Chapter 42 : Integrational Vectors for Genetic Manipulation in Bacillus subtilis

First generation of integrative vectors for B. subtilis. pJH10l and pHV32 are both derived from pBR322. The fragment carrying the cat gene is from pC194 ( 17 ). Restriction sites: B, BamHI; E, EcoKI; H, HindIII; P, PstI; S, SalI; Sp, SphI. oriE is the ColEl origin of replication for E. coli. AMP, ampicillin; TET, tetracycline; CAM, chloramphenicol.

Schematic representation of the integrative recombination event by the Campbell-type mechanism, which results in duplication of the region cloned into the plasmid. Bold lines represent the chromosome, and thin lines represent the plasmid; the open box represents the region of homology between plasmid and chromosome. CAM, chloramphenicol.

Second generation of integrative vectors. pJM103 and pJM113 ( 34a ) are derivatives of pUC19 ( 47 ), while pSGMU2 ( 11 ) derives from pUC13 ( 28 ). The fragment carrying the cat gene is from pC194, and the one carrying the kanamycin resistance gene is from pJHl ( 45 ). Plasmids pJM102 and pJM112, described in the text, differ from pJM103 and pJM113, respectively, in that they are derivatives of pUC18. The pUC18–19 MCS contains the following restriction sites: EcoRI, SstI, KpnI, SmaI, XmaI, BamHI,XbaI, SalI, AccI, HincI, PstI, SphI, and HindIII. The pUC13 MCS, compared with the pUC18–19 MCS, is missing the KpnI and SphI sites. Bg, BglII. AMP, ampicillin; CAM, chloramphenicol; KAN, kanamycin.

Integrative vectors carrying additional features. pBG6 ( 50 ) derives from pJH10l, in which the Clal-Ball region has been replaced by the Clal-Ball fragment from M13mpl9 that contains the M13 origin of replication, the lacZ gene, and the MCS. M13mpl9cat contains the cat gene from pC194 in the Avail site of M13mpl9. pGEM-3Zf(+)cat-l ( 50 ) was obtained from pGEM-3Zf(+) (Promega) by inserting a 1-kb fragment containing the cat gene originally associated with pC194 into the unique Ndel site filled in with Klenow polymerase; fl ori is the fl phage origin for single-stranded replication; PT7 is the promoter for E. coli phage T7, while PSP6 is the promoter for S. typhimurium phage SP6. pCT571 ( 15 ) is a low- to high-copy-number integrative vector: the low-copy-number state is controlled by the pSClOl replicon, while oriVRK2, when supplied with the trfA gene in trans, allows the plasmid to replicate at an elevated copy number. For the pUC19 MCS, see Fig. 3 . X, Xhol; Sm, Smal. All other restriction sites are as in Fig. 1 . AMP, ampicillin; CAM, chloramphenicol; KAN, kanamycin; TET, tetracycline.

Schematic representation of the use of integrative vectors in gene function analysis. The boundaries of the transcriptional unit are indicated by P for promoter and T for terminus, (a) When the cloned region contains one end of the transcriptional unit, integration gives rise to a heterologous but functional unit, (b) When the cloned region is internal to the transcriptional unit, integration results in gene disruption. See also the legend to Fig. 2 . CAM, chloramphenicol.

Antibiotic cassette vector derivatives of the Bluescript plasmid (Stratagene). The restriction site EcoRV used to construct the cassettes is pointed out by the arrow and is in parentheses to indicate its loss, which was due to the cloning. Directions of transcription of the antibiotic genes are indicated by arrows. The cat gene in pJM105A and pJM105C was a Sau3A-HpaII fragment from pC194 blunted with Klenow polymerase. The truncated cat gene in pJM105B was obtained as HindIII-Stul fragment from pJM105A recloned in a HindIII-EcoRV-cut Bluescript plasmid. The macrolide-lincosamide-streptogramin B resistance gene ermG was a HaeIII-BstNI Klenow-blunted fragment from pBD370 ( 30 ). The kanamycin resistance gene was recovered from plasmid pJHl ( 45 ) as a ClaI fragment whose ends were filled in with Klenow.

Plasmid rescue after chromosomal integration and cloning of adjacent sequences, (a) The EcoRI and HindIII sites on the plasmid belong to the vector MCS. (b) After plasmid integration, chromosomal DNA is extracted and then digested with a restriction enzyme that is unique in the plasmid in order to create fragments carrying the entire vector and adjacent sequences, (c) Digested chromosomal DNA is used at 10 µg/ml in a ligation mixture and then transformed into E. coli competent cells in order to recover the recircularized plasmid. See also the legend to Fig. 2 . CAM, chloramphenicol; H, HindIII; E, E?oRI.

Integrational vectors for lacZ fusion constructions. pJF751 and pJM783 promote lacZ fusions for, respectively, translational and transcriptional analyses after integration into the B. subtilis chromosome by the Campbell-type mechanism. pDH32, a derivative of pBGtrp ( 40 ), integrates via a double-crossover event in the amyE region and promotes lacZ transcriptional fusions. The lacZ gene in each of these vectors is a promoterless truncated version of the E. coli lacZ gene. The spoVG ribosome-binding sites (RBS) in pJM783 and pDH32 come from pTV32 ( 36 ) and are preceded by one translational stop codon in each of the three possible frames and by a small MCS. Restriction sites: E, EcoRI; Sm, SmaI; B, BamHI;S, SalI; P, PstI. AMP, ampicillin; CAM, chloramphenicol.

Integrative vectors for inducible systems. pDH87 and pDH88 derive from pSIl ( 48 ). They have origins of replication and ampicillin genes from pBR322, while the cat gene is from pC194. The multiple cloning site in pDH87 contains the following restriction sites: HindIII, XbaI, SalI, PstI, and SphI. The MCS of pDH88 is made of the following sites: HindIII, SmaI, XbaI, HpaI, BglII. ClaI, and SphI.PstI in pDH87 and HpaI in pDH88 are not unique ( 16 ).