Bacillus subtilis is a generally recognized as safe (GRAS) strain that has been widely used in industries including fodder, food, and biological control. In addition, B. subtilis expression system also plays a significant role in the production of industrial enzymes. However, its application is limited by its low sporulation frequency and transformation efficiency. Immense studies have been done on interpreting the molecular mechanisms of sporulation and competence development, whereas only few of them were focused on improving sporulation frequency and transformation efficiency of B. subtilis by genetic modification. The main challenge is that sporulation and competence development, as the two major developmental events in the stationary phase of B. subtilis, are regulated by the complicated intracellular genetic regulatory systems. In addition, mutual regulatory mechanisms also exist in these two developmental events. With the development of genetic and metabolic engineering, constructing genetic regulatory networks is currently one of the most attractive research fields, together with the genetic information of cell growth, metabolism, and development, to guide the industrial application. In this review, the mechanisms of sporulation and competence development of B. subtilis, their interactions, and the genetic regulation of cell growth were interpreted. In addition, the roles of these regulatory networks in guiding basic and applied research of B. subtilis and its related species were discussed.
Bacillus subtilis ; genetics ; physiology ; Gene Regulatory Networks ; Metabolic Engineering ; Spores, Bacterial ; physiology

Metabolic engineering has become a powerful tool for optimization of industrial fermentation processes. Metabolic engineering usually undergoes three steps: construction of a recombinant strain with improved properties, genetic and biochemical analysis of the strain, and identification of target for further improvement. Metabolic fluxes analysis is an important part of the biochemical analysis. Based on the law of mass conservation and assuming pseudo-steady-state for the intermediates in the metabolic pathways, we have quantitatively analyzed the time course of the flux distribution in Bacillus subtilis and used the data to reveal the nature of the so-called "40 hour" phenomenon in fermentation of guanosine, a key raw material for the synthesis of additives for human consumption and animal feeds. The phenomenon refers to the observation that guanosine production, which proceeds at high rate from 12 hour on, declines around 40 hour while consumption of glucose keeps increasing, leading to the lower yield of the nucleoside. Equations based upon the metabolic network of Bacillus subtilis consisted of EMP pathway, HMP pathway, TCA cycle, oxidative phosphorylation pathway and others reactions of the intermediates, was constructed. The equations were solved by using the quantitative data obtained in this study. The air flow and volume, concentration of oxygen and carbon dioxide in the exit-gas were monitored online; the concentration of biomass, glucose and guanosine was analyzed manually; and the concentration of acetate, citric acid, pyruvate, and 17 amino acids were HPLC quantified. The solutions of the equation were proved to be valid, as the experimental data on oxygen consumption agrees with that of predicted form the equation. The results indicated that at 40h of the fermentation process the flux of HMP pathway, which provides the precursor of the nucleoside, decreased while that of EMP pathway and the pathways that generate amino acids and organic acids increased. The shift correlated with the accumulation of NH4+ in the broth. The assimilation of NH4+ is an energy consuming process and could shift the metabolism to the energy generating EMP pathway. Accordingly, measures were taken to prevent the accumulation of NH4+. The interference indeed stopped the metabolism shift and boosted the guanosine production at 30 g/L, 70% higher than the level reported in literature.
Bacillus subtilis ; metabolism ; Fermentation ; physiology ; Guanosine ; metabolism ; Models, Theoretical ; Quaternary Ammonium Compounds ; metabolism

