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Chapter 42 : Integrational Vectors for Genetic Manipulation in Bacillus subtilis

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Abstract:

The development of integrational vectors has provided a versatile system for the advance of molecular genetic and mutagenic studies in . Over the past decade, the identification of genes of interest and the subsequent study of their structures and regulation have been greatly facilitated by the application of techniques for using integrational vectors. This chapter describes these techniques and the strategies used and vectors devised to investigate the mechanisms of gene function and regulation in . Based on findings, some integrational vectors carrying additional features were constructed. Several modifications have been introduced into pJHIOl in order to improve vector performance. In more recent years, a new series of integrational vectors has been developed by several laboratories. The first generally useful integrational vectors described were plasmids pHV32 and pJH10l. The presence in these plasmids of unique restriction sites allows the easy cloning of DNA fragments, and the detection of insertion can be monitored by disruption of the ampicillin or tetracycline resistance markers. The advantage of using integrational vectors is that obtaining the fusions does not necessarily result in gene disruption, and thus the pattern of regulation of essential genes can be monitored. The study of the transcriptional potential of a specific DNA fragment is best accomplished if the DNA fragment is placed in front of in a transcription-ally neutral region of the chromosome.

Citation: Perego M. 1993. Integrational Vectors for Genetic Manipulation in Bacillus subtilis, p 615-624. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch42

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Figures

Image of Figure 1
Figure 1

First generation of integrative vectors for . pJH10l and pHV32 are both derived from pBR322. The fragment carrying the gene is from pC194 ( ). Restriction sites: B, HI; E, I; H, dIII; P, I; S, I; Sp, I is the ColEl origin of replication for . AMP, ampicillin; TET, tetracycline; CAM, chloramphenicol.

Citation: Perego M. 1993. Integrational Vectors for Genetic Manipulation in Bacillus subtilis, p 615-624. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch42
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Image of Figure 2
Figure 2

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.

Citation: Perego M. 1993. Integrational Vectors for Genetic Manipulation in Bacillus subtilis, p 615-624. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch42
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Image of Figure 3
Figure 3

Second generation of integrative vectors. pJM103 and pJM113 ( ) are derivatives of pUC19 ( ), while pSGMU2 ( ) derives from pUC13 ( ). The fragment carrying the gene is from pC194, and the one carrying the kanamycin resistance gene is from pJHl ( ). 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: RI, IIIIHI,I, SalIIIII, and III The pUC13 MCS, compared with the pUC18–19 MCS, is missing the I and I sites. Bg, II. AMP, ampicillin; CAM, chloramphenicol; KAN, kanamycin.

Citation: Perego M. 1993. Integrational Vectors for Genetic Manipulation in Bacillus subtilis, p 615-624. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch42
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Image of Figure 4
Figure 4

Integrative vectors carrying additional features. pBG6 ( ) derives from pJH10l, in which the region has been replaced by the fragment from M13mpl9 that contains the M13 origin of replication, the gene, and the MCS. M13mpl9cat contains the gene from pC194 in the site of M13mpl9. pGEM-3Zf(+)cat-l ( ) was obtained from pGEM-3Zf(+) (Promega) by inserting a 1-kb fragment containing the gene originally associated with pC194 into the unique site filled in with Klenow polymerase; fl ori is the fl phage origin for single-stranded replication; PT7 is the promoter for phage T7, while PSP6 is the promoter for phage SP6. pCT571 ( ) 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 gene in allows the plasmid to replicate at an elevated copy number. For the pUC19 MCS, see Fig. 3 . X, Sm, All other restriction sites are as in Fig. 1 . AMP, ampicillin; CAM, chloramphenicol; KAN, kanamycin; TET, tetracycline.

Citation: Perego M. 1993. Integrational Vectors for Genetic Manipulation in Bacillus subtilis, p 615-624. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch42
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Figure 5

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.

Citation: Perego M. 1993. Integrational Vectors for Genetic Manipulation in Bacillus subtilis, p 615-624. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch42
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Image of Figure 6
Figure 6

Antibiotic cassette vector derivatives of the Bluescript plasmid (Stratagene). The restriction site RV 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 gene in pJM105A and pJM105C was a 3AII fragment from pC194 blunted with Klenow polymerase. The truncated gene in pJM105B was obtained as dIIIStul fragment from pJM105A recloned in a dIII-RV-cut Bluescript plasmid. The macrolide-lincosamide-streptogramin B resistance gene was a IIINI Klenow-blunted fragment from pBD370 ( ). The kanamycin resistance gene was recovered from plasmid pJHl ( ) as a I fragment whose ends were filled in with Klenow.

Citation: Perego M. 1993. Integrational Vectors for Genetic Manipulation in Bacillus subtilis, p 615-624. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch42
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Image of Figure 7
Figure 7

Plasmid rescue after chromosomal integration and cloning of adjacent sequences, (a) The RI and dIII 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 competent cells in order to recover the recircularized plasmid. See also the legend to Fig. 2 . CAM, chloramphenicol; H, dIII; E, RI.

Citation: Perego M. 1993. Integrational Vectors for Genetic Manipulation in Bacillus subtilis, p 615-624. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch42
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Image of Figure 8
Figure 8

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

Citation: Perego M. 1993. Integrational Vectors for Genetic Manipulation in Bacillus subtilis, p 615-624. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch42
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Image of Figure 9
Figure 9

Integrative vectors for inducible systems. pDH87 and pDH88 derive from pSIl ( ). They have origins of replication and ampicillin genes from pBR322, while the gene is from pC194. The multiple cloning site in pDH87 contains the following restriction sites: dIII, III and I. The MCS of pDH88 is made of the following sites: dIIIIIIIII and I.I in pDH87 and I in pDH88 are not unique ( ).

Citation: Perego M. 1993. Integrational Vectors for Genetic Manipulation in Bacillus subtilis, p 615-624. In Sonenshein A, Hoch J, Losick R (ed), and Other Gram-Positive Bacteria. ASM Press, Washington, DC. doi: 10.1128/9781555818388.ch42
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