Kinetics of Initiation of Germination of Bacillus pumilus Spores by Hydrostatic Pressure

The kinetics of initiation of germination and inactivation by hydrostatic pressure of phosphate-buffered Bacillus pumilus spores is shown to be a consecutive first-order process at 25 C. The effect of increasing pressure at constant temperature was studied, and rate constants were derived by using the criteria of heat resistance, refractility, and stainability. The calculated volume change of activation (DeltaVdouble dagger) was -139 +/- 6 cm(3)/mole for loss of heat resistance, -158 +/- 8 cm(3)/mole for the loss of refractility, and -153 +/- 4 cm(3)/mole for the change in permeability to dilute stains for the pressure range 800 to 1,010 atm at 25 C. It is suggested that the spore exists as a Donnan phase and that pressure triggers germination by influencing the equilibrium.     

Initiation of Germination and Inactivation of Bacillus pumilus Spores by Hydrostatic Pressure

The effect of hydrostatic pressures as high as 1,700 atm at 25 C on the heat and radiation resistance of Bacillus pumilus spores was studied. Phosphate-buffered spores were more sensitive to compression than spores suspended in distilled water. Measurements of the turbidity of suspensions, the viability, refractility, stainability, dry weight, and respiratory activity of spores, and calcium and dipicolinic acid release were made for different pressures and times. Initiation of germination occurred at pressures exceeding 500 atm and was the prerequisite for inactivation by compression. The rate of initiation increased with increasing pressure at constant temperature. This result is interpreted as a net decrease in the volume of the system during initiation as a result of increased solvation of the spore components.     

Characterization of Bacillus pumilus E601 Spores After Single Sublethal Gamma Irradiation Treatments

Eighteen survivor strains of Bacillus pumilus E601 have been isolated after single sublethal irradiation treatments with ⁶⁰Co. Primary isolation was based on the loss of motility and pellicle formation. However, with subsequent subcultivation, eight isolates reverted back to the standard of exhibiting motility and pellicle formation. Characteristics of the isolates include alterations in spore radiation resistance and in the amino acid requirements for spore germination and outgrowth. Other alterations in cultural and physiological characteristics were found. Three of the isolates were asporogenous.     

Candidate Genes That May Be Responsible for the Unusual Resistances Exhibited by Bacillus pumilus SAFR-032 Spores

The spores of several Bacillus species, including Bacillus pumilus SAFR-032 and B. safensis FO-36b, which were isolated from the spacecraft assembly facility at NASA''s Jet Propulsion Laboratory, are unusually resistant to UV radiation and hydrogen peroxide. In order to identify candidate genes that might be associated with these resistances, the whole genome of B. pumilus SAFR-032, and the draft genome of B. safensis FO-36b were compared in detail with the very closely related type strain B. pumilus ATCC7061^T. 170 genes are considered characteristic of SAFR-032, because they are absent from both FO-36b and ATCC7061^T. Forty of these SAFR-032 characteristic genes are entirely unique open reading frames. In addition, four genes are unique to the genomes of the resistant SAFR-032 and FO-36b. Fifty three genes involved in spore coat formation, regulation and germination, DNA repair, and peroxide resistance, are missing from all three genomes. The vast majority of these are cleanly deleted from their usual genomic context without any obvious replacement. Several DNA repair and peroxide resistance genes earlier reported to be unique to SAFR-032 are in fact shared with ATCC7061^T and no longer considered to be promising candidates for association with the elevated resistances. Instead, several SAFR-032 characteristic genes were identified, which along with one or more of the unique SAFR-032 genes may be responsible for the elevated resistances. These new candidates include five genes associated with DNA repair, namely, BPUM_0608 a helicase, BPUM_0652 an ATP binding protein, BPUM_0653 an endonuclease, BPUM_0656 a DNA cytosine-5- methyltransferase, and BPUM_3674 a DNA helicase. Three of these candidate genes are in immediate proximity of two conserved hypothetical proteins, BPUM_0654 and BPUM_0655 that are also absent from both FO-36b and ATCC7061^T. This cluster of five genes is considered to be an especially promising target for future experimental work.     

短小芽胞杆菌产碱性木聚糖酶发酵条件的研究

碱性木聚糖酶在碱性条件下催化水解木聚糖,广泛应用于造纸、纺织等领域.着重对短小芽胞杆菌M-11产碱性木聚糖酶的发酵条件进行初步的探索.研究了菌株的生长曲线、确定最佳接种龄为16 h、最佳接种量为1％;确定最适碳源浓度为7％、最适单一氮源为氯化铵、其浓度为1.0％、最适无机盐为氯化铁、其浓度为3 mmol/L;在此基础之上进行6因素3水平的正交试验,确定最适产酶培养基组成:麸皮5％,接种量3％,氯化铵1.2％,氯化铁3.5 mmol/L,硫酸镁0.03％,氯化钠5 mmol/L,磷酸氢二钾0.4％;最适培养条件:接种龄16 h,初始pH 8.0,温度37℃,300 mL摇瓶装液量50 mL,摇床转速220 r/min,发酵周期48 h.通过对发酵条件的优化使发酵液酶活达613 IU/mL.无机氮源为其最适氮源,因此短小芽胞杆菌M-11在碱性木聚糖酶的产品开发上优于短小芽胞杆菌M -26.     