The oxidative response of Bacillus sp F26 to different forms of reactive oxygen species (ROS) stress including H2O2, O2- * and OH * were investigated by using diverse generating source of ROS, which were characterized by synthesis of antioxidative enzymes. It was shown that the responses of cells to oxidative stress are largely dependent on species, mode (instantaneous and continual) and intensity of stress. Higher synthesis rate of catalase (CAT) is crucial for Bacillus sp F26 to resist H2O2 stress. The damage of H2O2 to cell was minor if CAT can efficiently decompose H2O2 entering into cell, furthermore, the response can stimulate cell growths and sugar consumption. Conversely, cell growth and synthesis of antioxidative enzymes are greatly inhibited when the intensity of H2O2 stress overwhelms the cell capability of clearing H2O2. Due to the difference in mode and effect on cells between O2- * and H2O2, higher synthesis rates of CAT and superoxide dismutase (SOD) couldn't guarantee cells to eliminate H2O2 and O2- * efficiently. Therefore, the toxicity to cells induced by intracellular O2- * is more severe than H2O2 stress. Unlike response to H2O2 and O2- *, OH stress significantly inhibited cell growth and synthesis of antioxidative enzymes due to the fact OH * is most active ROS. Our results indicated that Bacillus sp F26 will show diverse biological behaviour in response to H2O2, O2- * and OH * of stress due to the discrepancy in chemical property. In order to survive in oxidative stress, cells will timely adjust their metabolism to adapt to new environment including regulating synthesis level of antioxidative enzymes, changing rates of cells growth and substrate consumption.
Adaptation, Physiological ; Bacillus ; enzymology ; metabolism ; physiology ; Catalase ; biosynthesis ; Oxidative Stress ; physiology ; Reactive Oxygen Species ; analysis ; metabolism ; Superoxide Dismutase ; biosynthesis

To reveal the colonization characteristics in host of endophytic biocontrol bacteria NJ13 isolated from Panax ginseng, this study obtained the marked strain NJ13-R which was double antibiotic resistant to rifampicin and streptomycin through enhancing the method of inducing antibiotic. The colonization characteristics in ginseng and its biocontrol efficiency against Alternaria spot of ginseng in the field were studied. The results showed that the strain could colonize in root, stem and leaf of ginseng and the colonization amount was positive correlated with inoculation concentration. Meanwhile, the strain could infect and then transfer in different tissues of ginseng The colonization amount of strain in roots and leaves of ginseng increased first and then decreased. However, the tendency of colonization amount of strain in stems was ascend at first and then descend slowly, and was more than that in roots and leaves along with time, which had a preference to specific tissue of its host. In field experiment, the endophytic bacteria NJ13 was proved to be effective in controlling Alternaria leaf spot of ginseng. The biocontrol efficiency of fermentation broth at the concentration of 0.76 x 10(8) cfu x mL(-1) reached 75.62%, which was close to the controlling level (73.06%) of 0.67 mg x L(-1) 50% cyprodinil WG.
Alternaria ; physiology ; Antibiosis ; Bacillus ; growth & development ; isolation & purification ; physiology ; Endophytes ; growth & development ; isolation & purification ; physiology ; Panax ; growth & development ; microbiology ; Plant Diseases ; microbiology

Pasteuria penetrans will build-up faster where there is a high initial nematode density and can suppress root-knot nematode populations in the roots of tomato plants. The effect of different initial densities of nematode (Meloidogyne javanica) (150, 750, 1500, 3000) and P. penetrans infected females (F1, F3) densities (F0=control and AC=absolute control without nematode or P. penetrans inoculum) on the build-up of Pasteuria population was investigated over four crop cycles. Two major points of interest were highlighted. First, that within a confined soil volume, densities of P. penetrans can increase >100 times within 2 or 3 crop cycles. Second, from a relatively small amount of spore inoculum, infection of the host is very high. There were more infected females in the higher P. penetrans doses. The root growth data confirms the greater number of females in the controls particularly at the higher inoculum densities in the third and fourth crops. P. penetrans generally caused the fresh root weights to be higher than those in the control. P. penetrans has shown greater reduction of egg masses per plant at most densities. The effects of different initial densities of M. javanica and P. penetrans on the development of the pest and parasite populations were monitored. And no attempt was made to return the P. penetrans spores to the pots after each crop so the build-up in actual numbers of infected females and spores under natural conditions may be underestimated.
Animals ; Bacillus ; pathogenicity ; physiology ; Lycopersicon esculentum ; growth & development ; parasitology ; Pest Control, Biological ; methods ; Tylenchoidea ; growth & development ; microbiology ; Women

