Structure Elucidation and Biosynthetic Enzyme Characterization of Bacteriocins

• Author / Creator

  Acedo, Jeella Z

• Acidocin B (AcdB), a bacteriocin from Lactobacillus acidophilus M46 that was initially reported to be a linear peptide, was purified and shown to be circular based on MALDI-TOF MS and MS/MS sequencing. MS analysis further revealed that AcdB is comprised of 58 amino acid residues, instead of 59 residues as initially reported. The NMR solution structure of AcdB in sodium dodecyl sulfate micelles was solved, revealing that AcdB consists of four α-helices that are folded to form a globular bundle with a central pore. This is the first reported three-dimensional structure (3D) for a subgroup II circular bacteriocin. Comparison of the structure of AcdB to that of carnocyclin A, a subgroup I circular bacteriocin, highlighted the differences between the two subgroups. At least seven putative subgroup II circular bacteriocins were identified using BLAST, and sequence analysis revealed a highly conserved asparagine residue at the leader peptide cleavage sites, suggesting that an asparagine endopeptidase might be involved in their biosynthesis. Lastly, the biosynthetic gene cluster of AcdB was sequenced and characterized. Lacticin Q (LnqQ) and aureocin A53 (AucA) are leaderless bacteriocins (class IIc) from Lactococcus lactis QU 5 and Staphylococcus aureus A53, respectively. Their 3D NMR solution structures were determined, revealing that both peptides are composed of four α-helices that assume a saposin-like fold with a highly cationic surface and a hydrophobic core. The observed structural motif is remarkably similar to the overall fold of the two-component leaderless bacteriocins, enterocin 7A and 7B. Homology modeling showed that the aforementioned motif may be shared among broad-spectrum leaderless bacteriocins despite the variations in their sequence identities and lengths. The structures of LnqQ and AucA were also demonstrated to exhibit certain similarities to those of the circular bacteriocins. Activity assays showed that the two peptides, LnqQ and AucA, combined do not act synergistically and have different antimicrobial spectra and potency, suggesting that sequence disparities play a vital role in their modes of action. Carnobacteriocin X (CbnX) was originally reported as a single-peptide bacteriocin (class IId). However, sequence analysis and synergy assays revealed that CbnX belongs to a two-peptide bacteriocin (class IIb), with CbnY as its partner. CbnXY is the first two-peptide bacteriocin reported in carnobacteria. CbnX and CbnY are inactive individually, but elicit synergistic activity against closely related strains when combined. The NMR solution structures of CbnX and CbnY were elucidated and shown to strongly resemble the structures of other class IIb bacteriocins (i.e. LcnG, PlnEF, PlnJK). CbnX consists of an extended, amphipathic α-helix and a flexible C-terminus. CbnY has two α-helices (one hydrophobic, one amphipathic) connected by a short loop, and a cationic C-terminus. Binding studies showed that CbnX and CbnY do not interact directly, suggesting that a membrane-bound receptor may be required to mediate the formation of the CbnXY complex. Pneumococcin is a two-component lantibiotic, comprised of PneA1 and PneA2, from Streptococcus pneumoniae R6. Its biosynthetic machinery encodes a putative flavin-dependent reductase, named PneJB, which is likely involved in the formation of D-Ala and D-Abu. The activity of PneJB was investigated through the heterologous expression of pneumococcin biosynthetic proteins in Escherichia coli. Coexpression of the precursor peptides (PneA1 and PneA2) and the lantibiotic synthetase (PneM) with and without PneJB produced a mixture of partially modified substrates that could not be separated by RP-HPLC. To potentially address this issue, truncated precursor peptides were designed and cloned. The truncated peptides, however, could not be successfully expressed in E. coli. Hence, chemical synthesis of substrate analogues is currently being pursued. Other approaches to obtain the precursor peptides are presented herein. The PneJB enzyme was expressed and purified as a SUMO fusion protein, wherein the SUMO tag could be readily cleaved. The substrate analogues and the PneJB enzyme will consequently be used for in vitro PneJB activity assays and future crystallization trials.

• Subjects / Keywords

  • Antimicrobial
  • Bacteriocins
  • Peptides

• Graduation date

  Spring 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3833ND4P

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.