Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9

Abstract

Aims: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers. Methods and Results: Bacillus thuringiensis ssp. entomocidus HD9 produced in the culture supernatant an antimicrobial activity against Gram-positive bacteria including Listeria monocytogenes, one of four pathogenic Pseudomonas aeruginosa and several fungi. Production of the antibacterial activity, named entomocin 9, started during mid-logarithmic growth reaching its maximum at the early stationary phase. Entomocin 9 retained more than 72% of activity after incubation for 20 min at 121°C. Activity was lost after proteinase K treatment, it was stable in a pH range between 3 and 9, and resistant to lyophilization. After partial purification with ammonium sulphate precipitation followed by gel-filtration and anion-exchange chromatography, an active protein of ca 12AE4 kDa was isolated. The mode of action of entomocin 9 was bactericidal and caused cell lysis of growing cells. Despite the presence of a range of virulence related genes, including haemolysin BL, nonhaemolytic enterotoxin, cytotoxin K and several hydrolytic activities, B. thuringiensis HD9 was not toxic against Vero cells. Conclusions: Entomocin 9 is a novel heat-stable, bacteriocin produced by B. thuringiensis HD9. The absence of toxicity against Vero cells suggests the suitability of strain HD9 for a safe application in antimicrobial treatments. Significance and Impact of the Study: New finding on entomocin 9 would make B. thuringiensis attractive in biotechnological applications as an antimicrobial agent in agriculture and food industry.

Purification and partial characterization of an antimicrobial peptide produced by Bacillus sp. strain P45, a bacterium from the Amazon basin fish Piaractus mesopotamicus

“…(Table 1). The inhibitory spectrum of this substance is rather similar to that of some BLS, such as lichenin (Pattnaik et al, 2001), entomocin 9 (Cherif et al, 2003) and BLS P40 .…”

Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile

“…Complete amino acid sequencing using infusion tandem mass spectrometry indicated that each peptide is posttranslationally modified at its respective 21st, 25th, and 28th residues. Solution NMR studies on [ 13 C, 15 N] Trn-α and [ 13 C, 15 N]Trn-β were used to characterize these modifications. Analysis of multidimensional NOESY data shows that specific cysteines are linked to the α-carbons of the modified residues, forming three sulfur to α-carbon bridges.…”

“…lactis that is used commercially in food preservation, has a broad spectrum of activity against other lactic acid bacteria (LAB) and most Gram-positive organisms (8). Although bacteriocins produced by LAB are the most widely investigated (9, 10), bacteriocins are also produced by most other Gram-positive (11)(12)(13)(14)(15) and Gram-negative (16,17) bacterial species.…”

Identification of Three Novel <i>B. thuringiensis</i> Strains that Produce the Thuricin S Bacteriocin

“…In this work, bacteriocins produced by three Bt strains (HD9, HD110 and HD125) were studied. Entomocin 9, an anionic bacteriocin produced by strain HD9 and entomocin 110, a bacteriocin produced by strain HD110 were previously described (Cherif et al, 2003;Cherif et al, 2008) but none of them has been purified to homogeneity and fully characterized at the biochemical level.…”

“…The main activity of each family is within the group of species producing it (Reeves, 1965). In the past few years, bacteriocin production was reported for many B. thuringiensis (Bt) strains: thuricin from strain HD2 (Favret and Yousten, 1989); tochicin from strain HD868 (Paik et al, 1997); thuricin 7 from strain BMG1.7 (Cherif et al, 2001); thuricin 439A and thuricin 439B from strain B439 (Ahern et al, 2003); entomocin 9 from strain HD9 (Cherif et al, 2003); bacthuricin F4 from strain BUPM4 (Kamoun et al, 2005), thuricin 17 from strain NEB17 (Gray et al, 2006); thuricin S from strain HD198 (Chehimi et al, 2007;Chehimi et al, 2010); entomocin 110 from strain HD110 (Cherif et al, 2008) and thuricin CD from strain DPC 6431 (Rea et al, 2010). Among these bacteriocins, three were reported to share the conserved N-terminal amino acid sequence DWTXWSXL (Kamoun et al, 2005;Gray et al, 2006;Chehimi et al, 2007).…”