Isolation and characterization of the glnD gene of Gluconacetobacter diazotrophicus, encoding a putative uridylyltransferase/uridylyl-removing enzyme

Abstract

The glnD gene of Gluconacetobacter diazotrophicus was isolated by complementation of the Azotobacter vinelandii glnD ( nfrX) mutant strain MV17 using a pLAFR3 cosmid library. The 5 kb chromosomal DNA region encoding the glnD gene on cosmid pAD401 was identified by introduction of deletions as well as subcloning of restriction fragments followed by subsequent DNA sequencing. Three open reading frames were identified with the deduced amino acid sequence of ORF1 showing significant homologies to known GlnD proteins of other proteobacteria such as Sinorhizobium meliloti, Rhizobium tropici, Escherichia coli and Azotobacter vinelandii. A mutagenesis of the chromosomal glnD gene was carried out by insertion of an interposon carrying the kanamycin resistance gene of Tn 5. Mutants carrying the cassette inserted into a central region of glnD could not be isolated, while an interposon mutation at the 3′ end of glnD was successful. The resulting strain showed a prolonged generation time in complex growth medium and was unable to utilize ammonium as sole nitrogen source. This phenotype appears to be pleiotropic, since the addition of single amino acids to the minimal medium was not sufficient to allow growth. Furthermore, the glnD mutant was able to express nitrogenase under diazotrophic as well as repressing growth conditions.