Sensitivity to the two peptide bacteriocin plantaricin EF is dependent on CorC, a membrane-bound, magnesium/cobalt efflux protein

Abstract

Lactic acid bacteria produce a variety of antimicrobial peptides known as bacteriocins. Most bacteriocins are understood to kill sensitive bacteria through receptor-mediated disruptions. Here, we report on the identification of the Lactobacillus plantarum plantaricin EF (PlnEF) receptor. Spontaneous PlnEF-resistant mutants of the PlnEF-indicator strain L. plantarum NCIMB 700965 (LP965) were isolated and confirmed to maintain cellular ATP levels in the presence of PlnEF. Genome comparisons resulted in the identification of a single mutated gene annotated as the membrane-bound, magnesium/cobalt efflux protein CorC. All isolates contained a valine (V) at position 334 instead of a glycine (G) in a cysteine-β-synthase domain at the C-terminal region of CorC. In silico template-based modeling of this domain indicated that the mutation resides in a loop between two β-strands. The relationship between PlnEF, CorC, and metal homeostasis was supported by the finding that PlnEF-resistance was lost when PlnEF was applied together with high concentrations of Mg²⁺ , Co²⁺ , Zn²⁺ , or Cu²⁺ . Lastly, PlnEF sensitivity was increased upon heterologous expression of LP965 corC but not the G334V CorC mutant in the PlnEF-resistant strain Lactobacillus casei BL23. These results show that PlnEF kills sensitive bacteria by targeting CorC.

Keywords: Lactobacillus; antimicrobial resistance; bacteriocins; lactic acid bacteria; probiotics.

Figure 1

Spontaneous plantaricin EF (PlnEF) resistant mutants of LP965. Lactobacillus plantarum NCIMB 700965 (WT) and PlnEF‐resistant isolates were grown in MRS in the presence of 25 nM PlnEF or 1,000 nM PlnA. Red circles indicate LP965 growth in MRS without PlnEF. The avg ± SD of n = 3 replicates is shown

Figure 2

Effects of plantaricin EF (PlnEF) on cellular concentrations of ATP. Cells of LP965 and the PlnEF‐resistant isolate EF.A were initially energized with 10 mM glucose (arrow at 1 min). At 10 min (second arrow), 25 nM of PlnEF, 25 μM nisin, or water (NT) was added to each culture. The avg ± SD of n = 3 replicates is shown

Figure 3

Structural model of the LP965 CorC protein. (a) Top‐down view of proposed homodimer formed by tandem CBS domains. (b) Top‐down and (c) side‐views of (monomer) LP965 CorC protein region from Y216 to G351 shown in ribbon representation and colored by a rainbow scheme from N‐terminus region (blue) to C‐terminus region (red). All sidechains are shown in stick representation, except for G334, which is shown in space‐filling representation. Template‐based modeling was accomplished using PDB: 3OCO

Figure 4

Additive effects of plantaricin EF (PlnEF) and metals on Lactobacillus plantarum growth. WT LP965 and the EF‐resistant strain EF.A were incubated in MRS supplemented with 25 nM PlnEF or MRS with 25 nM PlnEF and the indicated metal salt. The avg ± SD of n = 3 replicates is shown

Figure 5

Heterologous expression of LP956 and EF.A CorC in L. casei BL23. L. casei was grown in MRS with 50 nM of plantaricin EF (PlnEF). pJIMDH1 contains corC from WT and pJIMDH2 contains CorC from strain EF.A. Red circles indicate BL23 growth in MRS without PlnEF. The avg ± SD of n = 3 replicates is shown

Figure A1

Effects of plantaricin EF (PlnEF) on intracellular concentrations of ATP. Cells of WT and PlnEF‐resistant isolates were initially energized with 10 mM glucose (arrow at 1 min). At 10 min (second arrow), 25 nM of PlnEF, 25 µM nisin, or water (NT) was added to each culture. The avg ± SD of n = 3 replicates is shown

Figure A2

Effects of plantaricin EF (PlnEF) on extracellular concentrations of ATP. WT and PlnEF‐resistant isolates of LP965 were initially energized with 10 mM glucose (arrow at 1 min). At 10 min (second arrow), 25 nM of PlnEF, 25 µM nisin, or water (NT) was added to each culture. The avg ± SD of n = 3 replicates is shown

Figure A3

LP965 CorC domain annotation. LP965 CorC amino acid sequnce was submitted to the TMHMM server (top) and the InterPro domain annotation server (bottom). The alphanumeric strings in purple are the InterPro domain identifiers. The alphanumeric strings in black are the domain identifier links to other databases

Figure A4

Alignment of CorC amino acid sequences. Amino acid sequences from LP965 and Oenococcus oeni were aligned using MUSCLE. Due to gaps in the alignment, the mutated glycine residue in LP965 (334) in this figure is in position 336

Figure A5

WT and EF.A metal sensitivity. WT and EF.A LP965 were grown in MRS or MRS supplemented with the indicated growth‐inhibiting concentration of metal salt