Abstract
It is difficult to genetically manipulate the medically and biotechnologically important genus Clostridium due to the existence of the restriction and modification (RM) systems. We identified and engineered the RM system of a model clostridial species, C. acetobutylicum, with the aim to allow the host to accept the unmethylated DNA efficiently. A gene CAC1502 putatively encoding the type II restriction endonuclease Cac824I was identified from the genome of C. acetobutylicum DSM1731, and disrupted using the ClosTron system based on group II intron insertion. The resulting strain SMB009 lost the type II restriction endonuclease activity, and can be transformed with unmethylated DNA as efficiently as with methylated DNA. The strategy reported here makes it easy to genetically modify the clostridial species using unmethylated DNA, which will help to advance the understanding of the clostridial physiology from the molecular level.
Highlights
Restriction and modification (RM) systems are widespread in bacteria and archea
The resulted vector, designated as pMTL008, were digested by MluI and self-ligated to generate a vector pMTL009 without RAM. This vector was transformed into C. acetobutylicum DSM1731, and the CAC1502 disrupted mutant was screened according the reported procedure [30]
The very low efficiency of genetically modifying Clostridium strains has greatly hampered the progress for studying the physiology of this medically and biotechnologically important genus [19]
Summary
Restriction and modification (RM) systems are widespread in bacteria and archea. Most RM systems comprise a DNA methyltransferase (MTase) and a restriction endonuclease (REase). The MTase enables recognition of ‘self’ DNA by methylation of specific nucleotides within particular DNA sequences whereas the REase enzymatically cleaves the ‘foreign’ unmodified DNA [3]. The RM system is a genetic barrier for gene transfer among bacteria or phage invasion [5,6,7]. It is a barrier for genetic manipulation such as conjugation [8] and transformation [9], which hampers the understanding and exploitation of many important bacteria through genetic manipulation
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