Extensive bacteriocin gene shuffling in the Streptococcus bovis/Streptococcus equinus complex reveals gallocin D with activity against vancomycin resistant enterococci

Abstract

Streptococcus gallolyticus LL009 produces gallocin D, a narrow spectrum two component bacteriocin with potent activity against vancomycin-resistant enterococci. Gallocin D is distinct from gallocin A, a separate two component bacteriocin produced by S. gallolyticus. Although the gene clusters encoding gallocin A and gallocin D have a high degree of gene synteny, the structural genes are highly variable and appear to have undergone gene shuffling with other streptococcal species. Gallocin D was analysed in laboratory-based experiments. The mature peptides are 3,343 ± 1 Da and 3,019 ± 1 Da and could be readily synthesized and display activity against a vancomycin resistant Enterococcus strain EC300 with a MIC value of 1.56 µM. Importantly, these bacteriocins could contribute to the ability of S. gallolyticus to colonize the colon where they have been associated with colorectal cancer.

Figure 1

Detection of the 3,021.29 Da gallocin D2 peptide by MALDI-TOF MS from colonies on a plate. Inset shows the antimicrobial activity of S. gallolyticus LL009 against L. bulgaricus LMG6901.

Figure 2

Organisation of the predicted operon encoding the bacteriocin, aligned to the operon of (A) type strain S. gallolyticus DSM 16,831, with below alignment of structural genes; gllA2 and gllD2 show 42% identity, gllA1 and gllD1 share 22% identity. (B) S. infantarius LP90 infantaricin ABCDEFG bacteriocin cluster. Genes in line with the same background colour are homologous and the amino acid percent identity is indicated.

Figure 3

Comparison of the operons of all strains analysed. Genes in line with the same background colour are homologous (> X% amino acid sequence identity indicated), while the white background represents genes for which no homologs have been found between strains; box 1 shows the immunity protein and infantaricin A in S. infantarius strains and immunity protein and the gallocin D variant in S. gallolyticus LL009; box 2 shows potential homologs for gallocin A.

Figure 4

(A) Mixing gallocin D and infantaricin A peptides indicates that any combination of the alpha and beta peptides results in activity, concentration in µM across the top. ‘Rugby ball’ shape seen between wells indicates where the peptides meet following diffusion into the media, showing complementary activity. (B) Alignment of sequences of the prepeptides from both species, the double glycine cleavage point is underlined.

Figure 5

(A) Sgg LL009 colonies showing inhibition of Sgg DPC6501 (indicator organism). (B) Well diffusion assay using synthesized gallocin D against Sgg DPC6501, concentration in µM indicated above each well.

Figure 6

Kill curve of VRE EC300 using 15.6 µM gallocin D. EC300 + samples contain bacteriocin (each representing a biological triplicate), EC300 has no bacteriocin (control).