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Several different plasmids have been used as cloning vectors in Bacillus subtilis, but many of them suffer from the disadvantage that they replicate via a single-stranded DNA intermediate (2). Several steps in the replication cycle render these plasmids highly susceptible to structural rearrangements, and these effects are often dramatically enhanced in recombinant plasmids (5). An alternative method to avoid the problem of the instability of recombinant plasmids in B. subtilis is to use integrative plasmids. Such plasmids are usually based on an Escherichia coli replicon (mostly pBR322 or one of its derivatives) and carry an antibiotic resistance marker gene that can be selected in B. subtilis and DNA sequences homologous to the B. subtilis chromosome. The most prominent and widely used systems are delivery plasmids which allow the insertion of any kind of genetic information into the bacterial chromosome. The amyE locus, coding for a nonessential -amylase, is used in most cases for ectopic integration. This system has been developed by Shimotsu and Henner (12) and contains in its simplest form an antibiotic resistance marker and a multiple cloning site sandwiched between the two halves of the amyE gene, designated amyE-front and amyE-back. Upon transformation of B. subtilis cells, both amyE sequences will recombine at their homologous sites, thereby stably inserting the DNA sequences in between amyE-front and amyE-back into the B. subtilis chromosome via a double-crossover event (12).

In some cases, it is appropriate to have two different integration sites available, e.g., to use two different expression systems to allow the study of gene regulation. Therefore, the lacA locus has been explored as an additional site for ectopic integration of DNA sequences. The lacA gene codes for -galactosidase (-Gal) and is weakly expressed, if at all, in B. subtilis (1). In addition, two different expression cassettes allowing the regulatable transcription of cloned genes have been developed.

Construction of the lacA delivery expression vector pAX01. The delivery expression vector pAX01 (Fig. 1A) was assembled from different plasmids and from chromosomal DNA of B. subtilis in the following way. From pBgaB (8), the ColE1-bla backbone (3,537 bp) was PCR amplified using primers ON1 and ON2 (Table 1), which are both flanked by EcoRV restriction sites, resulting in pK1. Next, 5' lacA (lacA-front; 500 bp; made with ON3 and ON4) and 3' lacA (lacA-back; 500 bp; made with ON5 and ON6) were generated by PCR using chromosomal DNA of B. subtilis 1012 as a template; both fragments were flanked by SmaI and ClaI sites on their 5' and 3' ends, respectively. In a triple-ligation reaction, both fragments were cloned into pK1, yielding pK2.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Delivery expression vectors. (A) pAX01. (B) pA-spac. Both vectors are derivatives of pBR322 which do not replicate in B. subtilis. All the DNA sequences located between lacA' and 'lacA allow integration into the chromosome of B. subtilis via a double-crossover event, resulting in selection for Ermr.

Construction of a lacA::spec chromosomal insertion mutation. Integration of pAX01 at the lacA locus can occur via a single- or a double-crossover event at the lacA locus. However, if a B. subtilis gene has been fused to the P_xylA promoter, the whole vector might recombine at the site of the B. subtilis gene. Integrations into the B. subtilis chromosome will result in Erm^r transformants, and experimental discrimination between these possibilities might become tedious and time-consuming. Therefore, we decided to construct a recipient strain allowing easy and fast discrimination between the various integration possibilities by screening. This was accomplished by insertion of a Spec^r marker within the chromosomal copy of lacA.

Construction of the IPTG-inducible expression vector pA-spac. The most widely used expression system in B. subtilis is based on the E. coli lac promoter, which is negatively controlled by the Lac repressor; in this system, the activity of the repressor protein is modulated by isopropyl--D-thiogalactopyranoside (IPTG). The E. coli-B. subtilis shuttle expression vector pREP9 is mentioned here as a paradigm (7). Using plasmid pDR66 as a template and oligonucleotides ON15 and ON16, the lac expression cassette, including the lacI gene and the lac promoter-operator region, was amplified and inserted into SacI-linearized vector pAX01, resulting in pA-spac (Fig. 1B).

