Thuringiensis Species Research Articles

Synthesis, spectral characterization, and biological studies of 3,5-disubstituted-1,3,4-oxadiazole-2(3H)-thione derivatives.

The reaction of 3,4-dichlorophenyl-1,3,4-oxadiazole-2( 3H )-thione with piperidine derivatives via Mannich reaction was used to generate eleven novel compounds in moderate to good yields. Synthesized molecules were characterized according to their structure with 1H NMR, 13C NMR and FT-IR spectral foundations, which were compatible with literature informations. Antimicrobial activity and cytotoxicity studies were done by disc diffusion and NCI-60 sulphordamine B assay methods. The antimicrobial test results revealed that synthesized compounds have better activity against gram-positive species than gram-negative ones. A total analysis of the antibacterial, antifungal, and antiyeast activity revealed that newly synthesized compounds were really active against Bacillus cereus , Bacillus ehimensis, and Bacillus thuringiensis species . For cytotoxicity, among three different cancer cell lines (HCT116, MCF7, HUH7) compounds 5c, 5d, 5e, 5f, 5g, 5i, 5j and 5k were seemed especially effective on HUH7 cancer cell line via moderate to good activity. More significantly, against liver carcinoma cell line (HUH7) most of the compounds of the series ( 5c-5g and 5i-5j ) have better IC50 values (IC50= 18.78 µM) than 5-Florouracil (5-FU) and also compound 5d possessed 10.1 µM value, which represents good druggable cytotoxic activity. Further, the molecules were also screened for in silico chemoinformatic and toxicity data to gather the predicted bioavailibity and safety measurements.

Endophytic Bacillus Bacteria Living in Sugarcane Plant Tissues and Telchin licus licus Larvae (Drury) (Lepidoptera: Castniidae): The Symbiosis That May Open New Paths in the Biological Control.

Bacteria of the genus Bacillus can colonize endophytically and benefit several crops including the control of some pest orders. In view of the benefits provided by these microorganisms and in order to find out an efficient biotechnological control for the giant borer, our interest in studying the microorganisms in symbiosis with sugarcane and the giant borer has arisen, since there is no efficient chemical or biological control method for this pest. Therefore, endophytic Bacillus strains were isolated from three sugarcane niches (apoplast fluid, central internode cylinder and roots) and also from the giant borer larvae living inside sugarcane varieties grown in the Northeast region of Brazil. The taxonomical characterization (16S rRNA) of 157 Gram-positive isolates showed that 138 strains belonged to the Bacillus genus. The most representative species were phylogenetically closely related to B. megaterium (11.5%) followed by B. safensis (10.8%), B. cereus (8.9%), B. oleronius (8.9%), B. amyloliquefaciens (7.0%), and B. pacificus (6.4%). BOX-PCR analyses showed very distinct band pattern profiles suggesting a great diversity of Bacillus species within the sugarcane niches and the digestive tract, while the B. cereus group remained very closely clustered in the dendrogram. According to XRE biomarker analysis, eleven strains (FORCN005, 007, 008, 011, 012, 014, 067, 076, 092, 093, and 135) correspond to B. thuringiensis species. Additional studies using conserved genes (glp, gmk, pta, and tpi) indicated that most of these strains were phylogenetically closely related to B. thuringiensis and may be considered different subspecies. In conclusion, this study suggests that the culturable Bacillus species are greatly diversified within the plant niches and showed Bacillus species in the digestive tract of the giant borer for the first time. These results open new perspectives to understand the role and functions played by these microorganisms in symbiosis with this pest and also the possibility of developing an efficient biological control method for the giant borer using strains identified as the B. thuringiensis species.

Selection of Bacillus thuringiensis against Pathogenic Nematodes Attacking Pepper Tree

Nine strains belonging to the Bacillus genus have been isolated from soil samples in Tien Phuoc district, Quang Nam province. They were capable of surviving at 42°C, and the Bl, B4, B7 and B9 strains could produce toxic crystals at this temperature. B7 had the most promising characteristics in terms of spore-forming ability. The result of the 16S rRNA gene sequencing showed that B7 belongs to the Bacillus thuringiensis species, which is known to effectively control root-knot Meloidogyne sp. nematodes attacking pepper tree. This study was aimed at evaluating the inhibitory effect of the bacterial isolate under study on root-knot eggs and juveniles. It was shown that the highest inhibitory activity of the cultures of the bacterial strains under study was observed at their concentration of 109cells/mL; in this case, up to 89.67% of nematode eggs and 100% of juveniles J2 were killed after 10 h of treatment. Bacillus thuringiensis, Meloidogyne sp., pepper tree, root-knot, nematodes We are grateful to the Danang University of Science and Education and Duy Tan University for supporting this study.

