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MessagePosté le: Jeu 18 Déc - 10:09 (2008) Répondre en citant

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MessagePosté le: Jeu 18 Déc - 10:09 (2008)

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The study of microorganisms in nature and their isolation is not always straight forward mainly due to the lack of a selective isolation process, overgrowth by competing microorganism, the presence of inhibitory substances and some organisms may enter a resting state or be viable but not culturable. Therefore, the efficiency of the existing isolation methods is conditioned by microbial interactions and the nature of the samples per se. As the innovative capacity of the biotechnology fields mostly relies on prokaryotic cells engineering, microbe hunters are in the process of finding more and better strategies to capture and culture brand new strains with potential applications in medicine and agriculture.
In this regard, Bacillus thuringiensis (Bt) is one of the most sought after entomopathogenic bacterium. It produces crystalline insecticidal proteins (ICPs) Cry and Cyt, formed in its steady stage as intracellular inclusions ([Bernhard et al., 1997] and [Crickmore et al., 1998]). The interest in this microorganism relies on its forthcoming potential as an economic, effective species-specific and environmentally safe pesticide (Han et al., 2006). Moreover, given its spore-forming capacity, this microorganism is found in many different habitats, particularly soils (Schnepf et al., 2005). Currently, more than 38 Bt insecticidal proteins have been described (Jensen et al., 2003). However, there are still many important agricultural pests that are not effectively controlled by the existing Bt strains.
At present, the isolation of Bt strains from soil samples is performed predominantly by two methodologies widely cited in the scientific literature ([Ohba and Aizawa, 1986], [Martin and Travers, 1989], [Lonc et al., 1997], [Theunis et al., 1998] and Valicente and Barret, 2003 F.H. Valicente and M.R. Barret, Bacillus thuringiensis Survey in Brazil: geographical distribution and insecticidal activity against Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), Neotrop. Entomol 32 (4) (2003), pp. 639–644.[Valicente and Barret, 2003]). The first, developed by the World Health Organization (WHO) (Ferreira da Silva et al., 2002), consists on a thermal shock treatment of a soil solution to kill vegetative bacteria and the [url:5c3d34e8e1=http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-4STB0B6-3&_user=6587154&_coverDate=10%2F31%2F2008&_alid=842445486&_rdoc=76&_fmt=high&_orig=search&_cdi=4932&_sort=d&_docanchor=&view=c&_ct=2581&_acct=C000064368&_version=1&_urlVersion=0&_userid=6587154&md5=9723a7d8ad4ce94df864d0285bb90c33#hit9#hit9]isolation of bacterial spores. The second method, developed by [/url:5c3d34e8e1]Travers et al. (1987) selectively inhibits the germination of Bt spores by sodium acetate and then other bacteria in vegetative form are also eliminated by a heat treatment. However, we found that these procedures do not always allow for the [url:5c3d34e8e1=http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-4STB0B6-3&_user=6587154&_coverDate=10%2F31%2F2008&_alid=842445486&_rdoc=76&_fmt=high&_orig=search&_cdi=4932&_sort=d&_docanchor=&view=c&_ct=2581&_acct=C000064368&_version=1&_urlVersion=0&_userid=6587154&md5=9723a7d8ad4ce94df864d0285bb90c33#hit10#hit10]isolation of Bt from soil samples. In this study an improved method to isolate Bt strains from soil was developed by performing dry-heat pre-treatment of soil samples.
Soil samples collected from five locations in southeastern Venezuela. were processed in triplicates using the WHO Bt isolating methodology (Ferreira da Silva et al., 2002). The methodology consists of mixing 1 g of soil sample in 10 ml of saline solution (0,85% (w/v) NaCl) by vortexing the sample vigorously for 2 min. One millimeter of this solution is then incubated at 80 °C for 12 min and then cooled on ice for 5 min and cultured using a spread plate method where the sample was serially diluted (10− 1–10− 3) and 0.1 ml were spread on Luria Broth Agar (1% (w/v) tryptone, 0,5% (w/v) yeast extract, 1% (w/v) NaCl, 1,5% (w/v) agar, pH 7.5) Chemicals were obtained from Alpha Biosciences, Baltimore, Maryland, USA. Bt like colonies were sporulated in water-agar plates (1.5% (w/v) agar) and then checked for the presence of crystals under a phase contrast microscope (Konus Campus BM-100FL) at 1000X. HD-1 Bacillus thuringiensis ssp kurstaki was used as control.
Since no Bt colonies were isolated with the methodology described above, a preheat treatment to the soil samples was introduced. Five grams of soil were wrapped in aluminum foil and incubated for 3, 5 and 7 h at 80 °C in a dry oven. Samples were then processed as described by WHO methodology. To determinate significant differences between the recovered of Bt with the different dry-heat treatments, a single-factor ANOVA test was performed using the software MINITAB version 14 (MINITAB Inc, State College, PA, USA). The data were tested to see if they fulfilled the homogeneity of variance assumption (Table 1).
Soil samples were initially dry-heat treated at 80 °C and then processed as indicates the WHO methodology, reported by Ferreira da Silva et al., 2002 S. Ferreira da Silva, J.M. Cabral and R. Gomes, Comparaçao entre três Métodos de Isolamento de Bacilos Entomopatogênicos, Circular Técnica del Ministerio de Agricultura, Pecuaria e Abastecimiento vol. 14 (2002), pp. 1–3.Ferreira da Silva et al. (2002). (n = 3).
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[url:5c3d34e8e1=http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-4STB0B6-3&_user=6587154&_coverDate=10%2F31%2F2008&_alid=842445486&_rdoc=76&_fmt=high&_orig=search&_cdi=4932&_sort=d&_docanchor=&view=c&_ct=2581&_acct=C000064368&_version=1&_urlVersion=0&_userid=6587154&md5=9723a7d8ad4ce94df864d0285bb90c33#tbl1#tbl1]View Within Article

