New integrative method to generate Bacillus subtilis recombinant strains free of selection markers

The novel method described in this paper combines the use of blaI, which encodes a repressor involved in Bacillus licheniformis BlaP beta-lactamase regulation, an antibiotic resistance gene, and a B. subtilis strain (BS1541) that is conditionally auxotrophic for lysine. We constructed a BlaI cassette containing blaI and the spectinomycin resistance genes and two short direct repeat DNA sequences, one at each extremity of the cassette. The BS1541 strain was obtained by replacing the B. subtilis P(lysA) promoter with that of the P(blaP) beta-lactamase promoter. In the resulting strain, the cloning of the blaI repressor gene confers lysine auxotrophy to BS1541. After integration of the BlaI cassette into the chromosome of a conditionally lys-auxotrophic (BS1541) strain by homologous recombination and positive selection for spectinomycin resistance, the eviction of the BlaI cassette was achieved by single crossover between the two short direct repeat sequences. This strategy was successfully used to inactivate a single gene and to introduce a gene of interest in the Bacillus chromosome. In both cases the resulting strains are free of selection marker. This allows the use of the BlaI cassette to repeatedly further modify the Bacillus chromosome.     

Vectors for regulated gene expression in the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans possesses an exceptional capacity to withstand the lethal and mutagenic effects of most form of DNA damage and has received considerable interest for use in both fundamental and applied research. Here we describe vectors that allow regulated expression of Deinococcal genes for functional analysis. The vectors contain the IPTG-regulated Spac system (Pspac promoter and lacI repressor gene), originally designed for Bacillus subtilis, that we have adapted to be functional in D. radiodurans. We show that the Spac system can control the expression of a lacZ reporter gene over two orders of magnitude depending on the inducer concentration and the copy number of the lacI regulatory gene. Furthermore, we demonstrate that the Spac system can be used to regulate the synthesis of a critical repair protein, such as RecA, resulting in a conditional mitomycin-resistant cell phenotype. We have also developed tools for the construction of conditional mutants where the expression of the target gene is regulated by an inducible promoter. The utility of these conditional gene inactivation systems is exemplified by the conditional lethal phenotype of a mutant expressing gyrA from the Pspac promoter.     

New Integrative Method To Generate Bacillus subtilis Recombinant Strains Free of Selection Markers

The novel method described in this paper combines the use of blaI, which encodes a repressor involved in Bacillus licheniformis BlaP β-lactamase regulation, an antibiotic resistance gene, and a B. subtilis strain (BS1541) that is conditionally auxotrophic for lysine. We constructed a BlaI cassette containing blaI and the spectinomycin resistance genes and two short direct repeat DNA sequences, one at each extremity of the cassette. The BS1541 strain was obtained by replacing the B. subtilis P_lysA promoter with that of the P_blaP β-lactamase promoter. In the resulting strain, the cloning of the blaI repressor gene confers lysine auxotrophy to BS1541. After integration of the BlaI cassette into the chromosome of a conditionally lys-auxotrophic (BS1541) strain by homologous recombination and positive selection for spectinomycin resistance, the eviction of the BlaI cassette was achieved by single crossover between the two short direct repeat sequences. This strategy was successfully used to inactivate a single gene and to introduce a gene of interest in the Bacillus chromosome. In both cases the resulting strains are free of selection marker. This allows the use of the BlaI cassette to repeatedly further modify the Bacillus chromosome.     

Cloning and expression of subtilisin amylosacchariticus gene

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.     

Intergenotic Transformation of the Bacillus subtilis Genospecies

A multiple auxotrophic derivative of Bacillus subtilis 168 (strain BR151 carrying lys-3, trpC2, metB10) was transformed with deoxyribonucleic acid (DNA) isolated from B. subtilis 168, Bacillus amyloliquefaciens H, B. subtilis HSR, Bacillus pumilus, and Bacillus licheniformis. Transformation with heterologous DNA occurred at a very low frequency for the three auxotrophic markers. Heterologous transformation to rifampin resistance was 100 to 1,000 times more efficient than transformation to prototrophy. Transformants from the various heterologous exchanges were used to prepare donor DNA. The fragment of integrated DNA from the heterologous (foreign) species, termed the "intergenote," was capable of transforming BR151 with an efficiency almost equal to that of homologous DNA. When BR151 DNA contained the Rfm(R) (rifampin resistance) intergenote from B. amyloliquefaciens H, the frequency of transformation was frequently greater than that of the homologous DNA. Accompanying this increased efficiency was a marked change in the physiology of the cells. The growth rate of the transformants carrying this intergenote was approximately one-half that of either parental strain. Thus, in a prokaryotic transformation system, adverse side effects can occur after incorporation of a segment of foreign DNA.     

