Article Figures & Data
Figures
- FIG 1
(A) Sequence alignment of subgroup II circular bacteriocins using Clustal W (36). Conserved, conservative, and semiconservative substitutions are indicated by asterisks, colons, and semicolons, respectively. The previously proposed N-terminal amino acid for acidocin B is in brackets (22). (B) Schematic representation of the acidocin B gene cluster. The solid arrow indicates the structural bacteriocin gene aciA; the hatched arrow indicates aciT, encoding an ATP-binding protein; open arrows indicate aciB, aciC, aciD, aciI, and aciE, encoding proteins containing putative transmembrane domains. (C) Acidocin B precursor peptide. The cleavage site during maturation is indicated.
- FIG 2
MALDI-TOF mass spectrum of acidocin B showing singly and doubly charged species consistent with an average molecular mass of 5,621.5 Da.
- FIG 3
CD profile of acidocin B in SDS (solid line) and DPC (dashed line) micelles. The peptide exhibited similar α-helical contents in both micelles.
- FIG 4
NMR solution structure of acidocin B (PDB code 2MWR): helix 1 is in blue, helix 2 is in purple, helix 3 is in cyan, and helix 4 is in orange. The arrow indicates the linkage of the N and C termini.
- FIG 5
(A) Solution structure of acidocin B showing the amphipathicity of the helices. Hydrophobic residues are in green, while hydrophilic residues are in white. The arrow indicates the linkage of the N and C termini. (B) Hydrophobic surface map as generated from PyMOL (34). (C) Electrostatic potential surface map calculated using the APBS functionality of the PDB2PQR (version 1.8) online pipeline (35). Cationic regions are shown in red, while anionic regions are in blue.
- FIG 6
Predicted structures of gassericin A and butyrivibriocin AR10 derived from homology modeling (SWISS-MODEL) (37) using the structure of acidocin B as the template. Basic residues are shown in red, and acidic residues are in blue. The arrow indicates the N- to C-terminal linkage.
- FIG 7
Multiple-sequence alignment of known and putative (indicated by source organisms) subgroup II circular bacteriocin precursors using Clustal W (36). The known circular bacteriocins are acidocin B, gassericin A, and butyrivibriocin AR10. The UniProt accession numbers are in parentheses. The region corresponding to leader peptide sequences is underlined, and the highly conserved asparaginyl cleavage site is marked by an asterisk. Conserved residues (similarity threshold of 80%) are highlighted in black.
Tables
- TABLE 1
Characteristics of predicted proteins encoded by the acidocin B gene clustera
Protein Size (aa) TM Function No. of identical amino acids/total (% identity) relative to gassericin A gene cluster homologs AciB 174 5 Unknown 173/174 (99.4) AciC 60 2 Unknown 60/60 (100) AciA 91 2 Acidocin B precursor 88/91 (96.7) AciD 162 4 Unknown, DUF95 family 160/162 (98.8) AciI 53 1 Immunity 53/53 (100) AciT 226 0 ATP-binding protein 225/226 (99.6) AciE 212 6 Membrane transporter 209/212 (98.6) ↵a aa, amino acids; TM, number of putative transmembrane domains.
- TABLE 2
MS/MS sequences of fragments of acidocin B after trypsin-chymotrypsin digestion
Fragment Linear sequence Observed m/z Charge Calculated m/z 1 --WIADQF 779.388 1 779.365 2 ---IADQFGIHL 507.276 2 1,013.534 3 ---IADQFGIHLATGT 672.360 2 1,343.688 4 ------------------KLLDAVASGASLGTAF 760.929 2 1,520.825 5 -------------------LLDAVASGASLGTAF 696.886 2 1,392.730 6 -----------------------------------AILGV 472.290 1 472.306 7 --------------------------------------GVTLPAW 743.447 1 743.401 8 ---------------------------------------------ALAAAGAL 657.446 1 657.386 9 IY---------------------------------------------AAAGALGATAA 1,120.619 1 1,120.592 Complete sequencea IYWIADQFGIHLATGTARKLLDAVASGASLGTAFAAILGVTLPAWALAAAGALGATAA ↵a Ala-17, Arg-18, and Ala-35 were inferred from the deduced amino acid sequence from DNA sequence analysis.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Chemical shift assignments for acidocin B (Table S1), structure calculation statistics for acidocin B (Table S2), RP-HPLC trace (Fig. S1), superimposition of the 20 lowest energy conformers calculated for acidocin B (Fig. S2), hydrophobic surface maps (Fig. S3), and molecular phylogenetic analysis (Fig. S4).
PDF, 1.7M
- Supplemental file 1 -
