Isolation and characterization of toluene-sensitive mutants from Pseudomonas putida IH-2000

Abstract

Two toluene-sensitive mutants were generated from Pseudomonas putida IH-2000, the first known toluene-tolerant isolate, by Tn5 transposon mutagenesis. These mutants were unable to grow in the presence of toluene (log P ow 2.8) but they could grow in medium overlaid with organic solvents having a log P ow value higher than that of toluene such as p-xylene (log P ow 3.1), cyclohexane (log P ow 3.4) and n-hexane (log P ow 3.9). The Tn5 transposable element knocked out a cyoB-like gene in one mutant and a cyoC-like gene in the other mutant. Seven open reading frames were found in a 5.5-kb region containing the cyoB-and cyoC-like genes of strain IH-2000. ORFs 3^7 showed significant identity to the cyoABCDE gene products of Escherichia coli, but ORFs 1 and 2 showed no significant homology to any protein reported so far. The growth patterns of the Tn5 mutants with the inactivated cyo-like gene were similar to that of the wild-type strain in the absence of organic solvents, although the doubling times were slightly longer than that of the wild-type strain. Our findings indicate that cyo is an important gene for toluene tolerance, although its role is still unclear. z

Expression of the Pseudomonas putida OCT Plasmid Alkane Degradation Pathway Is Modulated by Two Different Global Control Signals: Evidence from Continuous Cultures

“…When the oxygen supply becomes limiting, the levels of the cytochrome o ubiquinol oxidase decrease, and the function of this enzyme is replaced by other terminal oxidases with higher affinities for oxygen. The cytochrome o ubiquinol oxidase present in Pseudomonas species is believed to play a similar role (5,12,36). Given the strong influence of the cytochrome o ubiquinol oxidase on PalkB activity, we analyzed the effect of oxygen availability on PalkB expression.…”

“…In P. putida, the situation is less well understood. The different components of the oxidase are encoded in the cyoABCDE cluster (12). S1 nuclease protection assays allowed us to localize the promoter for the cyoA gene 319 nucleotides upstream from the translation start site.…”

Inactivation of Cytochrome o Ubiquinol Oxidase Relieves Catabolic Repression of the Pseudomonas putida GPo1 Alkane Degradation Pathway

“…The point of insertion of the mini-Tn5Sm in these strains was localized either by cloning and sequencing of the chromosomal DNA segment containing the Sm r determinant or by arbitrary PCR (see Materials and Methods). In mutant strain RCM4, the minitransposon was found inserted at an open reading frame showing 88% amino acid similarity to the cyoC gene of P. putida IH-2000 (28), which codes for subunit III (also known as subunit C) of cytochrome o ubiquinol oxidase (Fig. 2).…”

“…2). In mutant strains RCM18, RCM2A, and RCM117A, the minitransposon was found to interrupt an open reading frame showing 96% amino acid similarity to the cyoB gene of P. putida IH-2000 encoding subunit I (or subunit B) of the same cytochrome (28). These two genes form part of the cyo-ABCDE cluster, which encodes subunits II, I, III, and IV of the P. putida cytochrome o oxidase complex (also known as the cyo complex) and the heme o synthase (28, 36) (see Fig.…”

Transposon mutations in the flagella biosynthetic pathway of the solvent-tolerant Pseudomonas putida S12 result in a decreased expression of solvent efflux genes

“…In these mutants protondependent e¥ux genes, an ABC-transporter, and enzymes possibly involved in repair mechanisms of damaged membranes were found to be inactivated. Moreover, two toluene-sensitive mutants from P. putida IH-2000 were interrupted in cytochrome o, although the exact role of the cyo gene cluster in solvent tolerance has not been determined [30]. Surprisingly, toluene-sensitive mutants of P. putida DOT-T1 were isolated that were also interrupted in an operon for the £agella biosynthetic pathway (Segura, A. and Ramos, J.L., personal communication).…”