Cloning of an unstable spoIIA-tyrA fragment from Bacillus subtilis

A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA+) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.     

Far different levels of gene expression provided by an oriented cloning system in Bacillus subtilis and Escherichia coli

A gene expression system for both Bacillus subtilis and Escherichia coli was developed. The expression vector, pHASH102, produces any combination of promoter and open reading frame to be expressed based on the T-extended cloning method. Because the pHASH series vectors are designed to shuttle between the genome and a high copy plasmid in B. subtilis, the expression profiles of copy number dependence can be examined systematically. We demonstrated that vectors with Pr, Pspac, and PS10 promoters are suitable for the overexpression of GFPuv. Moreover, aadK encoding aminoglycoside 6-adenylyltransferase (a streptomycin-resistance gene) of B. subtilis was successfully overexpressed in both B. subtilis and E. coli. These highly expressed GFPuv and aadK genes can be used as a genetic marker for both organisms.     

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。     

A general method for the consecutive integration of single copies of a heterologous gene at multiple locations in the Bacillus subtilis chromosome by replacement recombination.

We have devised a two-step procedure by which multiple copies of a heterologous gene can be consecutively integrated into the Bacillus subtilis 168 chromosome without the simultaneous integration of markers (antibiotic resistance). The procedure employs the high level of transformability of B. subtilis 168 strains and makes use of the observation that thymine-auxotrophic mutants of B. subtilis are resistant to the folic acid antagonist trimethoprim (Tmpr), whereas thymine prototrophs are sensitive. First, a thymine-auxotrophic B. subtilis mutant is transformed to prototrophy by integration of a thymidylate synthetase-encoding gene at the desired chromosomal locus. In a second step, the mutant strain is transformed with a DNA fragment carrying the heterologous gene and Tmpr colonies are selected. Approximately 5% of these appear to be thymine auxotrophic and contain a single copy of the heterologous gene at the chromosomal locus previously carrying the thymidylate synthetase-encoding gene. Repetition of the procedure at different locations on the bacterial chromosome allows the isolation of strains carrying multiple copies of the heterologous gene. The method was used to construct B. subtilis strains carrying one, two, and three copies of the Bacillus stearothermophilus branching enzyme gene (glgB) in their genomes.     

Genetic analysis of an incomplete bio operon in a biotin auxotrophic strain of Bacillus subtilis natto OK2

We describe the genetic analysis of the bio operon of the biotin auxotrophic Bacillus subtilis natto OK2 strain. The OK2 strain would only cross-feed with the Escherichia coli bioB mutant and also grew well in medium containing dethiobiotin. Sequencing analysis revealed two significant genetic alterations in the bioW and bioF genes within the bio operon of the OK2 strain. Complementation analysis with B. subtilis 168 bio mutants demonstrated that only the bioB gene could complement, but other bio operon genes could not. A bio(+) transformant, isolated from an OK2 strain, has biotin autotrophy.     

Identification of Bacillus subtilis NRRL B-3275 as a Strain of Bacillus pumilus

The physiological and biochemical properties of a species of Bacillus previously identified as B. subtilis NRRL B-3275 (B-3275) were compared with those of seven strains of B. pumilus and five strains of B. subtilis. The biotin requirement of B-3275, its inability to hydrolyze starch, and its failure to reduce nitrate indicate that the organism is more closely related to the B. pumilus strains than to those of B. subtilis. Hybridization of deoxyribonucleic acid (DNA) from B-3275 with that of the strains of B. pumilus showed a binding efficiency (compared with the homologous reaction) of 58 to 99%, depending on the strain. Hybridization with the DNA from any of the strains of B. subtilis did not exceed 24%. DNA from B-3275 was unable to transform two amino acid auxotrophic markers to prototrophy in a highly competent strain of B. subtilis 168. We conclude that B-3275 is a strain of B. pumilus which we designate as B. pumilus NRRL B-3275.     

