Research Report

Identification of Three Novel B. thuringiensis Strains that Produce the Thuricin S Bacteriocin  

Sonia Chehimi1 , Ferid Limam2 , Isabelle Lanneluc1 , Franeois Delalande3 , Alain van Dorsselaer3 , Sophie Sable1
1. Littoral environnement et societes (LIENSs), Universite de La Rochelle, 17042 La Rochelle CEDEX 1, France
2. Laboratoire des substances bioactives, Centre de Biotechnologie de Borj-Cedria, BP 901-2050 Hammam-lif, Tunisia
3. Laboratoire de spectrometrie de masse bio-organique, Universite Louis Pasteur, 67087 Strasbourg CEDEX 2, France
Author    Correspondence author
Bt Research, 2012, Vol. 3, No. 2   doi: 10.5376/bt.2012.03.0002
Received: 10 Jan., 2012    Accepted: 02 Feb., 2012    Published: 20 Feb., 2012
© 2012 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Chehimi et al., 2012, Identification of Three Novel B. thuringiensis Strains that Produce the Thuricin S bacteriocin, Bt Research, Vol.3, No.2 3-10 (doi: 10.5376/bt.2012.03.0002)

Abstract

In the present work, three antibacterial substances produced by three Bacillus thuringiensis strains: Bacillus thuringiensis subsp. entomocidus HD9, Bacillus thuringiensis subsp. entomocidus HD110 and Bacillus thuringiensis subsp. tolworthy HD125 were used to study the bacteriocin diversity within Bacillus thuringiensis species. The inhibitory substances produced by these strains were purified to homogeneity by reverse phase high-performance liquid chromatography and were identified as bacteriocins. These molecules were all sensitive to proteinase K, heat-resistant and stable over a wide range of pH (3-10.5). Mass spectrometry ESI-TOF-MS analysis provided the monoisotopic masses of the three newly identified bacteriocins. The N-terminal amino acid sequences were also determined by Edman sequencing and by nanoESI-MS/MS experiments. Interestingly, the three newly identified bacteriocins shared the same molecular mass and the same N-terminal sequence with the anti-Listeria bacteriocin: thuricin S, produced by Bacillus thuringiensis subsp. entomocidus HD198 strain. Thereby, we demonstrated at the biochemical level and for the first time that four different Bacillus thuringiensis strains produce the same bacteriocin.

Keywords
Bacteriocin; Bacillus thuringiensis; Thuricin S

Bacteriocins are antimicrobial compounds ribosomally synthesized by various species of bacteria which affect growth and/or viability of species closely related to the producer (Reeves, 1965; Jack et al., 1995). Bacteriocins produced by Gram-negative bacteria are the first to be studied in depth, e.g. the colicins produced by Escherichia coli, are often larger than 20 kDa and inhibit the growth of closely related strains. Colicin V and microcins, a heterogeneous group of small peptides (<10 kDa) produced by Enterobacteriaceae strains, are exceptions. Microcins can be distinguished from colicins by their smaller size and the fact that their synthesis, non-lethal for the producer strains, is not SOS inducible (Papagianni, 2003). Gram-positive bacteria also produce bacteriocins most often smaller than 8 kDa, cationic, amphiphilic, membrane-permeabilizing peptides with very high specific activities (Papagianni, 2003; Maqueda et al., 2008).

Lactic acid bacteria (LAB) are part of the most well known and investigated producers of antimicrobial antagonists, especially bacteriocins. They are Grampositive, non-spore-forming, catalase-negative, acid tolerant, and strictly fermentative, with lactic acid as the major end product of sugar fermentation (Line et al., 2008). The best known and most fully studied bacteriocin from LAB is nisin, a lantibiotic produced by Lactococcus lactis which has been accepted by the world Health Organization as a preservative in food industry (Abee, 1995; Jack et al., 1995; Delves-Brougnton et al., 1996). 

The production of bacteriocins appears to be a wide spread phenomena. 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). 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). However, no previous work showed that different Bt strains produce the same bacteriocin.

Indeed, the aim of this work was to investigate the bacteriocin production within Bt species. For this purpose, bacteriocins from three different Bt strains HD125, HD 9 and HD110 were purified to homogeneity and characterized. Molecular masses and N-terminal amino acids sequences of the three newly identified bacteriocins were also determined.

1 Results 
1.1 Production of antimicrobial substances

The kinetics of production of the inhibitory substances produced by strain HD9, strain HD110 and strain HD125 were determined. The obtained results showedsome similarities: all the productions started during mid-logarithmic growth phases, reached their maximums at the early stationary phases and remained detectable up to 40 hours of incubation (Figure 1). However, some differences in their activities against the indicator strain Bt 10T were recorded: the substance produced by strain HD110 exhibited the highest inhibitory activity (150 AU.ml-1) compared to those produced by strains HD9 and HD125 (120 AU.ml-1 and 100 AU.ml-1, respectively) (Table 1).
 

