Expansion of growth substrate range in Pseudomonas putida F1 by mutations in both cymR and todS, which recruit a ring-fission hydrolase CmtE and induce the tod catabolic operon, respectively

Abstract

Expansion of growth substrate range in Pseudomonas putida F1 by mutations in both cymR and todS, which recruit a ring-fission hydrolase CmtE and induce the tod catabolic operon, respectively Pseudomonas putida F1 can assimilate benzene, toluene and ethylbenzene using the toluene degradation pathway, and can also utilize p-cymene via p-cumate using the p-cymene and p-cumate catabolic pathways. In the present study, P. putida F1 strains were isolated that were adapted to assimilate new substrates such as n-propylbenzene, n-butylbenzene, cumene and biphenyl, and the molecular mechanisms of genetic adaptation to an expanded range of aromatic hydrocarbons were determined. Nucleotide sequence analyses showed that the selected strains have mutations in the cymR gene but not in todF gene. The impairment of the repressor CymR by mutation led to the constitutive expression of CmtE, a meta-cleavage product hydrolase from the cmt operon. This study also showed that CmtE has a broad range of substrates and can hydrolyse meta-cleavage products formed from biphenyl and other new growth substrates via the toluene degradation pathway. However, the artificially constructed strain P. putida F1(cymR : : Tc r ) and a recombinant P. putida F1, which expressed CmtE constitutively, could not grow on the new substrates. The adapted strains possess the tod operon, which is induced by new growth substrates that are poor inducers of wild-type P. putida F1. When the todS gene from the adapted strains was introduced in a trans manner to P. putida F1(cymR : : Tc r ), the resulting recombinant strains were able to grow on biphenyl and other new substrates. This finding indicates that the TodS sensor was altered to recognize these substrates and this conclusion was confirmed by nucleotide sequence analyses. Amino acid substitutions were found in the regions corresponding to the receiver domain and the second PAS domain and their boundaries in the TodS protein. These results showed that P. putida F1 adapted strains capable of growth on n-propylbenzene, n-butylbenzene, cumene and biphenyl possess mutations to employ CmtE and to induce the tod catabolic operon by the new growth substrates. INTRODUCTIONPseudomonas putida F1 can assimilate benzene, toluene and ethylbenzene (Gibson et al., 1968). The enzymic pathway responsible for converting these aromatic hydrocarbons to TCA cycle intermediates is called the toluene degradation (tod) pathway (Finette et al., 1984;Gibson et al., 1990). This tod pathway is one of the cis-dihydrodiol pathways and has been well characterized biochemically and genetically. The catabolic genes of this pathway are clustered in a configuration of todXFC1C2BADEGIH (Fig. 1) (Lau et al., 1997;Menn et al., 1991;Wang et al., 1995;Zylstra & Gibson, 1989;Zylstra et al., 1988). The tod pathway consists of seven enzymic reactions and is initiated by a multicomponent toluene dioxygenase (encoded by todC1C2BA) (Zylstra & Gibson, 1989) that catalyses, for instance, the formation of cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene fro...

The TodS–TodT two-component regulatory system recognizes a wide range of effectors and works with DNA-bending proteins

“…This observation provided direct proof that toluene binds to the N-terminal sensor domain. At first sight, our data may appear inconsistent with the findings by Choi et al (4), who showed that TodS mutants within the C-terminal half had a wider effector specificity range. Although we demonstrated that toluene binds to the N-terminal supradomain, an indirect role of the C-terminal region in toluene sensing is likely.…”

“…On the basis of moderate sequence similarity with the heme-binding oxygen sensor FixL, the C-terminal supradomain of TodS was proposed to be involved in oxygen sensing (3). Choi et al (4) isolated TodS mutants that recognized aromatic compounds that are not effectors of the wild-type protein. Mutations were scattered along the C-terminal half of TodS.…”

Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

“…The authors concluded that TodS might actually be a dual sensor that is capable of sensing both toluene as a primary signal and oxidative stress in the cytoplasm (118). Isolation of TodS effector specificity mutants proved that the aromatic compounds directly interact with the sensor kinase (26). From amino acid similarity comparisons, it was concluded that TutC, TmoS, and StyS are homologous to TodS (35,205,264).…”

“…An intrinsic response regulator domain reminiscent of CheY (266) spans amino acids 443 to 570 and could be potentially phosphorylated by other signal-transducing proteins. Amino acids 34 to 153 contain a hypothetical chemical sensor domain, whereas the region between positions 592 and 735 is supposed to form the oxygen-sensing domain (26,118). Oxygen-sensing domains are found in various redox and light sensor proteins (248,276).…”

Aerobic and Anaerobic Toluene Degradation by a Newly Isolated Denitrifying Bacterium, Thauera sp. Strain DNT-1

“…The composition of the growth medium used follows: 1. 12 , and 10 mg of D-biotin (all in 1 liter of distilled water) (pH 7.0 to 7.1). Toluene (0.5 mM) was added as the sole carbon source, and KNO 3 (5 mM) was added when required.…”