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. 2009 Jan;37(1):e3.
doi: 10.1093/nar/gkn884. Epub 2008 Nov 12.

Improvement of bacterial transformation efficiency using plasmid artificial modification

Kazumasa Yasui  1 , Yasunobu Kano, Kaori Tanaka, Kunitomo Watanabe, Mariko Shimizu-Kadota, Hirofumi Yoshikawa, Tohru Suzuki
Affiliations

Improvement of bacterial transformation efficiency using plasmid artificial modification

Kazumasa Yasui et al. Nucleic Acids Res. 2009 Jan.

Abstract

We have developed a method to improve the transformation efficiency in genome-sequenced bacteria, using 'Plasmid Artificial Modification' (PAM), using the host's own restriction system. In this method, a shuttle vector was pre-methylated in Escherichia coli cells, which carry all the putative genes encoding the DNA modification enzymes of the target microorganism, before electroporation was performed. In the case of Bifidobacterium adolescentis ATCC15703 and pKKT427 (3.9 kb E. coli-Bifidobacterium shuttle vector), introducing two Type II DNA methyltransferase genes lead to an enhancement in the transformation efficiency by five orders of magnitude. This concept was also applicable to a Type I restriction system. In the case of Lactococcus lactis IO-1, by using PAM with a putative Type I methyltransferase system, hsdMS1, the transformation efficiency was improved by a factor of seven over that without PAM.

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Figures

Figure 1.
Figure 1.
The PAM concept. (A) The conventional method for the transformation of bacteria. The introduced shuttle vector is degraded by a restriction enzyme of the target bacterium. A small amount of vector survives and replicates in the target bacterium. (B) A PAM plasmid carries all the modification methylase genes of the target host. A shuttle vector plasmid is introduced to E. coli host, which had harboured the PAM plasmid (PAM host). The shuttle vector is methylated by the modification enzyme encoded by the genes on the PAM plasmid in the E. coli host. The shuttle vector is then extracted and introduced into the target host by electroporation. The shuttle vector is resistant against restriction enzymes and yields higher transformation efficiency. (C) The R–M system has a complicated structure, such as a gene cluster that includes subunits or unknown accessory genes. Alternatively, the PAM plasmid, containing a modification gene and also unknown parts, could be introduced into the transformant harbouring a shuttle vector. The restriction enzyme acts, but some copies of the plasmid could survive in the PAM host. The plasmid is then extracted and introduced the target bacterium.
Figure 3.
Figure 3.
Molecular structure of shuttle vector pKKT427. A Bifidobacterium–E. coli shuttle vector, pKKT427, was a modified pBRASTA101 replicon. This shuttle vector had been constructed by modification from a previously reported shuttle vector pBRASTA101, a composite plasmid of pUC18 and MCS and it excluded the β-galactosidase and ampicillin-resistant genes.
Figure 2.
Figure 2.
Construction of pPAM plasmids. (A) The B. adolescentis ATCC15703 genome includes two R–M clusters, BAD_1227–1234 and BAD_1279–1284. Red boxes show putative restriction genes. The blue boxes (BAD_1233 and BAD_1283) show putative methyltransferase genes. (B) The putative methyltransferase genes were amplified by PCR using primers as listed in Table 2. The PCR products were joined by in vitro homologous recombination to plasmid vector pBAD33, which had been cleaved by HincII, using the In-Fusion Dry-Down PCR cloning kit (Clonetech) to obtain pPAM plasmids. Overlap extension PCR was used for BAD_1233–1283. The pPAM1233–1283 plasmid was a constructed operon of BAD_1233 and BAD_1283. In the first PCR, the coding region of BAD_1233, which was added to the downstream 19 bases from the 5′-end of BAD_1283 was amplified. BAD_1283 was obtained in the same manner 20 bases from the 3′-end of BAD_1233. In the second PCR, the first PCR products were used as a DNA template and PMT1-F and PMT2-R primers were used. The amplified DNA fragment was ligated to the same vector and the plasmid pPAM1233–1283 then obtained.
Figure 4.
Figure 4.
Comparison of PAM effects on transformation efficiencies. (A–D) Bifidobacterium adolescentis ATCC15703 was transformed by electroporation using the PAM method. The plasmid pKKT427 was prepared from E. coli TOP10 carrying pPAM1233-1283 (A), pPAM1233 (B), pPAM1283 (C) or without pPAM plasmid (D). An alkaline-SDS method using purification by agarose gel electrophoresis was used to isolate the PAM plasmids which were then introduced into B. adolescentis ATCC15703 by electroporation, as described previously (6). The electroporated samples were 100 times diluted in (A–C), but not in D. (E) Schematic presentation of transformation efficiencies. Plasmid pKKT427 was prepared from the PAM host (blue), B. longum 105-A (green) or B. adolescentis ATCC15703. The numbers beside arrows indicate transformation efficiencies (CFU/µg DNA).
Figure 5.
Figure 5.
The transformation of Bifidobacterium was confirmed by plasmid isolation followed by agarose gel electrophoresis. Plasmids extracted from PAM host E. coli TOP10 harbouring pPAM1233–1283 (Lane 1) and from recombinant B. adolescentis ATCC15703 (Lane 2). Vector pKKT427 (Lane 3) and pPAM1233-1283 (Lane 4).
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