Biogenic Amine Production by and Phylogenetic Analysis of 23 Photobacterium Species.

Photobacterium species are members of the bacterial communities typically associated with scombrotoxin-forming fish. Reclassification and discovery of new Photobacterium species has caused confusion as to which species are capable of biogenic amine production. We analyzed histamine, cadaverine, and putrescine production by 104 Photobacterium strains representing 23 species. The presence of the genes for histidine decarboxylase ( hdc), lysine decarboxylase ( ldc), and ornithine decarboxylase ( odc) was determined by real-time or conventional PCR and whole genome sequencing. Significant histamine production (>200 ppm) was detected in five Photobacterium species: P. angustum, P. aquimaris, P. kishitanii, P. damselae, and P. phosphoreum. The hdc gene was detected in all of these histamine-producing species except P. phosphoreum. Cadaverine was produced by eight Photobacterium species: P. angustum, P. aquimaris, P. damselae, P. iliopiscarium, P. kishitanii, P. leiognathi, P. mandapamensis, and P. phosphoreum. Putrescine was produced by six Photobacterium species: P. angustum, P. aquimaris, P. kishitanii, P. leiognathi, P. mandapamensis, and Photobacterium sp. Cadaverine production correlated closely with the presence of the ldc gene, but putrescine production did not correlate closely with the presence of the odc gene. Characterization of the biogenic amine production by Photobacterium species will allow identification of these marine bacteria and help ensure that current guidelines account for mitigation of these bacteria.

Genomic analysis of a cardinalfish with larval homing potential reveals genetic admixture in the Okinawa Islands.

Discrepancies between potential and observed dispersal distances of reef fish indicate the need for a better understanding of the influence of larval behaviour on recruitment and dispersal. Population genetic studies can provide insight on the degree to which populations are connected, and the development of restriction site-associated sequencing (RAD-Seq) methods has made such studies of nonmodel organisms more accessible. We applied double-digest RAD-Seq methods to test for population differentiation in the coral reef-dwelling cardinalfish, Siphamia tubifer, which based on behavioural studies, have the potential to use navigational cues to return to natal reefs. Analysis of 11,836 SNPs from fish collected at coral reefs in Okinawa, Japan, from eleven locations over 3 years reveals little genetic differentiation between groups of S. tubifer at spatial scales from 2 to 140 km and between years at one location: pairwise FST values were between 0.0116 and 0.0214. These results suggest that the Kuroshio Current largely influences larval dispersal in the region, and in contrast to expectations based on studies of other cardinalfishes, there is no evidence of population structure for S. tubifer at the spatial scales examined. However, analyses of outlier loci putatively under selection reveal patterns of temporal differentiation that indicate high population turnover and variable larval supply from divergent source populations between years. These findings highlight the need for more studies of fishes across various geographic regions that also examine temporal patterns of genetic differentiation to better understand the potential connections between early life-history traits and connectivity of reef fish populations.

Life history of the symbiotically luminous cardinalfish Siphamia tubifer (Perciformes: Apogonidae).

Characteristics of the life history of the coral reef-dwelling cardinalfish Siphamia tubifer, from Okinawa, Japan, were defined. A paternal mouthbrooder, S. tubifer, is unusual in forming a bioluminescent symbiosis with Photobacterium mandapamensis. The examined S. tubifer (n = 1273) ranged in size from 9·5 to 43·5 mm standard length (LS ), and the minimum size at sexual maturity was 22 mm LS . The number of S. tubifer associated during the day among the spines of host urchins was 22·9 ± 16·1 (mean ± s.d.; Diadema setosum) and 3·6 ± 3·2 (Echinothrix calamaris). Diet consisted primarily of crustacean zooplankton. Batch fecundity (number of eggs; FB ) was related to LS by the equations: males (fertilized eggs) FB = 27·5LS - 189·46; females (eggs) FB = 31·3LS - 392·63. Individual mass (M; g) as a function of LS was described by the equation: M=9·74×10-5LS2·68. Growth, determined from otolith microstructure analysis, was described with the von Bertalanffy growth function with the following coefficients: L∞ = 40·8 mm LS , K = 0·026 day(-1) and t0 = 23·25 days. Planktonic larval duration was estimated to be 30 days. The age of the oldest examined individual was 240 days. The light organ of S. tubifer, which harbours the symbiotic population of P. mandapamensis, increased linearly in diameter as S. tubifer LS increased, and the bacterial population increased logarithmically with S. tubifer LS . These characteristics indicate that once settled, S. tubifer grows quickly, reproduces early and typically survives much less than 1 year in Okinawa. These characteristics are generally similar to other small reef fishes but they indicate that S. tubifer experiences higher mortality.