 

 

The main reason for this study was to improve our recovery methods for Bt from soils. In previous experiments, only Bacillus cereus (Bc) had been isolated (data not shown) using different Bt isolation methodologies. Fig. 1 shows the results obtained when soil was pre-treated with dry-heat for different lengths of time. Significant differences were observed in the number of Bt strains that were isolated from soil samples pre-treated for 5 h when compared to untreated samples. In general, it can be observed that the number of total bacteria growing after the treatment is approximately a tenth of the untreated soil. However, on samples with 5 h dry-heat treatment, 60% of the bacteria growing on plates were Bt compared to 0.43% observed for untreated soil.

 
 
 

Bacillus cereus is a ubiquitous soil bacterium near neighbor to Bt (Han et al., 2006) and other spore-forming bacteria. Other authors have also found that the concentration of Bt in certain kind of soils seemed to be very low. DeLucca et al. (1981) found that it is possible to find only one Bt for every two thousand Bacillus isolates; and more recently, Rampersad and Ammons, 2005 J.N. Rampersad and D.R. Ammons, A Bacillus thuringiensis isolation method utilizing a novel stain, low selection and high throughput produced atypical results, BMC Microbiol. 5 (2005), p. 52. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (2)Rampersad and Ammons (2005), based in a novel stain for the identification of Bt found only 79 Bt candidate isolates from over ten thousand colonies observed. It is also known that Bt strains can be distinguished from Bc by the presence of cry and cyt genes in large plasmids. Thus, if these plasmids are lost, these bacteria are phenotypically alike (Slamti and Lereclus, 2005).
Attributable to the fact that Bt was found in some soils only when they were pretreated with dry-heat it seems that the concentration of this bacterium in these soils could be low in comparison with other spore-forming bacteria including thermophiles and that Bt spores can be more resistant to high temperature exposure for larger periods of time than other bacteria. In fact, Nicholson et al. (2000) stated that spores of thermophiles do not have higher resistance to dry-heat than spores of mesophiles. Since the spores of Bacillus species are known to be the most resistant to extreme environments ([Driks, 1999], [Venkata et al., 2003] and [Du et al., 2005]), a stronger condition over the soil samples as the preheating step introduced can be used successfully. In short, a preliminary dry-heat treatment of soil samples largely enhanced the probabilities to isolate Bt strains. This change in the methodology is a relevant contribution to the available procedures for the selective isolation of Bt


