SUPPLEMENTAL METHODS

Plasmid construction. For production of the DdcY D,D-carboxypeptidase in E. faecium, the ddcY gene of E. faecium D344S was amplified by PCR using two pairs of oligonucleotides (5’-CTTCTGTTTTCCCTAAATATTCT with 5’-CATGAAAAGATCGTATAAGACAG and 5’-GATCCTTCTGTTTTCCCTAAATATTCT with 5’-AAAAGATCGTATAAGACAGTAGCTGT). The amplicons were mixed, denatured, renatured, and the resulting heteroduplexes were ligated with DNA of vector pJEH11 (1) digested with NcoI and BamHI.

For production of serine threonine kinase Stk in E. faecium, gene stk of E. faecium D344S was amplified with oligonucleotides (5’-AACCATGGGGATCGAGTTGGGAAAAAAATT and 5’-AAAGATCTCAGCACTTATTTTGAACTTTC), digested with NcoI and BglII (underlined), and cloned into pJEH11 digested with NcoI and BamHI. For production of a soluble fragment of Stk in E. coli, the 5’ portion of stk (nucleotides 1 to 1014), encoding the kinase domain plus juxtamembrane segment (Stk-DK; residues 1 to 338) was amplified with oligonucleotides (5’- AACCATGGGGATCGAGTTGGGAAAAAAATT and 5’-AAGGATCCTTTCTGTCCTTTCTTTTTAGG), digested with NcoI and BamHI, and cloned into pET2818 (1) (1) digested with the same enzymes.

For production of the serine threonine phosphatase StpA in E. faecium, gene stpA of E. faecium D344S was amplified with oligonucleotides (5’-AACCATGGGGCAGATTGAATATCAATCAG and 5’-AAGGATCCTACACTGTCCTCCTTATATTCG), digested with NcoI and BamHI (underlined), and cloned into pJEH11 digested with the same restriction endonucleases. For production of StpA in E. coli, the same amplicon was cloned into pET2818 digested with NcoI and BamHI.

Mass spectrometry analysis of protein phosphorylation. Recombinant Stk produced in E. coli (12 µg in 10 µl of 50 mM Tris-HCl containing 150 mM NaCl) was dialyzed against water. Five µl of dialyzed protein were mixed extemporaneously with 5 µl of acetonitrile and 1 µl of 1% formic acid and extemporaneously injected into the mass spectrometer (Qstar Pulsar I, Applied Biosystem) using rp-HPLC pumps at a flow rate of 0.05 ml/min (acetonitrile 50%, water 49.5 %, formic acid 0.5 %, per volume). Mass spectra were acquired in the positive mode with a capillary voltage of 5,200 V and a declustering potential of 20 V.

Purification of full length Stk from E. faecium D344S pJEH11Ωstk. Bacteria were grown overnight in brain heart infusion (BHI) broth containing gentamicin (128 µg/ml) at 37 °C. Bacteria were collected by centrifugation and resuspended in 50 mM Tris-HCl (pH 8.0) containing 300 mM NaCl and 1 mg/ml lysozyme. Bacteria were incubated 1 h at 37 °C and lysed by sonication. Triton X-100 (1%) was added to the lysates and the incubation was continued for 15 min at 4 °C. Cell debris were removed by centrifugation and Stk was purified by affinity and size-exclusion chromatographies (Superdex 200 HL26/60 column; GE Healthcare), as previously described for the catalytic domain of Stk, except that all buffers contained 10% glycerol and 0,1% Triton X-100.

1. Bellais S, Arthur M, Dubost L, Hugonnet JE, Gutmann L, van Heijenoort J, Legrand R, Brouard JP, Rice L, Mainardi JL. 2006. Aslfm, the D-aspartate ligase responsible for the addition of D-aspartic acid onto the peptidoglycan precursor of Enterococcus faecium. J. Biol. Chem. 281:11586-11594.

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