Site-directed mutagenesis, heterologous expression of cyanamide hydratase gene and antimicrobial activity of cyanamide.

Site-directed mutagenesis on a recombinant plasmid, pUC8, that contained the cah gene, was conducted and confirmed by sequence analysis. Single base substitution, G to A at nucleotide position 81 or T to C at nucleotide position 84 of cah gene does not change the amino acid sequence of cah enzyme but eliminates the HindIII site. The wild-type cah and its mutants were cloned and overexpressed in pQE-60 Escherichia coli expression system. Western blot analysis confirmed the production of 27.7-kDa cah enzyme by all the recombinants. The mutated cah gene devoid of HindIII site was used to generate a recombinant plant transformation vector (pCAMBIA-cah). Agrobacterium-mediated transformation was performed in Nicotiana tabaccum cv. Samsun plants by employing the leaf-disc method. The integration and expression of cah gene in transgenic plants were confirmed by polymerase chain reaction, Southern and Western blot analyses. Antimicrobial activity of cyanamide against phytopathogenic fungi and bacteria was determined. Cyanamide can be used as fertilizer as well as an antimicrobial salt against phytopathogenic fungi and bacteria. The present investigation reports the heterologous expression of the cah marker gene. Due to its innate ability to convert cyanamide to urea and the broad-spectrum antimicrobial activity of cyanamide, the cah gene can be used to facilitate plant growth promotion and biocontrol of phytopathogens.