Bacterial strains and plasmids used in this study.

<p>Ap^r: ampicillin resistance.</p><p>Cm^r: chloramphenicol resistance.</p

Induction of BlaPβ-lactamase of the BlaR1 L3 loop mutants.

*<p>The induction factor corresponds to the ratio between the β-lactamase quantity/A⁶⁰⁰ for induced culture and the β-lactamase quantity/A⁶⁰⁰ for uninduced culture.</p

Coomassie Blue-stained SDS-PAGE of partially purified inclusion bodies of wild-type and L3 loop mutants (A) and Zinc blot analysis (B).

<p>In A and B: 1: bovine carbonic anhydrase (AC) (∼30 kDa; 5 µg), was used as positive control and 2 to 6 are, respectively, wild type (WT), E²¹³A, H²¹²A/H²¹⁶A, D²⁵⁷ A, D²²¹ A and E²⁵³ A L3 loop mutants. For each gel 40 µg of inclusion bodies were loaded. The fluorographies were exposed at -70°C for 72 hours. M: molecular weight marker. The arrow indicates L3 loops.</p

Western blot analysis of membrane-bound BlaR1 produced by <i>B. subtilis</i> strains carrying plasmids harboring <i>blaR</i>1 mutants.

<p>Membrane proteins from induced (+) or uninduced (-) cultures were separatedon on SDS-PAGE. Purified BlaR-CTD antibodies were used for the Western blotting. Pre-stained protein molecular weight markers were used (M). The pinpoints an intense and non-specific band (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036400#pone-0036400-g006" target="_blank">Figure 6</a>). BlaR1 and BlaR1* point out, respectively, the full size and the activated <i>B. licheniformis</i> BlaR1 receptor. Except for mutant E²⁷⁴A, all other mutants exhibit non-inducible β-lactamase phenotype. The E²⁷⁴A mutant has the same profile as the wild-type (to compare see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036400#pone-0036400-g006" target="_blank">Figure 6</a>). For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036400#pone-0036400-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036400#pone-0036400-t002" target="_blank">2</a> and Experimental procedures.</p

Oligonucleotides used in this study.

<p>Modified codons are underlined and mutagenised bases are highlighted.</p

Sequence alignment of the amino-terminal domain of the proteins <i>B. licheniformis</i> 749/I BlaR1 (BlBlaR-NTD) and <i>S. aureus</i> RN4220 BlaR1 (SaBlaR-NTD) and <i>S. aureus</i> MRSA252 MecR (SaMecR-NTD).

<p>The conserved residues are highlighted in red and the loop and transmembrane segments are marked: the transmembrane segments (TM1 to 4) in orange; the extracellular loops B1 (M¹-P⁸) and L2 (P⁵³-S¹¹⁵) in green and the cytoplasmic loops L1 (K²⁷-T³⁵) and L3 (Y¹³⁴-K³²²) in blue. The site of cleavage, situated between the R²⁹³ and R²⁹⁴ in <i>S. aureus</i> BlaR1, is indicated by an arrow.</p

Western blot analysis of membranes of <i>B. subtilis</i> transformed with pMK4 (negative control), pDML995 (wild type), pDML1269 (E²¹³A mutant) or pDML3045 (R³⁰⁴A/R³⁰⁵A mutant).

<p>Membrane proteins from induced (+) or uninduced (−) cultures were separatedon on SDS-PAGE. Purified BlaR-CTD antibodies were used for the Western blotting. Pre-stained protein molecular weight markers were used (M). The indicates an intense and non-specific band. BlaR1 and BlaR1* highlight, respectively, the full size and the activated <i>B. licheniformis</i> BlaR1 receptor.</p

Induction of β-lactamase synthesis in presence (induced) or not (non-induced) of inducer (2.5 µg ml⁻¹ cephalosporin C) for the <i>B. subtilis</i> strains transformed with pDML995 (wild-type divergeon; •) or pDML3045 (mutation R³⁰⁴A/R³⁰⁵A;○).

<p>The level of the induction is expressed as an induction factor (IF, the ratio between the β-lactamase quantity/A⁶⁰⁰ for induced culture and the β-lactamase quantity/A⁶⁰⁰ for uninduced culture).</p

MALDI-TOF MS and LC-MS analysis of amylolysin.

<p>Mass spectrum of purified amylolysin sample was recorded in negative mode. Insert: LC-MS chromatograms of commercial lanthionine standard and amylolysin hydrolysate. Intensity (%, Y-scale) was recorded by setting the SQD mass analyzer on the specific mass of lanthionine (315 Da). </p

Additional file 1: of Enterococcus hirae LcpA (Psr), a new peptidoglycan-binding protein localized at the division site

Supplemental data. (DOCX 226 kb