SPINE

SPINE is a method to detect in vivo protein-protein interactions PubMed

A detailed protocol to detect the interaction between RocG and GltC:

1 litre of a B. subtilis culture was grown to an OD600 of approx. 1.0 and incubated with 0.6% formaldehyde ( 4% stock solution in PBS, pH 6.5!) for 20 minutes @ 37°C on a shaker. The cells were harvested and washed once in 1 X PBS pH 6.5. The pellets can then be stored @ -20 °C. The GltC protein was expressed carrying a Strep-tag and RocG expression was induced by arginine (PubMed). Expression of the Strep-tagged GltC protein allows to test the functionality of the protein. Crude extracts (10-15 ml) were prepared by using a French Press. After a centrifugation step for 1 h @ 27.000 g the clarified crude extracts were loaded onto a Streptactin sepharose column (0.5-1 ml matrix) to isolate the cross-linked protein complexes (the detailed procedure for protein purification is described in the IBA manual, http://www.iba-go.com/). After the purification of the protein complexes the crosslinks can be resolved by boiling the samples in Laemmli buffer for 10-15 minutes @ 95 °C (PubMed). A 12.5% SDS gel was loaded with the samples and the proteins were then visualized by silver-staining. The interaction partner/s were identified by mass spectroscopy and Western blotting.

Preparation of the formaldehyde stock solution (max. 4% in 1 X PBS pH 6.5): We use para-formaldehyde (a white powder; http://en.wikipedia.org/wiki/Paraformaldehyde). para-formaldehyde dissolves within approx. 20-30 minutes in 1 X PBS for @ 65 to 70 °C.

The sepharose matrix was purchased from the IBA company, Göttingen (http://www.iba-go.com/).

Relevant plasmids:

for use in B. subtilis (multicopy plasmids): pGP380, pGP382

for use in B. subtilis (chromosomal integration under the control of the native promoter): pGP1389

for use in E. coli: pGP172, pGP574

Biotin-containing proteins that are purified with the Strep-Tactin column

PycA, AccB

Studies that made use of SPINE:

Felix M P Mehne, Katrin Gunka, Hinnerk Eilers, Christina Herzberg, Volkhard Kaever, Jörg Stülke
Cyclic di-AMP homeostasis in bacillus subtilis: both lack and high level accumulation of the nucleotide are detrimental for cell growth.
J Biol Chem: 2013, 288(3);2004-17
[PubMed:23192352] [WorldCat.org] [DOI] (I p)

Alexander K W Elsholz, Kürsad Turgay, Stephan Michalik, Bernd Hessling, Katrin Gronau, Dan Oertel, Ulrike Mäder, Jörg Bernhardt, Dörte Becher, Michael Hecker, Ulf Gerth
Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis.
Proc Natl Acad Sci U S A: 2012, 109(19);7451-6
[PubMed:22517742] [WorldCat.org] [DOI] (I p)

Andrea Wünsche, Elke Hammer, Maike Bartholomae, Uwe Völker, Andreas Burkovski, Gerald Seidel, Wolfgang Hillen
CcpA forms complexes with CodY and RpoA in Bacillus subtilis.
FEBS J: 2012, 279(12);2201-14
[PubMed:22512862] [WorldCat.org] [DOI] (I p)

Frederik M Meyer, Matthieu Jules, Felix M P Mehne, Dominique Le Coq, Jens J Landmann, Boris Görke, Stéphane Aymerich, Jörg Stülke
Malate-mediated carbon catabolite repression in Bacillus subtilis involves the HPrK/CcpA pathway.
J Bacteriol: 2011, 193(24);6939-49
[PubMed:22001508] [WorldCat.org] [DOI] (I p)

Jens J Landmann, Ricarda A Busse, Jan-Hendrik Latz, Kalpana D Singh, Jörg Stülke, Boris Görke
Crh, the paralogue of the phosphocarrier protein HPr, controls the methylglyoxal bypass of glycolysis in Bacillus subtilis.
Mol Microbiol: 2011, 82(3);770-87
[PubMed:21992469] [WorldCat.org] [DOI] (I p)

Martin Lehnik-Habrink, Joseph Newman, Fabian M Rothe, Alexandra S Solovyova, Cecilia Rodrigues, Christina Herzberg, Fabian M Commichau, Richard J Lewis, Jörg Stülke
RNase Y in Bacillus subtilis: a Natively disordered protein that is the functional equivalent of RNase E from Escherichia coli.
J Bacteriol: 2011, 193(19);5431-41
[PubMed:21803996] [WorldCat.org] [DOI] (I p)

A K W Elsholz, K Hempel, S Michalik, K Gronau, D Becher, M Hecker, U Gerth
Activity control of the ClpC adaptor McsB in Bacillus subtilis.
J Bacteriol: 2011, 193(15);3887-93
[PubMed:21622759] [WorldCat.org] [DOI] (I p)

Frederik M Meyer, Jan Gerwig, Elke Hammer, Christina Herzberg, Fabian M Commichau, Uwe Völker, Jörg Stülke
Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: evidence for a metabolon.
Metab Eng: 2011, 13(1);18-27
[PubMed:20933603] [WorldCat.org] [DOI] (I p)

Martin Lehnik-Habrink, Henrike Pförtner, Leonie Rempeters, Nico Pietack, Christina Herzberg, Jörg Stülke
The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complex.
Mol Microbiol: 2010, 77(4);958-71
[PubMed:20572937] [WorldCat.org] [DOI] (I p)

Fabian M Commichau, Fabian M Rothe, Christina Herzberg, Eva Wagner, Daniel Hellwig, Martin Lehnik-Habrink, Elke Hammer, Uwe Völker, Jörg Stülke
Novel activities of glycolytic enzymes in Bacillus subtilis: interactions with essential proteins involved in mRNA processing.
Mol Cell Proteomics: 2009, 8(6);1350-60
[PubMed:19193632] [WorldCat.org] [DOI] (I p)

Fabian M Commichau, Christina Herzberg, Philipp Tripal, Oliver Valerius, Jörg Stülke
A regulatory protein-protein interaction governs glutamate biosynthesis in Bacillus subtilis: the glutamate dehydrogenase RocG moonlights in controlling the transcription factor GltC.
Mol Microbiol: 2007, 65(3);642-54
[PubMed:17608797] [WorldCat.org] [DOI] (P p)

The use of SPINE in other microbes:

Jens F Novak, Marit Stirnberg, Benjamin Roenneke, Kay Marin
A novel mechanism of osmosensing, a salt-dependent protein-nucleic acid interaction in the cyanobacterium Synechocystis Species PCC 6803.
J Biol Chem: 2011, 286(5);3235-41
[PubMed:21123179] [WorldCat.org] [DOI] (I p)