Conditional mutations in OutE and OutL block exoenzyme secretion across the Erwinia carotovora outer membrane

The phytopathogen Erwinia carotovora subspecies carotovora secretes pectinases and cellulase via the general secretory pathway, a process requiring at least 13 proteins encoded by the out gene cluster. By exploiting λ::Tn5, a generalised transducing phage (φKP) and localised mutagenesis of the out gene cluster, we have produced a histidine auxotroph and 19 new secretory mutants, including two (HJN1003 and HJN1004) which were conditional (temperature sensitive) for secretion. All of the mutants accumulated pectinases and cellulase in the periplasm, but in the case of HJN1003 and HJN1004, only at the restrictive temperature. HJN1003 and HJN1004 were complemented by the outE and outL wild-type genes, respectively, and both mutant alleles were cloned and sequenced to reveal single missense substitutions. HJN1003 carries an Arg¹⁶⁶ to His alteration in OutE and HJN1004 carries a Pro¹⁵⁹ to Leu alteration in OutL. Topology mapping of OutL using a β-lactamase probe confirmed that OutL is a type II bitopic trans-inner membrane protein and that the mutated Pro¹⁵⁹ residue in HJN1004 is located in the cytoplasmic domain of OutL. Hence, the secretion of exoenzymes across the outer membrane is critically dependent on the conformation of secretory components located at the cytoplasmic face of the inner membrane.