Host-controlled modification and restriction in Bacillus subtilis: Bsu 168-system and BsuR-system in B. subtilis 168.

Summary A Bsu168-specific restriction deficient (r ₁₆₈ ^- ) mutant of Bacillus subtilis Marburg 168 was transformed to be BsuR-specific restriction proficient (r _R ⁺ ) with B. subtilis R DNA as efficiently as the Bsu168-specific restriction proficient (r ₁₆₈ ⁺ ) parental strain (hsrM ⁺, hsdR ^-).We constructed r _R ⁺ m _R ⁺ r ₁₆₈ ⁺ m ₁₆₈ ⁺ strain (ISMR 4), r _R ⁺ m _R ⁺ r ₁₆₈ ^- m ₁₆₈ ⁺ strain (ISR 11) and r _R ⁺ m _R ⁺ r ₁₆₈ ^- m ₁₆₈ ^- strain (ISR 6) from strain 101 (r ₁₆₈ ⁺ m ₁₆₈ ⁺ ), strain 1012 (r ₁₆₈ ^- m ₁₆₈ ⁺ ) and strain RM125 (r ₁₆₈ ^- m ₁₆₈ ^- ), respectively by transformation with B. subtilis R DNA, and tested their restriction and modification activities on phage 105C. The results show that the sites recognized by Bsu168-specific restriction and modification enzymes and the sites recognized by BsuR-specific ones are not overlapping.We conclude that the Bsu168-modification and restriction system and the BsuR-modification and restriction system are controlled independently by two distinct sets of genes in the r _R ⁺ m _R ⁺ transformant of r ₁₆₈ ⁺ m ₁₆₈ ⁺ strain B. subtilis 168.     

极端耐热木聚糖酶基因在枯草芽孢杆菌中的整合表达

目的:为了在枯草芽孢杆菌中整合表达极端耐热木聚糖酶。方法:将嗜热网球菌(Dictyoglomus thermophilum)Rt46B.1的极端耐热木聚糖酶基因xynB通过穿梭载体pDL整合到B.subtilis168染色体上,使其实现表达。结果:极端耐热木聚糖基因在枯草芽孢杆菌中成功整合并表达。结论:基因工程菌B.subtilis168-xynB能外泌表达极端耐热木聚糖酶,且表达水平为0.732IU/mL,比在大肠杆菌中的高。酶学性质表明,此酶分子量约为24kD,其最适反应温度为85℃,最适反应pH值为6.5,且在弱碱性条件下稳定。     

Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168.

Summary Recombinant plasmids composed of Bacillus subtilis 168 leucine genes and a B. subtilis (natto) plasmid have been constructed in a recombination deficient (recE4) mutant of Bacillus subtilis 168. The process involved EcoRI fragmentation and ligation of a B. subtilis (natto) plasmid and a composite plasmid RSF2124-B · leu in which B. subtilis 168 leucine genes are linked to the R-factor RSF2124. A constructed plasmid (pLS102) was found to be composed of an EcoRI fragment derived from the vector plasmid and two tandemly repeated EcoRI fragments carrying the leucine genes. A derivative plasmid (pLS101 or pLS103) consisting of one molecule each of the EcoRI fragments was obtained by in vivo intramolecular recombination between the repeated leucine gene fragments in pLS102. pLS103 was cleaved once with BamNI, SmaI and HpaI. Insertion of foreign DNA (Escherichia coli plasmid pBR322) into the BamNI site inactivated leuA but not the leuC function which thus can serve as selective marker if the plasmid is used as vector in molecular cloning. The penicillin resistance carried in pBR322 was not functionally expressed in B. subtilis cells. By partial digestion of pLS103 with HindIII followed by ligation with T4-induced ligase, pLS107 was obtained which contained only one EcoRI site. However, insertion of exogenous DNA (pBR322) into this EcoRI site inactivated both leuA and leuC functions.     

Autolytic enzyme-deficient mutants of Bacillus subtilis 168.

Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. These mutants grow at normal rates as very long chains of unseparated cells. The length of the chains is directly related to the growth rates. They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology. Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter. Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6. Likewise, Microcococcus luteus walls at pH 5.6 and B. subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts. Thus, the activity of both autolytic enzymes in the mutants is depressed. The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation. The mutants did not produce more protease or amylase than did the wild type. They sporulate and the spores germinate normally. The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type. The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.     

Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport.

