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 in the alginate biosynthetic pathway, is highly regulated in Azotobacter vinelandii. We describe here the characterization of a Tn5 insertion mutant (AC28) 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 the ampD gene, encoding a cytosolic N-acetyl-anhydromuramyl-L-alanine amidase that participates in the intracellular recycling of peptidoglycan fragments. The ampE gene encodes a transmembrane protein, but the function of the protein 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 operon showed a phenotype similar to that of mutant AC28. The present work demonstrates that both alginate production and bacterial encystment are greatly influenced by the bacterial ability to recycle its cell wall.
Núñez
C
C
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca Morelos 62250, México.
Moreno
S
S
Cárdenas
L
L
Soberón-Chávez
G
G
Espín
G
G
D000464:Alginates / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002237:Carbohydrate Dehydrogenases / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics*; D009876:Operon; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D019895:Transposases; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000464:Alginates; C062461:AmpE protein, Bacteria; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006603:Hexuronic Acids; D008565:Membrane Proteins; D020723:Glucuronic Acid; D002237:Carbohydrate Dehydrogenases; C020562:GDPmannose dehydrogenase; D019895:Transposases; D001616:beta-Galactosidase; C062460:AmpD protein, Bacteria; D009238:N-Acetylmuramoyl-L-alanine Amidase