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. 2023 Jun 20;17(10):1564–1577. doi: 10.1038/s41396-023-01457-2

Fig. 2. LqqE1 is a T4E that could be translocated into P. fluorescens by L. enzymogenes using the T4ASS.

Fig. 2

A Schematic maps of LqqE1 and VirD4, a T4ASS-pathway-specific ATPase. B Bacterial two-hybrid assays showing LqqE1 bound to VirD4. pTRG-VirD4 and pBT-LqqE1 were co-expressed in E. coli XL-1-blue. “+”, positive control with co-expression of plasmids pBT-GacS and pTRG-GacS; “-”, negative control with co-expression of empty pTRG and pBT vectors. LB, a non-selective condition; +3AT+Str, a selective condition. C Pull-down assays showing LqqE1-FLAG, but not the GFP-FLAG control, directly interacted with VirD4T-His. Immunoprecipitation (IP) was carried out using anti-FLAG antibody and Western blot was performed using anti-FLAG and anti-His antibodies. D Schematic diagram of the LqqE1 translocation assay. Lysobacter enzymogenes OH11 carrying the Cre-LqqE1 hybrid protein served as a donor strain resistant to kanamycin and gentamicin, while P. fluorescens 2P24 harboring pBBR-loxP as a recipient strain resistant to kanamycin and chloromycetin. No parent grew on selective medium containing kanamycin, gentamicin, and chloromycetin. The translocation of Cre-LqqE1 hybrid protein to the recipient strain results in the excision, through recombination at the loxP sites, of the DNA fragment that reconstitutes a functional loxP-GentR translational fusion. Only the recombined strain can survive on selective medium. Yellow diamond, transcriptional terminator; sacB, levansucrase; GentR, gentamycin acetyltransferase. E Transfer of Cre-LqqE1 hybrid protein from strain OH11 to strain 2P24. As described in Materials and Methods, protein transfer was performed by using either WTOH11 or ΔvirD4OH11 expressing the designated protein fusions as the donor strains and strain 2P24 expressing the pBBR-loxP as the recipient strains. The data show the efficiency of recombination (CmRKmRGentR/CmRKmR CFU recovered; recombined/total recipients). ***p < 0.001. The results are expressed as mean ± SD. F Analysis of Cre recombination by gel electrophoresis. The pBBR-loxP DNA fragment after Cre recombination can be amplified to 761-bp in size with M13F/M13R primers, and 3015-bp in size can be amplified without recombination. All experiments were repeated at least three times.