Molecular and Functional Characterization of MobK Protein—A Novel-Type Relaxase Involved in Mobilization for Conjugational Transfer of Klebsiella pneumoniae Plasmid pIGRK

Abstract

Conjugation, besides transformation and transduction, is one of the main mechanisms of horizontal transmission of genetic information among bacteria. Conjugational transfer, due to its essential role in shaping bacterial genomes and spreading of antibiotics resistance genes, has been widely studied for more than 70 years. However, new and intriguing facts concerning the molecular basis of this process are still being revealed. Most recently, a novel family of conjugative relaxases (Mob proteins) was distinguished. The characteristic feature of these proteins is that they are not related to any of Mobs described so far. Instead of this, they share significant similarity to tyrosine recombinases. In this study MobK—a tyrosine recombinase-like Mob protein, encoded by pIGRK cryptic plasmid from the Klebsiella pneumoniae clinical strain, was characterized. This study revealed that MobK is a site-specific nuclease and its relaxase activity is dependent on both a conserved catalytic tyrosine residue (Y179) that is characteristic of tyrosine recombinases and the presence of Mg2+ divalent cations. The pIGRK minimal origin of transfer sequence (oriT) was also characterized. This is one of the first reports presenting tyrosine recombinase-like conjugative relaxase protein. It also demonstrates that MobK is a convenient model for studying this new protein family.

Salmonella Genomic Island 1 requires a self‐encoded small RNA for mobilization

“…The fact that the mobilization proteins are completely unrelated to those of the helper IncC plasmids raises the question of how the initiation complex of SGI1 is assembled on oriT and transported to T4SS of the helper plasmid. A similar question has been posed for the initiation complex of pIGRK, which is mobilized by the unrelated conjugation system of RP4 (Nowak et al., 2021 ). It was previously shown that the IncC helper‐encoded relaxase TraI is not necessary for SGI1 mobilization, but increases the transfer rate of the island with orders of magnitude (Kiss et al., 2019 ).…”

“…MpsA and MpsB, the two mob SGI1 ‐encoded proteins essential for SGI1 mobilization are related to the recombinases of the λ integrase family and not to relaxases. MpsA, as a first example among IEs, has been suggested to act as an atypical relaxase of SGI1‐family elements, similar to TcpM and MobK, the Tyr‐recombinase‐related relaxase proteins of plasmids pCW3 and pIGRK, respectively (Nowak et al., 2021 ; Wisniewski et al., 2016 ). The other two putative proteins encoded by ORFs S021 and S022 appear unrelated to any known proteins.…”

“…The newest group of relaxases includes enzymes related to the Tyr‐recombinases. Currently, only two plasmid‐derived proteins, TcpM and MobK, the relaxases of pCW3 and pIGRK, respectively, and MpsA protein, the presumed relaxase of the Salmonella IME, SGI1, belong to this group (Kiss et al., 2019 ; Nowak et al., 2021 ; Wisniewski et al., 2016 ).…”

“…MpsA and two plasmid‐borne relaxases, TcpM and MobK, appear to be Tyr‐recombinase‐like proteins, which is mostly based on the similarity of their catalytic domains to the DNA_BRE_C domains with the conserved catalytic pentad RK(H/Y)YRH. However, they are not related to each other and the genetic context of the respective mob regions is also different, except that the oriT s are located upstream of the relaxase genes (Kiss et al., 2019 ; Nowak et al., 2021 ; Wisniewski et al., 2016 ). The three proteins lack the core DNA‐binding domain that is obligatory to the classical Tyr‐recombinases suggesting that they bind to their cognate oriT s differently.…”

“…The three proteins lack the core DNA‐binding domain that is obligatory to the classical Tyr‐recombinases suggesting that they bind to their cognate oriT s differently. In the case of SGI1, MpsB may be an accessory protein involved in DNA binding as it resembles the core‐binding domains of integrases (Kiss et al., 2019 ; Nowak et al., 2021 ); however, similar accessory proteins have not yet been identified on pCW3 and pIGRK.…”