Biology Reference
In-Depth Information
apparent that CitC is more abundant than CitZ. This is consistent with the absolute
cellular abundance for these two proteins under these conditions determined previ-
ously (
Buescher
et al.
, 2012
).
9
CONCLUDING REMARKS
The development of fluorescent proteins as tools for imaging protein dynamics
in situ
has revolutionised our understanding of microbial cell biology. Today, many
system packages are available from all the major microscope suppliers that enable
high-quality imaging of fluorescently tagged microbial cells. For the more demand-
ing user, bespoke systems can also be readily assembled. In this chapter, we have
outlined and highlighted basic principles and approaches to constructing and imag-
ing fluorescent protein fusions in some of the most common model microbes, and
many of these approaches are transferable to other organisms. Once the basics have
been mastered, there are now many systems available that enable more technically
demanding studies such as protein-protein interaction (FRET/FLIM), 3D reconstruc-
tion, super-high resolution (e.g. DeltaVision OMX) and single-molecule tracking
using total internal reflection microscopy.
Acknowledgements
This work was supported by grants from the Australian Research Council (ARC),
National Health and Medical Research Council (NHMRC) and European Union.
References
Buescher, J. M., Liebermeister, W., Jules, M., Uhr, M., Muntel, J., Botella, E., Hessling, B.,
Kleijn, R. J., Le Chat, L., et al. (2012). Global network reorganization during dynamic
adaptations of Bacillus subtilis metabolism.
Science
335, 1099-1103.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994). Green fluores-
cent protein as a marker for gene expression.
Science
263, 802-805.
Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996). FACS-optimized mutants of the green
fluorescent protein (GFP).
Gene
173, 33-38.
Datsenko, K. A. and Wanner, B. L. (2000). One-step inactivation of chromosomal genes in
Escherichia coli K-12 using PCR products.
Proc. Natl. Acad. Sci. U.S.A.
97, 6640-6645.
Davies, K. M., Dedman, A. J., van Horck, S., and Lewis, P. J. (2005). The NusA:RNA poly-
merase ratio is increased at sites of rRNA synthesis in Bacillus subtilis.
Mol. Microbiol.
57,
366-379.
de Berardinis, V., Vallenet, D., Castelli, V., Besnard, M., Pinet, A., Cruaud, C., Samair, S.,
Lechaplais, C., Gyapay, G., et al. (2008). A complete collection of single-gene deletion
mutants of Acinetobacter baylyi ADP1.
Mol. Syst. Biol.
4, 174.
