Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.     

Construction of a new shuttle expression vector for Bacillus subtilis and Escherichia coli by using a polycistronic system

A shuttle vector has been constructed by fusing the Bacillus subtilis trimethoprim-resistance-carrying (TpR) plasmid pNC601 with the Escherichia coli plasmid pBR322. The resultant plasmid pNBL1 can replicate in both B. subtilis and E. coli, conferring Tp resistance on both cells and ampicillin resistance (ApR) on E. coli. The B. subtilis dihydrofolate reductase operon (dfr) on pNC601 and therefore on pNBL1 consists of the thymidylate synthase B gene (thyB) and the TpR-dihydrofolate reductase gene lacking the C-terminal seven codons (designated as drfA' as compared with the complete dfrA gene). A direct-expression vector pNBL3 has been constructed by inserting synthetic oligodeoxynucleotides containing a Bacillus ribosome-binding site (RBS) and the ATG codon downstream from dfrA' on pNBL1. When the E. coli lacZ gene was placed downstream from the dfrA' gene in pNBL3, efficient synthesis of beta-galactosidase was observed in both cells, showing that the polycistronic expression system is suitable for directing expression of heterologous genes. Translational efficiency of the lacZ gene on pNBL3 was further examined in B. subtilis by changing the sequence upstream from lacZ. Unlike the results previously reported [Sprengel et al., Nucleic Acids Res. 13 (1985) 893-909], when RBS was present, the high level of lacZ expression was preserved irrespective of spacing between the stop codon of the upstream dfrA' gene and the start codon of the downstream lacZ gene. However, in the absence of RBS, the spacing between both genes affected lacZ expression. That is, translational coupling of dfrA'-lacZ was observed, although the translational efficiency was very low.     

A general method for fusion of the Escherichia coli lacZ gene to chromosomal genes in Bacillus subtilis

A series of plasmids has been constructed that can be used to fuse the beta-galactosidase gene (lacZ) of Escherichia coli to chromosomal genes of Bacillus subtilis. Insertion of the lacZ gene is facilitated by the use of a selectable chloramphenicol acetyl-transferase (cat) gene. The latter is included, along with the lacZ gene, in a single DNA fragment or 'cartridge' that can be removed from the plasmid with a variety of different restriction endonucleases. Methods applicable to any cloned B. subtilis gene are described that enable the lac-cat cartridge to be inserted at specific sites, or at random, directly into the B. subtilis chromosome in a single step. These single-copy chromosomal fusions can be readily transferred, by selection for chloramphenicol resistance, to a temperate phage such as phi 105, to permit a more extensive genetic analysis of the expression of the target gene. Alternatively, the lac-cat cartridge and flanking DNA sequences can be transferred into different genetic backgrounds by transformation. These techniques have been used to construct, in a single step, lac fusions to genes in the sporulation operons spoIIA and spoVA.     

Cloning vehicles for the homologous Bacillus subtilis host-vector system

A series of Bacillus subtilis plasmids was constructed which carry either the leu region or both the leu and the dihydrofolate reductase (DHFR) regions of the B. subtilis chromosome. The DHFR-coding gene was derived from a trimethoprim resistant (Tmpr) B. subtilis strain, and cells harboring the DHFR plasmid showed resistance to trimethoprim (Tmp). One such leu+tmpr plasmid, pTL12, was found to be useful for cloning DNA fragments at the BamHI, EcoRI, BglII and XmaI sites. It was also shown that insertion of DNA fragments at the BamHI and XmaI sites of pTL12 inactivated the leuA gene function (insertional inactivation) but not tmpr, indicating that cells carrying recombinant plasmids can be detected easily by selecting Leu-Tmpr colonies. Combination of B. subtilis 168 and plasmid pTL12 should serve as an efficient homologous cloning system in B. subtilis.     

The Bacillus subtilis acyl lipid desaturase is a delta5 desaturase

Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions delta5, delta7, and delta9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B. subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B. subtilis is located exclusively at the delta5 position, regardless of the growth temperature and the length chain of the fatty acids.     

Genetic analysis and overexpression of lipolytic activity in Bacillus subtilis.

The previously cloned Bacillus subtilis lipase gene (lip) was mapped on the chromosome and used in the construction of a B. subtilis derivative totally devoid of any lip sequence. Homologous overexpression was performed in this strain by subcloning the lip open reading frame on a multicopy plasmid under the control of a strong gram-positive promoter. A 100-fold overproducing strain was obtained, which should facilitate purification of the secreted protein. Furthermore, the delta lip strain BCL1050 constitutes an ideal host for the cloning of heterologous lipase genes.     

