Cloning, sequence, and transcriptional regulation of the operon encoding a putative N-acetylmannosamine-6-phosphate epimerase (nanE) and sialic acid lyase (nanA) in Clostridium perfringens.
Clostridium perfringens can obtain sialic acid from host tissues by the activity of sialidase enzymes on sialoglycoconjugates. After sialic acid is transported into the cell, sialic acid lyase (NanA) then catalyzes the hydrolysis of sialic acid into pyruvate and N-acetylmannosamine. The latter is converted for use as a biosynthetic intermediate or carbohydrate source in a pathway including an epimerase (NanE) that converts N-acetylmannosamine-6-phosphate to N-acetylglucosamine-6-phosphate. A 4.0-kb DNA fragment from C. perfringens NCTC 8798 that contains the nanE and nanA genes has been cloned. The identification of the nanA gene product as sialic acid lyase was confirmed by overexpressing the gene and measuring sialic acid lyase activity in a nanA Escherichia coli strain, EV78. The nanA gene product was also shown to restore growth to EV78 in minimal medium with sialic acid as the sole carbon source. By using Northern blot experiments, it was demonstrated that the nanE and nanA genes comprise an operon and that transcription of the operon in C. perfringens is inducible by the addition of sialic acid to the growth medium. The Northern blot experiments also showed that there is no catabolite repression of nanE-nanA transcription by glucose. With a plasmid construct containing a promoterless cpe-gusA gene fusion, in which beta-glucuronidase activity indicated that the gusA gene acted as a reporter for transcription, a promoter was localized to the region upstream of the nanE gene. Primer extension experiments then allowed us to identify a sialic acid-inducible promoter located 30 bp upstream of the nanE coding sequence.
Walters
D M
DM
Department of Microbiology and Immunology, University of Tennessee, Memphis, Memphis, Tennessee 38163, USA.
Stirewalt
V L
VL
Melville
S B
SB
D001426:Bacterial Proteins; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009876:Operon; D007652:Oxo-Acid-Lyases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D002238:Carbohydrate Epimerases; C120657:N-acetylmannosamine-6-phosphate epimerase