We studied the overproduction of catalase (CAT) by Bacillus sp.WSHDZ-01 by oxidative stress via the feeding of ethanol and the pulse addition of H2O2. By adding 2.0% (V/V) ethanol to the culture broth, the intracellular CAT activity reached 11 151 U/mL, which was 2.5 times than that of the control (4 450 U/mL in flask). By adding 0.3% (V/V) H2O2, more extracellular CAT secreted to the culture broth, and the ratio of extracellular CAT to the total CAT increased to 27%. Based on these results, an oxidative stress strategy combining the ethanol feeding and the pulse addition of H2O2 was developed. With this strategy, the ratio of extracellular CAT to the total CAT reached 82.5%, increased by 18.6% than that of the control (without ethanol and H2O2 addition). CAT production increased to 28 990 U/mL, which was 95.5% higher than the control (14 830 U/mL in 3 L fermentor). The fermentation time decreased to 42 h, which was much shorter than that of adding ethanol or H2O2, and CAT productivity reached 470 U/(mL x h) while the control achieved 396.4 U/(mL x h).
Bacillus subtilis ; drug effects ; enzymology ; physiology ; Catalase ; biosynthesis ; Culture Media ; pharmacology ; Ethanol ; pharmacology ; Hydrogen Peroxide ; pharmacology ; Oxidative Stress

OBJECTIVETo investigate the toxicity of indigenous Bacillus thuringiensis (B. thuringiensis)isolates from Malang City for controlling Aedes aegypti (Ae. aegypti) larvae.

METHODSSoil samples were taken from Purwantoro and Sawojajar sub-districts. Bacterial isolation was performed using B. thuringiensis selective media. Phenotypic characteristics of the isolates were obtained with the simple matching method. The growth and prevalence of spores were determined by the Total Plate Count method, and toxicity tests were also performed on the third instar larval stage of Ae. aegypti. The percentage of larval mortality was analysed using probit regression. The LC50 was analysed by ANOVA, and the Tukey HSD interval was 95%.

RESULTSAmong the 33 selected bacterial isolates, six were obtained (PWR4-31, PWR4-32, SWJ4-2b, SWJ4-4b, SWJ-4k and SWJ5-1) that had a similar phenotype to reference B. thuringiensis. Based on the dendrogram, all of the bacterial isolates were 71% similar. Three isolates that had a higher prevalence of reference B. thuringiensis were PWR4-32, SWJ4-4b and SW5-1, of which the spore prevalence was 52.44%, 23.59%, 34.46%, respectively. These three indigenous isolates from Malang City successfully killed Ae. aegypti larvae. The PWR4-32 isolates were the most effective at killing the larvae.

CONCLUSIONSSix indigenous B. thuringiensis isolates among the 33 bacterial isolates found in the Sawojajar and Purwantoro sub-districts were toxic to the third instar larvae of Ae. aegypti. The PWR4-32 isolates were identical to the reference B. thuringiensis and had 88% phenotype similarity. The PWR4-32 isolates had the highest spore prevalence (52.44%), and the early stationary phase occurred at 36 h. The PWR4-32 isolates were the most effective at killing Ae. aegypti larvae (LC50-72 h=2.3×10(8) cells/mL).

