Kinetics of stringent response revealed by microarray analysis

Stringent response is immensely complex and many different proteins are targeted by ppGpp. Different targets have different susceptibility to ppGpp - K_D of binding or K_i of inhibition. Therefore as stringent response progresses and ppGpp concentration changes, different targets will be engaged sequentially, in this way orchestrating changes in the cellular physiology.

Studying the development of stringent response in time is a tricky business. Temporal resolution in one key component, ability following many parameters (many targets) is the other one. Microarrays seem to be the way (Traxler 2006, Traxler 2008, Durfee 2008, Traxler 2011 and Balsalobre 2011).

Here are the main points from Traxler 2011. They did microarray analysis of four strains: wt E. coli K-12-derivative MG1655, ppGpp⁰, Lrp- and RpoS- in MG1655 background. The nature of the wt is important - L12 derivatives are special in that sense that they exhibit stringent response upon L-Valine and L-isolucine overdose, and the latter is used to induce the stringent response in Traxler 2011.

In these strains they follow the effects of two global regulators, Transcription factor Lrp (leucine responsive protein) and RpoS AKA Sigma 38, a specialized σ-factor.

It turnes out that ppGpp regulates both the feed-back loop regulated by Lrp and the feed-forward loop regulated by RpoS (for discussion of how these motifs work see Alon 2007). Moreover, Lrp regulon is induced much earlier than RpoS.

So how is one molecule - ppGpp - evokes two temporally separated responses (early Lrp and late RpoS)? The obvious idea is that these two regulons have different sensitivity to ppGpp, thus the more sensitive one is induced earlier (Lrp) and the less sensitive one is induced later (RpoS). Combining microarray data with measurements of ppGpp concentrations in vivo confirmed this hypothesis.

Rendering Lrp operon inactive in turn disrupts normal progression of the stringent response: both wt and Lpr- strains rapidly accumulate ppGpp, but during the later stage wt cells lower the ppGpp concentration and resume growth, whereas Lrp- strain retains high ppGpp concentration is unable to adapt to stringent conditions. Functional RpoS (which works on the later stages of stringent response) does not help - you have to come prepared, i.e. Lrp regulon should do its job.

However powerful, microarray-based techniques inherently average out the cell-to-cell variability which plays important role in the stringent response as shown by the systems biology investigations.

References:

Matthew F. Traxler, Vineetha M. Zacharia, Stafford Marquard, Sean M. Summers, Huyen-Tran Nguyen, S. Elizabeth Stark and Tyrrell Conway. Discretely calibrated regulatory loops controlled by ppGpp partition gene induction across the ‘feast to famine’ gradient in Escherichia coli. Molecular Microbiology (2011) doi:10.1111/j.1365-2958.2010.07498.x

Matthew F. Traxler, Sean M. Summers, Huyen-Tran Nguyen, Vineetha M. Zacharia, G. Aaron Hightower, Joel T. Smith and Tyrrell Conway. The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli. Molecular Microbiology (2008) 68(5), 1128–1148

Carlos Balsalobre. Concentration matters!! ppGpp, from a whispering to astrident alarmone. Molecular Microbiology (2011) doi:10.1111/j.1365-2958.2010.07521.x

Matthew F Traxler, Dong-Eun Chang, Tyrrell Conway. Guanosine 3',5'-bispyrophosphate coordinates global gene expression during glucose-lactose diauxie in Escherichia coli. PNAS 2006 vol. 103 (7) pp. 2374-9

Tim Durfee, Anne-Marie Hansen, Huijun Zhi, Frederick R. Blattner and Ding Jun Jin. Transcription Profiling of the Stringent Response in Escherichia coli. J. of Bacteriol. 2008, p. 1084-1096, Vol. 190, No. 3

R. I. Leavitt, H. E. Umbarger. Isoleucine and valine metabolism in Escherichia coli. XI. Valine inhibition of the growth of Escherichia coli strain K-12. J. Bacteriol. 1962 vol. 83 pp. 624-30

Uri Alon. Network motifs: theory and experimental approaches. Nat Rev Genet. 2007 vol. 8 (6) pp. 450-61

Brinkman AB, Ettema TJ, de Vos WM, van der Oost J. The Lrp family of transcriptional regulators. Mol Microbiol. 2003 48(2) pp. 287-94

Mendeley group on stringent response