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.

Keywords: Shewanella; Shewanella putrefaciens; defense system; genetic manipulation; genomic islands.

Fig 1

Three defense islands in S. putrefaciens CN32. (a) Genomic comparison of CN32 (CP000681) with W3-18-1 (CP000503) and ANA-3 (NC_008577) using BRIG showing the locations of the defense islands CGI48, GI_trmA, and MGI_trmE on the circular genome. Sequence-based synteny analysis of the CN32, W3-18-1, and ANA-3 genomes using Mauve to locate the positions of the two ends of GI_trmA (b) and MGI_trmE (c). The integration locus trmA and trmE were indicated. Mapping of the integration locus of GI_trmA (d) and MGI_trmE (e) in CN32, W3-18-1, and ANA-3. Open reading frames with putative functions are shown in different colors.

Fig 2

The excision of GI_trmA and MGI_trmE in CN32. The excision rate of GI_trmA (a) and MGI_trmE (b) when the corresponding excisionase Xis was overexpressed or when the hns gene was deleted in CN32 . The data are shown as the mean ± SD. For biological replicates, n = 4. Statistical analysis was performed using an unpaired t-test, “ns” denotes no significant difference, **** indicates P < 0.0001.

Fig 3

Construction and verification of mutant strains ΔGI_trmA and ΔMGI_trmE. (a) A schematic diagram illustrating the construction of ΔMGI_trmE using Xis₃₉₉₁ over-expression. (b) PCR verification of ΔMGI_trmE. (c) A schematic diagram illustrating the construction of ΔGI_trmA using Xis₃₅₂₂ over-expression in the toxin gene deletion mutant ΔparE₃₂. (d) PCR verification of ΔparE₃₂ using the indicated primers in c. (e) PCR verification of ΔGI_trmA.

Fig 4

GI_trmA and MGI_trmE can help host bacteria defense against phages and plasmids. Representative images (a) and the quantification (b) of the efficiency of plating (EOP) of phages T1, T4, T5, T7, and EEP infection in engineered strain BW25113/pGI_trmADs (n = 5), BW25113/pMGI_trmEDs (n = 3), and control strain BW25113/pBBR1Cm (n = 5). The defense systems in GI_trmA and MGI_trmE with locus tag accessions cloned in the engineered bacteria are indicated. (c-f) Conjugation efficiency of plasmid pBBR1Cm, pMMD207, pMBLCas9, and pSC189 to strains ΔGI_trmA and ΔMGI_trmE, compared with CN32 wild type. “ND” stands for “not detectable,” indicating that the conjugation efficiency for samples CN32 and ΔCGI48 in panels e and f was below the limit of detection (1 × 10⁻⁸). For statistical analysis, the value of 1 × 10⁻⁸ was used. The data are shown as the mean ± SD. For biological replicates: panels c-f, n = 4. Statistical analysis was performed using an unpaired t-test, “ns” denotes no significant difference; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001).

Fig 5

The defense systems in GI_trmA and MGI_trmE are widely distributed in gram-negative bacteria. Sequence comparison of the defense systems in GI_trmA (a) and MGI_trmE (b) with their homologous defense systems in their corresponding genomic islands. The predicted length of the genomic islands and their integration sites was shown. Open reading frames with putative functions are shown in different colors.