Mutations in genes involved in the flagellar export apparatus of the solvent-tolerant Pseudomonas putida DOT-T1E strain impair motility and lead to hypersensitivity to toluene shocks

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium. Random mutagenesis with mini-Tn5-'phoA-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo -4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol). The mutant strain e xhibited patterns of tolerance or sensitivity to a number of antibiotics, d etergents, and chelating agents similar to those of the wild-type strain. T he mutation in this strain therefore seemed to specifically affect toluene tolerance. Cloning and sequencing of the mutation revealed that the mini-Tn 5-'phoA-Km was inserted within the fliP gene, which is part of the fliLMNOP QRflhBA cluster, a set of genes that encode flagellar structure components. FliP is involved in the export of flagellar proteins, and in fact, the P. putida fliP mutant was nonmotile. The finding that, after replacing the mut ant allele with the wild-type one, the strain recovered the wild-type patte rn of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance. An flhB knockout mutant, another gene component of the' flagellar export apparatus, was also nonmotile and hypersensitive to t oluene. In contrast, a nonpolar mutation at the fliL gene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yiel ded a nonmotile yet toluene-resistant strain. The results are discussed reg arding a possible role of the flagellar export apparatus in the transport o f one or more proteins necessary for toluene tolerance in P. putida DOT-T1E to the periplasm.