1. Building Polar Protein Networks in Alphaproteobacteria
Taylor Clawson, Haley Ehrle, Jacob Guidry, and Grant Bowman
University of Wyoming Department of Molecular Biology
Abstract
Rod-shaped bacteria exhibit polar
asymmetry during growth and cell
division
Figure 1: Caulobacter
crescentus cells.
Cytoplasmic regulatory
proteins have been
labeled with genetically
encoded fluorescent
protein tags.
PopZ (Polar organizing protein Z)
accumulates at Caulobacter cell poles
Bowman et al. 2008
Figure 2: An electron micrograph of a Caulobacter cell pole,
showing PopZ localization by immuno-gold labeling.
Caulobacter PopZ is required for the
localization of polar regulatory proteins
Figure 3: In wild-type cells, the regulatory proteins DivJ, CckA,
and SpmX accumulate in polar foci. In DpopZ knockout cells,
these proteins are mostly diffuse. To date, 12 polar proteins are
known to be de-localized in DpopZ knockout cells .
Multispecies alignment of PopZ
homologs in select Alphaproteobacteria
Figure 4. The amino acid sequences of PopZ in Caulobacter
crescentus (CC), Agrobacterium tumefaciens (AT) and Brucella
abortus (BA) are aligned with respect to regions of amino acid
conservation, as indicated by blue shading. PopZ has not been
functionally characterized outside of Caulobacter crescentus. Its
role in supporting cell organization in pathogenic bacteria is
unknown.
How do we identify PopZ binding partners?
Figure 5. Escherichia coli can be used as a heterologous expression
system for identifying PopZ binding partners, using candidate
proteins from any species. Due to its intrinsic capacity for self-
assembly, PopZ will accumulate at a cell pole. GFP-tagged
candidate proteins are screened for PopZ binding activity by co-
expressing them with mCherry-PopZ. A binding protein will co-
localize with the polar focus of PopZ .
Brucella and Caulobacter PopZ have
common polar organizing properties
Figure 6. Heterologous E. coli expression assay. Top panels: In the
absence of PopZ, Caulobacter ParB-GFP exhibits diffuse localization.
Co-expression of Caulobacter PopZ and Caulobacter ParB
demonstrates binding between these proteins. Bottom left panels: Co-
expression of Brucella PopZ with Caulobacter proteins ParB and ChpT.
The Brucella homolog of PopZ is able to interact with these proteins.
Bottom right panels: Co-expression of Brucella PopZ and Brucella
pole-localized virulence proteins BtaE and BtaF, showing the absence
of PopZ binding. Right panel: A complete list of Caulobacter polar
proteins that were shown to interact with Brucella PopZ in this assay.
Agrobacterium and Caulobacter use histidine
kinases to regulate the cell cycle
Figure 7. Histidine kinase signaling
and its influence on gene
expression. In Caulobacter, DivJ
and Ccka (shown in Figure 3) are
polar histidine kinases that are
localized to the cell poles by
interaction with PopZ.
Figure 8. Agrobacterium produces four histidine kinases that have similarity
with Caulobacter DivJ. Their localization in Agrobacterium is unknown.
Agrobacterium DivJ retains polar localization
in the absence of PopZ
Figure 10. A time-lapse series of PopZ-deficient Agrobacterium cells (DpopZ),
which are expressing DivJ-GFP (green). DivJ-GFP remains stably localized to
the pole. Interestingly, multiple buds of cell wall growth arise from the
opposite pole, suggesting that the regulation of bud formation is defective in
this strain.
Agrobacterium PdhS1 is localized to
the pole opposite from PopZ
Figure 11. Agrobacterium cells expressing mCherry-PopZ (red)
and PdhS1-GFP (green). Multiple stages of the cell cycle are
shown, including newly formed daughter cells (asterisks) following
asymmetric cell division.
A model of polar histidine kinase
localization during the Agrobacterium
cell cycle
Figure 12. Agrobacterium divides asymmetrically. DivJ and PdhS1
are localized to the pole opposite from PopZ, and this pole
appears to be a developmental end-point for polar differentiation.
Summary
• Brucella and Caulobacter PopZ have common polar
organizing properties.
• Agrobacterium PopZ exhibits asymmetric polar
localization.
• The Agrobacterium histidine kinases DivJ and PdhS1
are localized to the pole opposite from PopZ.
• In Agrobacterium, DivJ does not require PopZ for polar
localization.
• In Agrobacterium, PopZ appears to be important for
pinpointing the site of polar bud growth.
Some bacteria exhibit robust sub-cellular organization, with
regulatory proteins, structural elements, and other factors
specifically targeted to cell poles. Among the class
Alphaproteobacteria, a polar organizing protein called PopZ is
thought to be involved in the localization of many polar proteins by
either direct or indirect means. However, this model of PopZ as a
central polar organizing protein is limited by our knowledge of
PopZ function in only one organism. This research focuses on
determining PopZ binding partners in two additional
Alphaproteobacterial species, Agrobacterium tumefaciens and
Brucella abortus. To do this, we are using two different
experimental methods. In Agrobacterium, we are expressing
candidate polar regulatory proteins as GFP-fusions and asking if
they are localized to cell poles and if they are mis-localized in a
DpopZ knockout strain. To look for PopZ binding proteins in
Brucella, we are co-expressing Brucella PopZ with candidate
binding proteins in E. coli and looking for direct binding
interactions in this heterologous context. Thus far we have
identified two pole-localized kinase signaling proteins in
Agrobacterium, and we have used these markers to illustrate
robust polar asymmetry in this species. We have also shown that
Brucella PopZ interacts with the same cell cycle regulatory
proteins as its Caulobacter homolog.
Figure 9. A time-lapse series of dividing Agrobacterium cells, which are
expressing mCherry-PopZ (red) and DivJ-GFP (green). Agrobacterium PopZ
exhibits dynamic localization to the new pole after cell division. Agrobacterium
DivJ accumulates at the old pole opposite from PopZ, where it remains
localized through the cell cycle.
Agrobacterium DivJ is localized to the pole
opposite from PopZ