of each localization pattern in panels A to H. Bar, 3 μm.

FIG 7

FtsZ-GFP localization in ΔpodJ_At cells. FtsZ-GFP (green) localizes in unipolar (A), bipolar (B), subpolar (C), unipolar and subpolar (D), polar cluster (E), unipolar and Z-ring (F), Z-ring (G), contracting Z-ring (H), multiple-Z-ring (I), and multiple-sidewall-focus (J) patterns in ΔpodJ_At cells. Percentages of ΔpodJ_At cells (n = 252) and WT (12) cells (in parentheses) showing different FtsZ-GFP localizations are shown beneath representative images. Localizations seen in notably higher percentages of WT cells than ΔpodJ_At cells are highlighted by red rectangles, and localizations seen in notably higher percentages of ΔpodJ_At cells than WT cells are highlighted by blue rectangles. Bar, 3 μm.

FIG 8

The ΔpodJ_At strain produces nongrowing and small anucleate cells. (A) The arrow indicates a cell that does not grow or divide, and the arrowhead indicates a spherical minicell. (B) FM4-64 and DAPI staining. Some small ΔpodJ_At cells do not stain with DAPI (arrows). Bars, 3 μm.