Identification of the origin of transfer (oriT) and DNA relaxase required for conjugation of the integrative and conjugative element ICEBs1 of Bacillus subtilis

Abstract

Integrative and conjugative elements (ICEs), also known as conjugative transposons, are mobile genetic elements that can transfer from one bacterial cell to another by conjugation. ICEBs1 is integrated into the trnS-leu2 gene of Bacillus subtilis and is regulated by the SOS response and the RapI-PhrI cell-cell peptide signaling system. When B. subtilis senses DNA damage or high concentrations of potential mating partners that lack the element, ICEBs1 excises from the chromosome and can transfer to recipients. Bacterial conjugation usually requires a DNA relaxase that nicks an origin of transfer (oriT) on the conjugative element and initiates the 5'-to-3' transfer of one strand of the element into recipient cells. The ICEBs1 ydcR (nicK) gene product is homologous to the pT181 family of plasmid DNA relaxases. We found that transfer of ICEBs1 requires nicK and identified a cis-acting oriT that is also required for transfer. Expression of nicK leads to nicking of ICEBs1 between a GC-rich inverted repeat in oriT, and NicK was the only ICEBs1 gene product needed for nicking. NicK likely mediates conjugation of ICEBs1 by nicking at oriT and facilitating the translocation of a single strand of ICEBs1 DNA through a transmembrane conjugation pore.

FIG. 1.

Effect of deletions in ICEBs1 on transfer and mobilization. (A) The genetic map of ICEBs1, indicating genes as open arrows and the flanking 60-bp repeats at attL and attR as thin black rectangles. The vertical dotted lines indicate the region of ICEBs1 oriT. (B to E) Thin lines below the map of ICEBs1 indicate the regions of ICEBs1 between attL and attR that are present in the various ΔICEBs1 mutations. Open spaces represent regions that are missing. Mating efficiencies are indicated to the right. Donor cells were induced with MMC and mixed with recipient strain CAL264, an ICEBs1⁰ recipient strain that expresses int from the Pspank promoter. Donor strains either contained the indicated ΔICEBs1 allele alone (thrC⁺) or also carried an immobilized ICEBs1 at thrC {thrC325::[ICEBs1(ΔattR::tet)]} that supplied all of the ICEBs1 excision and conjugation functions in trans but is unable to excise due to the deletion of attR. Mating efficiency was calculated as the percentage of transconjugant CFU per donor cell. The mean from at least two independent assays is reported. Mating efficiencies for the Δ(rapI-phrI)342::kan donor strain ranged from 0.81% to 3.7% in six independent assays and gave a mean of 2.0% with a standard deviation of 1.2%. Except for donor strains that gave no detectable transconjugants, mating efficiencies for other donor strains had similar amounts of variability. (E) Thick lines indicate that two derivatives of ΔICEBs1-205::kan carry an ∼0.8-kb oriT fragment from the ydcQ-nicK region (wild-type fragment, solid; mutant fragment, dashed).

FIG. 2.

NicK-dependent nicking of ICEBs1 between the GC-rich inverted repeat in oriT. (A and B) Primer extension products generated using end-labeled CLO76 and B. subtilis genomic DNA are shown along with DNA sequencing reactions (GATC). (A) Lanes 1 to 3, nicK⁺ (JMA168); lane 4, ΔnicK306 (CAL306); lane 5, ΔnicK306 Pxis-nicK (CAL346). Strains contained the IPTG-inducible Pspank(hy)-rapI and were grown without IPTG (lane 2) or with IPTG for 1 h (lanes 1 and 3 to 5). (B) Lanes 1 and 4, control ICEBs1⁺ Pspank(hy)-rapI (JMA168); lanes 2 and 5, ICEBs1⁰ Pspank(hy)-nicK488 (CAL502); lanes 3 and 6, ICEBs1⁰ Pspank(hy)-nicK477 (CAL501). Strains were grown for 1 h with (lanes 1 to 3) or without (lanes 4 to 6) IPTG. (C) Diagram of the double-stranded DNA sequence showing the nicK start codon (ATG in box), inverted repeats (horizontal lines), location of the nic site (vertical arrow), and base pairs conserved in the Tn916 oriT region (uppercase). (D) Alignment of the top strands of the conserved sequence in ICEBs1, Tn916, ICESt1, and ICESt3. A gap in the top two sequences indicates that ICEBs1 and Tn916 have one less base pair than ICESt1 and ICESt3 in the intervening region. Dashes indicate identity with ICEBs1 sequence. The lines and arrow are as in panel C.