Cd2+ and Mn2+ accumulation was studied with wild-type Bacillus subtilis 168 and a Cd2+-resistant mutant. After 5 min of incubation in the presence of 0.1 microM 109Cd2+ or 54Mn2+, both strains accumulated comparable amounts of 54Mn2+, while the sensitive cells accumulated three times more 109Cd2+ than the Cd2+-resistant cells did. Both 54Mn2+ and 109Cd2+ uptake, which apparently occur by the same transport system, demonstrated cation specificity; 20 microM Mn2+ or Cd2+ (but not Zn2+) inhibited the uptake of 0.1 microM 109Cd2+ or 54Mn2+. 54Mn2+ and 109Cd2+ uptake was energy dependent and temperature sensitive, but 109Cd2+ uptake in the Cd2+-resistant strain was only partially inhibited by an uncoupler or by a decrease in temperature. 109Cd2+ uptake in the sensitive strain followed Michaelis-Menten kinetics with a Km of 1.8 microM Cd2+ and a Vmax of 1.5 mumol/min X g (dry weight); 109Cd2+ uptake in the Cd2+-resistant strain was not saturable. The apparent Km value for the saturable component of 109Cd2+ uptake by the Cd2+-resistant strain was very similar to that of the sensitive strain, but the Vmax was 25 times lower than the Vmax for the sensitive strain. The Km and Vmax for 54Mn2+ uptake by both strains were very similar. Cd2+ inhibition of 54Mn2+ uptake had an apparent Ki of 3.4 and 21.5 microM Cd2+ for the sensitive and Cd2+-resistant strains, respectively. Mn2+ had an apparent Ki of 1.2 microM Mn2+ for inhibition of 109Cd2+ uptake by the sensitive strain, but the Cd2+-resistant strain had no defined Ki value for inhibition of Cd2+ uptake by Mn2+.     

Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy

L-asparaginase (EC 3.5.1.1, ASN) exhibits great commercial value due to its uses in the food and medicine industry. In this study, we reported the enhanced expression of type II ASN from Bacillus subtilis 168 in B. subtilis WB600 through a combined strategy. First, eight signal peptides (the signal peptide of the ASN, ywbN, yvgO, amyE, oppA, vpr, lipA, and wapA) were used for ASN secretion in B. subtilis by using Hpa II promoter, respectively. The signal peptide wapA achieved the highest extracellular ASN activity (28.91 U/mL). Second, Hpa II promoter was replaced by a strong promoter, P43 promoter, resulting in 38.1 % enhanced ASN activity. By two rounds of error-prone PCR mutation, the P43 promoter variants with remarkably enhanced strength (D7, E2, H6, B2, and F3) were identified. B2 (−28: A → G, −13: A → G) achieved ASN activity up to 51.13 U/mL. Third, after deletion of the N-terminal 25-residues, ASN activity reached 102.41 U/mL, which was 100 % higher than that of the intact ASN. At last, the extracellular ASN of the B. subtilis arrived at 407.6 U/mL (2.5 g/L of ASN protein) in a 3-L bioreactor by using a fed-batch strategy. The purified ASN showed maximal activity at 65 °C and its half-life at 65 °C was 61 min. The K _m and k _cat of the ASN were 5.29 mM and 54.4 s⁻¹, respectively. To the best of our knowledge, we obtained the highest yield of ASN in a food-grade host ever reported, which may benefit the industrial production and application of ASN.

     

Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis

A novel phytase gene ( phyL) was cloned from Bacillus licheniformis by multiple steps of degenerate and inverse PCR. The coding region of the phyL gene was 1,146 bp in size and a promoter region of approximately 300 bp was identified at the upstream sequence. This gene, together with a phytase gene ( 168phyA) identified in the B. subtilis strain 168 genome by a homology search, was cloned and over-expressed in B. subtilis using a phi105MU331 prophage vector system. Up to 35 units of phytase/ml were secreted into the culture media; and mature enzymes of around 44-47 kDa were purified for characterization. Both phytases exhibited broad temperature and pH optima and showed high thermostability. Of the two, the phytase encoded by phyL exhibited higher thermostability, even at a lower calcium concentration, as it was able to recover 80% of its original activity after denaturation at 95 degrees C for 10 min. With their neutral pH optima and good temperature stabilities, these Bacillus phytases are good candidates for animal feed applications and transgenic studies.     

