Describing the non-canonical restriction-modification type II system (RMII) in pseudomonas putida PaW85

Abstract

Bacteriophages are the most abundant biological entities on Earth and exert a major selective pressure on bacterial populations. To counteract phage infection, bacteria have evolved a diverse arsenal of anti-phage defense mechanisms, among which Restriction-Modification (RM) systems are the most prevalent. RM systems protect the host by methylating self-DNA at specific recognition sequences while cleaving incoming foreign DNA that lacks the corresponding modification. This thesis focuses on a non-canonical type II RM system (RMII) encoded in Pseudomonas putida PaW85, a biotechnologically important soil bacterium. The RMII locus comprises a putative restrictase (R, PP_3988) and two distinct methylases (M1, PP_3989 and M2, PP_5651). Using phage susceptibility assays with an expanded CEPEST phage collection, the system was confirmed to reduce infection efficiency of phages from genus clusters G16 and G17 by up to 200-fold. To determine the functional contribution of each methylase, deletion strains lacking either or both methylases were constructed and used for restrictase expression experiments. Both methylases were shown to be important, which refers to redundancy. Bioinformatic and structural analyses show that the M1 and M2 are unrelated, probably coupled together by horizontal gene transfer. M1 is a cytosine methyltransferase with structural similarity to methylases of known type IIA(S) systems, while M2 is evolutionarily unrelated to M1 and shares structural similarity with adenine methyltransferases. These findings characterize a non-canonical RMII system in P. putida PaW85 and provide a foundation for future biochemical studies of the restrictase and for informed genomic engineering of this industrially relevant bacterium for possible biotechnological applications.