The nitrile hydratase (NHase) of Pseudomonas chlororaphis B23, which is composed of two subunits, a and IA, catalyzes the hydration of nitrile compounds to the corresponding amides. The NHase gene of strain B23 was cloned into Escherichia coli by the DNA-probing method with the NHase gene of Rhodococcus sp. strain N-774 as the hybridization probe. Nucleotide sequencing revealed that an amidase showing significant similarity to the amidase of Rhodococcus sp. strain N-774 was also coded by the region just upstream of the subunit a-coding sequence. In addition to these three proteins, two open reading frames, P47K and OrfE, were found just downstream of the coding region of subunit P. The direction and close locations to each other of these open reading frames encoding five proteins (amidase, subunits a and j1, P47K, and OrfE, in that order) suggested that these genes were cotranscribed by a single mRNA. Plasmid pPCN4, in which a 6.2-kb sequence covering the region coding for these proteins is placed under control of the lac promoter, directed overproduction of enzymatically active NHase and amidase in response to addition of isopropyl-o-D-thiogalactopyranoside.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell extract showed that the amount of subunits a and , of NHase was about 10% of the total cellular proteins and that an additional 38-kDa protein probably encoded by the region upstream of the amidase gene was also produced in a large amount. The 38-kDa protein, as well as P47K and OrfE, appeared to be important for efficient expression of NHase activity in E. coli cells, because plasmids containing the NHase and amidase genes but lacking the region coding for the 38-kDa protein or the region coding for P47K and OrfE failed to express efficient NHase activity.
Physiological characteristics of salt tolerance in centipedegrass (Eremochloa ophiuroides (Munro) Hack.) were investigated by comparing ion uptake, osmotic regulation, osmolytes, oxidative stress and antioxidant enzymes of salt‐tolerant cultivar ‘TifBlair’ and salt‐sensitive ‘Common’. Treatments at 0–150 mmol L−1 NaCl were conducted for 10 days. Growth was higher in ‘TifBlair’ than in ‘Common’ at 25 and 50 mmol L−1 NaCl, and was depressed in both cultivars at above 75 mmol L−1 NaCl. No significant difference in Na and Cl concentrations in the shoot and roots was observed between the cultivars, although ‘TifBlair’ was more tolerant to Na and Cl than ‘Common’. Osmotic pressure, glycinebetaine and free proline concentrations in the shoot increased in both cultivars with an increase in NaCl concentration; the increase was higher in ‘TifBlair’. Malondialdehyde concentration, an index of oxidative damage, in the shoot increased in both cultivars with an increase in NaCl concentration; the increase was not significantly different between the cultivars. On the contrary, hydrogen peroxide concentration was higher in ‘TifBlair’ than in ‘Common’. ATPase activity of the shoot plasmalemma increased in ‘TifBlair’ with an increase in NaCl concentration, while decreased in ‘Common’ at 50 mmol L−1 NaCl. The activity of antioxidant enzymes (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase) tended to be higher in ‘TifBlair’ than in ‘Common’. The results suggest that adjustment of osmotic pressure by accumulation of compatible solutes and exclusion of Na and Cl, and elimination of activated oxygen by induction of antioxidant enzymes are responsible for the salt tolerance of centipedegrass ‘TifBlair’.
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