Physiological Role Of The Isofuctional Salicylate Hydroxylase Gene-nahU In Mineralization Of Naphthalene By Pseudomonas Putida ND6

Naphthalene is a common environmental pollutant, composed of most simple two benzene rings. P. putida ND6, which was isolated from Tianjin southern effluents, can degrade naphthalene of 2 g/L in minimal medium by 98% in 48 hours. As most of the degrading bacteria, the naphthalene catabolic genes of ND6 strain were located in plasmid. This strain contains two plasmids, the one related to naphthalene degradation is 101 858 bp plasmid pND6-1. Plsmid pND6-1 has been sequenced and annotated, and the copy number is two determined by quantitative real-time PCR. Sequence analysis indicated that P. putida ND6-mediated naphthalene degradation occurred via mechanisms similar to those described for other naphthalene-degrading strains. The naphthalene-degrading genes in these strains are organized in two operons:the upper pathway operon (nahAaAbAcAdBFCED) encoding enzymes involved in the conversion of naphthalene to salicylate and the lower pathway operon (nahGTHINLOMKJ) encoding enzymes necessary for the conversion of salicylate to tricarboxylic acid cycle intermediates via the metacleavage pathway.P. putida ND6 is unique in that it possesses two isofunctional salicylaldehyde dehydrogenase genes nahF and nahV in addition to two isofunctional salicylate hydroxylase genes nahG and nahU. In this study, the biological function of nahU in P. putida ND6 was studied.Using homologous recombination method, nahU-mutated strain P. putida ND6-ΔΔU and nahG-mutated strain P. putida ND6-AAG were constructed, respectively. By comparing the respective mutants to the parental strain with respect to growth and naphthalene-degrading rates, we found that the naphthalene-degrading rates differed between the wild-type and mutant strains, that is, naphthalene-degrading rates was lower in the ND6-ΔΔU than that in wild-type ND6, but naphthalene-degrading rates in ND6-AAG was the similar in wild-type ND6. At the mRNA and protein level, the nahU expression levels in the mutants were lower compared to levels observed in the wild-type strain, but the nahG expression levels in the mutants were higher than expression levels in the wild-type ND6; Furthermore, no matter cultured with any carbon source, salicylate hydroxylase activitives of the wild-type ND6 were higher than ND6-ΔΔU, but lower than ND6-ΔΔG. In conclusion, the presence of the additional salicylate hydroxylase NahU may be physiologically advantageous to the host strain ND6, which facilitates degradation of naphthalene through increasing the salicylaldehyde transformation efficiency, and reduces lethality when deletion mutations happened in the classical salicylate hydroxylase gene nahG.