Search for Cry proteins expressed by Bacillus spp. genomes, using hidden Markov model profiles.

This report focuses on a systematic search for Cry proteins in Bacillus spp. other than B. thuringiensis by analyzing reported Bacillus spp. genomes, using conserved sequences from the C-terminal half of reported Cry proteins in hidden Markov model profiles. A high-throughput model based on the use of HMMER and CD-HIT tools was designed, which identified Cry proteins. This model was used on 857 reported Bacillus spp. genomes, where 174 Cry protein sequences were identified, mostly, as expected, in B. thuringiensis genomes but, interestingly, 42 were identified on other species. Despite including 89 species of Bacillus in the HMMER analysis, Cry protein sequences were found only in genomes from species within the B. cereus group. According to the species registered at the NCBI database containing each genome, this group was formed by 18 non-B. thuringiensis strains. However, when sequences in those genomes were analyzed by multilocus sequence typing, the number of non-B. thuringiensis strains increased to 39, indicating that as many as 119 Cry protein sequences were found in four non-B. thuringiensis species. Therefore, dispersion of Cry proteins is much wider and frequent than previously thought, questioning its role in nature.

A plasmid-borne Rap-Phr system regulates sporulation of Bacillus thuringiensis in insect larvae.

The entomopathogen Bacillus thuringiensis species harbours numerous plasmids essentially studied for their involvement in pathogenicity, as Cry-plasmids. The life cycle of B. thuringiensis in the insect host is regulated by the sequential activation of quorum sensing systems to kill, survive and sporulate. In this study, we characterize a new quorum sensing system belonging to the Rap-Phr family. The Rap8-Phr8 system is borne by the pHT8_1 plasmid, a small cryptic plasmid from the B. thuringiensis var. kurstaki HD73 strain. Our results demonstrate that the Rap8 protein inhibits sporulation and biofilm formation through the Spo0A pathway. The Rap8 activity is inhibited by the mature Phr8 heptapeptide YAHGKDI. The key residues specific for the Rap phosphatase activity are conserved in Rap8 suggesting a common mode of action. Interestingly, we show that the Rap8-Phr8 system is specifically required for regulating sporulation of B. thuringiensis in insect larvae. This system may allow the bacteria to exert a tight control of the sporulation process in the host cadaver for optimizing the multiplication, the survival and the dissemination of the bacteria. Thus, our results suggest that pHT8_1 provides advantages for adaptation and evolution of B. thuringiensis in its ecological niche.

CHEMOTAXONOMIC FEATURES AND PLASMID PROFILES OF AEROBIC AND FACULTATIVE ANAEROBIC SPORE-FORMING BACTERIA FROM VEGETABLES

Aim. To set the systematic position and some molecular genetic characteristics of potential agents of food poisoning and spoilage microorganisms residual vegetable products – aerobic and facultative anaerobic spore-forming bacteria in their hemotaxonomic and plasmid profiles. Methods. The fatty acid analysis of the investigated strains was carried out by gas chromatography using the system identification of microorganisms MIDI Sherlock (MIDI, USA). The species belonging of bacilli strains that according to chromatography of fatty acids had high, but close similarity indices by types of Bacillus cereus and B. thuringiensis, was confirmed by conducting PCR with specific primers. Plasmid DNA isolation was performed by the modified Jensen’s method. Results. The analysis of the results of gas chromatography showed that content of branched fatty acids in the investigated strains was from 54 to 85% of the total fatty acid cell pool, including saturated and unsaturated fatty acids and their branched structural isomers with a predominance of iso-C15:0 and antеizo -C15:0 fatty acids. It was also characterized for them a high content of antеizo-C17:0 and iso-C17:0 fatty acids. Mostly strains of Bacillus cereus, Lysinibacillus sphaericus and B. pumilus contained plasmids of different sizes, which fell into two groups: the small plasmids from 2000 to 12000 bp and megaplazmids to 200000 bp. Conclusion. The investigated strains of bacteria due their chemotaxonomic features have belonged to 4 genera of Bacillales order: Bacillus, Lysinibacillus, Paenibacillus, Brevibacillus with the predominance of the first group of rRNA representatives and the dominance of B.subtilis strains. The results of the fatty acid biomarking of Bacillus cereus strains have been confirmed by PCR with the group- and species-specific primers for the samples that according to fatty acids chromatography had high but similar indices of similarity between B. cereus and B. thuringiensis species. The peculiarities of fatty acid and plasmid profiles of investigated microorganisms can be used as an auxiliary key for taxonomic differentiation of bacillary contaminants – potential pathogens of foodborne diseases, damage and residual microbiota of vegetable production in the South of Ukraine.