 
Figure 1 Kinetics of production of the antibacterial substances produced by B. thuringiensis

 
Table 1 Inhibitory activity of the supernatants during purification steps


1.2 Purification

After filter-sterilisation and Sep-Pak chromatography of the supernatants, the eluted fractions from Sep-Pak C18 cartridges were tested for antibacterial activity. The 40% acetonitrile fractions of all the culture supernatants provided the largest zone of inhibition and were consequently subjected to a C18 reverse phase HPLC column. Interestingly, all the HPLC chromatograms showed the presence of a single and symmetric peak eluted at 76% acetonitrile. 

1.3 Characteristics of the newly identified bacteriocins
The sensitivity of the HPLC purified inhibitory compounds to proteolytic enzymes as proteinase K, pepsine, papaïn and trypsin showed that all the antibacterial activities were completely lost after proteinase K treatement. All the newly identified bacteriocins were also shown to be stable over a wide pH range (3-9), resistant to lyophilization and stable when kept at 4℃ and after storage at -20℃ for more than one year. Additionally, heat treatment showed that these bacteriocins were heat-stable since all of them retained their activities after boiling for 10 min. The complete loss of the inhibitory activities was observed after treatment at 121℃. 

The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) against the indicator strain Bt 10T were determined (Table 1). Amazingly, we found that the MIC and MBC of the different bacteriocins were identical (1.43 and 2.86 µmol/L, respectively). These results led us to conclude that the three newly identified bacteriocins exerted a high bactericidal effect on this strain. 

1.4 Molecular mass determination
The molecular masses of the different peptides were determined by MALDI-TOF-MS analysis (Table 2). We noticed that each mass spectrum revealed the presence of different masses with a major peak at 3160.78 Da for strain HD9 bacteriocin, at 3160.62 Da for strain HD110 bacteriocin and at 3160.67 Da for strain HD125 bacteriocin. However, NanoESI-MS analysis showed that the monoisotopic mass (M) of each peptide was 3137.61±0.3 Da. Fortunately the HPLC results showed a single peak for each peptide. These results led us to conclude that the secondary masses obtained by MALDI-TOF-MS analysis were not representative of different peptides and that they were the product of ionization and/or oxidation procedures.
 

 
Table 2 Bacteriocin masses determined by both MALDI-TOF-MS and ESI-TOF-MS analysis

 
1.5 Determination of the amino acid sequences

Both Edman and NanoESI-MS/MS experiments were used to determine the sequences of the HPLC-purified peptides produced by strains HD9, HD110 and HD125. As indicated in Table 3, the different analysis showed the following sequence: D1WTXWSXLVXAACSVELL18 for each peptide. Despite numerous attempts, no additional sequence data were obtained. Three of these 18 amino acids could not be unambiguously identified, and were designated with X. These unidentified amino acids gave a high chromatographic signal that had no correlation with known amino acids. 

 
Table 3 Bacteriocins sharing the conserved N-terminal sequence: DWTXWSXL


1.6 Plasmidic content of the different strains
To check that strains HD9, HD110, HD125 and HD198 were different, we extracted the plasmids from these bacteria. We also tried to extract plasmid(s) three times from 10T, but we were never able to obtain DNA. For the other strains, we got plasmids of high molecular weight. To see if they were different, we digested them with two restriction enzymes (EcoRâ… , BamHâ… ). The most complex restriction patterns were obtained with EcoRâ… . All strains showed different patterns with this enzyme unless the HD110 pattern which was not quite clear and looked like HD9. The BamHâ…  digestion showed that they were different (Figure 2).

 
Figure 2 Electrophoresis of plasmids digested with EcoRâ… or BamHâ…  (1% agarose gel)


2 Discussion

Bacillus is an interesting genus to investigate for antimicrobial activity, sinceBacillus species produce a large number of peptide antibiotics with various chemical structures (von Döhren, 1995). 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 recorded kinetics of production of the inhibitory substances produced by HD9, HD110 and HD125 strains seemed to be similar although the differences recorded for their specific activities against the indicator strain Bt 10T. The kinetics of production were all different from those reported for tochicin,bacthuricin F4 and cerein 7B (Paik et al., 1997; Kamoun et al., 2005; Oscáriz et al., 2006), which were essentially produced during their exponential growth phases. They were also different from thuricin and cerein productions, which were detected at the beginning of the stationary growth phase and decreased after the induction of sporulation (Favret and Yousten, 1989: Naclerio et al., 1993). However, the kinetics of production of the bacteriocin-like inhibitory substances produced by strains HD9,HD110 and HD125 were similar to those described for thuricin 7, cerein 8A, the bacteriocin-like inhibitory substance produced by B. licheniformis strain P40 and thuricin S (Cherif et al., 2001; Bizani and Brandelli, 2002; Bizani et al., 2005; Cladera-Olivera et al., 2004; Chehimi et al., 2007).

