Localization of GFP-MtgA in wild-type ponA⁺ ponB⁺ strain LMC500 (A) and ponA(ts) ponB strain EJ801 (B) grown in LB medium at 28°C, both harboring pDML2005. The arrows point to cells with fluorescent signals at the division site. Photographs were taken with a cooled AxioCam MRm (Zeiss) mounted on an Zeiss Axio Imager.Z1 fluorescent microscope through a EC Plan-Neofluar 100 × 1.3 oil immersion objective in bright field and fluorescence using filter set 37 (400- to 500-nm band pass excitation and 460- to 560-nm band pass emission; Zeiss). Images were analyzed using the AxioVision Rel. 4.5 (Zeiss) software as previously described (24). Bar, 5 μm.

Interaction between MtgA and PBP3, FtsW, FtsN, or itself by the Cya bacterial two-hybrid assay. DHM1 transformants producing T18-T25 (1), T18-MtgA-T25 (2), T18-T25-MtgA (3), T18-MtgA-T25-Lpp-PBP3 (4), T18-Lpp-PBP3-T25-MtgA (5), T18-PBP1B-T25-PBP3 (6), T18-PBP3-T25-PBP1B (7), T18-MtgA-T25-PBP3 (8), T18-PBP3-T25-MtgA (9), T18-MtgA-T25-FtsN (10), T18-FstN-T25-MtgA (11), T18-MtgA-T25-FstW (12), T18-FtsW-T25-MtgA (13), and T18-MtgA-T25-MtgA (14) were used. (A) Transformants were grown at 30°C for 30 h on LB agar plates containing 0.5 mM IPTG, 40 μg/ml X-Gal, 50 μg/ml ampicillin, and 20 μg/ml chloramphenicol. (B) Quantitative analysis of the β-galactosidase activity from transformants grown in LB medium in the presence of 0.4 mM IPTG for 16 h at 30°C was carried out as described previously (17). Data are averages of three independent experiments.