Mannanase Components from Bacillus pumilus

beta-d-Mannanase from Bacillus pumilus was purified into two components, A and B, which exhibited homogeneity on polyacrylamide gel electrophoresis. These components had molecular weights of 55,000 (A) and 37,000 (B); carbohydrate contents of 15.3% (A) and 7.2% (B); specific activities of 78 (A) and 1,616 (B) U/mg; pH optima of 5.5 to 6.9 (A) and 6.0 (B); and half-lives of 60 (A) and 21 (B) min at 70 degrees C.     

短小芽孢杆菌的遗传转导

短小芽孢杆菌(Bacillus pumilus)噬菌体PP5在碱性蛋白酶生产菌珠B.pumilus 289中能进行普遍性转导。PP5对于B.pumilus 289的营养标记的转导频率为10~(-6)转导子/PFU。对于B.pumilus 1037和B.pumilus 289之间的链霉素抗性标记的转导频率为10~(-4)至10~(-7)转导子/PFU。从而建立了一个新的B.pumilus遗传转导系统。     

Differentiation of Bacillus pumilus and Bacillus safensis Using MALDI-TOF-MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.     

Extracellular ribonuclease from Bacillus pumilus]

The extracellular ribonuclease (RNAse Bp) was isolated from the cultural medium filtrate of Bacillus pumilus by ammonium sulfate precipitation and two stages of ion-exchange chromatography on carboxymethyl- and phospho-cellulose columns. The amino acid composition and N-terminal amino acid residue have been determined. The kinetic parameters of cleavage reaction of synthetic polynucleotides have been measured. According to their structural homology RNAse Bp has been shown to be similar to RNAses Ba and Bi. Catalytic properties of the enzyme are very close to RNAse Bi.     

Sporulation-converting bacteriophages for Bacillus pumilus.

Thirty-three sporulation-converting bacteriophages for Bacillus pumilus NRS576 were assigned to two apparently unrelated groups on the basis of morphology and antiserum neutralization. Bacterial sporulation mutants responded similarly (conversion or nonconversion) to representatives of both phage groups. Evidence is presented indicating that PMB1 and related phages specify a restriction and/or modification system.     

Peptide Synthesis by Extracts from Bacillus subtilis Spores

Cell-free peptide synthesis by extracts from vegetative cells and spores of Bacillus subtilis was analyzed and compared. The initial rate of phenylalanine incorporation in a polyuridylate-directed system was found to be in a similar range for the two extracts. However, spore extracts frequently incorporated less total phenylalanine as did the vegetative cell system. Optimal conditions for amino acid incorporation by spore extracts were found to be similar to those of vegetative cell extracts. Polyphenylalanine synthesis was stimulated by preincubation of both extracts prior to the addition of polyuridylic acid (poly U) and labeled phenylalanine. Both systems showed a dependence on an energy-generating system and were inhibited by chloramphenicol and puromycin. Ribonuclease, but not deoxyribonuclease, inhibited the reaction significantly. The presence of methionine transfer ribonucleic acid (tRNA(F)) and methionyl-tRNA(F) transformylase was demonstrated in spore extracts. An analysis of several aminoacyl-tRNAs in spores revealed that the relative amounts of these tRNAs were similar to those found in vegetative cells. Only lysine tRNA was found to be present in relatively greater amounts in spores. These results indicate that dormant spores of B. subtilis contain the machinery for the translation of genetic information.     

An efficient cyanide-degrading Bacillus pumilus strain

A Gram-positive, aerobic, endospore-forming bacterium was isolated by an enrichment technique for the ability to degrade cyanide and was identified as a Bacillus pumilus strain. The bacterium rapidly degraded 100 mg l-1 of free cyanide in the absence of added inorganic and organic substances. The ability to degrade cyanide was linked to the growth phase and was not exhibited before late exponential/early stationary phase. Cyanide-degrading activity could not be induced before this time by the addition of 20 mg cyanide l-1. Production of the cyanide-degrading activity required 0.01 mg Mn2+ l-1 and did not occur at Mn2+ concentrations below 0.002 mg l-1. Cyanide-degrading activity was intracellular and cell-free extracts rapidly degraded cyanide.     

利用生物可降解海藻酸钙微球保护苏云金杆菌晶体与芽胞免受紫外线降解

采用基于注射挤压器的液滴形成技术制备包裹了苏云金杆菌晶体和芽胞的海藻酸钙凝胶微球.通过调节该装置的活塞重量和空气压力,获得了平均直径为20μm的微球.SDS-PAGE分析与平板菌落涂布实验表明,凝胶微球可有效减少紫外线对苏云金杆菌晶体和芽胞的损伤作用.利用小菜蛾进行的毒力生测发现,凝胶微球可有效防止紫外线引起的晶体和芽胞杀虫毒力的下降.本研究的液滴形成技术也可适用于其它微球包裹过程.     

