Caracterización morfológica y bioquímica de Bacillus thuringiensis Bt-EM14

Abstract

The bacterial strain Bt-EM14 was found in water samples collected from the “Pitch Lake”, located in the Trinidad Island at the Caribbean Sea. Such strain is a Gram-positive, aerobic, spore former, able to synthesize an intracellular proteinic crystal. According with such features, Bt-EM14 was considered initially as a Bacillus thuringiensis strain. However, observations by optical microscopy using negative staining revealed the presence of a capsule surrounding the bacterial body. Such structure is normally found in Bacillus anthracis, but do not in B. thuringiensis, although both bacterial species are genetically closed. The aim of the present study was to identify the bacterial strain Bt-EM14, by using conventional biochemical test for Gram-positive spore-formers, including some commercial kits as the API 50CHB and the BBL-Crystal ones. Results showed that Bt-EM14 belonged to the B. cereus group. Other assays as the profile of 13 extracellular hydrolytic enzymes, as well the utilization of a set of carbohydrates as carbon sources, showed that Bt-EM14 had a similar behaviour with respect to the B. thuringiensis typical strains included in the assays. Another aspect of the research was to determine whether Bt-EM14 was a new strain of B. anthracis, able to produce intracellular proteinic crystals. Therefore, its ability to produce hemolysins, the presence of functional flagella for movility, and its resistance to !-lactam antibiotics, was investigated. In addition, the pathogenicity of Bt-EM14 on a mice model was also studied. Results showed that the studied bacterium is not a pathogenic one, which it is either a B. anthracis strain. With respect to the capsulate of Bt-EM14, its composition and characteristics were studied by thin-layer chromatography, polyacrylamide gel electrophoresis and infrared spectroscopy. Results showed that such cellular structure is formed by polyglutamic acid and its molecular weight and characteristics are similar to the capsule found in some wild type strains of B. subtilis. Related to the parasporal crystals produced by Bt-EM14, such proteinic bodies were purified by a discontinuous gradient ultracentrifugation technique, and then analyzed using PAGESDS electrophoresis. The crystals showed to be composed by at least two different proteins, whose molecular weights were 43 and more 66 KDa. The crystals showed an irregular, nonpreviously described shape, the crystals were observed by scanning electronic microscopy. Their size is close to one micron. Another aspect of the present study concerns to the sporulation cycle, and the formation of the intracellular crystals, by means of transmission electron microscopy. The well-known B. thuringiensis HD-1 strain was included as a reference. The results showed that Bt-EM14 presents the seven typical stages during its sporulation, as occurred with the reference strain. The conclusion of the present study is that considering the presence of the paraesporal proteinic crystal, and as well the biochemical and extracellular enzymes profiles, Bt-EM14 is a rare capsulated strain of B. thuringiensis.