The integrative expression vectors pAX01 and pA-spac express -Gal from the lacA locus in a regulatable way. In order to prove that both integrative expression vectors work properly, the bgaB gene, coding for heat-stable -Gal (4), was inserted into both vectors. With pAX01, bgaB was generated from plasmid pBgaB (8) using ON17 and ON18, both flanked with BamHI sites. The BamHI-treated amplicon was then inserted into BamHI-linearized pAX01 to result in pAX01-BgaB. With pA-spac, the bgaB gene was generated from the same template using ON19 and ON20 and ligated into the SalI-SphI-cleaved vector (pA-spac-BgaB). Next, the two transcriptional fusions were recombined independently at the lacA locus using strain IHA01, and the correct integration was verified by Southern blotting (strains IHA01-Xyl-BgaB and IHA01-Spac-BgaB).

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Induction of -Gal activity. Bacteria were grown at 37°C in Luria-Bertani medium containing the appropriate antibiotic (erythromycin at 1 µg/ml or/and spectinomycin at 100 µg/ml) and were induced by either IPTG or xylose at the early exponential growth phase. -Gal activities were determined at 55°C as described previously (8) using o-nitrophenyl--D-galactopyranoside as a substrate and are given as units per milligram of protein. All assays were repeated at least three times and yielded comparable results; the data from one experiment are shown. Growth of B. subtilis strains IHA01-Spac-BgaB (A), IHA01-Xyl-BgaB (B), and IHA13 (C) either in the absence of an inducer () or after the addition of an inducer () was recorded. -Gal activities in the absence or presence of the inducer are as indicated, and the time point of addition of the inducer is marked by a vertical arrow. OD600, optical density at 600 nm; t, time.

The hrcA gene integrated at lacA regulates the expression of a transcriptional fusion inserted at amyE. To test for the usage of both integration loci in a single strain, the hrcA repressor gene (11) was integrated at the lacA locus in strain AM19, where the operator recognized by HrcA was fused to bgaB at the amyE locus. hrcA was generated using oligonucleotides ON21 and ON22 as primers and B. subtilis 1012 chromosomal DNA as a template. The BamHI-flanked amplicon was ligated into BamHI-linearized and dephosphorylated vector pAX01, resulting in pAX-hrcA. The operon fusion was transferred into strain AM19 devoid of its own hrcA gene and carrying a transcriptional fusion between the promoter-operator region of the dnaK operon and bgaB, resulting in strain IHA13. -Gal activity was then measured in the absence and in the presence of the inducer xylose. While in the absence of xylose there was considerable expression of the bgaB gene (60 to 80 U) due to the absence of an active HrcA repressor (Fig. 2C), this activity dropped to about 10 U upon the addition of 0.5% xylose (Fig. 2C). Again, upon prolonged incubation of the cells in the presence of 0.5% xylose, -Gal activity began to rise; this effect could be prevented by using 2% xylose (data not shown). This experiment clearly demonstrates that regulated expression from one chromosomal locus will influence transcription at a distant one.

Conclusions. (i) The lacA locus has been selected as an alternative site for ectopic integration of foreign DNA sequences. This gene codes for -Gal and is very weakly expressed. For the easy screening of integrants, a mutated strain was constructed which carries a spectinomycin resistance cassette within lacA (lacA::spec; strain IHA01). Another strain was constructed containing, in addition, a genetically marked amyE locus (lacA::spec amyE::cat; strain IHA02). (ii) Two versatile delivery expression vectors were constructed (pAX01 and pA-spac) and tested by measuring the activity of a reporter gene fused to the regulatable promoters. (iii) To demonstrate communication between the lacA and amyE loci, the xylose-inducible hrcA repressor gene was integrated at lacA and at the promoter-operator region recognized by that repressor and fused to the bgaB reporter gene at amyE. While -Gal activity was low in the presence of an inducer, it increased significantly in its absence. (iv) The two recipient strains and the two expression vectors can be ordered from the Bacillus Genetic Stock Center (http://bacillus.biosci.ohio-state.edu). The DNA sequences of the two expression vectors and their plasmid maps can be found at http://btbgn1.bio.uni-bayreuth.de/lsgenetik/frames.htm.