Genetic Analysis in Bacillus pumilus by PBS1-Mediated Transduction

Bacteriophage PBS1 mediates generalized transduction in Bacillus pumilus NRRL B-3275 (BpB1). Transduction frequencies for single auxotrophic markers are of the order of 10(-4) transductants per plaque-forming unit in crude phage lysates. The characteristics of PBS1 propagated on BpB1 and the properties of the system of transduction are similar to those reported for PBS1 propagated on Bacillus subtilis. By transduction, eight amino acid auxotrophic markers in BpB1 have been oriented into two linkage groups. One group contains the auxotrophic markers arginine A, leucine, and phenylalanine, and the other group contains the markers lysine, serine, tryptophan, isoleucine-valine, and isoleucine. The nature and relative order of the markers within each linkage group suggest that the arrangement of genes in these areas of the chromosome of BpB1 is similar to the arrangement of phenotypically comparable genes in two linkage groups (defined by PBS1 transduction) in B. subtilis. However, transduction of any of the above cited markers in BpB1 to prototrophy with PBS1 propagated on B. subtilis 168 could not be demonstrated. In addition to BpB1, seven other strains of B. pumilus can be infected with PBS1. Transduction has been demonstrated in three of these strains.     

Biochemical Studies of Two Bacillus pumilus Plasmids

Bacillus pumilus NRS 576 harbored an estimated two copies per chromosome of a covalently closed, circular (CCC) deoxyribonucleic acid (DNA) molecule, the 576 plasmid. The 576 plasmid has a buoyant density of 1.698 g/cm(3) and a molecular weight of about 28 x 10(6). Plasmid copy number remained about the same in both exponentially growing and stationary-phase cells. Spontaneous variants of NRS 576 that formed spores at an elevated frequency were designated as W mutants. W mutants appeared to have lost the 576 plasmid on the basis of the following: W mutants (38 tested) lacked detectable CCC DNA, and the majority of the plasmid homologous sequences in bulk NRS 576 DNA were absent from bulk W mutant DNA. B. pumilus ATCC 7065 harbored at least 10 copies per chromosome of a CCC DNA element, the 7065 plasmid. The 7065 plasmid has a buoyant density of 1.696 g/cm(3) and a molecular weight of about 6 x 10(6). Although the copy number of the plasmid appeared to remain the same in exponentially growing and stationary-phase cells, an additional CCC form of higher molecular weight was detected in stationary-phase cells.     

Increased Radiation Resistance of Vegetative Bacillus pumilus

A 4.5-fold increase in vegetative cell radiation resistance of Bacillus pumilus E601, the internationally recognized biological standard for irradiation sterilization, was obtained by the repeated passage of resistant survivors through successive sublethal doses of (60)Co irradiation. This increase in resistance was accompanied by a corresponding increase in spore resistance through the seventh irradiation passage. By the fifteenth passage, the ability for spore formation was lost. Other effects noted by the successive irradiation dosages included loss of motility and pellicle formation, and changes in the Gram reaction, cell morphology, and colonial morphology. Increased resistance was also accompanied by an increased nutritional requirement for specific amino acids. Radiation resistance was not transferred from vegetative cells to spores.     

Improving the catalytic efficiency of Bacillus pumilus CotA-laccase by site-directed mutagenesis

Bacterial laccases are potential enzymes for biotechnological applications because of their remarkable advantages, such as broad substrate spectrum, various reactions, high thermostability, wide pH range, and resistance to strongly alkaline environments. However, the use of bacterial laccases for industrialized applications is limited because of their low expression level and catalytic efficiency. In this study, CotA, a bacterial laccase from Bacillus pumilus, was engineered through presumptive reasoning and rational design approaches to overcome low catalytic efficiency and thermostability. L386W/G417L, a CotA double-mutant, was constructed through site-directed mutagenesis. The catalytic efficiency of L386W/G417L was 4.3 fold higher than that of wild-type CotA-laccase, but the thermostability of the former was decreased than that of the latter and other mutants. The half-life (t _1/2) of wild-type and G417L were 1.14 and 1.47 h, but the half-life of L386W/G417L was only 0.37 h when incubating the enzyme at 80 °C. Considering the high catalytic efficiency of L386W/G417L, we constructed L386W/G417L/G57F, another mutant, to improve thermostability. Results showed that the half-life of L386W/G417L/G57F was 0.54 h when incubating the enzyme at 90 °C for 2 h with about 34% residual activity, but the residual activity of L386W/G417L was less than 40% when incubating the enzyme at 90 °C for 5 min. L386W/G417L was more efficient in decolorizing various industrial dyes at pH 10 than other mutants. L386W/G417L/G57F also exhibited an efficient decolorization ability. L386W/G417L/G57F is appropriate for biotechnological applications because of its high activity and thermostability in decolorizing industrial dyes. CotA-laccase may be further subjected to molecular modification and be used as an enhancer to improve decolorization efficiency for the physical and chemical treatment of dye wastewater.