DNA data storage technology may supersede conventional chip or magnetic data storage medium, providing long-term stability, high density, and sustainable storage. Due to its error-correcting capability, DNA data stored in living organisms exhibits high fidelity in information replication. Here we report the development of a Bacillus chassis integrated with an inducible artificially assembled bacterial chromosome to facilitate random data access. We generated three sets of data in the form of DNA sequences using a rudimentary coding system accessible by the regulatory promoter. Sporulated Bacillus harboring the genes were used for long-term storage, where viability assays of spores were subjected to harsh environmental stresses to evaluate the data storage stability. The data accuracy remained above 99% after high temperature and oxidative stress treatment, whereas UV irradiation treatment provided above 96% accuracy. The developed Bacillus chassis and artificial chromosome facilitate the long-term storage of larger datum volume by using other DNA digital encoding and decoding programs.

The manganese oxidase complex, Mnx, from Bacillus sp. PL-12 contains a multicopper oxidase (MCO) and oxidizes dissolved Mn(II) to form insoluble manganese oxide (MnO2) mineral. Previous kinetic and spectroscopic analyses have shown that the enzyme\'s mechanism proceeds through an activation step that facilitates formation of a series of binuclear Mn complexes in the oxidation states II, III, and IV on the path to MnO2 formation. We now demonstrate that the enzyme is inhibited by first-row transition metals in the order of the Irving-Williams series. Zn(II) strongly (Ki ~ 1.5 μM) inhibits both activation and turnover steps, as well as the rate of Mn(II) binding. The combined Zn(II) and Mn(II) concentration dependence establishes that the inhibition is non-competitive. This result is supported by electron paramagnetic resonance (EPR) spectroscopy, which reveals unaltered Mnx-bound Mn(II) EPR signals, both mono- and binuclear, in the presence of Zn(II). We infer that inhibitory metals bind at a site separate from the substrate sites and block the conformation change required to activate the enzyme, a case of allosteric inhibition. The likely biological role of this inhibitory site is discussed in the context of Bacillus spore physiology. While Cu(II) inhibits Mnx strongly, in accord with the Irving-Williams series, it increases Mnx activation at low concentrations, suggesting that weakly bound Cu, in addition to the four canonical MCO-Cu, may support enzyme activity, perhaps as an electron transfer agent.

OBJECTIVE: We investigated the potential cooperative effects of carotenoid-producing Bacillus aquimaris SH6 and nonpigmented Bacillus subtilis SH23 on white-leg shrimp growth and health.
RESULTS: SH6, SH23 and a combination of both spores (1 × 106  CFU per g pellet) were administered in shrimp. The growth rate (2·36% day-1 ), red-colour score (25) and astaxanthin concentration (3·5 µg g-1 shrimp) were maximum in two-spore-administered shrimp. Immune-related Rho mRNA expression level and phenoloxidase and superoxidase dismutase activities were higher in two-spore-administered shrimp than in control shrimp, with Rho mRNA expression level being 55-fold higher in two-spore-administered shrimp than in SH6-administered shrimp and phenoloxidase activity being 1·2-fold higher in two-spore-administered shrimp than in SH23-administered shrimp. Although live SH6 count was 2·7-fold lower, SH6 germination level was 3·5-fold higher in the combination group than in SH6 group.
CONCLUSIONS: When both SH6 and SH23 spores were administered, SH6 spore germination was enhanced and cooperative improvement was seen in growth, astaxanthin level and red-colour score of white-leg shrimp; however, immune-related parameters were induced in a noncooperative manner.
CONCLUSIONS: This is the first report showing the cooperative probiotic activities of Bacillus strains and their possible mechanisms in a shrimp model.

