Transcriptional repression of the poly(3-hydroxybutyrate) depolymerase in Ralstonia pickettii T1 by a tetR-like gene

Abstract

Ralstonia pickettii T1 secretes a poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZ) and a 3-hydroxybutyrate (3HB)-oligomer hydrolase, and extracellularly degrades PHB to produce 3HB. However, it is not clear how the expression of phaZ is regulated. In this study, the mechanism by which phaZ expression is controlled in R. pickettii T1 was examined using a mutant made by the random insertion of a transposon, Tn5. The mutant produced a larger amount of PhaZ than the wild type in nutrient broth or a minimal salt (SM) medium supplemented with succinate. However, there was essentially no difference in the activity or amount of PhaZ in the culture supernatant between the wild type and mutant when the two were grown on 3HB. The gene disrupted by the insertion of Tn5 (epdR) was cloned and its nucleotide sequence was determined. In a BLAST search, epdR showed a high degree of similarity to genes for TetR transcriptional regulators of several bacteria. The introduction of epdR into the wild type and mutant grown on the three media described above decreased the amount of PhaZ. These results indicated EpdR to be involved in the repression of phaZ in R. pickettii T1. A quantitative RT-PCR analysis indicated that mRNA levels corresponded with the activity detected and the amounts of PhaZ in the wild type and mutant. Furthermore, the amount of epdR transcript was inversely proportional to the amount of phaZ transcript. In addition, the existence of a positive element acting on phaZ expression was suggested, because in the mutant lacking EpdR, the amount of phaZ transcript varied in cells grown in SM-3HB, SM-succinate or nutrient broth. Based on the above results, a model for the regulation of PhaZ expression in R. pickettii T1 is proposed.