Eukaryotic protein kinase takes a role in bacterial cytokinesis and morphology

Research Areas:

Mitosis, Cell fusion, Pneumococcus, fluorescence microscopy, immunofluorescence, GFP, RFP, BADA

Imaging Needs:

Time-lapse microscopy and red/green fluorescence co-localization

Imaging System (fluorescence and immunofluorescence microscopy):
  • FtsZ tagged with GFP or RFP
  • Peptidoglycan tagged with BADA or GFP
  • Zeiss AxioObserver Z1 microscope
  • 100× NA 1.46 objective lens
  • Hamamatsu ORCA-R2 C10600 CCD camera
  • AxioVision (Carl Zeiss) and ImageJ (NIH) software
Imaging System (time-lapse microscopy):
  • Nikon Ti-E/B automated inverted epifluorescence microscope
  • Nikon Perfect Focus System
  • CFI Plan Fluor DLL 100× oil NA1.3 phase contrast objective
  • Semrock filter set for GFP (Ex: 482BP35; DM: 506; Em: 536BP40)
  • Nikon Intensilight 130 W High-Pressure Mercury Lamp
  • Hamamatsu ORCA-R2 CCD camera and ImagEM-1K EM-CCD camera
  • Image capture every 5 minutes with exposure time: 2 seconds; camera gain: 50; and light attenuation with neutral-density filters: 25%
  • Image processing in Nikon Nis-Elements AR software
Measuring molecular events in living organisms

Recent techniques enable comparison of in vitro results with observations in living organisms. Learn more in Illuminating Activity—In Vitro and In Vivo. Read now.

THE QUESTION

How do bacteria coordinate cell division and establish cell shape?

Whether round, rod-like, curved or spiral, each bacterial strain presents a characteristic shape. Cross-strain differences in morphology are thought to involve specific variations in the mechanisms of cell growth, division and chromosome segregation.

THE BARRIERS

In recent years, researchers have found that many bacteria contain serine/threonine protein kinases of a type previously associated with eukaryotes. These kinases seem to play a role in bacterial cell division, but their actions and regulation are unknown.

THE SOLUTION

Interplay of the Serine/Threonine-Kinase StkP and the Paralogs DivIVA and GpsB in Pneumococcal Cell Elongation and Division
Aurore Fleurie, Sylvie Manuse, Chao Zhao, Nathalie Campo, Caroline Cluzel, Jean-Pierre Lavergne, Céline Freton, Christophe Combet, Sébastien Guiral, Boumediene Soufi, Boris Macek, Erkin Kuru, Michael S. VanNieuwenhze, Yves V. Brun, Anne-Marie Di Guilmi, Jean-Pierre Claverys, Anne Galinier, and Christophe Grangeasse
PLoS Genet. Apr 2014; 10(4): e1004275. PMCID: PMC3983041.

To examine the possible involvement of one such kinase in cytokinesis and the establishment of bacterial morphology, Fleurie et al.3 studied the interactions of the cell-division paralogs DivIVA and GpsB with the serine/threonine kinase StkP in Streptococcus pneumoniae.

The bacterial culprit behind pneumonia, S. pneumoniae, or pneumococcus, has a readily identifiable ellipsoid shape. Using membrane staining, phase-contrast and electron microscopy, Fleurie and colleagues showed that deletion of the divIVA gene stunted elongation, resulting in shorter, rounder cells. GpsB knockout had the opposing effect of excess cell elongation—an effect suppressed by divIVA deletion—while inhibiting cell division.

The authors took a closer look at the cytokinetic machinery of dividing pneumococci, using fluorescence and immunofluorescence microscopy captured on a Hamamatsu ORCA-ER CCD camera. The team stained the site of ongoing cell division, a center of peptidoglycan synthesis, by labeling peptidoglycans with Bodipy-FL containing fluorescent D-amino acid (BADA). GpsB knockout mutants showed an altered, helical pattern of BADA labeling, as compared with the band shape revealing Z-ring formation in wild-type cells. Similarly, sites of synthesis for DivIVA as well as the Z-ring protein FtsZ showed this helix pattern.

Two-color fluorescence microscopy with the ORCA-ER camera confirmed co-localization of peptidoglycans labeled with green fluorescent protein (GFP) and FtsZ tagged with red fluorescent protein (RFP). Through time-lapse microscopy using Hamamatsu ORCA-R2 and ImageEM cameras, the authors traced formation of the Z-spiral during ΔGpsB mutant cell elongation. Interestingly, protein interaction experiments showed that GpsB and DivIVA interact with FtsZ only indirectly, through another cell division protein, EzrA.

The authors conclude that DivIVA and GpsB play opposing roles in regulation of pneumococcus division and elongation, coordinating peptidoglycan synthesis and incorporation into the Z-ring through the actions of EzrA. The kinase StkP plays a key role, catalyzing the phosphorylation of DivIVA in a GpsB-dependent fashion. Together, the three cell division proteins and serine/threonine kinase help to actuate cell elongation and assembly of the cell division machinery in S. pneumoniae.

THE POSSIBILITIES

Fleurie et al. relied on the high resolution and speed and Hamamatsu’s ORCA-R2 and ImageEM cameras to co-localize fluorescently tagged cell division proteins and track their assembly in growing bacteria. Learn how Takeaki Ozawa and colleagues use Hamamatsu’s ImageEM camera to detect receptor binding in vivo using luminescence in place of fluorescence. Read more in Illuminating Activity—in vitro and in vivo.

References

  1. Fleurie A, et al. Interplay of the Serine/Threonine-Kinase StkP and the Paralogs DivIVA and GpsB in Pneumococcal Cell Elongation and Division. PLoS Genet. Apr 2014; 10(4): e1004275. PMCID: PMC3983041.
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