SMC-like Wadjet system prevents plasmid transfer into Clostridium cellulovorans

Abstract

This study demonstrates the impact of a Structure Maintenance of Chromosome (SMC)-like Wadjet system on the horizontal gene transfer of plasmids by conjugation to a recipient that naturally containing such a system for the first time. A Clostridium cellulovorans mutant with dramatically improved efficiency to receive plasmid DNA by conjugation was isolated and sequenced. Three spontaneous chromosomal deletions included a type II restriction-modification system, a putative CRISPR system, and a cluster of ORFs named jetABCD encoding a putative Wadjet system. Since nearly nothing is known about the role of naturally occurring Wadjet systems in their native host bacteria, markerless chromosomal deletion of jetABCD in the C. cellulovorans wildtype strain 743B was achieved and the effect on conjugative plasmid uptake was studied. The transconjugation frequency of the jetABCD mutant was increased by about five orders of magnitude compared to wildtype C. cellulovorans recipient cells. Bioinformatic analysis of genome sequences of the Bacillota phylum revealed near-complete mutually exclusive possession of either plasmids < 40 kb or jetABCD genes, indicating high efficiency of Wadjet systems in small plasmid prevention in bacteria. Importantly, the implications of this study go beyond the case of C. cellulovorans. Our study demonstrates that the eradication of Wadjet systems can dramatically improve the uptake of recombinant plasmids and thereby enhance genetic engineering of bacterial strains of interest for biotechnological applications. KEY POINTS: • Native Wadjet system inhibits plasmid transfer by conjugation in C. cellulovorans • Deleting jetABCD increased plasmid uptake by about five orders of magnitude • Possession of Wadjet systems efficiently block plasmid maintenance in Bacillota.

Keywords: Clostridium cellulovorans; CRISPR-Cas; Plasmid defense system; Restriction-modification system; Transconjugation; Wadjet system.

Fig. 1

Improvement of transconjugation efficiency in a spontaneous mutant of C. cellulovorans. a Selection plate with merely few C. cellulovorans transconjugant colonies (marked with arrows) after a representative mating experiment with E. coli NEB10B-pMTL85131/placORMI as donor and the C. cellulovorans 743B wildtype strain as recipient. b Selection plate after mating with E. coli NEB10B-pMTL85131/placORMI as donor and the C. cellulovorans mutant strain ΔengE/2 as recipient. c Comparison of the genome sequence of C. cellulovorans 743B WT and the mutant strain ΔengE/2 by dot plot analysis (Cabanettes and Klopp 2018). The three deletion regions A, B and C that were found to be missing in the chromosome of the ΔengE/2 mutant strain are marked with orange ellipses. d Chromosomal location of deletion A. e Chromosomal location of deletion B. f Chromosomal location of deletion C

Fig. 2

Spontaneous deletion of the cas locus. a The gDNA of wildtype C. cellulovorans 743B was isolated after 3 to 17 consecutive sub-cultivations after the strain was obtained from DSMZ (P3, P4, P6, P7, P8, P11, and P17). The cas locus was amplified using primers D5 and D6, resulting in a 6 kb product, while no product was expected when the locus was spontaneously deleted (lanes 1–15, the PCR was performed in technical duplicates). The integrity of the gDNA was controlled using primers D7 and D8 to amplify the Clocel_4007-4008 locus (lanes 16–22). b C. cellulovorans 743B chromosomal cas locus. The identity of the ORF cas8 is hypothetical and requires further confirmation. The binding sites of the check primers Del5 and Del6 are indicated with arrows. The confirmed deletion of C. cellulovorans ΔengE/2 (see Fig. 1e) is shown in grey

Fig. 3

Alignment of JetD amino acid sequence segments. The protein sequences of JetD proteins from Bacillus cereus Q1, Pseudomonas aeruginosa PA14, C. cellulovorans 734B, Clostridium kluyveri JZZ, and Clostridium beijerincki DJ123 were aligned using Jalview’s Muscle algorithm with default settings. Conserved amino acids are shown in blue, and the experimentally confirmed essential residues of the JetD enzymes from Bacillus cereus Q1 and Pseudomonas aeruginosa PA14 are indicated with arrows

Fig. 4

Deletion of the putative Wadjet gene cluster jetABCD in the C. cellulovorans 743B wildtype strain. a PCR analysis with primers D1 and D2 either confirmed the chromosomal deletion of jetABCD (4.3 kb) or showed the wildtype genotype (10.7 kb) (lanes 1–5). Confirmation of plasmid curing was done by PCR with primers D3 and D4 targeting cas9 (4.1 kb) (lanes 6–10). Lanes 1 and 6, C. cellulovorans ΔjetABCD K1; lanes 2 and 7, C. cellulovorans ΔjetABCD K2; lanes 3 and 8, C. cellulovorans ΔjetABCD K4; lanes 4 and 9, C. cellulovorans 743B wildtype; lanes 5 and 10, pNick_delJetABCD-sg1 plasmid. b C. cellulovorans 743B chromosomal Wadjet locus. The check primers Del1 and Del2 are indicated with arrows. The confirmed deletion of C. cellulovorans ΔjetABCD is shown in black

Fig. 5

Conjugation efficiency of plasmid pMTL83151 into C. cellulovorans ΔjetABCD. Panels a and b show selection plates after mating of pMTL83151-bearing E. coli donor strains with C. cellulovorans ΔjetABCD as recipient. a Mating experiment using E. coli NEB10B-pMTL85131 (unmethylated plasmid) as donor and C. cellulovorans ΔjetABCD as recipient. b Mating experiments were performed using E. coli NEB10B-pMTL85131/placORMI (plasmid methylated in vivo via expression of a C. cellulovorans-born methyltransferase of a type II RM system) as donor and C. cellulovorans ΔjetABCD as recipient. c Comparison of the conjugation efficiency of different C. cellulovorans strains using unmethylated plasmid (black bar) and methylated plasmid (striped bar). The efficiency values are given as number of transconjugants per recipient cell with standard deviation of five independent experiments for ΔjetABCD (n = 5, mean ± SD). *Data for WT and ΔTypeII are taken from Schöllkopf et al. (2025). The experimental conditions (see Materials and Methods) were the same in all experiments

Fig. 6

Co-occurrence of jetA, jetB, jetC orthologs and plasmid/jetD ortholog-bearing Bacillota strains with sequenced genomes