Inactivation of the ampDE operon increases transcription of algD and affects morphology and encystment of Azotobacter vinelandii

Transcription of algD, encoding GDP-mannose dehydrogenase, the key enzyme i n the alginate biosynthetic pathway, is highly regulated in Azotobacter vin elandii. We describe here the characterization of a Tn5 insertion mutant (A C28) which shows a higher level of expression of an algD::lacZ fusion. AC28 cells were morphologically abnormal and unable to encyst. The cloning and nucleotide sequencing of the Tn5-disrupted locus in AC28 revealed an operon homologous to the Escherichia coli ampDE operon. Tn5 was located within th e ampD gene, encoding a cytosolic N-acetyl-anhydromuramyl-L-alanine amidase that participates in the intracellular recycling of peptidoglycan fragment s. The ampE gene encodes a transmembrane protein, but the function of the p rotein is not known. We constructed strains carrying ampD or ampE mutations and one with an ampDE deletion. The strain with a deletion of the ampDE op eron showed a phenotype similar to that of mutant AC28. The present work de monstrates that both alginate production and bacterial encystment are great ly influenced by the bacterial ability to recycle its cell wall.