Co-expression of a prophage system and a plasmid system in Bacillus subtilis

A dual expression system for overexpressing two proteins by a single cell strain has been developed in Bacillus subtilis. This dual expression system combines the phi105MU331 prophage system and a plasmid system within a single cell. Protein expression by the prophage system is heat inducible, while that of the plasmid system is constitutive. Three candidate genes, BPN, BT, and amyE, all of Bacillus origin, were used as test models. Seven strains (BPN, BT, AMY, BS168K, MU331K, BPNK, and BTK) were constructed to investigate the influences of the prophage system and the plasmid system on each other, and to compare the efficiency of the individual expression systems with that of the dual expression system. Individually, the yield of the plasmid system is higher than that of the prophage system, which could be attributed to the constitutive nature of the expression of the plasmid system. Nonetheless, for the dual expression strains, the expression of two enzymes in a single fermentation run can reduce costs in facilities, manpower, and utilities. Fed-batch fermentation of BPNK strains confirmed the feasibility of applying this dual expression system in industrial-scale production.     

Characterization of chromosome and plasmid transformation in Bacillus subtilis using gently lysed protoplasts

Competent cells of Bacillus subtilis were transformed with DNA from gently lysed protoplasts. Significant linkages among markers separated by distances of approximately 2.3% of the total chromosome were found, which have not been detected for conventional transformation. In comparison to previous reports, enhanced plasmid transformation was observed [4.0×10⁷ transformants per g DNA (one transformant per 5×10⁴ molecules added)], when competent cells were transformed with DNA from lysed protoplasts harboring pUB110.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Construction and characterization of a novel maltose inducible expression vector in Bacillus subtilis

A maltose-inducible expression vector in Bacillus subtilis has been developed and characterized. The vector permitted β-galactosidase expression at a high level (maximum activity, 8.16 U/ml) when induced and its expression was markedly repressed by glucose. Using this vector, we successfully expressed the other two genes, bioA and vgb. This thus provided a potential expression system for cloned genes in B. subtilis.     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.     

Design of an expression vector and its application to heterologous protein expression in Bacillus subtilis

An expression vector, pUBEX, was constructed for extracellular production of heterologous proteins in Bacillus subtilis using a polyhistidine tag on the C-terminal sequence, providing an efficient and easy purification of the protein. A CII protein, a member of Bowman–Birk protease inhibitors, which was expressed as an inactive protein in Escherichia coli, was successfully expressed in Bacillus subtilis using the pUBEX vector and was purified to 6.4 mg l^–1 by the immobilized metal affinity chromatography.     

An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

Background

The Bacillus subtilis genome (BGM) vector is a novel cloning system based on the natural competence that enables B. subtilis to import extracellular DNA fragments into the cell and incorporate the recombinogenic DNA into the genome vector by homologous recombination. The BGM vector system has several attractive properties, such as a megabase cloning capacity, stable propagation of cloned DNA inserts, and various modification strategies using RecA-mediated homologous recombination. However, the endogenous RecA activity may cause undesirable recombination, as has been observed in yeast artificial chromosome systems. In this study, we developed a novel BGM vector system of an inducible recA expression BGM vector (iREX), in which the expression of recA can be controlled by xylose in the medium.

Results

We constructed the iREX system by introducing the xylose-inducible recA expression cassette followed by the targeted deletion of the endogenous recA. Western blot analysis showed that the expression of recA was strictly controlled by xylose in the medium. In the absence of xylose, recA was not expressed in the iREX, and the RecA-mediated recombination reactions were greatly suppressed. By contrast, the addition of xylose successfully induced RecA expression, which enabled the iREX to exploit the same capacities of transformation and gene modifications observed with the conventional BGM vector. In addition, an evaluation of the stability of the cloned DNA insert demonstrated that the DNA fragments containing homologous sequences were more stably maintained in the iREX by suppressing undesirable homologous recombination.

Conclusions

We developed a novel BGM vector with inducible recA expression system, iREX, which enables us to manipulate large DNA fragments more stably than the conventional BGM vector by suppressing undesirable recombination. In addition, we demonstrate that the iREX can be applied to handling the DNA, which has several homologous sequences, such as multiple-reporter expression cassettes. Thus, the iREX expands the utility of the BGM vector as a platform for engineering large DNA fragments.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1425-4) contains supplementary material, which is available to authorized users.     

FT-IR spectroscopic characteristics of differently cultivated Bacillus subtilis

Bacillus subtilis is an aerobic endospore forming bacterium widely spread in different environments. Because it represents a biological agent of some health relevance, its rapid detection and identification is highly desirable. By using FT-IR spectroscopy for this purpose slightly different characteristics were obtained from cell mass grown in differently composed cultural media, and harvested in different phases of bacterial growth. If cultivated uniformly, i.e., 24h at 30 degrees C in a minimum-strength nutrient broth, cell mass of B. subtilis delivered a well differentiated spectrum with major absorption bands of nucleic acid structures at 3300cm(-1), cell wall constituents at 3000-2800cm(-1), proteinaceous structures at 1660, 1544 and 1235cm(-1), and some aliphatic structural units at 1080cm(-1). Attenuated total reflectance, and absorption/transmission scanning techniques, delivered structurally identical spectra but those obtained by the former technique were more expressed.     

Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

A Bacillus amyloliquefaciens neutral protease gene was cloned and expressed in Bacillus subtilis.The chromosomal DNA of B. amyloliquefaciens strain F was partially digested with restriction endonuclease Sau3AI, and 2 to 9 kb fragments isolated were ligated into the BamHI site of plasmid pUB110. Then, B. subtilis strain 1A289 was transformed with the hybrid plasmids by the method of protoplast transformation and kanamycin-resistant transformants were screened for the formation of large halo on a casein plate. A transformant that produced a large amount of an extracellular neutral protease harbored a plasmid, designated as pNP150, which contained a 1.7 kb insert.The secreted neutral protease of the transformant was found to be indistinguishable from that of DNA donor strain B. amyloliquefaciens by double immunodiffusion test and SDS-polyacrylamide gel electrophoresis.The amount of the neutral protease activity excreted into culture medium by the B. subtilis transformed with pNP150 was about 50-fold higher than that secreted by B. amyloliquefaciens. The production of the neutral protease in the transformant was partially repressed by addition of glucose to the medium.     

The mmgA gene from Bacillus subtilis encodes a degradative acetoacetyl-CoA thiolase

Early in sporulation, the mother cell compartment of Bacillus subtilis transcribes the mother cell metabolic gene (mmg) operon. The gene mmgA was assigned by other workers using sequence homology as an acetyl-CoA acetyltransferase [E.C. 2.3.1.9]. The gene was overexpressed in Escherichia coli, and the protein was purified by Ni²⁺-affinity chromatography. However, the expected MmgA-catalyzed biosynthesis of acetoacetyl-CoA from acetyl-CoA was undetectable by a standard UV assay, HPLC, and mass spectrometry. These methods indicated a preference for the reverse degradative thiolytic reaction, with a k _cat of 80 s⁻¹, and a K _m of 70 and 50 μM for CoA and acetoacetyl-CoA, respectively.     

Plasmid transfer in bacilli by a self-transmissible plasmid p19 from a Bacillus subtilis soil strain

The cryptic 95-kb plasmid p19 of the Bacillus subtilis 19 soil strain promotes the transfer of a small kanamycin resistance plasmid pUB110. To facilitate direct selection for p19 transfer, a plasmid derivative carrying the chloramphenicol resistance gene was constructed. The frequency of transfer of the large plasmid between cells of B. subtilis 19 approached 100% but was more than two orders of magnitude lower when the strain B. subtilis 168 was a recipient. However, when the restriction-deficient strain B. subtilis 168 was a recipient, the transfer efficiency was almost completely recovered. The effectiveness of pUB110 mobilization was virtually not altered in all these cases. pC194 was not mobilized by p19. The kinetics of p19 conjugative transfer is also presented.     

基因串联策略强化枯草芽孢杆菌终止子的功能及其在基因表达中的应用

在底盘微生物中使用高性能终止子能够显著增强基因终止效率,维持新合成mRNA的稳定性,提升外源基因表达性能.而目前缺乏专一用于枯草芽孢杆菌的终止子元件,限制了复杂功能基因电路的设计.[目的]在枯草芽孢杆菌中挖掘新的高性能终止子,并进一步重新设计,丰富适用于这一底盘的高性能人工终止子.[方法]将枯草芽孢杆菌终止子和枯草芽孢...     

Low copy number plasmid vectors for gene cloning and for monitoring gene expression

Abstract Derivatives of an IncW incompatibility plasmid with a low copy number are described which can be used for gene cloning or for analysing gene expression in conditions similar to those found in the host chromosome. Gene expression can be monitored after construction of operon or protein fusions with the lacZYA operon and measurement in Escherichia coli of the β-galactosidase activity.     

Subspecies-specific distribution of intervening sequences in the Bacillus subtilis prophage ribonucleotide reductase genes

A collection of 212 gram-positive bacilli isolated from natural habitats was screened for the presence of intervening sequences (introns and intein-coding sequences) in the SPbeta prophage-related ribonucleotide reductase genes bnrdE and bnrdF. Three novel configurations were identified on the basis of the presence of (i) intervening sequences in bnrdE and bnrdF, and (ii) an ORF in the bnrdE-bnrdF spacer. Analysis of the cell wall genetic determinants as well as of the incorporation of radio-labelled glycerol into cell wall allowed newly and previously identified B. subtilis strains with different configurations of bnrdE/bnrdF intervening sequences to be assigned to one of two subspecies. Strains apparently belonging to the subsp. subtilis contain three intervening sequences many of which are associated with the putative homing endonuclease activity. Strains of the subsp. spizizenii contain only one or two ORF-less group I introns. Introns occupying bnrdF are confined to the subspecies subtilis.     

Efficient secretory expression of an alkaline pectate lyase gene from Bacillus subtilis in E. coli and the purification and characterization of the protein

The gene encoding pectate lyase (PL) from Bacillus subtilis WSHB04-02 was amplified by PCR, fused with a periplasmic secretion signal peptide sequence, pelB, from pET22b(+), cloned and expressed in Escherichia coli cells using a temperature control vector, pHsh. The recombinant E. coil was grown in a 5 l fermentor. PL was secreted in broth at 22 U l⁻¹ after 20 h when temperature was increased from 30°C to 42°C. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. It was optimally active at pH 9.4 and 50°C over 30 min. Analysis of polygalacturonic acid (PGA) degradation products by electrospray ionization (ESI)-mass spectrometry (MS) indicated that PL produced a mixture of unsaturated oligo-galacturonides including unsaturated tri-galacturonic acid and unsaturated bi-galacturonic acid but not unsaturated mono-galacturonic acid.     

Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis

We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene (DeltaaprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the DeltaaprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30 degrees C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of DeltaaprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins.