Biological Function And Transcriptional Regulatory Model Of Salicylaldehyde Dehydrogenase NahV In Naphthalene-degrading Pseudomonas Putida ND6

Pseudomonas putida ND6, which was isolated from Tianjin southern effluents, can degrade naphthalene of2g/L in minimal medium by98%in48hours. As most other naphthalene-degrading strains, the naphthalene-degrading genes of P. putida ND6are located on the large10,1858base pair (bp) pND6-1plasmid which has been sequenced and annotated. 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 meta-cleavage pathway.P. putida ND6is 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 and regulation model of NahV in P. putida ND6were studied.Plasmid copy number (PCN) is the number of the plasmid copy included in one strain. The PCN of plasmid pND6-1in P. putida ND6is two determined by quantitative real-time PCR. This is the first report of copy number of plasmid that the size is more than100kb.Using homologous recombination method, nahF-mutated strain P. putida ND6-AAF and nahv-mutated strain P. putida ND6-△△V 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 considerably lower in both the ND6-△△F and ND6-AAV than that in wild-type ND6. At the mRNA and protein level, the nahF or nah V expression levels in the ND6-△△V or ND6-△△F mutant were higher compared to levels observed in the wild-type strain. Furthermore, the salicylaldehyde dehydrogenase activities were considerably lower in both the nah V and nahF mutants compared to wild-type ND6when cultured with naphthalene or salicylate as the sole carbon source. In conclusion, the presence of the additional salicylaldehyde dehydroxylase NahV may be physiologically advantageous to the host strain ND6, which facilitates degradation of naphthalene through increasing the salicylaldehyde transformation efficiency, prevents salicylaldehyde toxic effect and reduces lethality when deletion mutations happened in the classical salicylaldehyde dehydroxylase NahF.nahR-mutated strain P. putida ND6-△△R was also constructed to compare the differences between mutant and wild-type strain at mRNA and salicylaldehyde dehydrogenase activities level. We can detect the expression of nahR in P. putida ND6, whenever cultured with any carbon source, and the expression of nahR increased with the addition of inducer salicylate. Neither nahF nor nahV gene expressed constitutively. NahR binds to the promoter first. In the presence of inducer salicylate, NahR shifts its conformation to interact with RNA polymerase and help it bind to the promoter to initiate transcription.