RESUMEN
AIMS: To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats. METHODS AND RESULTS: Germination was studied by feeding germ-free rats with spores of a B. thuringiensis strain harbouring a plasmid encoding green fluorescent protein (GFP), which enabled quantification of germinated bacteria by flow cytometry. To study in vivo conjugation, germ-free rats were first associated with a B. thuringiensis recipient strain and after 1 week an isogenic donor strain harbouring the conjugative plasmid pXO16 was introduced. Both strains were given as spores and transfer of pXO16 was observed from the donor to the recipient strain. CONCLUSIONS: Bacillus thuringiensis is able to have a full life cycle in the intestine of gnotobiotic rats including germination of spores, several cycles of growth and sporulation of vegetative cells. For the first time conjugative plasmid transfer in a mammalian intestinal tract was shown between two B. thuringiensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of B. thuringiensis are used worldwide to combat insect pests, and this study brings new insights into the nature of B. thuringiensis showing the potential of the bacteria to germinate and transfer DNA in the mammalian intestinal tract.
Asunto(s)
Bacillus thuringiensis/fisiología , Conjugación Genética , ADN Bacteriano/genética , Vida Libre de Gérmenes , Intestinos/microbiología , Animales , Chlorocebus aethiops , Femenino , Citometría de Flujo , Masculino , Modelos Animales , Ratas , Ratas Sprague-Dawley , Esporas Bacterianas , Células VeroRESUMEN
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 degrees 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 12.4 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.
Asunto(s)
Bacillus thuringiensis/patogenicidad , Bacteriocinas/aislamiento & purificación , Animales , Bacillus cereus/genética , Bacillus thuringiensis/metabolismo , Bacteriocinas/biosíntesis , Bacteriocinas/farmacología , Chlorocebus aethiops , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Liofilización , Genes Bacterianos , Calor , Concentración de Iones de Hidrógeno , Microbiología Industrial , Pruebas de Sensibilidad Microbiana , Peso Molecular , Reacción en Cadena de la Polimerasa , Células Vero , VirulenciaRESUMEN
Recent investigations have shown that members of the Bacillus cereus group carry genes which have the potential to cause gastrointestinal and somatic diseases. Although most cases of diseases caused by the B. cereus group bacteria are relatively mild, it is desirable to be able to detect members of the B. cereus group in food and in the environment. Using 16S rDNA as target, a PCR assay for the detection of B. cereus group cells has been developed. Primers specific for the 16S rDNA of the B. cereus group bacteria were selected and used in combination with consensus primers for 16S rDNA as internal PCR procedure control. The PCR procedure was optimized with respect to annealing temperature. When DNA from the B. cereus group bacteria was present, the PCR assay yielded a B. cereus specific fragment, while when non-B. cereus prokaryotic DNA was present, the consensus 16S rDNA primers directed synthesis of the PCR products. The PCR analyses with DNA from a number of non-B. cereus confirmed the specificity of the PCR assay.
Asunto(s)
Bacillus cereus/aislamiento & purificación , Bacillus cereus/genética , Cartilla de ADN , ADN Bacteriano/genética , ADN Ribosómico/genética , Microbiología de Alimentos , Infecciones por Bacterias Grampositivas/microbiología , Oryza/microbiología , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/análisisRESUMEN
Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereus and B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes in B. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.
Asunto(s)
Bacillus cereus/clasificación , Bacillus thuringiensis/clasificación , Proteínas Bacterianas/genética , Enterotoxinas/genética , Reacción en Cadena de la Polimerasa/métodos , Bacillus cereus/genética , Bacillus cereus/aislamiento & purificación , Bacillus cereus/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/aislamiento & purificación , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/metabolismo , Southern Blotting , ADN Bacteriano/análisis , ADN Bacteriano/genética , Diarrea/microbiología , Enterotoxinas/metabolismo , Infecciones por Bacterias Grampositivas/microbiología , Proteínas Hemolisinas , HumanosRESUMEN
The gfp-tagged Pseudomonas fluorescens biocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3- to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.
Asunto(s)
Hordeum/microbiología , Proteínas Luminiscentes/metabolismo , Pseudomonas fluorescens/fisiología , Proteínas Fluorescentes Verdes , Microscopía Confocal , Pseudomonas fluorescens/crecimiento & desarrolloRESUMEN
Extraction and purification of bacteria from soil by the Nycodenz gradient centrifugation procedure described by Bakken and Lindahl (1995; Recovery of bacterial cells from soil. In: van Elsas, J.D., Trevors, J.T. (Eds.), Nucleic Acids in the Environment: Methods and Applications. Springer Verlag, Berlin, pp. 9-27) were compared to soil slurry extractions. Bacterial communities from four different soils were described by the bacterial abundance, CTC-reducing capacity, culturability and the community level physiological profiles (CLPP) in BIOLOG GN plates. A significant loss of both total and culturable number of bacteria g(-1) soil dry weight were found after extraction and purification of cells. The origin of soil influenced the yield of cells and a difference between the four soils and an interaction between the soils and extraction procedure were found. The culturability and the CLPP were different between the four soils but were unaffected by the extraction procedure. The bacterial community obtained after extraction and purification thus represented the same fraction of the indigenous bacterial community.
Asunto(s)
Bacterias/crecimiento & desarrollo , Bacterias/aislamiento & purificación , Ecosistema , Microbiología del Suelo , Centrifugación por Gradiente de Densidad , Medios de CultivoRESUMEN
Genetically engineered Pseudomonas sp. strain B13(FR1) was released into laboratory-scale marine ecosystem models (microcosms). Survival of the introduced population in the water column and the sediment was determined by plating on a selective medium and by quantitative competitive PCR. The activity of the released bacteria was determined by in situ hybridization of single cells with a specific rRNA-targeting oligonucleotide probe. Two microcosms were inoculated with 10(6) cells ml-1, while an uninoculated microcosm served as a control. The number of Pseudomonas sp. strain B13(FR1) cells decreased rapidly to ca. 10(2) cells ml-1 within 2 days after the release, which is indicative of grazing by protozoa. Three days after the introduction into seawater, cells were unculturable, but PCR continued to detect cells in low numbers. Immediately after the release, the ribosomal content of Pseudomonas sp. strain B13(FR1) corresponded to a generation time of 2 h. The growth rate decreased to less than 0.04 h-1 in 5 days and remained low, probably because of carbon limitation of the cells. Specific amendment of the microcosms with 10 mM 4-chlorobenzoate resulted in a rapid increase of the growth rate and an exponentially increasing number of cells detected by PCR, but not in resuscitation of the cells to a culturable state. The release of Pseudomonas sp. strain B13(FR1) into the microcosms seemed to affect only the indigenous bacterioplankton community transiently. Effects on the community were also apparent from the handling of water during filling of the microcosms and the amendment with 4-chlorobenzoate.