High Production of Thermostable beta-Galactosidase of Bacillus stearothermophilus in Bacillus subtilis

By cloning the beta-galactosidase gene of Bacillus stearothermophilus IAM11001 (ATCC 8005) into Bacillus subtilis, enzyme production was enhanced 50 times. beta-Galactosidase could be purified to 80% homogeneity by incubating the cell extract of B. subtilis at 70 degrees C for 15 min, followed by centrifugation to remove the denatured proteins. Because of its heat stability and ease of production, beta-galactosidase is suitable for application in industrial processes.     

Possibility of using fusion proteins for detection of nonsense mutations and frame-shift mutations in BRCA1 gene

The aim of this study was to evaluate the possibility of detecting nonsense and frame-shift mutations in exon 11 of brca1 gene by constructing fusion open reading frame (ORF) "exon 11 ORF-alpha-peptide of beta-galactosidase". The ability/inability of this newly constructed ORF to cause alpha-complementation in E. coli delta M15gal cells transformed by the plasmid with the ORF may reflect the absence/presence of nonsense and frame-shift mutations in the studied fragment. A single ORF fragment of exon 11 of brca1 gene--LacZ' gene was designed in pGEN7Zf plasmid, the plasmid was shown to cause Lac+ phenotype in E. coli delta M15gal. Four frame-shift deletion mutations were introduced into exon 11 sequence in the plasmid. Surprisingly, the frame-shift deletion mutations did not influence the ability of plasmids to induce Lac+ phenotype in E. coli delta M15gal in 3 cases and only one deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E. coli delta M15gal. We suppose that the phenomenon can be explained by the alpha-peptide translation reinitiation from inframe ATG codons situated within the exon 11 sequence. Seven inframe ATG sequences were found in exon 11, at least two in-frame ATG-containing fragments were demonstrated to cause reinitiation. On the other hand, the only deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E. coli delta M15gal did not leave LacZ' in-frame ATG in econ 11 sequence. We conclude that it is possible to detect frame-shift mutations by in-frame cloning with the LacZ' reporter gene, but this possibility is strongly impeded by the reinitiation of alpha-peptide translation from the in-frame ATG codons within the studied sequence.     

The Bacillus subtilis spoIIA locus is expressed at two times during sporulation

Abstract The Bacillus subtilis spoIIA and spoIIAB genes were fused to the Escherichia coli lacZ gene on a novel integrational plasmid vector. The constructs were integrated into the B. subtilis chromosome, and used to show that the spoIIA locus was expressed at two times during sporulation.     

Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.     

Vectors that facilitate the replacement of transcriptional lacZ fusions in Streptococcus mutans and Bacillus subtilis with fusions to gfp or gusA

Plasmid vectors have been constructed for Streptococcus mutans and Bacillus subtilis that make possible rapid replacement of the widely used reporter gene lacZ (encoding beta-galactosidase) with either gfp (encoding green fluorescent protein) or gusA (encoding beta-glucuronidase). The lacZ-->gfp replacement vectors greatly facilitate the analysis of the spatial location of gene expression in biofilms of S. mutans and in sporulating B. subtilis. The lacZ-->gusA replacement vectors facilitate the comparison of two promoters within the same organism. A vector is also described that enables gusA to be replaced with gfp in B. subtilis.     

Molecular cloning and expression in Escherichia coli of two modification methylase genes of Bacillus subtilis

Two modification methylase genes of Bacillus subtilis R were cloned in Escherichia coli by using a selection procedure which is based on the expression of these genes. Both genes code for DNA-methyltransferases which render the DNA of the cloning host E. coli HB101 insensitive to the BspRI (5'-GGCC) endonuclease of Bacillus sphaericus R. One of the cloned genes is part of the restriction-modification (RM) system BsuRI of B. subtilis R with specificity for 5'-GGCC. The other one is associated with the lysogenizing phage SP beta B and produces the methylase M.BsuP beta BI with specificity for 5'-GGCC. The fragment carrying the SP beta B-derived gene also directs the synthesis in E. coli of a third methylase activity (M.BsuP beta BII), which protects the host DNA against HpaII and MspI cleavage within the sequence 5'-CCGG. Indirect evidence suggests that the two SP beta B modification activities are encoded by the same gene. No cross-hybridization was detected either between the M.BsuRI and M.BsuP beta B genes or between these and the modification methylase gene of B. sphaericus R, which codes for the enzyme M.BspRI with 5'-GGCC specificity.