Characterization of a periplasmic 3':5'-cyclic nucleotide phosphodiesterase gene, cpdP, from the marine symbiotic bacterium Vibrio fischeri.
Vibrio fischeri, a marine bacterium that forms a bioluminescent symbiosis with certain fish and squids, exhibits the unusual attribute of growth on 3':5'-cyclic AMP (cAMP), apparently through the activity of a 3':5'-cyclic nucleotide phosphodiesterase (3':5'-CNP) with exceptionally high activity. The V. fischeri 3':5'-CNP is located in the periplasm, a novel cellular location for this enzyme in bacteria. To gain insight into the physiological function of this enzyme, we cloned the gene (designated cpdP) encoding it from V. fischeri MJ-1. This is the first bacterial 3':5'-CNP gene to be cloned. Sequencing and analysis of the 1.26-kb cpdP locus revealed a single open reading frame specifying a protein of 330 amino acid residues, including a 22-amino-acid leader peptide. The putative cpdP promoter contained a reasonable -10 promoter region (TATTAT) but contained no obvious -35 region; instead, a 12-bp inverted repeat (TTAAATATTTAA) occurred just upstream of this location. A possible rho-independent transcriptional terminator with a calculated free energy of -21.2 kcal.mol-1 (ca. -88.7 kJ.mol-1) followed the CpdP protein coding sequence. The predicted subunit molecular weight of 33,636 for the mature CpdP protein (36,087 less 2,451 for the leader peptide) was consistent with the molecular weight of 34,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the CpdP protein exhibited 30.3% identity with that of the low-affinity 3':5'-CNP (PDE1) of Saccharomyces cerevisiae and 33.6% identity with that of the extracellular 3':5'-CNP of Dictyostelium discoideum. The residue identities clustered in two regions, residues 100 to 146 and 238 to 269, which contained 30 of the 33 amino acids conserved in all three proteins, 4 of which were histidines. A gene replacement mutant of V. fischeri MJ-1 containing a 0.45-kb BglII deletion within the cpdP gene lacked periplasmic 3':5'-CNP activity and did not grow on cAMP, confirming for V. fischeri the relationship among cpdP, synthesis of the periplasmic 3':5'-CNP, and growth on cAMP. The mutant exhibited no obvious sensitivity to high extracellular concentrations of cAMP (5 and 10 mM), suggesting that the enzyme does not play a role in defense against extracellular cAMP.
Dunlap
P V
PV
Biology Department, Woods Hole Oceanographic Institution, Massachusetts 02543.
Callahan
S M
SM
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004926:Escherichia coli; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology*
D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1