Photobacterium angustum and Photobacterium kishitanii, Psychrotrophic High-Level Histamine-Producing Bacteria Indigenous to Tuna.

Scombrotoxin fish poisoning (SFP) remains the main contributor of fish poisoning incidents in the United States, despite efforts to control its spread. Psychrotrophic histamine-producing bacteria (HPB) indigenous to scombrotoxin-forming fish may contribute to the incidence of SFP. We examined the gills, skin, and anal vents of yellowfin (n = 3), skipjack (n = 1), and albacore (n = 6) tuna for the presence of indigenous HPB. Thirteen HPB strains were isolated from the anal vent samples from albacore (n = 3) and yellowfin (n = 2) tuna. Four of these isolates were identified as Photobacterium kishitanii and nine isolates as Photobacterium angustum; these isolates produced 560 to 603 and 1,582 to 2,338 ppm histamine in marine broth containing 1% histidine (25°C for 48 h), respectively. The optimum growth temperatures and salt concentrations were 26 to 27°C and 1% salt for P. kishitanii and 30 to 32°C and 2% salt for P. angustum in Luria 70% seawater (LSW-70). The optimum activity of the HDC enzyme was at 15 to 30°C for both species. At 5°C, P. kishitanii and P. angustum had growth rates of 0.1 and 0.2 h(-1), respectively, and the activities of histidine decarboxylase (HDC) enzymes were 71% and 63%, respectively. These results show that indigenous HPB in tuna are capable of growing at elevated and refrigeration temperatures. These findings demonstrate the need to examine the relationships between the rate of histamine production at refrigeration temperatures, seafood shelf life, and regulatory limits.

Symbiosis initiation in the bacterially luminous sea urchin cardinalfish Siphamia versicolor.

To determine how each new generation of the sea urchin cardinalfish Siphamia versicolor acquires the symbiotic luminous bacterium Photobacterium mandapamensis, and when in its development the S. versicolor initiates the symbiosis, procedures were established for rearing S. versicolor larvae in an aposymbiotic state. Under the conditions provided, larvae survived and developed for 28 days after their release from the mouths of males. Notochord flexion began at 8 days post release (dpr). By 28 dpr, squamation was evident and the caudal complex was complete. The light organ remained free of bacteria but increased in size and complexity during development of the larvae. Thus, aposymbiotic larvae of the fish can survive and develop for extended periods, major components of the luminescence system develop in the absence of the bacteria and the bacteria are not acquired directly from a parent, via the egg or during mouth brooding. Presentation of the symbiotic bacteria to aposymbiotic larvae at 8-10 dpr, but not earlier, led to initiation of the symbiosis. Upon colonization of the light organ, the bacterial population increased rapidly and cells forming the light-organ chambers exhibited a differentiated appearance. Therefore, the light organ apparently first becomes receptive to colonization after 1 week post-release development, the symbiosis is initiated by bacteria acquired from the environment and bacterial colonization induces morphological changes in the nascent light organ. The abilities to culture larvae of S. versicolor for extended periods and to initiate the symbiosis in aposymbiotic larvae are key steps in establishing the experimental tractability of this highly specific vertebrate and microbe mutualism.

LuxR- and acyl-homoserine-lactone-controlled non-lux genes define a quorum-sensing regulon in Vibrio fischeri.

