super-resolution-imaging-cytokinetic-z-ring-live-bacteria-using-fast

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

University of Technology, Sydney

We have established a plasmid-based system that enables tightly controlled gene expression and the generation of GFP fusion proteins in Staphylococcus aureus simply and rapidly. This system takes advantage of an Escherichia coli-S. aureus shuttle vector that contains the replication region of the S. aureus theta-mode multiresistance plasmid pSK41, and is therefore a stable low-copy-number plasmid in the latter organism. This vector also contains a multiple cloning site downstream of the IPTG-inducible Pspac promoter for insertion of the gene of interest. Production of encoded proteins can be stringently regulated in an IPTG-dependent manner by introducing a pE194-based plasmid, pGL485, carrying a constitutively expressed lacI gene. Using GFP fusions to two essential proteins of S. aureus, FtsZ and NusA, we showed that our plasmid allowed tightly controlled gene expression and accurate localization of fusion proteins with no detrimental effect on cells at low inducer concentrations. At higher IPTG concentrations, we obtained sixfold overproduction of protein compared with wild-type levels, with FtsZ-GFP-expressing cells showing lysis and delocalized fluorescence, while NusA-GFP showed only delocalized fluorescence. These results show that our system is capable of titratable induction of gene expression for localization or overexpression studies.

A simple plasmid-based system that allows rapid generation of tightly controlled gene expression in Staphylococcus aureus.

FtsZ is a tubulin-like GTPase that is the major cytoskeletal protein in bacterial cell division. It polymerizes into a ring, called the Z ring, at the division site and acts as a scaffold to recruit other division proteins to this site as well as providing a contractile force for cytokinesis. To understand how FtsZ performs these functions, the in vivo architecture of the Z ring needs to be established, as well as how this structure constricts to enable cytokinesis. Conventional wide-field fluorescence microscopy depicts the Z ring as a continuous structure of uniform density. Here we use a form of super resolution microscopy, known as 3D-structured illumination microscopy (3D-SIM), to examine the architecture of the Z ring in cells of two Gram-positive organisms that have different cell shapes: the rod-shaped Bacillus subtilis and the coccoid Staphylococcus aureus. We show that in both organisms the Z ring is composed of a heterogeneous distribution of FtsZ. In addition, gaps of fluorescence were evident, which suggest that it is a discontinuous structure. Time-lapse studies using an advanced form of fast live 3D-SIM (Blaze) support a model of FtsZ localization within the Z ring that is dynamic and remains distributed in a heterogeneous manner. However, FtsZ dynamics alone do not trigger the constriction of the Z ring to allow cytokinesis. Lastly, we visualize other components of the divisome and show that they also adopt a bead-like localization pattern at the future division site. Our data lead us to propose that FtsZ guides the divisome to adopt a similar localization pattern to ensure Z ring constriction only proceeds following the assembly of a mature divisome.

3D-SIM super resolution microscopy reveals a bead-like arrangement for FtsZ and the division machinery: implications for triggering cytokinesis.

Spatial regulation of cell division in bacteria has been a focus of research for decades. It has been well studied in two model rod-shaped organisms, Escherichia coli and Bacillus subtilis, with the general belief that division site positioning occurs as a result of the combination of two negative regulatory systems, Min and nucleoid occlusion. These systems influence division by preventing the cytokinetic Z ring from forming anywhere other than midcell. However, evidence is accumulating for the existence of additional mechanisms that are involved in controlling Z ring positioning both in these organisms and in several other bacteria. In some cases the decision of where to divide is solved by variations on a common evolutionary theme, and in others completely different proteins and mechanisms are involved. Here we review the different ways bacteria solve the problem of finding the right place to divide. It appears that a one-size-fits-all model does not apply, and that individual species have adapted a division-site positioning mechanism that best suits their lifestyle, environmental niche and mode of growth to ensure equal partitioning of DNA for survival of the next generation.

Andrew T. F. Liew

The ithree Institute

University of Technology, Sydney

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Andrew T. F. Liew

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The ithree Institute

University of Technology, Sydney

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

The ithree Institute