This study was designed to assess the efficiency of eight extraction methods regarding their ability to release superficial (exogenous) and intracellular (endogenous) DNA from B. cereus spores for subsequent analysis by quantitative PCR (qPCR). B. cereus spore suspensions were subjected to both commercial DNA extraction kits and mechanical DNA extraction methods. The spores were observed by transmission electron microscopy to evaluate any damage caused during extraction. The efficiency of both extraction and purification were assessed using a qPCR assay targeting the bclA gene. Most of the extraction methods assessed, except the passage through the French press or the use of the QIAamp DNA Blood Mini kit without 95°C treatment, allowed the amplification of significant amounts of DNA. By using propidium monoazide, which is a photoreactive DNA-binding dye, the presence of non-negligible amounts of amplifiable DNA at the spore surface was highlighted. A further set of extraction assays was then performed on spores previously treated with PMA. The results of this study show that both superficial and intracellular spore DNA can be released by extraction methods to a greater or lesser extent and then further amplified by qPCR. The Precellys extraction allowed the detection of both intracellular and superficial DNA, the DNeasy Blood & Tissue kit the specific detection of intracellular DNA, while the Instagene kit detected only superficial DNA. Of the methods tested in this study, the Precellys extraction was the most efficient in terms of further DNA detection.
CONCLUSIONS: In order to verify the presence or absence of B. cereus spores in food or on surfaces in the food environment, the use of an efficient extraction method is required, followed by a qPCR analysis on the DNA released. Conversely, in order to quantify the population of Bacillus spores, any superficial DNA must be blocked, e.g. with PMA, prior to intracellular DNA extraction and further amplification.

In this paper, we report a high-throughput biological method to prepare spore-based monodisperse microparticles (SMMs) and then form the nanocomposites of CdTe quantum dot (QD)-loaded SMMs by utilizing the endogenous functional groups from Bacillus spores. The SMMs and QD-incorporated spore microspheres (QDSMs) were characterized by using transmission electron microscopy, high-resolution transmission electron microscopy, fluorescence microscopy, fluorescence and UV-visible absorption spectroscopy, zeta potential analysis, Fourier-transform infrared spectroscopy, potentiometric titrations, X-ray photo-electron spectroscopy. The thermodynamics of QD/SMM interaction and antigen/QDSM interaction was also investigated by isothermal titration microcalorimetry (ITC). Fluorescent QDSMs coded either with a single luminescence color or with multiple colors of controlled emission intensity ratios were obtained. Green QDSMs were used as a model system to detect porcine parvovirus antibody in swine sera via flow cytometry, and the results demonstrated a great potential of QDSMs in high-throughput immunoassays. Due to the advantages such as simplicity, low cost, high throughput and eco-friendliness, our developed platform may find wide applications in disease detection, food safety evaluation and environmental assessment.

Spore-forming bacterial strains were isolated from chicken gastrointestinal tracts to develop a heat-stable feed supplement that promotes weight gain in broilers. Seven Bacillus strains having more than 90% sporulation were screened from the isolates and identified to be closely related with Bacillus subtilis and Bacillus licheniformis. Of the seven strains, B. subtilis CH16 was selected to develop a feed supplement for broilers, because it formed 100% heat-stable spores, grew rapidly at 42°C and quickly formed a biofilm. In large-scale trials in broilers (n ≥ 1150 per group), the group fed CH16 (3 × 10(6) CFU g(-1) pellet) showed higher average daily gain (ADG = 61·16) and lower food conversion ratio (FCR = 1·696) than did the group fed B. licheniformis CH22 (ADG = 57·10 and FCR = 1·792), the group fed B. subtilis HU58 (ADG = 51·90 and FCR = 1·868), BioPlus group (ADG = 59·32 and FCR = 1·807) and the control group (ADG = 56·02 and FCR = 1·880). In conclusion, CH16 spores significantly increased ADG by 9·17% and reduced FCR by 9·79% in broilers. The result supports the use of B. subtilis CH16 of chicken intestinal origin as a feed supplement that promote weight gain in broilers. Significance and impact of the study: This study reports screening of Bacillus strains isolated from chicken gastrointestinal tracts for development of a feed supplement that promote weight gain in broilers. Of the seven Bacillus isolates with high sporulation efficiency (≥90%), Bacillus subtilis CH16 strain showed the best growth and biofilm formation at body temperature of broilers (42°C). In large-scale trials in broilers (n ≥ 1150 per group), CH16 spores induced a 9·17% increase in daily weight gain (ADG) and a 9·79% reduction in FCR while the commercial BioPlus(®) YC induced only a 5·89% increase in ADG and a 3·88% reduction in FCR.