Comparative genomics of extrachromosomal elements in Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis subsp. israelensis is one of the most important microorganisms used against mosquitoes. It was intensively studied following its discovery and became a model bacterium of the B. thuringiensis species. Those studies focused on toxin genes, aggregation-associated conjugation, linear genome phages, etc. Recent announcements of genomic sequences of different strains have not been explicitly related to the biological properties studied. We report data on plasmid content analysis of four strains using ultra-high-throughput sequencing. The strains were commercial product isolates, with their putative ancestor and type B. thuringiensis subsp. israelensis strain sequenced earlier. The assembled contigs corresponding to published and novel data were assigned to plasmids described earlier in B. thuringiensis subsp. israelensis and other B. thuringiensis strains. A new 360 kb plasmid was identified, encoding multiple transporters, also found in most of the earlier sequenced strains. Our genomic data show the presence of two toxin-coding plasmids of 128 and 100 kb instead of the reported 225 kb plasmid, a co-integrate of the former two. In two of the sequenced strains, only a 100 kb plasmid was present. Some heterogeneity exists in the small plasmid content and structure between strains. These data support the perception of active plasmid exchange among B. thuringiensis subsp. israelensis strains in nature.

Bacillus thuringiensis B1(2015b) is a Gram-Positive Bacteria Able to Degrade Naproxen and Ibuprofen.

A Gram-positive bacterium, designated as strain B1(2015b), was isolated from the soil of the chemical factory “Organika-Azot” in Jaworzno, Poland. On the basis of 16S rRNA gene sequence analysis, the isolated strain was classified as a Bacillus thuringiensis species. Strain B1(2015b) is able to degrade ibuprofen and naproxen, however, these compounds are not sufficient carbon sources for this strain. In the presence of glucose, Bacillus thuringiensis B1(2015b) degrades ibuprofen and naproxen with higher efficiency. Twenty milligrams per liter of ibuprofen was degraded within 6 days and 6 mg l−1 of naproxen was removed within 35 days. Simultaneously, the growth of the bacterial culture was observed. The obtained results suggest that Bacillus thuringiensis B1(2015b) appears to be a powerful and useful tool in the bioremediation of non-steroidal anti-inflammatory drugs-contaminated environment.

Bioinformatics Analysis of NprR-NprX Quorum-Sensing System of <i>Bacillus thuringiensis</i> Isolates from the Papaloapan Region, Oaxaca-Mexico

Quorum sensing is a chemical communication process that bacteria use to regulate collective behaviors. In Gram-positive bacteria, oligopeptides (called autoinducers) are the signaling molecules to elicit quorum sensing. In Bacillus thuringiensis, NprR is a transcriptional regulator whose activity depends on the NprX signalling peptide. Bacillus thuringiensis is closely related to Bacillus cereus and Bacillus anthracis. The principal difference between them is that Bacillus thuringiensis is the only one that produced Cry protein. The aim of this study is to explore the relation of nprR and 16S rRNA genes in Bacillus thuringiensis. Phylogenetic trees of nucleotide sequences of nprR and 16S rRNA genes were built. Sequences of fourteen new isolates from Papaloapan region were included in those phylogenetic trees. In order to identify the isolates, a simple and fast methodology considering the Cry protein formation was used. The 16S rRNA phylogenetic tree allows identify eight isolates as Bacillus thuringiensis and the others as Bacillus spp. The nprR phylogenetic tree does not match with the 16S rRNA phylogenetic tree. This confirms that nprR is not a molecular marker for evolution. Most of the new isolates have the same NprR sequence (WTSDIVG). However, the SKPDIVG is the most common NprR sequence in thuringiensis species.

Identification of Three Novel <i>B. thuringiensis</i> Strains that Produce the Thuricin S Bacteriocin

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.

