FIG 1.

Role and regulation of int and 85-86-xis in the mobilization of MGIVchHai6. (A) Schematic genetic map of MGIVchHai6 drawn to scale. The left and right junctions (attL and attR) within the host chromosome are indicated by blue ticks at the extremities. ORFs with similar function are color coded as indicated in the figure. Green flags indicate the position and orientation of predicted AcaCD binding sites (13). The black flag indicates the position and orientation of the P_int promoter. The insertion sites of In36A1 integron and Tn6310 transposon are shown. The gene numbers correspond to the last digits of the respective locus tags in GenBank accession no. AXDR01000001. ACSSuTmT, resistance to ampicillin, chloramphenicol, spectinomycin/streptomycin, sulfamethoxazole, trimethoprim, and tetracycline; Hg, mercury resistance. (B) Mobilization assays of MGI^Kn or its Δint, Δxis, Δ86, or Δ85 mutants were carried out using E. coli GG56 (Nx) bearing pVCR94^Sp as the donor strain, and CAG18439 (Tc) as the recipient strain. Complementation assays were performed in the donor (D) or recipient (R) strain by expressing the missing gene from P_BAD on pBAD-int or pBAD-xis. An “×” indicates that the transfer frequency was below the detection limit (<10⁻⁷). Bars represent the means ± standard errors of the means (error bars) from three independent experiments. Statistical analyses were carried out on the logarithm of the values using a one-way analysis of variance (ANOVA), followed by Dunnett’s multiple-comparison test with the wild-type (WT) MGI^Kn as the control. Statistical significance is indicated as follows: ****, P < 0.0001; ns, not significant. (C) β-Galactosidase activities of P_int, P₈₅, and P₈₄ transcriptionally fused to lacZ. Colonies were grown on LB agar with or without arabinose to induce acaCD expression from pacaCD. (D) Induction levels of P_int, P₈₅, and P₈₄ in response to AcaCD. β-Galactosidase assays were carried out using the strains of panel C. Ratios between the enzymatic activities in Miller units for the arabinose-induced versus noninduced strains containing pacaCD are shown. The AcaCD-regulated promoter P_traHs of SGI1 served as a positive control, and cells devoid of pOPlacZ served as a negative control. The bars represent the means ± standard errors of the means (error bars) from two independent experiments.