The luminescence (lux) operon (luxICDABEG) of the symbiotic bacterium Vibrio fischeri is regulated by the transcriptional activator LuxR and two acyl-homoserine lactone (acyl-HSL) autoinducers (the luxI-dependent 3-oxo-hexanoyl-HSL [3-oxo-C6-HSL] and the ainS-dependent octanoyl-HSL [C8-HSL]) in a population density-responsive manner called quorum sensing. To identify quorum-sensing-regulated (QSR) proteins different from those encoded by lux genes, we examined the protein patterns of V. fischeri quorum-sensing mutants defective in luxI, ainS, and luxR by two-dimensional polyacrylamide gel electrophoresis. Five non-Lux QSR proteins, QsrP, RibB, AcfA, QsrV, and QSR 7, were identified; their production occurred preferentially at high population density, required both LuxR and 3-oxo-C6-HSL, and was inhibited by C8-HSL at low population density. The genes encoding two of the QSR proteins were characterized: qsrP directs cells to synthesize an apparently novel periplasmic protein, and ribB is a homolog of the Escherichia coli gene for 3,4-dihydroxy-2-butanone 4-phosphate synthase, a key enzyme for riboflavin synthesis. The qsrP and ribB promoter regions each contained a sequence similar to the lux operon lux box, a 20-bp region of dyad symmetry necessary for LuxR/3-oxo-C6-HSL-dependent activation of lux operon transcription. V. fischeri qsrP and ribB mutants exhibited no distinct phenotype in culture. However, a qsrP mutant, in competition with its parent strain, was less successful in colonizing Euprymna scolopes, the symbiotic host of V. fischeri. The newly identified QSR genes, together with the lux operon, define a LuxR/acyl-HSL-responsive quorum-sensing regulon in V. fischeri.

Aposymbiotic culture of the sepiolid squid Euprymna scolopes: role of the symbiotic bacterium Vibrio fischeri in host animal growth, development, and light organ morphogenesis.

The sepiolid squid Euprymna scolopes forms a bioluminescent mutualism with the luminous bacterium Vibrio fischeri, harboring V. fischeri cells in a complex ventral light organ and using the bacterial light in predator avoidance. To characterize the contribution of V. fischeri to the growth and development of E. scolopes and to define the long-term effects of bacterial colonization on light organ morphogenesis, we developed a mariculture system for the culture of E. scolopes from hatching to adulthood, employing artificial seawater, lighting that mimicked that of the natural environment, and provision of prey sized to match the developmental stage of E. scolopes. Animals colonized by V. fischeri and animals cultured in the absence of V. fischeri (aposymbiotic) grew and survived equally well, developed similarly, and reached sexual maturity at a similar age. Development of the light organ accessory tissues (lens, reflectors, and ink sac) was similar in colonized and aposymbiotic animals with no obvious morphometric or histological differences. Colonization by V. fischeri influenced regression of the ciliated epithelial appendages (CEAs), the long-term growth of the light organ epithelial tubules, and the appearance of the cells composing the ciliated ducts, which exhibit characteristics of secretory tissue. In certain cases, aposymbiotic animals retained the CEAs in a partially regressed state and remained competent to initiate symbiosis with V. fischeri into adulthood. In other cases, the CEAs regressed fully in aposymbiotic animals, and these animals were not colonizable. The results demonstrate that V. fischeri is not required for normal growth and development of the animal or for development of the accessory light organ tissues and that morphogenesis of only those tissues coming in contact with the bacteria (CEAs, ciliated ducts, and light organ epithelium) is altered by bacterial colonization of the light organ. Therefore, V. fischeri apparently makes no major metabolic contribution to E. scolopes beyond light production, and post-embryonic development of the light organ is essentially symbiont independent. J. Exp. Zool. 286:280-296, 2000.

Acylhomoserine lactone synthase activity of the Vibrio fischeri AinS protein.

Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone from an acyl-acyl carrier protein and S-adenosylmethionine. Another V. fischeri gene, ainS, directs the synthesis of N-octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the Vibrio harveyi LuxM protein. The luxM gene is required for the synthesis of N-(3-hydroxybutyryl)-L-homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of N-octanoylhomoserine lactone from S-adenosylmethionine and either octanoyl-acyl carrier protein or, to a lesser extent, octanoyl coenzyme A. With the exception that octanoyl coenzyme A served as an acyl substrate for the MBP-AinS fusion protein, the substrates for and reaction kinetics of the MBP-AinS fusion protein were similar to those of the several LuxI family members previously studied. We conclude that AinS is an acylhomoserine lactone synthase and that it represents a second family of such enzymes.

Quorum regulation of luminescence in Vibrio fischeri.

