Characterization, N-terminal sequencing and classification of Tolworthcin 524: A bacteriocin produced by Bacillus thuringiensis subsp. tolworthi

Abstract

Bacteriocins synthesized by entomopathogenic Bacillus thuringiensis are gaining attention owing to their inhibitory effects against a wide variety of pathogenic bacteria. In the present study, we purified and characterized Tolworthcin 524, a bacteriocin synthesized by B. thuringiensis subsp. tolworthi, and compared it with other bacteriocins synthesized by B. thuringiensis. Tolworthcin 524 was separated and purified from the secretome of B. thuringiensis by fast protein liquid chromatography with a gel filtration column to obtain yields of 17% and a specific activity of ∼3600U/mgprotein. The purified product showed two peptides of ∼9 and 6kDa with antimicrobial activity in a gel-screening assay. The purified product was analyzed by two-dimensional electrophoresis and the resolved peptides of ∼9 and 6kDa with isoelectric points of ∼8 were sequenced. Partial sequences (METPVVQPR and DWTCWSCLVCAACS) were obtained suggesting that the ∼9 and 6kDa correspond to the prebacteriocin and mature Tolworthcin 524, respectively. Sequences showed high identity with Thurincin H and Thuricin 17 and had a conserved motif with other bacteriocins of B. thuringiensis. Based on sequence data, Tolworthcin 524 was classified in subclass II.2 (Thuricin-like peptides) of the Bacillus bacteriocin classification scheme. The larger peptide did not harbor a sequence suggestive of a signal peptide neither did it contain the double-glycine (GG) motif characteristic of the secretion leader recognized by the ABC transport system. Implications of these properties in Tolworthcin 524 secretion are discussed.

Biomanufacturing process for the production of bacteriocins from Bacillaceae family

“…A maximum production of cerein 8A was achieved at fermentation temperature of 30 °C and between pH 6.5 and 9.0 (Dominguez et al 2007). Slightly different temperature has been reported for maximum yield of tolworthcin 524 which is at 28 °C, BLIS An6 at 30 °C and cerein 8A at temperature between 22 and 34 °C (Dominguez et al 2007;Pacheco-Cano et al 2014;Huang et al 2016;Ayed et al 2015). The production of the bacteriocin Bb obtained at 32 °C was higher as compared to that of elevated temperature in the range of 37-40 °C (Saleem et al 2009).…”

“…In this group, thuricin 17, tolworthcin 524 and thuricinlike peptide do not possess the glycine-glycine motif characteristic of the secretion leader peptides (8-27 amino acids) recognized by ABC transportation system (Pacheco-Cano et al 2014;Dominguez et al 2007). Tolworthcin 524 and other thuricin-like peptide share the preserved motif DWTXWSXL in amino acid sequences (Pacheco-Cano et al 2014). In particular, the N-terminal part of the pre-bacteriocin form of thuricin 17 is similar to those of the tolworthcin 524 partial sequences, MET-PVVQPR (Pacheco-Cano et al 2014).…”

“…Although some B. thuringiensis are excluded, a number of similar characterized bacteriocins are currently categorized under thuricin-like peptides. These include thricin S, thuricin HD2, thuricin 7, cerein MRX1, entomocin 9, thuricin 17, tochicin HD868, tolworthcin 524 and bacthuricin F4 (Cherif et al 2008;Pacheco-Cano et al 2014). In this group, thuricin 17, tolworthcin 524 and thuricinlike peptide do not possess the glycine-glycine motif characteristic of the secretion leader peptides (8-27 amino acids) recognized by ABC transportation system (Pacheco-Cano et al 2014;Dominguez et al 2007).…”

“…These include thricin S, thuricin HD2, thuricin 7, cerein MRX1, entomocin 9, thuricin 17, tochicin HD868, tolworthcin 524 and bacthuricin F4 (Cherif et al 2008;Pacheco-Cano et al 2014). In this group, thuricin 17, tolworthcin 524 and thuricinlike peptide do not possess the glycine-glycine motif characteristic of the secretion leader peptides (8-27 amino acids) recognized by ABC transportation system (Pacheco-Cano et al 2014;Dominguez et al 2007). Tolworthcin 524 and other thuricin-like peptide share the preserved motif DWTXWSXL in amino acid sequences (Pacheco-Cano et al 2014).…”

“…The effectiveness of thuricin CD against the clinical hypervirulent C. difficile isolates, makes it a good therapeutic agent to treat gastrointestinal problems associated with C. difficile (Rea et al 2010;Mathur et al 2017). The bacteriocins such as kenyacin 404, morricin 269, kurstacin 287, tolworthcin 524 and entomocin 420 from B. thuringiensis have also demonstrated positive effect against infectious pathogens in udder tissues as well as for gastrointestinal syndromes, emetic, throat infections, scarlet fever, septicaemia, pneumonia and urinary tract infections (Mathur et al 2017;Pacheco-Cano et al 2014;Salazar-Marroquín et al 2016).…”