Cells were disrupted by three passages using a French pressure cell (SLM Aminco, Silver Spring, MD) at 100 MPa and soluble fractions were cleared from cell debris and membranes by ultracentrifugation at 135,000 × g at 4°C for 1 h. The supernatant (soluble extract) was added to a 0.2-ml StrepTactin Superflow column (IBA, Göttingen, Germany) operated by gravity flow. The column was washed five times

with 400 μl of buffer W to remove unbound proteins, and the tagged protein was eluted by the addition of 600 μl (6 × 100 μl) of buffer W supplemented with 2.5 mM D-desthiobiotin. Relevant fractions were pooled and concentrated using a centrifugal filter device (Amicon Ultra 0.5 ml, 3 K). Western immunoblot and peptide mass fingerprinting Proteins were resolved by either standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or native PAGE in commercial gradient 4-20% polyacrylamide gels (Bio-Rad, selleck products Hercules, California, USA), and were transferred onto Immobilon-P membrane filters (Millipore, Bedford, MA, USA) as previously described [48]. HupL, HupK and HypB proteins were detected immunologically using antisera raised against R. leguminosarum HupL (1:400 dilution), HupK (1:100 dilution) and HypB (1:2,000 dilution). Blots were developed by using a secondary goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate and a chromogenic substrate

(bromochloroindolyl phosphate-nitro blue tetrazolium) as recommended by the manufacturer (Bio-Rad Laboratories, Inc. Hercules, CA, USA). For HupFST identification we used HCS assay StrepTactin conjugated to alkaline phosphatase (1:2,500; IBA, Göttingen, Germany). Immunoblot analyses were performed with 60 μg and 20 μg (total protein) of vegetative cells and bacteroids crude

extracts, respectively, for HupL, or 10 μg for HypB detection. For purification of HupFST protein and study of interactions, Silibinin immunoblot analysis was performed with 4 μg of protein from pooled eluate fractions and 60 μg of protein from soluble fraction samples. For identification of complexes by peptide mass fingerprinting, 20 μg (total protein) of pooled desthiobiotin-eluted fractions from bacterial cultures of R. leguminosarum UPM1155(pALPF4, pPM501) were resolved in native 4–20% gradient polyacrylamide gels. Then, gels were stained by Coomassie brilliant blue G-250, and bands were excised and sent to the CBGP proteomics facility for analysis by mass spectrometry on a Kratos MALDI-TOF MS apparatus (Kratos Analytical, Manchester) after trypsin digestion. Peptide profile was compared to MASCOT database supplemented with sequences from UPM791 hup/hyp gene products. Acknowledgements We thank Julia Kehr for her excellent help in protein identification by peptide mass fingerprinting. This work has been funded by research projects from Spain’s Ministerio de Ciencia y Tecnología (BIO2010-15301 to J.P.), from Comunidad de Madrid (MICROAMBIENTE-CM to T.R.A.), and from Fundación Ramón Areces (to J.I.). A.B.