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MessagePosté le: Jeu 18 Déc - 13:41 (2008) Répondre en citant

Production of bacteriocins or bacteriocin-like substances was described in many species of the genus Bacillus including B. subtilis, B. coagulans, B. cereus, B. thuringiensis, B. megaterium and others (Tagg et al., 1976; Jack et al., 1995). The best studied are subtilin and coagulin. Subtilin is a lantibiotic produced by B. subtilis ATCC 6633 (Klein et al., 1993; Klein and Etian, 1994) similar to nisin. Coagulin, secreted by B. coagulans I (4), is an anti-listerial bacteriocin (cystibiotic) that belongs to the pediocin family (Hyronimus et al., 1998) and homologous to pediocin AcH/Pa1 produced by Pediococcus acidilactici (Le Marrec et al., 2000).


Bacillus thuringiensis is widely used as biopesticide in agriculture for the control of many insect pathogens. It is characterized by the production of crystal proteins (delta-endotoxins) with a specific activity against insects (Beegle and Yamamoto, 1992), nematodes, mites and protozoa (Schnepf et al., 1998). Moreover, a number of extracellular compounds are produced by B. thuringiensis, including phospholipases, chitinases (Liu et al., 2002), proteases, beta-exotoxins, vegetative insecticidal proteins and antibiotic compounds with antifungal activity (Stabb et al., 1994; Hansen and Hendriksen, 2001). These biotechnological features together with the safe worldwide use of B. thuringiensis for over three decades on many agricultural crops for protection from several insect parasites, argue strongly that the microorganism can be harnessed for application in agriculture and industry for food and feed production.


As synergistic factors acting together with other metabolites, bacteriocins produced by B. thuringiensis have attracted a growing interest in the last decade because of the possible biotechnological application. A number of B. thuringiensis bacteriocins have been partially characterized such as, thuricin produced by B. thuringiensis subsp. thuringiensis HD2 (Favret and Yousten, 1989), thuricin B439 produced by B. thuringiensis B439 (Ahern et al., 2003), tochicin produced by B. thuringiensis subsp. tochigiensis HD868 (Paik et al., 1997), bacthuricin F4 from a local isolate of B. thuringiensis (Kamoun et al., 2005) and, from the phylogenetically very close species B. cereus, cerein 7 produced by B. cereus BC7 (Oscariz et al., 1999) and a bacteriocin-like inhibitory substance produced by the type strain B. cereus ATCC 14579T (Risøen et al., 2004).


In previous works, we have characterized two new bacteriocins from B. thuringiensis; thuricin 7 produced by a local strain, B. thuringiensis BMG1.7 and entomocin 9 produced by B. thuringiensis subsp. entomocidus HD9 ([Cherif et al., 2001] and Cherif et al., 2003 A. Cherif, S. Chehimi, F. Limem, B.M. Hansen, N.B. Hendriksen, D. Daffonchio and A. Boudabous, Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis subsp. entomocidus HD9, J. Appl. Microbiol. 95 (2003), pp. 990–1000. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (24)[Cherif et al., 2003]). The aim of this study is the characterization and the partial purification of a new bacteriocin produced by the bacteriocinogenic subspecies entomocidus. Entomocin 110 from Bacillus thuringiensis HD110 is described and compared with the other characterized bacteriocins of B. thuringiensis and B. cereus.


Materials and methods
Bacterial strains, culture conditions and bacteriocin production
The bacteriocin producer strain used in this study is Bacillus thuringiensis subsp. entomocidus HD110 obtained from the US Department of Agriculture, IL, USA. This strain and the other Bacillus strains were maintained at −70 °C in nutrient broth supplemented with 30% glycerol. Working cultures were propagated in tryptic soy broth (TSB) with shaking at 30 °C. The bacterial strains used as indicator organisms were obtained from different collections and were grown using the appropriate media and temperatures as indicated in Table 1.




















