Concerted action of NIC relaxase and auxiliary protein MobC in RA3 plasmid conjugation

Abstract

Summary Conjugative transfer of the broad‐host‐range RA3 plasmid, the archetype of the IncU group, relies on the relaxase NIC that belongs to the as yet uncharacterized MOBP4 subfamily. NIC contains the signature motifs of HUH relaxases involved in Tyr nucleophilic attack. However, it differs in the residue involved in His activation for cation coordination and was shown here to have altered divalent cation requirements. NIC is encoded in the mobC‐nic operon preceded directly by oriT, where mobC encodes an auxiliary transfer protein with a dual function: autorepressor and stimulator of conjugative transfer. Here an interplay between MobC and NIC was demonstrated. MobC is required for efficient NIC cleavage of oriT in supercoiled DNA whereas NIC assists MobC in repression of the mobC‐nic operon. A 7‐bp arm of IR3 (IR3a) was identified as the binding site for NIC and the crucial nucleotides in IR3a for NIC recognition were defined. Fully active oriTRA3 was delineated to a 47‐bp DNA segment encompassing a conserved cleavage site sequence, the NIC binding site IR3a and the MobC binding site OM. This highly efficient RA3 conjugative system with defined requirements for minimal oriT could find ample applications in biotechnology and computational biology where simple conjugative systems are needed.

The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation

“…Several auxiliary proteins of conjugative plasmids of Gram- origin are described to contain a RHH motif. These include, TraY and TraM of F plasmid, TrwA of R388, VirC2 of Agrobacterium tumefaciens , NikA of R64, TraJ of RP4, MobC of RSF1010, MbeC of ColE1, MobC of RA3 ( Bowie and Sauer, 1990 ; Zhang and Meyer, 1997 ; Moncalian and De la Cruz, 2004 ; Ragonese et al, 2007 ; Yoshida et al, 2008 ; Lu et al, 2009 ; Varsaki et al, 2009 ; Godziszewska et al, 2016 ). For some of them structure-based mutational analyses have demonstrated the importance of the RHH motif in oriT binding as well as relaxase recruitment ( Yoshida et al, 2008 ; Lu et al, 2009 ).…”

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

“…It poses an alpha-helical structure and resembles the CB domain of bacteriophage TR integrases. Both MpsA and MpsB are indispensable for CT and MpsA most likely binds DNA through MpsB [ 23 ]. No mpsB equivalent genes were identified in pIGRK and pCW3, suggesting that MobK and TcpM bind DNA differently.…”

“…The authors speculated that TcpM could recognize DNA sequences with a partial identity to pCW3 oriT . Alternatively, additional accessory protein(s), interacting with TcpM, is (are) needed to increase TcpM (as well as pIGRK MobK) specificity [ 23 , 24 , 28 ].…”

“…Representatives of the MOB T family belong to Rep_ trans nucleases [ 22 ]. A common feature of listed enzymes is a generation of single-strand cleavage in an oriT sequence [ 3 , 23 ]. Mobs remain covalently attached to the DNA 5′-end (except MOB C proteins [ 21 ]), leading to initiation of RCR replication from free 3′-OH DNA end.…”

Transcriptional Organization of the Stability Module of Broad-Host-Range Plasmid RA3, from the IncU Group

“…Our studies on plasmid RA3, the archetype of the IncU group, have revealed that this conjugative, low-copy-number, broad-host-range plasmid has a mosaic modular structure with functional modules intertwined by the global regulatory network ( 24 , 28 – 31 , 81 ). The present project focused on the expression of the RA3 stability module, comprising a unidirectionally transcribed set of ten ORFs split apparently into five differently regulated operons ( 24 ).…”