Purification, amino acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus thuringiensis

Abstract

Aims: Purification and characterization of a new bacteriocin, Bacthuricin F4 of Bacillus thuringiensis. Methods and Results: A newly isolated B. thuringiensis subsp. kurstaki strain BUPM4, was shown to produce a novel bacteriocin named Bacthuricin F4. The highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. Bacthuricin F4 could be purified by a two-step procedure: ammonium sulphate precipitation of protein from culture supernatant followed by a reverse phase chromatography. Upon purification, the specific activity was increased 100-fold. This bacteriocin was heat-stable up to 70°C and resisted up to pH 3AE0. Bacthuricin F4 was sensitive to proteases demonstrating its proteinaceous nature. Its molecular mass, determined by mass spectrometry was 3160AE05 Da. Direct N-terminal sequencing of Bacthuricin F4 revealed the following sequence: DWTXWSXL. The latter was unique in the databases. Bacthuricin F4 was active against Bacillus species while it had little or no effect on Gram-negative bacteria. Conclusions: A strain BUPM4 of B. thuringiensis subsp. kurstaki, was shown to produce a new bacteriocin named Bacthuricin F4 of both new molecular mass (3160AE05 Da) and new amino acid terminal sequence. This is, to our knowledge, the first bacteriocin exhibiting such characteristics reported to be produced by B. thuringiensis. Significance and Impact of the Study: The bacteriocin produced by the B. thuringiensis strain BUPM4 respond to both criteria of thermostability and stability to low pHs. Thus, it could be used for the control of the related species of Bacillus harmful for agricultural products.

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

“…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). The production of which more than one bacteriocin by the same strain has already been described for many Bacillus strains (Oscáriz et al, 1999;Ahern et al, 2003;Wu et al, 2005).…”

Isolation of the Bacillus thuringiensis plasmid carrying Bacthuricin F4 coding genes and evidence of its conjugative transfer

“…This bacterium produces many virulent factors such as chitinase [6,7] vegetative insecticidal proteins [8], and bacteriocins [9,10]. Bacteriocins are extracellularly released peptides or proteins affecting growth of closely related bacteria and several pathogenic species [9]. Genetic determinants responsible for bacteriocin activity are usually grouped on one or two operons encoding structural proteins involved in its synthesis and secretion as well as immunity proteins protecting the producer from its own bacteriocin [11].…”

“…Bacteriocin genes could be located on plasmids such as bacteriocin 32 produced by Enterococcus strains [12] on chromosomes such as mesarcidin [13], or on conjugative transposons, as in the case of lacticin 481 [14]. B. thuringiensis strain BUPM4 is known for its ability to produce a bacteriocin, called Bacthuricin F4 (BF4), which could be potentially used for the control of harmful related Bacillus species in agricultural products [9]. The search for genes impaired in BF4 synthesis has been carried out in our laboratory via the insertional transposon mutagenesis approach, which is based on the capability of the mini-Tn10 to insert into different sites in B. thuringiensis chromosomes or plasmids [9].…”

“…B. thuringiensis is the most known insecticidal bacterium worldwide for the control of various plant pests. This bacterium produces many virulent factors such as chitinase [6,7] vegetative insecticidal proteins [8], and bacteriocins [9,10]. Bacteriocins are extracellularly released peptides or proteins affecting growth of closely related bacteria and several pathogenic species [9].…”

“…B. thuringiensis strain BUPM4 is known for its ability to produce a bacteriocin, called Bacthuricin F4 (BF4), which could be potentially used for the control of harmful related Bacillus species in agricultural products [9]. The search for genes impaired in BF4 synthesis has been carried out in our laboratory via the insertional transposon mutagenesis approach, which is based on the capability of the mini-Tn10 to insert into different sites in B. thuringiensis chromosomes or plasmids [9]. A non-bacteriocin producing mutant (MB1) was obtained by insertion mutagenesis, in which the mini-Tn10 transposon had targeted a plasmidic gene involved in BF4 synthesis (ORF1) (accession number FJ499396) [10].…”