To confirm the presence of the antibacterial agent in the culture supernatant, the active strain B. thuringiensis subsp. entomocidus HD110 was cultivated overnight in TSB at 30 °C with vigorous shaking. The culture was centrifuged (10000g for 15 min) and filtered through a 0.22 μm filter. The well-diffusion method (Jack et al., 1995) was used to examine the antibacterial activity of the culture supernatant against the indicator organism B. thuringiensis subsp. kurstaki HD1. Plates were incubated for 24 h at 30 °C before diameters of inhibition zones were measured. The specific activity per ml of culture (AU ml−1) was calculated as the reciprocal of the highest dilution of the bacteriocin-containing sample that still gave a detectable zone of inhibition in a well-diffusion test against B. thuringiensis HD1.


For bacteriocin production, B. thuringiensis subsp. entomocidus HD110 was inoculated (1% v/v) into 500 ml of sterile TSB and incubated at 30 °C for 50 h on a rotary shaker. Two-milliliter samples were aseptically removed every 2 h. Cell growth was monitored spectrophotometrically (A600) and bacteriocin activity of the culture supernatant was evaluated by the well-diffusion method (Jack et al., 1995) and expressed as the inhibition zone diameter (mm) and as specific activity (AU ml−1) (Fig. 1).


Preliminary characterization of entomocin 110
For bacteriocin characterization studies, supernatant samples of B. thuringiensis subsp. entomocidus HD110 were collected from 16 h and 48 h-cultures. Sensitivity of the antibacterial agent to proteolytic enzymes (proteinase K, trypsine and papaine) was determined by incubation of cell-free supernatant samples for 1 h at 37 °C with proteinase K (1 mg ml−1 in 100 mM Tris–HCl buffer, pH 7.5), pepsine (1 mg ml−1 in 100 mM Tris–HCl buffer, pH 3) or in the presence of papaine at 40 °C (1 mg ml−1 in 50 mM phosphate buffer, pH 5). Following incubation, the enzymes were heat inactivated for 3 min at 100 °C. For each test, untreated bacteriocin plus buffer, bacteriocin plus buffer treated 5 min at 100 °C, buffer alone and enzymes solutions served as controls. Thermal stability of the antibacterial activity was determined by incubation of aliquots (500 μl) of cell-free supernatant at 55 °C, 65 °C, 75 °C, 85 °C, 95 °C for 30 min or at 121 °C for 20 min. The activity of bacteriocin at different pH values was estimated after storage of the culture supernatant for 1 day at 4 °C in buffers ranging from pH 3 to pH 9. The effect of several organic solvents was evaluated after incubation of the bacteriocin for 1 h at 25 °C with 10% (v/v) acetone, ethanol, butanol, toluene and methanol. Bacteriocin-containing samples were prepared from bacterial cultures grown on different media [brain heart infusion, BHI; medium T3: 0.3% (w/v) tryptone, 0.2% (w/v) tryptose, 0.0002% (w/v) MnSO4·7H2O, 0.002% (w/v) MgSO4·7H2O, 0.14% (w/v) Na2HPO4·12H2O and 0.12% (w/v) NaH2PO4·H2O; LB broth; TSB] to evaluate the effect of the growth medium on bacteriocin production. The residual activity was measured by the well-diffusion test, using B. thuringiensis HD1 as indicator strain. All the experiments were done in duplicate.


Partial purification of entomocin 110
Partially purified entomocin 110 was obtained as follows. Bacillus thuringiensis subsp. entomocidus HD110 was grown in one liter liquid culture until early stationary phase (16 h) or late stationary growth phase (48 h), then cells were removed by centrifugation (10 000g for 30 min) and the supernatants were precipitated with ammonium sulphate at 80% saturation at 4 °C for 24 h. The protein precipitates were obtained by centrifugation at 10 000g for 30 min. The precipitate samples were dissolved in 30 ml of 20 mM Tris–HCl-buffer pH 7.5 and dialysed two times against four liters of the same buffer for 24 h in Spectra-Por no. 3 dialysis tubing (molecular weight cut-off, 3500; Spectum, Los Angeles, CA, USA). The resulting solutions of partially purified entomocine 110 obtained from 16 and 48 h cultures were designated respectively as PP110-16 and PP110-48. Both PP110 samples were then extracted with butanol for 2 h (2:3 ratio of butanol:sample) with gentle shaking. Organic fractions were then recovered, butanol evaporated and the protein dissolved (1/100 of the original volume) in 20 mM Tris–HCl pH 7.5. These final protein solutions were called PPB110-16 and PPB110-48. For each purification step, entomocin 110 activity was tested by the well diffusion test against B. thuringiensis HD1.


