A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria.
Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-fou...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-09-01
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| Series: | PLoS Genetics |
| Online Access: | http://europepmc.org/articles/PMC3459991?pdf=render |
| _version_ | 1818215201452851200 |
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| author | Guoqiang Zhang Wenzhao Wang Aihua Deng Zhaopeng Sun Yun Zhang Yong Liang Yongsheng Che Tingyi Wen |
| author_facet | Guoqiang Zhang Wenzhao Wang Aihua Deng Zhaopeng Sun Yun Zhang Yong Liang Yongsheng Che Tingyi Wen |
| author_sort | Guoqiang Zhang |
| collection | DOAJ |
| description | Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography-Mass Spectrometry (LC-MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform. |
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| institution | Directory Open Access Journal |
| issn | 1553-7390 1553-7404 |
| language | English |
| last_indexed | 2024-12-12T06:32:19Z |
| publishDate | 2012-09-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS Genetics |
| spelling | doaj.art-d297bf77079e402dba4ea85cec9eef9f2022-12-22T00:34:33ZengPublic Library of Science (PLoS)PLoS Genetics1553-73901553-74042012-09-0189e100298710.1371/journal.pgen.1002987A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria.Guoqiang ZhangWenzhao WangAihua DengZhaopeng SunYun ZhangYong LiangYongsheng CheTingyi WenGenetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography-Mass Spectrometry (LC-MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform.http://europepmc.org/articles/PMC3459991?pdf=render |
| spellingShingle | Guoqiang Zhang Wenzhao Wang Aihua Deng Zhaopeng Sun Yun Zhang Yong Liang Yongsheng Che Tingyi Wen A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria. PLoS Genetics |
| title | A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria. |
| title_full | A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria. |
| title_fullStr | A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria. |
| title_full_unstemmed | A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria. |
| title_short | A mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction barrier of bacteria. |
| title_sort | mimicking of dna methylation patterns pipeline for overcoming the restriction barrier of bacteria |
| url | http://europepmc.org/articles/PMC3459991?pdf=render |
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