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.
We report the isolation and characterization of a new bacteriocin, thuricin S, produced by the Bacillus thuringiensis subsp. entomocidus HD198 strain. This antibacterial activity is sensitive to proteinase K, is heat-stable, and is stable at a variety of pH values (3-10.5). The monoisotopic mass of thuricin S purified by high performance liquid chromatography, as determined with mass spectrometry ESI-TOF-MS, is 3137.61 Da. Edman sequencing and NanoESI-MS/MS experiments provided the sequence of the 18 N-terminal amino acids. Interestingly, thuricin S has the same N-terminal sequence (DWTXWSXL) as bacthuricin F4 and thuricin 17, produced by B. thuringiensis strains BUPM4 and NEB17, respectively, and could therefore be classified as a new subclass IId bacteriocin.
Different methods were used to elucidate the mode of action of thuricin S, a new class IId bacteriocin produced by Bacillus thuringiensis subsp. entomocidus HD198. According to cell viability tests, thuricin S was shown to exert a bactericidal effect on the sensitive cells of Bacillus thuringiensis subsp. darmastadiensis 10T. The use of the fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide as an indicator proved that thuricin S interacts with the cytoplasmic membrane to dissipate the transmembrane potential. It was also demonstrated that thuricin S acts as a pore-forming bacteriocin, since it allows the nonpermeable stain propidium iodide to enter the cells. The loss of membrane integrity and the morphological changes in sensitive cells were visualized by scanning electron microscopy.
Pears have great importance in Tunisia for their desirable taste and commercial value. Until 2012, the pear cultivation was protected against fire blight by the application of a rigorous quarantine system. Fire blight, caused by Erwinia amylovora, was outbreak in Tunisia in the spring of 2012 and has spread rapidly through the most important pear growing regions destroying several hundred hectares of pear plantations. Therefore, the total pear production has decreased from 60,000 metric tons in 2011 to less than 20,000 metric tons in 2016. In this study, collected data of pear culture and surveys were carried out during four years (2012)(2013)(2014)(2015)(2016) in the main pear growing areas to evaluate the current situation of the disease in the country particularly in the damaged regions of the lower valley of Medjerda (Manouba, Ben Arous, Bizerte, and Beja). Samples collected from symptomatic trees were processed for the isolation and identification of the causal agent using microbiological and molecular techniques. The results indicate that the disease had destroyed more than 5500 hectares among a total of 8400 hectares of pear plantations area. Both provinces Manouba and Ben Arous were the most affected by fire blight disease resulting in the eradication of 350 and 325 hectares of pear plantations, i.e., 100% and 98% of the total infected area, respectively. All control attempts, including sanitary measures, the application of mineral oil and copper, growth regulators and biological control have failed to limit the spread of the disease. The presence of pathogen in the prospected regions was confirmed by pathogenicity and molecular tests, which are compatible with the symptoms observed throughout the surveys. The pear cultivation in Tunisia is threatened by fire blight due to the restriction tolerance of the available varieties and the climatic conditions favoring the staggered flowering of the species. Quarantine measures must be implemented to prevent the spread of this disease in a new disease-free areas.
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