Protein synthesis in Bacillus subtilis. I. Hydrodynamics and in vitro functional properties of ribosomes from B. subtilis W168.

On the basis of their sedimentation properties, the ribosomal particles in crude extracts of Bacillus subtilis W168 are characterized as pressure-sensitive couples, pressure-resistant couples, or non-associating subunits. Pressure-sensitive couples dissociate into subunits, yielding a peak at 60 S in the gradient profile, on sedimentation at high speed in the presence of 10 to 15 mm-Mg²⁺. Under the same conditions, pressure-resistant couples sediment at 70 S. Under certain conditions, pressure-resistant couples apparently aggregate, possibly in 70 S · 70 S dimers. Procedures are described for the isolation of pressure-sensitive couples from B. subtilis. The isolated couples are shown by chemical fixation experiments to require approximately twice the Mg²⁺ concentration required by Escherichia coli couples to remain associated at atmospheric pressure.All three types of B. subtilis ribosome incorporate amino acids into acid-insoluble material in the presence of B. subtilis cellular RNA, B. subtilis ribosomal salt wash fraction, and E. coli post-ribosomal supernatant. Overall incorporation, dependence on added RNA, and dependence on salt wash fraction are greatest with pressure-sensitive couples. The products of protein synthesis in vitro stimulated by total B. subtilis RNA appear to be a low molecular weight subset of the proteins synthesized most abundantly in vivo. Incubation of pressure-sensitive couples with cellular RNA from B. subtilis, fMet-tRNA_f^Met, ribosomal salt wash fraction and GTP results in their conversion to pressure-resistant couples, with concomitant and stoichiometric binding of fMet-tRNA to the 70 S species. It is concluded that in B. subtilis as in E. coli, pressure-sensitive couples are “vacant”, while pressure-resistant couples are “complexed” with messenger RNA. fMet-tRNA-bearing complexed couples are interpreted as initiation complexes in which ribosomes have bound mRNA, presumably at initiation sites. Their formation in vitro is strictly dependent on RNA, salt wash fraction and fMet-tRNA when vacant ribosomal couples are used.     

Bidirectional chromosome replication in Bacillus subtilis 168.

Density transfer analysis of deoxyribonucleic acid from Bacillus subtilis 168 thy spores germinating in 5-bromouracil medium shows the order of replication of genetic markers to be: purA16, cysA14, sacA, ctrA, (narB, arol), dal, (hisA1, purB6), (tre-12, thr-5), (argA, aroG, argC4), (metC, leu-8, pheA), (ura-1, aroD), lys-1, (trpC, metB, ilvA, citB, citK, gltA). The precise order of transfer of markers within parentheses could not be determined in these experiments. Taken together with new PBS1 transduction data presented here and in the accompanying paper of J. Lepesant-Kejzlarová, J.-A. Lepesant, J. Walle, A. Billaut, and R. Dedonder (1975), the results can be resolved in terms of a symmetric, fully bidirectional mode of chromosome replication with a replication origin close to the purA16 marker and a terminus in the region of the gltA, citK loci, diametrically opposed to the origin. A new genetic map of the B. subtilis 168 chromosome is presented.     

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r^-m^-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec ⁺ strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r⁺ or r^- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r^-m^- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.     

Isolation of acetyl esterase mutants of Bacillus subtilis 168.

Five mutants of Bacillus subtilis 168 defective in an intracellular esterase activity were identified. By polyacrylamide gel electrophoresis, four of the mutants were shown to lack esterase B activity, and the fifth lacked esterase A activity. All of the back-crossed esterase mutants were able to sporulate at wild-type frequency and produce exoprotease(s) and antibiotic(s). No difference in motility could be attributed to the esterase mutation. PBS1 transduction analysis showed all the esterase B mutations to be linked to the hisA marker.     

Structural differentiation of the Bacillus subtilis 168 cell wall.

Exponential-growth-phase cultures of Bacillus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on the cell surface is indicative of regions of extreme hydrolytic activity in which alpha-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially growing B. subtilis cells contains regions of structural differentiation.     

Potassium Content During Growth and Sporulation in Bacillus subtilis

Vegetative and sporulating cells of Bacillus subtilis retain a higher level of internal potassium than do nonsporulating stationary-phase cells. The addition of manganese to nonsporulating stationary-phase cells, at concentrations required for sporulation, rapidly stimulates uptake and net accumulation of potassium and induces sporulation.