Functional analysis of the ocnE gene involved in nicotine-degradation pathways in Ochrobactrum intermedium SCUEC4 and its enzymatic properties.

The SCUEC4 strain of Ochrobactrum intermedium is a newly isolated bacterium that degrades nicotine can use nicotine as the sole carbon source via a series of enzymatic catalytic processes. The mechanisms underlying nicotine degradation in this bacterium and the corresponding functional genes remain unclear. Here, we analyzed the function and biological properties of the ocnE gene involved in the nicotine-degradation pathways in strain SCUEC4. The ocnE gene was cloned by PCR with total DNA of strain SCUEC4 and used to construct the recombinant plasmid pET28a-ocnE. The overexpression of the OcnE protein was detected by SDS-PAGE analysis, and study of the function of this protein was spectrophotometrically carried out by monitoring the changes of 2,5-dihydroxypyridine. Moreover, the effects of temperature, pH, and metal ions on the biological activities of the OcnE protein were analyzed. The optimal conditions for the biological activities of OcnE, a protein of approximately 37.6 kDa, were determined to be 25 °C, pH 7.0, and 25 µmol/L Fe2+, and the suitable storage conditions for the OcnE protein were 0 °C and pH 7.0. In conclusion, the ocnE gene is responsible for the ability of 2,5-dihydroxypyridine dioxygenase. These findings will be beneficial in clarifying the mechanisms of nicotine degradation in O. intermedium SCUEC4.

Cloning and identification of opa22, a new gene involved in nodule formation by Mesorhizobium huakuii.

Using Tn5-sacB insertion mutagenesis, 3000 mutants were obtained from Mesorhizobium huakuii 7653R. Eight nodulation-defective mutants were screened by plant nodulation experiments. The DNA sequences of the contiguous region from the Tn5 insertion site were determined by thermal asymmetric interlaced PCR. A new gene was cloned and designated opa22, as judged from its structural and functional homology. Sequence analysis indicated that opa22 was composed of 774 nucleotides and encoded a protein of 257 amino acids. RPS-BLAST analysis of the Opa22 protein showed a sequence similarity (88.9%) to the opacity protein and related to surface antigens of the bacterial outer membrane, which can mediate various pathogen-host cell interactions and promote invasion. Our results from root hair curling experiments suggested that expression of the opa22 gene might occur at the stage of infection thread formation and nodule development. The complement stain HK24 was able to restore the nodule forming ability of the mutant.