Functional genomics and proteomic analysis of gentisate degradation in Pseudomonas alcaligenes NC1MB 9867

Abstract

Pseudomonas alcaligenes NCIMB 9867 (P25X) was postulated to harbor isofunctional (constitutively-expressed or inducible) enzymes for the gentisate pathway. To date, only the gene encoding the constitutively-expressed gentisate dioxygenase (GDO I) had been characterized.Proteome analysis of P25X mutant G56 (a GDO I mutant) and mutant G54 (a I?54 mutant) in response to gentisate showed that a protein spot was postulated to be the inducible GDO II in P25X and had I?54 dependent expression pattern and that I?54 played a global regulatory role in the expression of a wide variety of genes in P25X. Nineteen heat shock proteins (Hsp) were identified when P25X cells were cultured under high growth temperature and gentisate exposure.Via the reverse genetic technology, an hbz gene cluster containing 9 genes was cloned, including the hbzE encoding the GDOII and the hbzR encoding a putative LysR-type transcriptional regulator. The recombinant GDO II was biochemically characterized.