Supporting Information Foucault et al. 10.1073/pnas.1006855107 SI Materials and Methods KpnI plus BamHI and cloned under the control of the Ptet pro- Bacterial Strains, Plasmids, and Growth Conditions. The origin and moter of pAT890(pAT29ΩPtet) digested by the same enzymes to properties of bacterial strains and plasmids are summarized in generate plasmid pAT891(pAT29ΩPtet lucR)(Table S1). Escherichia coli Table S1. Top10 (Invitrogen) was used as a host pAT895(pGhost9Ωint′P lucR int″). To integrate the luciferase gene for recombinant plasmids, and kanamycin (50 μg/mL) was the tet in the chromosome of JH2-2::I, an int′Ptet lucR int″ fragment was selective agent for cloning PCR products in the pCR-Blunt E. coli constructed in the thermosensitive vector pGhost9 (Fig. S1). vector (Invitrogen). TG1 RepA (1) was used as a host for The int′ fragment amplified with the intB1 and intB2 primers constructions in the pGhost9 vector (2). Ampicillin (75 μg/mL), μ μ (Table S2) from total DNA of JH2-2::I was inserted in the XhoI spectinomycin (60 g/mL), and erythromycin (150 g/mL) were and PstI sites of pCR-Blunt to generate plasmid pAT892(pCR- added to culture media to prevent loss of plasmids derived from BluntΩint′). pGEX-6P-2ΩlucR, pAT29, and pGhost9, respectively. Strains The Ptet lucR insert of pAT891(pAT29ΩPtetlucR) amplified were grown in brain heart infusion broth or agar (Difco) at 37 °C. with primers Ptet2 and LucR-rev was cloned downstream from int′ Plasmid Constructions. The plasmids were constructed as follows. between the PstI and BamHI restriction sites of plasmid Ωint′ Ωint′ Construction of pAT886 and pAT889. pAT886(pCR-BluntΩvanX ). pAT892(pCR-Blunt ) to generate pAT893(pCR-Blunt B[H116A,D123A] P lucR The vanXB gene of the vanB operon was inactivated by two mu- tet ). The int″ fragment amplified with primers intB3 and intB4 and tations, leading to H116AandD123A substitutions in the active site total DNA of JH2-2::I as a template was cloned in the BamHI of the VanXB D,D-dipeptidase (3), by successive PCR amplifica- and HindIII sites downstream from the int′PtetlucR insert of tions. A SmaI restriction site was added in primers VanXB123 and Ω int′PtetlucR VanXB123R(Table S2) to screen for mutated vanXB(H116A,D123A). plasmid pAT893(pCR-Blunt ) to generate pAT894 Ωint′Ptet lucRint″ The vanXB gene of JH2-2::I was first amplified with the VanXB (pCR-Blunt ). int′PtetlucRint″ Ωint′ plus VanXB123R and VanXB123 plus VanXBR primers each con- The 2,989-bp insert of pAT894(pCR-Blunt PtetlucRint″ taining the D123A mutation (Table S2) and, in a second step, the ) was digested with XhoI and NotI, cloned in pGhost9 resulting PCR products were amplified with VanXB plus VanXBR digested with the same enzymes to generate plasmid pAT895 Ωint′PtetlucRint″ to obtain mutated vanXB(D123A). To introduce the second muta- (pGhost9 ), and its sequence was redetermined tion, vanXB(D123A) was amplified with primers VanXB plus using a dye-labeled ddNTP Terminator Cycle Sequencing Kit VanXB116R and VanXB116 plus VanXBR containing the H116A (Beckman Coulter) and a CEQ 2000 automated sequencer (Beck- mutation (Table S2), and the two products were amplified with man Coulter). VanXB plus VanXBR to generate vanXB(H116A,D123A), which was inserted in pCR-Blunt to generate plasmid pAT886(pCR- Strains Construction. In Gram-positive bacteria, pGhost9 (2), which BluntΩvanXB[H116A,D123A])(Table S1). replicates at 28 °C but is lost above 37 °C, allowed construction of Enterococcus faecalis pAT889(pGhost9ΩvanB′vanXB[H116A,D123A]360 bp). The thermosensi- JH2-2::I derivatives by insertional in- tive pGhost9 vector (Table S1) was used to integrate mutated activation. Transformants were selected at the permissive tem- vanXB(H116A,D123A) in the wild gene. Construction of pAT889 perature (28 °C) on M17 plates containing 10 μg/mL of (pGhost9ΩvanB′vanXB[H116A, D123A]360 bp) was performed in erythromycin and 0.5% glucose. One