Inhibitory spectrum and mode of action of entomocin 110
The inhibitory spectrum was determined by the well-diffusion assay. Cell-free supernatant of Bacillus thuringiensis subsp. entomocidus HD110 culture or PP110-16 were tested against several strains, including Gram-positive and Gram-negative bacteria. All strains used as indicator organisms were previously subcultured in their growth agar medium before propagation in liquid medium and inoculation of the soft agar. The presence of the inhibition zone was determined after 24 h of incubation in the appropriate conditions (Table 1).


The mode of action of entomocin 110 was investigated by adding filtered PP110-16 (200 AU ml−1) to a log-phase culture of B. thuringiensis HD1 grown in 50 ml TSB medium. The indicator culture was incubated at 30 °C and at different time intervals samples were taken to determine the O.D. at 600 nm and the viable cell (CFU ml−1) on TSA plates by the standard plates counting methods (Fig. 2).


Direct detection of entomocin 110 activity and molecular weight determination
To estimate the molecular mass of the bioactive peptide in PPB110 solution, sodium dodecyl sulphate-polyacrylamide gel electrophoresis using Tris–tricine buffer system (Tricine-SDS-PAGE) was carried out as described by Schägger and von Jagow, 1987 H. Schägger and G. von Jagow, Tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa, Analyt. Biochem. 166 (1987), pp. 368–379. Abstract | Article |  PDF (1422 K) | View Record in Scopus | Cited By in Scopus (6086)Schägger and von Jagow (1987). Polyacrylamide concentration in the stacking and separating gel were 3.9% and 16.5%, respectively. Duplicate samples of both PPB110-16 and PPB110-48 (20 μl) were loaded simultaneously with the following standard proteins: bacitracin (1423 Da), insulin (β chain) (3496 Da), aprotinin (6512 Da), α-lactalbumin (14437 Da), myoglobin (16950 Da) and triosephosphate isomerase (26625 Da). Electrophoresis was conducted at a constant voltage of 50 V for one hour and 100 V for five hours. After electrophoresis, the gel was cut vertically and the first part containing PPB110-16, PPB110-48 and standard proteins was stained with Coomassie brilliant blue G-250 (Sigma-Aldrich, Steinheim, Germany) to determine bacteriocin molecular weight (Fig. 3a). The second part containing PPB110 samples was assayed for direct detection of activity as described previously (Cherif et al., 2001). Briefly, the gel part was fixed for 1 h (25% isopropanol and 10% acetic acid), washed for 30 min with sterile distilled water, transferred to a TSA (tryptic soya agar) plate and overlaid with 5 ml of soft TSA (0.7% w/v) inoculated with a culture of the indicator organism B. thuringiensis HD1. The Petri dish was incubated at 30 °C for 24 h and observed for the presence of an inhibition zone (Fig. 3b).








Cross tests and plasmid profiles
Active bacteriocin-containing culture supernatants from the bacteriocin producer strains B. thuringiensis BMG1.7 and HD9 ([Cherif et al., 2001] and [Cherif et al., 2003]), HD868 (Paik et al., 1997), HD2 (Favret and Yousten, 1989 M.E. Favret and A.A. Yousten, Thuricin: the bacteriocin produced by Bacillus thuringiensis, J. Invertebr. Pathol. 53 (1989), pp. 206–216. Abstract | Article |  PDF (3239 K) | View Record in Scopus | Cited By in Scopus (24)Favret and Yousten, 1989), B. cereus BC7 (Oscariz et al., 1999) and B. cereus 14579T (Risøen et al., 2004) were used to perform cross tests towards B. thuringiensis HD 110. Plasmidic DNA was extracted by the rapid alkaline extraction protocol with a lysozyme concentration of 8 mg ml−1 as described previously (Sambrook et al., 1989). DNA was visualized following electrophoresis in 1% (w/v) agarose gel in Tris–acetate buffer and staining with ethidium bromide (0.5 μg ml−1).


