Isolation and Sequence Analysis of the GlnKamtB 1 amtB 2 Gene Cluster, Encoding a P II Homologue and Two Putative Ammonium Transporters, from Pseudomonas stutzeri A15

By PCR, using primers based on heterologous amtB genes, an amtB sequence of Pseudomonas stutzeri A15 was amplified. This DNA fragment was used as a probe in Southern hybridisation experiments and resulted in the isolation and sequence analysis of a 6017 u bp genomic fragment of P. stutzeri A15 containing glnKamtB 1 amtB 2. GlnK codes for a homologue of the nitrogen regulatory P II protein, amtB 1 and amtB 2 encode putative ammonium transporters. Whereas a glnKamtB gene cluster is common among bacteria, a tandem repeat of ammonium transporter genes has not been reported before. Apart from the presence of a second amtB gene, the gene organisation on this 6 u kbp fragment is very similar to a particular region in the genome of Pseudomonas aeruginosa PAO1, relatively closely related to P. stutzeri. Furthermore, the amtB 1 gene shows the highest similarity with P. aeruginosa amtB, whereas the amtB 2 gene is more closely related to cyanobacterial amtB genes, which are reported to be monocistronically transcribed and not clustered with glnK homologues. Upstream of glnK, NtrC and RpoN recognition sites can be observed. In the intergenic region of glnKamtB 1 amtB 2 no terminators nor extra promoter sequences were observed, indicating that glnKamtB 1 amtB 2 is possibly transcribed as a nitrogen regulated operon.