Prokaryotic expression and biological activities of the hemolysin BL subunit of a pathogenic Bacillus cereus of cattle origin / 生物工程学报

Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.

Isolation of a foodborne Bacillus cereus strain and its effect on intestinal mucosal immunity-associated factors and gut microbial community in mice / 生物工程学报

Bacillus cereus is a common foodborne pathogen. Accidently eating food contaminated by B. cereus will cause vomiting or diarrhea, and even death in severe cases. In the present study, a B. cereus strain was isolated from spoiled rice by streak culture. The pathogenicity and drug resistance of the isolated strain were analyzed by drug sensitivity test and PCR amplification of virulence-associated gene respectively. Cultures of the purified strain were injected intraperitoneally into mice to examine their effects on intestinal immunity-associated factors and gut microbial communities, to provide references for the pathogenic mechanism and medication guidance of these spoilage microorganisms. The results showed that the isolated B. cereus strain was sensitive to norfloxacin, nitrofurantoin, tetracycline, minocycline, ciprofloxacin, spectinomycin, clindamycin, erythrocin, clarithromycin, chloramphenicol, levofloxacin, and vancomycin, but resistant to bactrim, oxacillin and penicillin G. The strain carries seven virulence-associated genes including hblA, hblC, hblD, nheA, nheB, nheC and entFM, which are involved in diarrhea-causing toxins production. After infecting mice, the isolated B. cereus strain was found to cause diarrhea in mice, and the expression levels of immunoglobulins and inflammatory factors in the intestinal mucosae of the challenged mice were significantly up-regulated. Gut microbiome analysis showed that the composition of gut microbial community in mice changed after infection with B. cereus. The abundance of the uncultured_bacterium_f_Muribaculaceae in Bacteroidetes, which is a marker of body health, was significantly decreased. On the other hand, the abundance of uncultured_bacterium_f_Enterobacteriaceae, which is an opportunistic pathogen in Proteobacteria and a marker of dysbacteriosis, was significantly increased and was significantly positively correlated with the concentrations of IgM and IgG. These results showed that the pathogenic B. cereus carrying diarrhea type virulence-associated gene can activate the immune system by altering the composition of gut microbiota upon infection.

Variation analysis of bacterial polyhydroxyalkanoates production using saturated and unsaturated hydrocarbons

ABSTRACT Polyhydroxyalkanoates (PHA) are efficient, renewable and environment friendly polymeric esters. These polymers are synthesized by a variety of microbes under stress conditions. This study was carried out to check the suitability of waste frying oil in comparison to other oils for economical bioplastic production. Six bacterial strains were isolated and identified as Bacillus cereus (KF270349), Klebsiella pneumoniae (KF270350), Bacillus subtilis (KF270351), Brevibacterium halotolerance (KF270352), Pseudomonas aeruginosa (KF270353), and Stenotrophomonas rhizoposid (KF270354) by ribotyping. All strains were PHA producers so were selected for PHA synthesis using four different carbon sources, i.e., waste frying oil, canola oil, diesel and glucose. Extraction of PHA was carried out using sodium hypochlorite method and maximum amount was detected after 72 h in all cases. P. aeruginosa led to maximum PHA production after 72 h at 37 °C and 100 rpm using waste frying oil that was 53.2% PHA in comparison with glucose 37.8% and cooking oil 34.4%. B. cereus produced 40% PHA using glucose as carbon source which was high when compared against other strains. A significantly lesser amount of PHA was recorded with diesel as a carbon source for all strains. Sharp Infrared peaks around 1740-1750 cm-1 were present in Fourier Transform Infrared spectra that correspond to exact position for PHA. The use of waste oils and production of poly-3hydroxybutyrate-co-3hydroxyvalerate (3HB-co-3HV) by strains used in this study is a good aspect to consider for future prospects as this type of polymer has better properties as compared to PHBs.

Biosynthesis of gold nanoparticles by two bacterial and fungal strains, Bacillus cereus and Fusarium oxysporum, and assessment and comparison of their nanotoxicity in vitro by direct and indirect assays

Background: Although nanoparticles (NPs) have many advantages, it has been proved that they may be absorbed by and have toxic effects on the human body. Recent research has tried to evaluate and compare the nanotoxicity of gold nanoparticles (AuNPs) produced by two types of microorganisms in vitro by two different methods. AuNPs were produced by Bacillus cereus and Fusarium oxysporum, and their production was confirmed by visible spectral, transmission electron microscope, and X-ray diffraction (XRD) analyses. The human fibroblast cell line CIRC-HLF was treated with AuNPs, and the induced nanotoxicity was measured using direct microscopic and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Results: The results showed that the produced AuNPs had a maximum absorbance peak around 510­530 nanometer (nm), with spherical, hexagonal, and octagonal shapes and average sizes around 20­50 nm. The XRD results confirmed the presence of GNPs in the microbial culture supernatants. An MTT assay showed that GNPs had dose-dependent toxic effects, and microscopic analysis showed that GNPs induced cell abnormalities in doses lower than the determined half-maximal inhibitory concentrations (IC50s). Conclusions: In conclusion, the biologically produced AuNPs had toxic effects in the cell culture, and direct techniques such as microscopic evaluation instead of indirect methods such as MTT assay were more useful for assessing the nanotoxicity of the biologically produced AuNPs. Thus, the use of only MTT assay for nanotoxicity evaluation of AuNPs is not desirable.

Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus

Background: Glycine oxidase (GO), a type of D-amino acid oxidase, is of biotechnological interest for its potential in several fields. In our previous study, we have characterized a new glycine oxidase (BceGO) from Bacillus cereus HYC-7. Here, a variant of N336K with increased the affinity against all the tested substrate was obtained by screening a random mutant library of BceGO. It is observed that the residue N336 is invariable between its homogeneous enzymes. This work was aimed to explore the role of the residue N336 in glycine oxidase by site-directed mutagenesis, kinetic assay, structure modeling and substrate docking. Results: The results showed that the affinity of N336H, N336K and N336R increased gradually toward all the substrates, with increase in positive charge on side chain, while N336A and N336G have not shown a little significant effect on substrate affinity. The structure modeling studies indicated that the residue Asn336 is located in a random coil between -J-18 and a-10. Also, far-UV CD spectra-analysis showed that the mutations at Asn336 do not affect the secondary structure of enzyme. Conclusion: Asn336 site was located in a conserved GHYRNG loop which adjoining to substrate and the isoalloxazine ring of FAD, and involved in the substrate affinity of glycine oxidase. This might provide new insight into the structure-function relationship of GO, and valuable clue to redesign its substrate specificity for some biotechnological application.