Results
Bacteriocin production
A total of 120 Bacillus thuringiensis strains obtained from different culture collections were examined for the production of bacteriocins. The production of bacteriocin-like inhibitory substances was confirmed in the supernatant of 16 strains. Two of these substances have been characterized: thuricin 7 produced by a local isolate B. thuringiensis BMG1.7 (Cherif et al., 2001) and entomocin 9 produced by B. thuringiensis subsp. entomocidus HD9 (Cherif et al., 2003). In this study Bacillus thuringiensis subsp. entomocidus HD110 was selected because of its high antagonism toward indicator organisms and its close phylogenetic relationship with the other bacteriocin producer strain B. thuringiensis entomocidus, strain HD9.


Using B. thuringiensis HD1 as an indicator strain, secretion of the bacteriocin-like inhibitory substance, called entomocin 110, was shown to start at the exponential growth phase and reach its maximum at the early stationnary phase. Entomocin 110 activity was shown to be stable until 50 h of incubation (Fig. 1). When diluted samples of entomocin 110-containing culture supernatants were used, a progressive decrease in the diameter of the inhibition zone of the indicator strain was observed without the appearance of plaques which would indicate the absence of bacteriophage activity.


Characterization of entomocin 110 and mode of inhibition
The effect of several media on the production of antibacterial activity was evaluated. TSB and LB media were shown to be more favorable for the production of entomocin 110, in comparison with BHI, NB and T3 medium.


To test the effect of proteolytic enzymes, the entomocin 110-containing culture supernatants collected after 16 and 48 h of incubation were tested for their sensitivity to papain, trypsin and proteinase K, and residual activity were measured by the well-diffusion test against the indicator strain B. thuringiensis HD1. In both culture supernatants tested, entomocin 110 activity was completely lost only after proteinase K treatment at 1 mg ml−1 indicating its proteinaceous nature. For thermostability studies, samples of entomocin 110 (16 and 48 h culture supernatants) were incubated for 30 min at different temperatures and residual activities were measured. Entomocin 110 was shown to be heat resistant, retaining 53 % of its activity after 20 min of incubation at 121 °C. Incubation of entomocin 110 at a pH ranging from 3 to 9 and with 10% (v/v) organic solvents (acetone, ethanol, butanol, toluene and methanol), did not affect the activity. Results of this preliminary characterization indicated that inhibitory activity of the culture supernatant from late growth phase of B. thuringiensis HD110 corresponded to entomocin 110 activity.


To elucidate the mode of action of entomocin 110, cells of the indicator organism B. thuringiensis HD1 in exponential growth phase (6 h of incubation), were exposed to 200 AU of the bacteriocin. After 1 h of incubation, a decrease of the indicator strain viable cell number of (CFU ml−1) and optical density (O.D600) was observed, suggesting that the effect of entomocin 110 is both bactericidal and bacteriolytic (Fig. 2).


Inhibition spectrum of entomocin 110
The antibacterial spectrum of entomocin 110 was determined by assaying Gram-positive and Gram-negative bacteria with the well-diffusion method (Jack et al., 1995), (Table 1). Entomocin 110 had a relatively wide inhibition spectrum. It inhibited the growth of 32 of the 36 tested indicator strains of the Bacillus cereus group (B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides and B. weihenstephanensis). The tested strains of B. coagulans, B. megaterium, Paenibacillus alvei (4 strains), Paenibacillus larvae (17 strains), Paenibacillus polymyxa, Lactococcus lactis and Listeria monocytogenes were inhibited by entomocin 110. No activity was detected against Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes and towards the Gram-negative strains (Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica) tested.