Aedes ; microbiology ; Animals ; Bacillus thuringiensis ; isolation & purification ; physiology ; Biological Control Agents ; Indonesia ; Insecticides ; Larva ; microbiology ; Lethal Dose 50 ; Mosquito Control

In this study， an endophytic bacteria strain BZJN1 was isolated from Atractylodes macrocephala， and identified as Bacillus subtilis by physiological and biochemical tests and molecular identification. Strain BZJN1 could inhibit the growth of mycelia of Ceratobasidium sp. significantly， and the inhibition rate was more than 70%. The mycelium growth deformity with bulge as spherical and partially exhaustible in apex or central with microscopic observation. The inhibitory rates under 3% and 6% concentrations of the cell free fermentation were 22.7% and 38.7% expectively. The field test proved that the control efficacy of treatment of 1×10⁸ cfu·mL⁻¹ is 75.27% and 72.37% after 10 and 20 days. All the treatments of strain BZJN1 was able to promote the growth of A. macrocephala， the treatment of 1×10⁸ cfu·mL⁻¹ could able to increase the yield to 14.1%.
Atractylodes ; microbiology ; Bacillus subtilis ; physiology ; Basidiomycota ; pathogenicity ; Biological Control Agents ; Endophytes ; classification ; isolation & purification ; Plant Diseases ; microbiology ; prevention & control

The Astragalus membranaceus root rot disease,a soil-borne disease,has become increasingly severe in Shanxi province.This study was aimed at getting antagonistic Bacillus with excellent bio-control effects,and determining its effects on bacterial communities in root zone soil. With Fusarium solani and F. acuminatum as the target,antagonistic Bacillus was selected through such tests as living body dual culture,antifungal effect of bacteria-free filtrate,mycelia growth inhibition in vitro and control effect in detached roots,and identified with morphology,physio-biochemical characteristics and 16 S r DNA sequence analysis. The results showed that the Bacillus strain SXKF16-1 had obvious antifungal effect. The diameter of inhibition zone of its bacteria-free filtrate to F. solani and F. acuminatum was( 25. 90±1. 18) mm and( 25. 86±1. 85) mm respectively,and showed a lasting inhibition effect to mycelia growth. The disease index of the protective treatment and that of the cure treatment in detached roots test to F. solani and F. acuminatum were( 37. 50±8. 58),( 41. 67±4. 90) and( 25. 00±8. 33),( 38. 89±9. 62) respectively,both being significantly different( P<0. 05) from that of the control. The strain SXKF16-1 was identified as Bacillus atrophaeus. The B. atrophaeus SXKF16-1 showed significantly inhibition effect to pathogen causing root rot and could increase the bacterial diversity in root zone soil. It has potential to be developed as a special biocontrol agent.
Astragalus Plant/microbiology* ; Bacillus/physiology* ; Biological Control Agents ; Fusarium/pathogenicity* ; Plant Diseases/prevention & control* ; Plant Roots/microbiology* ; Soil Microbiology

OBJECTIVETo explore the germination effects of Bacillus anthracoides spores germinant to nutrient germinant.

METHODSHeat factors and nutrient germinant were used to stimulate the Bacillus anthracoides spores and to germinate. Ultraviolet spectrophotometer was used to measured the A value of spore solution in the wavelength of 600 nm. Accrding to the A value, the germination rates in different condition. Transmission electron microscope was used to observe the ultrastructure changes of spores.

RESULTSThe rate of germination effects were 68.0% under 6 mmol/L inosine at 37 degrees C, pH 7.9; 74.5% under 70 mmol/L L-alanine at 30 degrees C, pH 8.9; and 85.6% under 6 mmol/L inosine and 70 mmol/L L-alanine at 37 degrees C, pH 8.2. Under transmission electron microscope, the germinated spores' coat and cortex were brokendown and degraded with its core completely exposed.

CONCLUSIONUnder suitable environment, the nutrient germinant with inosine and L-alanine might be helpful for germinating the bacillus anthracoides spores.

Alanine ; pharmacology ; Bacillus anthracis ; drug effects ; metabolism ; physiology ; Bacterial Proteins ; metabolism ; Culture Media ; Inosine ; pharmacology ; Spores, Bacterial ; drug effects ; metabolism ; physiology