Response of Bacillus cereus ATCC 14579 to challenges with sublethal concentrations of enterocin AS-48

BackgroundEnterocin AS-48 is produced by Enterococcus faecalis S48 to compete with other bacteria in their environment. Due to its activity against various Gram positive and some Gram negative bacteria it has clear potential for use as a food preservative. Here, we studied the effect of enterocin AS-48 challenges on vegetative cells of Bacillus cereus ATCC 14579 by use of transcriptome analysis.ResultsOf the 5200 genes analysed, expression of 24 genes was found to change significantly after a 30 min treatment with a subinhibitory bacteriocin concentration of 0.5 μg/ml. Most of up-regulated genes encode membrane-associated or secreted proteins with putative transmembrane segments or signal sequences, respectively. One operon involved in arginine metabolism was significantly downregulated. The BC4206-BC4207 operon was found to be the most upregulated target in our experiments. BC4206 codes for a PadR type transcriptional regulator, while BC4207 codes for a hypothetical membrane protein. The operon structure and genes are conserved in B. cereus and B. thuringiensis species, but are not present in B. anthracis and B. subtilis. Using real-time qPCR, we show that these genes are upregulated when we treated the cells with AS-48, but not upon nisin treatment. Upon overexpression of BC4207 in B. cereus, we observed an increased resistance against AS-48. Expression of BC4207 in B. subtilis 168, which lacks this operon also showed increased resistance against AS-48.ConclusionBC4207 membrane protein is involved in the resistance mechanism of B. cereus cells against AS-48.

Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil.

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cry1 genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.

A phylogenetic analysis of Bacillus thuringiensis serovars by RFLP-based ribotyping

To determine the 23S and 5S rRNA gene fingerprints in order to reveal phylogenetic relationships among Bacillus thuringiensis strains. Eighty-six B. thuringiensis strains which include 80 serovar type strains, five intraserovar strains and a non-serotypeable strain, wuhanensis, were tested. Total DNA was digested with EcoRI and HindIII. The 23S and 5S rRNA gene restriction fragment length polymorphisms showed 82 distinctive ribopatterns. The dendrogram generated by numerical analysis showed 10 phylogenetic groups and six ungrouped serovars at the 95.5% DNA relatedness rate. A second dendrogram was constructed using a combination of the data from this study and from a previous study on 16S rRNA gene fingerprinting. It revealed eight distinct phylogenetic groups and three ungrouped serovars at the 94% DNA relatedness rate. This method permitted the classification and positioning of a wide variety of B. thuringiensis strains on a phylogenetic tree. Bacillus thuringiensis strains appear to be relatively homogeneous and to share a high degree of DNA relatedness. This study contributes a further step to the definition of valid taxonomic sublevels for the B. thuringiensis species.

A comparative study of the action of six crystalliferous bacteria on the cabbage looper, Trichoplusia ni

The present study was initiated to compare the effects of six Bacillus crystalliferous bacteria on the cabbage looper, Trichoplusia ni. The six bacteria, Bacillus thuringiensis thuringiensis, Bacillus thuringiensis galleria, Bacillus thuringiensis alesti, Bacillus thuringiensis sotto, Bacillus entomocidus entomocidus, and Bacillus finitimus, represented the various strains of Bacillus crystalforming bacteria. These bacteria were studied for their effects on cessation of feeding, pupation, mortality, and pathological effects on the midgut of the cabbage-looper larvae. The cabbage looper appeared to be most affected by the four B. thuringiensis species, especially B. thuringiensis galleria and B. thuringiensis thuringiensis. Of the four B. thuringiensis species, only the two highest concentration levels (Level 4 containing 5,000,000 viable spores/ml of medium and Level 5 containing 50,000,000 viable spores/ml of medium) caused effects in all aspects of this study. B. thuringiensis galleria and B. thuringiensis thuringiensis at the third level (containing 500,000 viable spores/ml of medium) caused some deleterious effects on the larvae. The lower three concentrations of B. thuringiensis alesti and B. thuringiensis sotto (Level 1 containing no viable spores, Level 2 containing 50,000 viable spores/ml of medium and Level 3), the two lower levels of B. thuringiensis galleria and B. thuringiensis thuringiensis, and all the levels of B. entomocidus entomocidus and B. finitimus caused little or no visible effects on the third- and fifth-instar larvae.