Partial purification of entomocin 110 and molecular weight determination
Partial purification of entomocin 110, collected from 16 and 48 h culture supernatants, was carried out by ammonium sulphate precipitation at 80% of saturation, dialysis and butanol extraction, resulting in two active products, PPB110-16 and PPB110-48. Purification steps determined a relatively low increase in specific bacteriocin activity from 200 AU ml−1 in the culture supernatants to 1000 AU ml−1 in the PPB samples. Direct detection of entomocin 110 activity following SDS-PAGE with the Tris–glycine buffer system (Laemmli, 1970) showed a molecular mass of about 7.5 kDa (data not shown). Taking into account that separation of peptides and proteins less than 10–15 kDa is inappropriate in the traditional Laemmli discontinuous gel system, SDS-PAGE in a Tris–tricine buffer system was used. Tris–tricine SDS-PAGE, revealed some contaminating proteins in both PPB samples (Fig. 3a). Direct on-gel detection of entomocin 110 activity in PPB110-16 and PPB110-48 showed the same inhibition zone, of the indicator strain B. thuringiensis HD1, associated with a protein band with an apparent molecular mass of about 4.8 kDa, as estimated by calculating the different rf values of standard proteins (Fig. 3b).


Cross test of the bacteriocin producers strains and plasmid profiles
To confirm that entomocin 110 is a new bacteriocin produced by B. thuringiensis strain HD110, cross-inhibition tests using active culture supernatant fractions from producer strains were performed and plasmidic profiles were compared. Among the Bacillus producer strains tested, B. cereus Bc7 (Oscariz et al., 1999) and B. thuringiensis subsp. tochigiensis HD868 (Paik et al., 1997) were sensitive to entomocin 110, while B. thuringiensis BMG1.7, B. thuringiensis subsp. entomocidus HD9 and B. thuringiensis subsp. thuringiensis HD2 were resistant to the bacteriocin. Interestingly, B. thuringiensis subsp. entomocidus HD110 was inhibited by entomocine 9, thuricin 7 and tochicin (Table 2), which indicates the novelty of entomocin 110. Moreover, chromosomal determinants seem to encode for entomocin 110 production since B. thuringiensis HD110 did not harbor any detectable plasmid frequently physically associated with bacteriocins (Fig. 4).








Discussion
From a collection of bacteriocin-like producer strains, an antimicrobial activity-producing strain was selected because of its high antagonism towards all other B. thuringiensis strains. The antibacterial activity produced by B. thuringiensis subsp. entomocidus HD110 was detected in the culture supernatant by the well-diffusion method (Jack et al., 1995) and it was named entomocin 110. Entomocin 110 was classified as a bacteriocin owing to its properties. The inhibitory activity of entomocin 110 was totally lost after proteinase K treatment, thereby revealing its proteinaceous nature and its similarity to the other bacteriocins produced by B. thuringiensis HD2, HD868, BMG1.7, HD9 and BUPM4, all susceptible to proteinase K. Entomocin 110 was stable in a pH range between 3 and 9 and retained 53% of its activity after autoclaving. Thermal and pH stability are also common traits of Bacillus bacteriocins, for example entomocin 9 was shown to retain 72% of its activity after autoclaving (Cherif et al., 2003).


The inhibitory activity of entomocin 110 was recovered in the culture supernatant at the mid logarithmic growth phase and during the stationary phase (Fig. 1). Interestingly, entomocin 110 activity was stable after 50 h of incubation. To confirm the real nature of the inhibitory agent present in the culture supernatant at a very late growth phase, proteinase K and physico-chemical treatments showed that the inhibitory factor present at this growth phase corresponded to entomocin 110.