Luminescence in Vibrio fischeri is controlled by a population density-responsive regulatory mechanism called quorum sensing. Elements of the mechanism include: LuxI, an acyl-homoserine lactone (acyl-HSL) synthase that directs synthesis of the diffusible signal molecule, 3-oxo-hexanoyl-HSL (V. fischeri autoinducer-1, VAI-1); LuxR, a transcriptional activator protein necessary for response to VAI-1; GroEL, which is necessary for production of active LuxR; and AinS, an acyl-HSL synthase that catalyzes the synthesis of octanoyl-HSL (VAI-2). The population density-dependent accumulation of VAI-1 triggers induction of lux operon (luxICDABEG; genes for luminescence enzymes and for LuxI) transcription and luminescence by binding to LuxR, forming a complex that facilitates the association of RNA polymerase with the luxoperon promoter. VAI-2, which apparently interferes with VAI-1 binding to LuxR, operates to limit premature luxoperon induction. Hierarchical control is imposed on the system by 3':5'-cyclic AMP (cAMP) and cAMP receptor protein (CRP), which are necessary for activated expression of luxR. Several non-lux genes in V. fischeri are controlled by LuxR and VAI-1. Quorum regulation in V. fischeri serves as a model for LuxI/LuxR-type quorum sensing systems in other gram-negative bacteria.

Laboratory Culture of the Sepiolid Squid Euprymna scolopes: A Model System for Bacteria-Animal Symbiosis.

The small Hawaiian sepiolid Euprymna scolopes, with its symbiotic luminous bacterium Vibrio fischeri, was cultured through one complete life cycle in 4 months. Paralarval squid hatchlings were actively planktonic for the first 20-30 days, after which they settled and assumed the typical adult mode of nocturnal activity and diurnal quiescence. Squids were aggressive predators that preferred actively swimming prey up to 2-4 times their size; the only diet that yielded good survival and rapid growth for paralarvae was large adult mysids. Survival to settlement was 73% on this diet, whereas it was 0%-17% on controls and three other diets. Paralarvae initially lacked both detectable luminescence and V. fischeri cells in their incipient light organs; all remaining stages produced luminescence, and their light organs were colonized by apparently pure cultures of > 105 V. fischeri typical of E. scolopes symbiont strains. Survival from settlement to sexual maturity was 76%. Mating and egg laying commenced at 2 months, yet attempts to culture the next laboratory generation of hatchlings were not as successful. The results indicate that the host organism of this symbiosis can soon be cultured with consistency through its brief life cycle, thus opening new avenues of research into developmental aspects of this symbiosis.

Modulation of luminescence operon expression by N-octanoyl-L-homoserine lactone in ainS mutants of Vibrio fischeri.

Population density-dependent expression of luminescence in Vibrio fischeri is controlled by the autoinducer N-3-oxohexanoyl-L-homoserine lactone (autoinducer 1 [AI-1]), which via LuxR activates transcription of the lux operon (luxICDABEG, encoding the putative autoinducer synthase [LuxI] and the luminescence enzymes). We recently identified a novel V. fischeri locus, ainS, necessary for the synthesis of a second autoinducer, N-octanoyl-L-homoserine lactone (AI-2), which via LuxR can activate lux operon transcription in the absence of AI-1. To define the regulatory role of AI-2, a luxI ainS double mutant was constructed; in contrast to the parental strain and a luxI mutant, the luxI ainS mutant exhibited no induction of luminescence and produced no detectable luminescence autoinducer, demonstrating that V. fischeri makes no luminescence autoinducers other than those whose synthesis is directed by luxI and ainS. A mutant defective only in ainS exhibited accelerated luminescence induction compared with that of the parental strain, indicating that AI-2 functions in V. fischeri to delay luminescence induction. Consistent with that observation, the exogenous addition of AI-2 inhibited induction in a dose-dependent manner in V. fischeri and Escherichia coli carrying the lux genes. AI-2 did not mediate luxR negative autoregulation, alone or in the presence of AI-1, and inhibited luminescence induction in E. coli regardless of whether luxR was under the control of its native promoter or a foreign one. Increasing amounts of AI-1 overcame the inhibitory effect of AI-2, and equal activation of luminescence required 25- to 45-fold-more AI-2 than AI-1. We conclude that AI-2 inhibits lux operon transcription. The data are consistent with a model in which AI-2 competitively inhibits the association of AI-1 with LuxR, forming a complex with LuxR which has a markedly lower lux operon-inducing specific activity than that of AI-1-LuxR. AI-2 apparently functions in V. fischeri to suppress or delay induction at low and intermediate population densities.

