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

Characterization of the RFB137 mutant.

<p>The RP-HPLC chromatogram of concentrated supernatant of GA1 is in red and that of the <i>ΔamlA</i> RFB137 mutant is in black. Insert: Antimicrobial activity of concentrated supernatant (10 µl) from GA1 and RFB137 strains on LB agar plates seeded with <i>M. luteus</i> ATCC 9341.</p

Interation with lipid

<p>II. (A) Direct interaction of amylolysin with lipid II. Both compounds were incubated in DMSO/1-octanol mixture (60/40 v/v) for 1 hour at room temperature before being subjected to TCL using a butanol/acetic acid/pyridine/water mixture (15/3/12/10, v/v/v/v) as mobile phase. (B) Inhibition of the transglycosylation reaction. Amylolysin, lipid II and glycosyltransferase were incubated for one hour before being subjected to TLC using a mixture of methanol/chloroform/ammoniac/water (88/48/10/1, v/v/v/v) as mobile phase. [¹⁴C] lipid II was detected with a Molecular Imager FX system. Direction of solvent migration during TLC is indicated by the arrow.</p

Characterization of the amylolysin gene cluster.

<p>(<b>A</b>). The gene coding for the amylolysin prepeptide (amlA) is colored in grey. The other genes are marked with the following patterns: immunity (amlEF) in blue, regulation (amlKR) in yellow, modification (amlM) in green and transport in orange. Stripes arrow corresponds to the Kan construct used for <i>amlA</i> disruption. Putative promoters and terminators are indicated by vertical arrows and red symbol, respectively. (<b>B</b>) Primary structure of <i>amlA</i> gene. Predicted -10box and -35box are in red, ribosome binding site (RBS) is in blue and predicted terminator of <i>amlKR</i> cluster is in green. The four-glycine stretch is underlined and the highly conserved motif CTLTXEC is in bold blue. (C) Amino acid alignment of AmlA with LanA1 peptides of the two-peptide lantibiotics haloduracin (HalA1, DAB04173) and lacticin 3147 (LtnA1, O87236). Conserved amino acids are in bold blue; those forming the CTLTXEC motif are in bold and underlined. The thioether bridging patterns represents that of HalA1.</p

Structure model surface representations of (a) the <i>H. jecorina</i> Endo T structure, colored in green and (b) <i>Streptomyces plicatus</i> Endo F1 (PDB ID 2EBN), colored in blue.

<p>The extended β1α1, β6α6 and β7α7 loops of <i>H. jecorina</i> EndoT are colored in gold. The C-terminal peptide in Endo F1 is colored in red. The octasaccharide found bound in the ligand complex structure of <i>E. meningoseptica</i> Endo F3 (PDB ID 1EOM) has been modeled in the active site of Endo T. Figure prepared with the program PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040854#pone.0040854-Brnger1" target="_blank">[52]</a>.</p

Structure based sequence alignment of the four GH family 18 proteins that possess endo-N-acetyl-β-d-glucosaminidase type activity, and with known three-dimensional structure.

<p>The important active site residues are highlighted with a green background. The secondary structure assignment (boxes), indicated on top of the sequence alignment, is rainbow colored according to the residue number, starting with blue at the N-terminus and ending with red at the C-terminus. The shown aligned sequences are (from the top); <i>Elizabethkingia meningoseptica</i> Endo F3 (PDB ID 1EOM, Uniprot access code P36913), <i>Hypocrea jecorina</i> EndoT (PDB ID 4AC1, Uniprot access code C4RA89); <i>Streptomyces plicatus</i> Endo H (PDB ID 1EDT, Uniprot access code P11797.1); <i>Elizabethkingia meningoseptica</i> Endo F1 (PDB ID 2EBN, Uniprot access code P36911.1), and <i>Bacteroides thetaiotaomicron</i> Endo BT (PDB ID 3POH, Uniprot access code Q8A0N4). The glycosylated Asn in the sequons of <i>H. jecorina</i> EndoT are shaded grey. Yellow shaded amino acids are the C-terminal residues observed in the respective crystal structures.</p

Phylogenetic tree of GH family 18 ENGases, group B.

<p>The phylogenetic tree is based on an amino acid sequence alignment (CLUSTALX) and was constructed by neighbour joining. Bootstrap values are based on 1000 replications and nodes that have bootstrap support above 70% are indicated with the percentage. The tree is rooted with fungal GH family 18 chitinases belonging to the same subgroup. Previously characterized ENGases are indicated with an asterisk*. Boxes indicate proteins belonging to micro-organisms of the same order as described in the text.</p

Cartoon representation overlay of the superimposed structures of; (a) <i>H. jecorina</i> Endo T, colored in green, and <i>S. plicatus</i> Endo H (PDB ID 1EDT), colored in blue.

<p>The hairpin loop of Endo T, colored in gold, is shorter than the corresponding loop of Endo H, colored in red; (b) <i>H. jecorina</i> Endo T, colored in green, and <i>E. meningoseptica</i> Endo F1 (PDB ID 2EBN), colored in blue. The hairpin loop of Endo T, colored in gold, is shorter than the corresponding loop of Endo F1, colored in red. The loop at the C-terminal of Endo F1 is also colored in red; (c) <i>H. jecorina</i> Endo T, colored in green, and <i>E. meningoseptica</i> Endo F3 (PDB ID 1EOM), colored in red. The hairpin loop is completely missing in the structure of <i>E. meningoseptica</i> Endo F3; (d) <i>H. jecorina</i> Endo T, colored in green, and <i>B. thetaiotaomicron</i> Endo BT (PDB ID 3POH). Figure prepared with the program PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040854#pone.0040854-Brnger1" target="_blank">[52]</a>.</p

Data collection and processing statistics.

a<p>Beamline at MAX-lab, Lund, Sweden.</p>b<p>Numbers in parentheses are for the highest resolution bins.</p>c<p>R_merge = Σ_hkl Σ_i|I – < I >|/Σ_hkl Σ_i | I |.</p

Electron density of the zinc atom bound in the active site of <i>H. jecorina</i> Endo T structure.

<p>The zinc atom, modeled in dual conformation, is shown in grey spheres and the surrounding water molecules that are involved in the coordination spheres of zinc are shown in red spheres. Two water molecules coordinating the zinc atom in the active site have also been modeled in dual confirmation. The displayed maximum likelihood/_σA weighted 2F_obs−F_calc electron density map, contoured at 1.0 σ level (0.38 e/ų), is shown in greyish-blue. Figure prepared with the program PyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040854#pone.0040854-Brnger1" target="_blank">[52]</a>.</p