Entomocin 110 showed a bactericidal and bacteriolytic effects towards sensitive strains which indicate that the bacteriocin induces autolysis of susceptible cells resulting in massive cell wall degradation and cell lysis. This was previously described for class I bacteriocins, nisin and Pep5, which were shown to release two cell wall hydrolyzing enzymes (an N-acetylmuramoyl-l-alanine amidase and an N-acetylglucosaminidase) from the cell wall intrinsic inhibitors by a cation exchange-like process, resulting in apparent enzyme activation and rapid lysis of the staphylococcal sensitive cells (Bierbaum and Sahl, 1985; Bierbaum and Sahl, 1987). It is well known that the mode of inhibition of bacteriocins depends on the available bio-concentration, and on the nature and the physiological stage of the target strain. In general bacteriocins of Bacillus display a bactericidal and bacteriolytic effects, while enterocins for example have only a bactericidal effect (Foulquié Moreno et al., 2003).


The spectrum of activity of entomocin 110 (Table 1) resulted relatively wide because it inhibited many of the Gram-positive bacteria tested. This is similar to the spectrum of activity of thuricin, cerein 7 and entomocin 9 but it is much wider than those of thuricin, cerein and tochicin, whose inhibitory spectra only include closely related species. Moreover, cross inhibition tests with the other B. thuringiensis and B. cereus bacteriocin producers indicated the novelty of entomocin 110, also with respect to entomocin 9 (Table 2). An interesting feature of entomocin 110 is the activity against Paenibacillus larvae, the causative-agent of American foulbrood disease, the most serious and fatal bacterial disease of honey bee larvae (Neuendorf et al., 2004). These results provide us with novel tools for antimicrobial therapy, which is particularly urgent at a time when Paenibacillus larvae has developed resistance mechanisms to almost all antibiotics known.


The antimicrobial activity of partially purified proteins from the culture supernatant was associated with a single band with an apparent molecular mass of about 4.8 kDa (Fig. 3). The same mass was observed in culture supernatant after 16 and 48 h indicating that the observed antibacterial activity in the very late stationary growth phase corresponded to the same bacteriocin, entomocin 110 and is characterized by a very good physical stability. The entomocin 110 molecular mass is different from thuricin, tochicin, thuricin 7, cerein, cerein 7, thuricin 439A, thuricin 439B, entomocin 9 and bacthuricin F4 having molecular weights of 950 kDa (native form), 10.5, 11.6, 9, 3.94, 2.9 and 2.8 kDa respectively. Differences between bacteriocin producer strains could be observed also in the plasmid profile that did not show apparent plasmids in HD110 (Fig. 4).


In conclusion, one novel bacteriocin, named entomocin 110 is produced by B. thuringiensis entomocidus HD110. Compared with the other bacteriocins from Bacillus cereus group, entomocin 110 is distinguished by its biochemical and physical properties, molecular mass, spectrum of activity, the kinetics of production and the genetic background of the producer strain. Entomocin 110 was not affected by organic solvents which would indicate that activity does not require lipid moieties as it was proposed for class IV bacteriocins such as leuconocin S. The low-molecular weight (smaller than 10 kDa), heat-stability and anti-listerial activity seem to fit the criteria of a class II bacteriocin (Héchard and Sahl, 2002 and references therein).


The characterization of the antimicrobial activity of B. thuringiensis subsp. entomocidus HD110 would give a potentially interesting application for improving bacterial and/or insect biocontrol by coupling with other antagonistic factors such as antifungal compounds and delta-endotoxins. Bacteriocins produced by B. thuringiensis could find a role in bacterial biocontrol, synergizing the N-acyl-homoserine lactonase enzymes that quench the quorum-sensing signals implicated in the virulence expression of several pathogenic and phytopathogenic gram-negative bacteria ([Dong et al., 2000] and Dong et al., 2002 Y.H. Dong, A.R. Gusti, Q. Zhang, J.L. Xu and L.H. Zhang, Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species, Appl. Environ. Microbiol. 68 (2002), pp. 1754–1795.[Dong et al., 2002]).


sidali
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MessagePosté le: Mer 24 Déc - 04:26 (2008) Répondre en citant

biouided;tu cherches une traduction ou quoi?


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MessagePosté le: Jeu 8 Jan - 17:05 (2009) Répondre en citant

sidali a écrit:
biouided;tu cherches une traduction ou quoi?


Je pose la même question ! Je voulais éditer le sujet, mais je n'y ai rien compris Mr. Green

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