Genetic mapping in Bacillus subtilis 168 of the aadK gene which encodes aminoglycoside 6-adenylyltransferase

Abstract Bacillus subtilis 168 has an aadK gene, which encodes aminoglycoside 6-adenylyltransferase, a streptomycin-modifying enzyme, on its chromosome. To characterize the aadK gene, we con tructed a B. subtilis 168 strain that carried the chloramphenicol resistance gene near the aadK on the chromosome and an aadK deletion mutant using an integration technique. The aadK gene was mapped between azlB and pheA on the chromosome of B. subtilis 168. The aadK deletion mutant was slightly more susceptible to streptomycin than the original strain. The result indicates that the aadK gene contributes low-level resistance to streptomycin in B. subtilis 168.     

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.     

Cloning and characterization of Bacillus subtilis homologs of Escherichia coli cell division genes ftsZ and ftsA.

The Bacillus subtilis homolog of the Escherichia coli ftsZ gene was isolated by screening a B. subtilis genomic library with anti-E. coli FtsZ antiserum. DNA sequence analysis of a 4-kilobase region revealed three open reading frames. One of these coded for a protein that was about 50% homologous to the E. coli FtsZ protein. The open reading frame just upstream of ftsZ coded for a protein that was 34% homologous to the E. coli FtsA protein. The open reading frames flanking these two B. subtilis genes showed no relationship to those found in E. coli. Expression of the B. subtilis ftsZ and ftsA genes in E. coli was lethal, since neither of these genes could be cloned on plasmid vectors unless promoter sequences were first removed. Cloning the B. subtilis ftsZ gene under the control of the lac promoter resulted in an IPTGs phenotype that could be suppressed by overproduction of E. coli FtsZ. These genes mapped at 135 degrees on the B. subtilis genetic map near previously identified cell division mutations.     

Cry1Ac基因载体构建及其在枯草芽孢杆菌中的杀虫活性表达

根据苏云金芽孢杆菌Bacillus thuringiensis HD-73基因Cry1Ac和枯草芽孢杆菌Bacillus subtilis木糖诱导型启动子PxylR序列, 分别设计2对特异引物Cry1Ac F/R和Pxy F/R,扩增获得了完整的启动子PxylR和Cry1Ac基因序列,进一步以上述产物混合物为模板,以Pxy F/Cry1Ac R作引物进行重迭PCR,获得了载体PxylR-Cry1Ac,经SphⅠ和BamHⅠ完全酶切后,将PxylR-Cry1Ac插入大肠杆菌-苏云金芽孢杆菌穿梭载体pHT315,重组表达质粒pCry1Ac315转化枯草芽孢杆菌感受态细胞。工程菌株质粒酶切电泳分析、SDS-PAGE电泳分析和杀虫生物活性测定结果证实了Cry1Ac基因的导入及其在枯草芽孢杆菌JAAS01D中的有效表达。     

Generation of an Escherichia coli lysA targeted deletion mutant by double cross-over recombination for potential use in a bacterial growth-based lysine assay

AIMS: To generate a stable Escherichia coli lysine auxotroph for the lysine bioavailability assay. METHODS AND RESULTS: An E. coli lysine auxotrophic strain was constructed by deleting the entire lysA gene and replacing it with a gene that confers resistance to ampicillin (bla). The linear DNA contained 50 bp homologous sequence of upstream of lysA in one end and 50 bp of downstream of lysA in the other end. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The deltalysA::bla strain exhibited a linear response to lysine supplementation and can be used for quantifying lysine.     

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.     

Analysis of mutants of Bacillus subtilis, auxotrophic for lysine

A number of B. subtilis mutants auxotrophic for lysine has been isolated and mapped in relation to the flanking ser2 marker. One of the mutants has been found to have a mutation in lys A gene coding for DAP-decarboxylase activity. The expression of DAP-decarboxylase is dependent on the product of lys R gene that is not linked with lysine genes cluster.