The purified bacteriocins from strain HD9, strain HD110 and strain HD125 were shown to be heat resistant and stable to a wide range of pH as many bacteriocins produced by Bt spp. (Cherif et al., 2001; Ahern et al., 2003; Gray et al., 2006). The molecular masses determined by mass spectrometry showed that the purified bacteriocins shared the same molecular mass (3137.61 Da) as thuricin S (Chehimi et al., 2007). 

Amino acid sequencing provided the 18 N-terminal amino acids of each peptide. The obtained sequences (Table 3) were compared with protein sequences from different databases and homology of 100% was recorded with the thuricin S sequence (UniProt Knowledgebase, accession No.P84763). The identified bacteriocins from strains HD9, HD110 and HD125 also shared the 18 N-terminal amino acids with thuricin 17 (Gray et al., 2006) and cerein MRX1 (Sebei et al., 2007), except for the non-identified amino acids. Such strong similarities among bacteriocins, in particular in their N-terminal peptide sequences was already described for lactic acid bacteria (Contreras et al., 1997; Mc Auliffe et al., 2001). We also noticed that the provided amino acid sequences for thuricin 17 and cerein MRX1 differed only by one amino acid substitution at position 10 (Val for thuricin 17; Cys for cerein MRX1). Similarly, nisin A differs from nisin Z by the substitution of asparagine residue instead of histidine at position 27 (Mulders et al., 1991; Noonpakdee et al., 2003). 

Bacthuricin F4 and thuricin 17 were isolated only once (Kamoun et al., 2005; Gray et al., 2006) from Bt. Similarly, cerein MRX1 was isolated only once from B. cereus spp. (Sebei et al., 2007) whereas thuricin S was isolated from four different Bt strains (HD9, HD110, HD125 and HD198). Furthermore, we noticed that the four producing strains contained plasmids, whereas strain10T, which did not produce thuricin S, did not seem to possess plasmids. 

We also demonstrated that in addition to the anionic bacteriocin: entomocin 9, strain HD9 produced a second bacteriocin which in this case is cationic. The production of more than one bacteriocin by the same strain have already been described for many Bacillus strains (Oscáriz et al., 1999; Ahern et al., 2003; Wu et al., 2005; Rea et al., 2010). 

Many bacteriocins as nisin (Choi et al., 2000; Noonpakdee et al., 2003; Rodriguez et al., 1995), sakacin P (Moretro et al., 2000) and aureocin A70 (Ceotto et al., 2009) were reported to be produced by different bacterial strains of the same species. However, no previous paper has reported the production of the same bacteriocin by different strains of Bt species. Furthermore, we noticed that despite the same culture conditions (inoculum, medium, temperature, incubation period), the recorded inhibitory activities differed from one strain to the other. Similarly, different strains and/or species could produce the same bacteriocin and bacteriocin production is strain dependent (Moretro et al., 2000).

As a conclusion to this work, we demonstrate at the biochemical level and for the first time, that different strains of Bt species produce the same bacteriocin. This bacteriocin, previously named thuricin S, is heat resistant, stable to a wide range of pH and inhibits not only the food-borne pathogene L. monocytogenes and the food poisoning bacterium B. cereus but also many Gram-negative pathogens as Salmonella enterica subsp. enterica ser. Cholerae and P. aeruginosa

Bacteriocins produced by Btcould find a role in bacterial biocontrol, synergizing the N-acyl-homoserine lactanase enzymes that quench the quorum-sensing signals of several pathogenic and phytopathogenic Gram-negative bacteria (Dong et al., 2002). In addition to its use as a biological insecticide, more informations about bacteriocins produced by Bt and on their potential practical use as an antagonistic toward the phytopathogen bacteria as Pseudomonas syringae would make this species much more interesting.

3 Material and Methods
3.1 Bacterial strains and culture media

In this study, we used three strains, from the Bacillus Genetic Stock Center collection (Columbus, USA), Bt subsp. entomocidus HD 9 (HD9), Bt subsp. entomocidus HD110 (HD110), and Bt subsp. tolworthi HD125 (HD125). Bt subsp. darmastadiensis 10T ( Bt 10T) was used as the sensitive indicator for antibacterial activity detection. All strains were grown in tryptic soy broth (TSB) at 30℃. 

3.2 Antibacterial activity detection and kinetics of production
Bacillus thuringiensis strains (HD9, HD110 and HD125) were tested for the presence of antibacterial activity by an agar spot assay (Cherif et al., 2001) on tryptic soy agar (TSA). Bacteriocin activity of each cell free supernatant, obtained by centrifugation (10 000 xg, 30 min, 4℃), was assayed by agar well-diffusion method (Jack et al., 1995). Bacteriocin activity, expressed in arbitrary units (AU), was calculated as the reciprocal of the highest dilution of a bacteriocin preparation giving an inhibition zone. Each strain was cultivated in TSB medium at 30℃ on a shaker, and growth was monitored at an optical density of 600 nm. Samples of each culture supernatant were collected at different times, and tested for the presence of inhibitory activity against the indicator strain. Tests were performed in duplicate.

3.3 Bacteriocin purification
Each Bt strain was grown overnight at 30℃ on a shaker in 1 000 mL of TSB medium. The cell-free supernatants (CFS) obtained by centrifugation (10 000×g, 30 min, 4℃) were filter-sterilized using 0.22 µm Millex-GV filters (Millipore Corp., Billerica, Massachusetts), and loaded (1 mL/min) onto Sep-Pak Plus Environmental C18 cartridges (Waters Corp., Milford, Massachusetts). After the different elution steps (50% methanol, 30%, 40%, 60% and 100% acetonitrile; flow rate 4 mL/min), all fractions were tested for antibacterial activity against the indicator strain Bt 10T. Then, the Sep-Pak fractions providing the largest inhibition zones were subjected to C18 reversed-phase high performance liquid chromatography (HPLC). The inhibitory compounds were eluted from the column (C18, 5 mm, 300 Å ; Waters Corp.) using an acetonitrile gradient (40%–100%) in 0.1% aqueous trifluoroacetic acid (0.8 mL/min). Absorbance was monitored at 215 nm. 

3.4 Effects of enzymes, temperature and pH

Purified molecules were treated by proteinase K, pepsin, papaïn and trypsin at a final concentration of 1 mg/mL in buffers recommended by the supplier. They were also heat-treated at 60℃, 80℃, 100℃ and 121℃ for 10 min. The effect of pH on the antimicrobial activity was determined according to Oscáriz and Pisabarro (2000). 

3.5 Determination of the molecular masses

Each bacteriocin sample (0.5 µL) was co-crystallized with a-cyano-4-hydroxycinnamic acid matrix before MALDI mass measurements. Analyses were performed using an UltraflexTM TOF/TOF (Bruker Daltonics, Bremen, Germany). 

For NanoMS/MS experiments, 2 μL aliquots of each bacteriocin fraction were loaded into a metal coated nano spray capillary (Protana, Odense, Denmark) and rinsed with H2O/CH3CN/HCOOH (50/50/5). Experiments were carried out using a hybrid quadrupole orthogonal acceleration time-of-flight tandem mass spectrometer (Q-TOF II, Waters) fitted with a z-spray nanoelectrospray (nanoES) source. Data processing and amino acid sequence analysis were performed with MaxEnt3 and MassLynx/PepSeq 3.5 software (Micromass), respectively.

3.6 Amino acid sequencing 
The amino acid sequences of the different bacteriocins were determined by automated Edman degradation on an Applied Biosystems model 492cLC protein sequencer.

3.7 Plasmid extraction and digestion
DNA was extracted with the QIAGEN Plasmid Midi Kit, with a protocol adapted to Gram positive bacteria. Strains were grown over-night in 100 mL Miller’s Luria broth (LB) at 30℃ with shaking. Cells were harvested by centrifugation at 3 000 g for 15 min at 4℃. Each bacterial pellet was resuspended in 10 mL buffer P1 from the kit, 5 mg/mL lysozyme were added, and cells were incubated at 37℃ for 30 min. Then, we followed the QIAGEN Plasmid Midi Kit protocol.

Each plasmid (1 µg) was digested at 37℃ for 4h with 40 U EcoRâ…  or BamHâ… in their specific buffer, supplied with the enzymes (Promega). An electrophoresis was carried out on 1% agarose gels. These assays were performed three times and the results were reproducible.

Authors’ contributions
CS carried out the design of the study, experiments, data analysis and the draft of the paper. LI, DF and DA participated in the experiment design, data analysis and manuscript preparation. LF and SS assisted experimental management, data analysis and reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgements
We dedicate this work to the memory of Dr. Anne-Marie Pons. This work was financially supported by the Agence Universitaire de la Francophonie (AUF) and by a grant from the United Nations Educational, Scientific, and Culture Organization (UNESCO) to Dr. Sonia Chehimi. 

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