Identification of a novel botulinum neurotoxin gene cluster in Enterococcus

Abstract

The deadly neurotoxins of Clostridium botulinum (BoNTs) comprise eight serotypes (A-G; X). The neurotoxin gene cluster encoding BoNT and its accessory proteins includes an operon containing an ntnh gene upstream of the boNT gene. Another operon contains either ha (haemagglutinin) or orfX genes (of unknown function). Here we describe a novel boNT gene cluster from Enterococcus sp. 3G1_DIV0629, with a typical ntnh gene and an uncommon orfX arrangement. The neurotoxin (designated putative eBoNT/J) contains a metallopeptidase zinc-binding site, a translocation domain and a target cell attachment domain. Structural properties of the latter suggest a novel targeting mechanism with consequent implications for application by the pharmaceutical industry. This is the first complete boNT gene cluster identified in a non-clostridial genome.

Keywords: Clostridium; Enterococcus; botulinum neurotoxin; eBoNT/J.

Figure 1

Neurotoxin gene clusters of (A) Enterococcus sp. 3G1_DIV0629; (B1) C. botulinum Group I ha types A, B; C. botulinum Group II type B; C. botulinum Group III (*Group III neurotoxin gene clusters position the gene for transcription factor botR upstream of ha70); C. botulinum Group IV (the type G neurotoxin gene cluster swaps the positions of ha70 and ha34). (B2) Group I orf‐X types A, F († Group I neurotoxin gene clusters include botR at this position); Group II types E, F. (B3) boNT/X. (B4) Weissella oryzae SG25. Purple regions in ntnh‐like refer to Big 3 domains that are not present in the ntnh‐like gene. Note that the putative Weisella neurotoxin gene is not associated with ntnh, ha or orfx accessory genes.

Figure 2

(A,B) SplitsTree plots of BoNT and NTNH protein sequences respectively. TeNT protein is not associated with an NTNH accessory protein. Both plots show that BoNT/X and eBoNT/J and their associated NTNH are more closely related to each other than they are to all other proteins. (C,D). SimPlot analyses of BoNT and NTNH protein domain sequences, respectively, using eBoNT/J and its NTNH as seeds; main functional domains of each eBoNT/J cluster protein are outlined above. The closer relatedness of BoNT/X to eBoNT/J continues throughout its length; however, the NTNH associated with eBoNT/J is almost equally distant from all other examples.

Figure 3

Structure prediction and comparison of putative eBoNT/J toxin and its associated NTNH to BoNT/A and BoNT/A complexed with its NTNH, respectively.(A) Superposition of the predicted structure of putative eBoNT/J (gold) with the crystal structure of an inactive BoNT/A (blue Protein Data bank ID: 3V0C). Positions of the zinc‐binding site (HELCH) and target cell‐binding motif (SAWY) are indicated by green ovals. (B) Superposition of the predicted structure of the eBoNT/J NTNH (gold) with that determined for BoNT/A (red) complexed with its own NTNH (blue; Protein Data bank ID: 3V0B).

Figure 4

Phylogenetic tree of Enterococcus, showing close relatedness of Enterococcus sp 3G1_DIV0629 to E. faecium T110.