colony of each transformant three steps (Fig. S1). The 360-bp intergenic region down- was inoculated into 50 mL of M17 broth without antibiotic and stream from vanXB (Fig. S1) was amplified from total DNA containing 0.5% glucose and was incubated for 2 h at 28 °C. The of JH2-2::I with the VanXBorf7 and VanXBorf7R primers culture was then shifted to a nonpermissive temperature (42 °C) and inserted between the BamHI and HindIII sites of pAT886 for 2 h, and integrants after a first recombination event were se- ΩvanX (pCR-Blunt B[H116A,D123A]) to generate pAT887(pCR- lected at 42 °C on M17 agar containing erythromycin (10 μg/mL). ΩvanX Blunt B[H116A,D123A]360 bp) (Table S1). Plasmid excision by a second recombination event was favored by The VB and VBR primers were used to amplify the 250-bp vanB′ vanB vanX subculturing at 28 °C in the absence of erythromycin, and plasmid fragment of the gene upstream from B (Fig. S1). The loss was screened for by plating at 42 °C on M17-glucose followed KpnI and XhoI sites of the primers allowed directional cloning of lucR vanX by replica plating on erythromycin. The gene under the this fragment upstream from B[H116A,D123A] in pAT887 to P ΩvanB′vanX control of the strong tet promoter was introduced in the integrase generate pAT888(pCR-Blunt B[H116A, D123A]360 bp) int (Table S1). gene of JH2-2::S, JH2-2::I, and JH2-2::C1, resulting in stabi- lization of the transposon (Fig. S1). Integration of lucR did not The 1,219-bp insert of pAT888 containing vanB′vanXB[H116A, alter the resistance phenotype nor the D,D-dipeptidase activitiy of D123A] and the 360 bp was digested with XhoI plus NotI and cloned in the same sites of pGhost9, leading to pAT889 (Fig. S1). the strains, and the bioluminescent derivatives displayed growth Construction of pAT891 and pAT895. Plasmid pAT891 was constructed rates similar to those of their nonbioluminescent counterparts first to further generate pAT895(pGhost9Ωint′PtetlucRint″)(Fig. (Table 1 in main text). S1 and Table S1). Pulsed-Field Gel Electrophoresis and Southern Hybridization. Geno- pAT891(pAT29ΩPtetlucR). The Ptet promoter of the tet(M) tetracy- cline resistance gene was amplified from BM4138 (JH2-2::Tn916, mic DNA from recipient JH2-2 and transconjugant JH2-2::I em- our laboratory collection) total DNA with primers Ptet1 and PtetR bedded in agarose plugs was digested overnight at 27 °C with 25 U (Table S2) containing, respectively, EcoRI and KpnI sites, and the of SmaI, and fragments were separated on a 0.8% agarose gel PCR product was cloned in the same sites of shuttle vector pAT29 using a CHEF-DRIII apparatus (Bio-Rad) as previously described (4), leading to plasmid pAT890(pAT29ΩPtet)(Table S1). The lucR (6). The DNA fragments were transferred to a nitrocellulose luciferase gene was amplified from plasmid pGEX-6P-2ΩlucR (5) membrane and hybridized to an [α-32P]-labeled vanB probe ob- with the LucR and LucR-rev primers containing KpnI and BamHI tained by amplification of an internal portion of the vanB gene with restriction sites, respectively. The PCR product was digested by primers VB7 and VB8 and JH2-2::I total DNA as a template. Foucault et al. www.pnas.org/cgi/content/short/1006855107 1of4 Measurement of VanXB D,D-Dipeptidase Activity. Activity of VanXB doglycan synthesis, and incubation was continued for 15 min to in the supernatant fraction (Fig. S2) was assayed by measuring allow accumulation of peptidoglycan precursors that were extracted the D-Ala released from substrate hydrolysis (D-Ala-D-Ala, 6.56 and analyzed by HPLC (Fig. S2) as previously described (7). mM) through coupled indicator reactions using D-amino acid oxidase and horseradish peroxidase (7). Bioluminescence Quantification. As already observed for E. coli (8), there was a linear correlation (R2 = 0.99) between the cfu counts and Analysis of Peptidoglycan Precursors. 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