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

Abstract

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.

Keywords: Agrobacterium cell cycle; PodJ; PopZ; bacterial cell division; polar growth.

Fig. S1.

Time-lapse microscopy of A. tumefaciens expressing FtsZ-GFP. Top and third rows, fluorescence images, where a single cell is outlined by a white dotted line. A second cell at right angles is not highlighted or described in the text. Second and fourth rows, combined fluorescence and brightfield images. Arrowheads indicate the growth pole. (Scale bar = 1 μm.)

Fig. 1.

Time-lapse microscopy of A. tumefaciens coexpressing FtsZ-RFP and FtsA-GFP. First row, FtsA-GFP. Second row, FtsZ-RFP. Third row, overlay of GFP and RFP. Fourth row, brightfield. The second frame (20 min) shows FtsZ beginning to form the Z-ring, when FtsA is still located at the growth pole. Cell outlines shown as dotted white lines. Arrowheads indicate growth poles. (Scale bar = 1 μm.)

Fig. 2.

FtsA and FtsZ in Z-rings. (A) 3D SIM of FtsA-GFP at the growth pole, and at the midcell where it forms a ring (Inset). (B) 3D-SIM of FtsZ-GFP shows a typical Z-ring in side view and inset. (C) Occasionally double FtsZ-rings were observed by deconvolution fluorescence microscopy (black arrows); i and ii show different cells with double Z-rings. (D) 3D-SIM of a double Z-ring (white arrows); i, ii, and iii are different views of the same Z-ring (s). Cell outlines shown as dotted white lines. All images taken in cells with wide Z-rings before contraction at division. Arrowheads, growth poles. (Scale bars = 1 μm.)

Fig. S2.

Alignment of PopZ_Cc (Cc1319) with PopZ_At (Atu1720). Over the regions that align, Cc1319 is 23% identical and 33% identical plus similar to Atu1720. Amino acid sequences predicted to form α-helices (highlighted in yellow) were identified in ref. for Cc1319 and predicted by Quick2D (toolkit.tuebingen.mpg.de/quick2_d) for Atu1720. Vertical lines indicate intervals of 10 amino acids. *, identical amino acids; :, strongly similar amino acids; ., weakly similar amino acids.

Fig. 3.

PopZ_At and PodJ_At localization. Time-lapse microscopy of A. tumefaciens expressing PopZ_At-GFP (A) or GFP-PodJ_At (B) shows PopZ_At localizes to new growth poles, whereas PodJ_At localizes to the old pole during the early part of the cell cycle and then also to the growth pole later in the cell cycle. Fluorescence images (with cell outlines as white dashed lines) are shown above overlays of fluorescence and brightfield images. Arrowheads indicate growth poles. (Scale bar = 1 μm.) (C) Demograph of cells expressing PopZ_At-GFP (n = 500). (D) Demograph of cells expressing GFP-PodJ_At (n = 400). Demographs are oriented with the growth pole on the right, using FM4-64 old pole-specific labeling (10) as a reference. Red triangle indicates the predicted growth pole–old pole transition marked by accumulation of PodJ.

Fig. S3.

Expression of A. tumefaciens PopZ_At and PodJ_At in E. coli. (A) Diffuse localization of PopZ_At-GFP. (B) Polar and midcell localization of GFP-PodJ_At. i, ii, and iii are different cells expressing the same proteins. (Scale bar = 1 μm.)

Fig. S4.

Alignment of PodJ_Cc (Cc2045) with PodJ_At (Atu0499). Over the regions that align, Cc2045 is 23% identical and 35% identical plus similar to Atu0499. Domains indicated were taken from ref. for Cc2045. For Atu0499, coiled coil domains with a 90% or higher probability (window size = 21 amino acids) were identified by COILS (ref. ; embnet.vital-it.ch/software/COILS_form.html), transmembrane domain by topcons.net, Sel-1 like repeats by Conserved Domain Architecture Retrieval Tool (CDART, www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi), and the peptidoglycan (PG) binding domain by CDART. Note, the three Cc2045 Sel-1 like repeats are contiguous, and are distinguished by bolded text of the second repeat. *, identical amino acids; :, strongly similar amino acids; ., weakly similar amino acids.

Fig. 4.

Simultaneous monitoring of PopZ_At and PodJ_At during the cell cycle. Time-lapse microscopy of A. tumefaciens coexpressing PopZ_At-GFP and RFP-PodJ_At. (A) First row, PopZ_At-GFP. Second row, RFP-PodJ_At. Third row, simultaneous imaging of PopZ_At-GFP and RFP-PodJ_At. Cell outlines are indicated by white dotted lines in fluorescence images. Fourth row, fluorescence of PopZ_At-GFP and RFP-PodJ_At coexpression, overlaid on brightfield images. (B) Simultaneous imaging of PopZ_At-GFP and RFP-PodJ_At by 3D-SIM. PopZ_At-GFP localizes to the growth pole and GFP-PodJ_At to the old pole during the early polar growth phase of the cell cycle (i). When the cell is longer and close to division, PodJ_At also localizes to the extreme tip of the growth pole (red arrow, ii), whereas PopZ_At is subpolar (green arrow, ii). Arrowheads indicate growth poles. (Scale bar = 1 μm.)

Fig. 5.

L,D-transpeptidase Atu0845 primarily localizes to the new pole. (A) Time-lapse microscopy of A. tumefaciens expressing Atu0845-sfGFP. Top row and third row, fluorescence images. Cell outlines shown as dotted white lines. Second row and fourth row, overlay of fluorescence and brightfield images. (B and C) 3D SIM of Atu0845-GFP. Atu0845 appears strongly at the growth pole, and weakly at the old pole. Cell polarity is determined by FM4-64 preferential labeling of the old pole versus the growth pole (10). White arrow shows Atu0845 closely associated with the membrane (B). Arrowheads indicate growth poles. (Scale bar = 1 μm.)

Fig. 6.

Summary of FtsA, FtsZ, PopZ_At, PodJ_At, and Atu0845 localization during the A. tumefaciens cell cycle. (A) FtsA and FtsZ colocalize (yellow) at the beginning of the cell cycle. FtsZ (red) leaves the growth pole (GP) to begin to form the Z-ring before FtsA (green). LDT Atu0845 localizes (brown shading) to regions of active PG synthesis at the GP and later at the midcell. (B) Localization of PopZ_At (green) and PodJ_At (red). Colocalization indicated by (yellow) and curved arrow indicates the transition of the GP into an old pole (OP). See text for details.