Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon

Abstract

Due to their role in methane production, methanoarchaea are of high ecological relevance and genetic systems have been ever more established in the last two decades. The system for protein expression in Methanosarcina using a comprehensive shuttle vector is established; however, details about its replication mechanism in methanoarchaea remain unknown. Here, we report on a significant optimisation of the rather large shuttle vector pWM321 (8.9 kbp) generated by Metcalf through a decrease in its size by about 35% by means of the deletion of several non-coding regions and the ssrA gene. The resulting plasmid (pRS1595) still stably replicates in M. mazei and—most likely due to its reduced size—shows a significantly higher transformation efficiency compared to pWM321. In addition, we investigate the essential gene repA, coding for a rep type protein. RepA was heterologously expressed in Escherichia coli, purified and characterised, demonstrating the significant binding and nicking activity of supercoiled plasmid DNA. Based on our findings we propose that the optimised shuttle vector replicates via a rolling circle mechanism with RepA as the initial replication protein in Methanosarcina. On the basis of bioinformatic comparisons, we propose the presence and location of a double-strand and a single-strand origin, which need to be further verified.

Teaching old dogs new tricks: genetic engineering methanogens

“…Shuttle vectors are genetic elements (i.e., DNA or plasmids) that are capable of replication and maintenance in multiple hosts by using multiple origins of replication, with each replication sequence specific to one host. E. coli is the most common partner host for shuttle vectors in methanogens because of its versatile and well-developed genetic techniques ( 62 ). Replicating vectors can also be useful for introducing genetic constructs without any residual host genome modifications, as discussed further in “Markerless genetic exchange.”…”

“…A method for high-efficiency liposome-mediated transformation has been developed for M. acetivorans C2A and M. barkeri Fusaro ( 101 ). Furthermore, a liposome-mediated transformation protocol for Methanosarcina mazei strain Gӧ1 has been developed ( 97 ), which has been used in subsequent studies to insert a shuttle vector into M. mazei ( 62 ), and to develop the first functional genetic system for a virus of methanogens, MetSV ( 102 ).…”