Efficient genetic manipulation of Shewanella through targeting defense islands.

Abstract

The Shewanella genus is widely recognized for its remarkable respiratory adaptability in anaerobic environments, exhibiting potential for bioremediation and microbial fuel cell applications. However, the genetic manipulation of certain Shewanella strains is hindered by defense systems that limit their genetic modification in biotechnology processes. In this study, we present a systematic method for predicting, mapping, and functionally analyzing defense islands within bacterial genomes. We investigated the genetically recalcitrant strain Shewanella putrefaciens CN32 and identified several defense systems located on two genomic islands integrated within the conserved chromosomal genes trmA and trmE. Our experimental assays demonstrated that overexpression of excisionases facilitated the excision of these islands from the chromosome, and their removal significantly enhanced the genetic manipulation efficiency of S. putrefaciens CN32. Further analysis revealed that these defense islands are widespread across various Shewanella strains and other gram-negative bacteria. This study presents an effective strategy to circumvent genetic barriers and fully exploit the potential of Shewanella for environmental and microbial engineering applications.

Importance: Efficiently modifying bacterial genomes is critical for advancing their industrial applications. However, bacteria in complex environments naturally develop defense mechanisms in response to bacteriophages and exogenous DNA, which pose significant challenges to their genetic modification. Several methods have emerged to tackle these challenges, including in vitro methylation of plasmid DNA and targeting specific restriction-modification (R-M) and CRISPR loci. Nevertheless, many bacteria harbor multiple, often uncharacterized defense mechanisms, limiting these strategies. Our study differs from previous approaches by specifically targeting defense islands-clusters of defense systems located within mobile genetic elements. Here, we investigated Shewanella putrefaciens CN32 and identified two key defense islands responsible for these protective functions. By selectively deleting these defense islands, we significantly enhanced the efficiency of genetic manipulation in S. putrefaciens. Our findings not only demonstrate a promising strategy for improving genetic engineering in Shewanella but also suggest broader applicability across other bacterial species. This work opens new opportunities for optimizing microbial processes in biotechnology, highlighting the potential of defense island-targeted genetic modification.