Figure 1.

Construction and verification of an M. barkeri ΔechABCDEF mutant. (A) Ech1 was constructed by transformation of M. barkeri to Pur^R with linearized plasmid carrying the Δech1∷pac-ori-aph mutation. Recombination (dotted lines) between homologous sequences on the plasmid and chromosome resulted in replacement of the ech operon with the mutation. A small fragment of echF remains in the mutant. (B) Mutant (Ech1) and wild-type (WT) chromosome structure was verified by DNA hybridization as described. The predicted sizes of hybridizing bands (base pairs) are shown in parentheses: lanes 1 and 8, DNA markers; lane 2, Ech1-HindIII (1938, 2018); lane 3, WT-HindIII (2011, 4540); lane 4, Ech1-EcoRV (3076, 3984); lane 5, WT- EcoRV (2132, 3551); lane 6, Ech1-EcoRI (3912, >4000); lane 7, WT-EcoRI (2283, >6526). (C) EchE and HdrD were detected by Western blot as described (8). Lane 1, 5 μg of Ech1 cell extract; lane 2, 5 μg of Ech1 membrane protein; lane 3, 0.1 μg of purified Hdr (control); lane 4, 0.1 μg of purified Ech (control); lane 5, 5 μg of WT cell extract; lane 6, 5 μg of WT membrane protein. The molecular mass of subunit EchE is about 39 kDa, the molecular mass of HdrD is 43 kDa. The migration of molecular mass standards is shown on the left.