AinS and a new family of autoinducer synthesis proteins.

In Vibrio fischeri, the autoinducer N-3-oxohexanoyl-L-homoserine lactone (AI-1) governs the cell density-dependent induction of the luminescence operon via the LuxR transcriptional activator. The synthesis of AI-1 from bacterial metabolic intermediates is dependent on luxI. Recently, we found a second V. fischeri autoinducer molecule, N-octanoyl-L-homoserine lactone (AI-2), that in E. coli also activates the luminescence operon via LuxR. A locus independent of luxI was identified as being required for AI-2 synthesis. This 2.7-kb ain (autoinducer) locus was characterized by transposon insertion mutagenesis, deletion and complementation analysis, and DNA sequencing. A single 1,185-bp gene, ainS, was found to be the sole exogenous gene necessary for the synthesis of AI-2 in Escherichia coli. In addition, a V. fischeri ainS mutant produced AI-1 but not AI-2, confirming that in its native species ainS is specific for the synthesis of AI-2. ainS is predicted to encode a 45,580-Da protein which exhibits no similarity to LuxI or to any of the LuxI homologs responsible for the synthesis of N-acyl-L-homoserine lactones in a variety of other bacteria. The existence of two different and unrelated autoinducer synthesis genes suggests the occurrence of convergent evolution in the synthesis of homoserine lactone signaling molecules. The C-terminal half of AinS shows homology to a putative protein in Vibrio harveyi, LuxM, which is required for the synthesis of a V. harveyi bioluminescence autoinducer. Together, AinS and LuxM define a new family of autoinducer synthesis proteins. Furthermore, the predicted product of another gene, ainR, encoded immediately downstream of ainS, shows homology to LuxN, which is similarly encoded downstream of luxM in V. harveyi and proposed to have sensor/regulator functions in the bioluminescence response to the V. harveyi auto inducer. This similarity presents the possibility that AI-2, besides interacting with LuxR, also interacts with AinR under presently unknown conditions.

Purification and properties of periplasmic 3':5'-cyclic nucleotide phosphodiesterase. A novel zinc-containing enzyme from the marine symbiotic bacterium Vibrio fischeri.

The 3':5'-cyclic nucleotide phosphodiesterase (CNP) of Vibrio fischeri, due to its unusual location in the periplasm, allows this symbiotic bacterium to utilize extracellular 3':5'-cyclic nucleotides (e.g. cAMP) as sole sources of carbon and energy, nitrogen, and phosphorus for growth. The enzyme was purified to apparent homogeneity by a four-step procedure: chloroform shock, ammonium sulfate precipitation, and chromotography on DEAE-Sephacel and Cibacron Blue 3GA-agarose. The active enzyme consists of a single polypeptide with a mass of 34 kDa. At 25 degrees C, it has a pH optimum of 8.25, a Km for cAMP of 73 microns, and a Vmax of 3700 mumol of cAMP hydrolyzed/min/mg protein (turnover number of 1.24 x 10(5)/min). The specific activity of the V. fischeri enzyme is approximately 20-fold greater than that of any previously characterized CNP when comparisons of activity are made at the same assay temperature. Activity increases with temperature up to 60 degrees C. The CNP contains 2 atoms of zinc/monomer, and zinc, copper, magnesium, and calcium can restore activity of the apoenzyme to varying degrees. The exceptional specific activity of the enzyme and its unusual location in the periplasm support proposals that the enzyme enables the bacterium to scavenge 3':5'-cyclic nucleotides in seawater and that the enzyme plays a role in cAMP-mediated host-symbiont interactions.

Thyroid disease and abnormal thyroid function tests in women with eating disorders and depression.

Forty-two female patients with an eating disorder and major depression were compared with 48 female patients with major depression in a retrospective chart study for the prevalence of thyroid disease and laboratory thyroid function abnormalities in the absence of thyroid disease. Eating disorder patients, aged 30-80 years, had a significantly higher incidence in thyroid diseases (53%) then those with major depression alone (17%). The incidence of thyroid disease did not differ between the two groups among patients aged 11-29 years. Abnormal thyroid screening values occurred in 40% of euthyroid eating disorder patients and 34% of those with major depression. While the overall prevalence of thyroid disease in depressed females (15%) was similar to that in the general population (10.5%), thyroid disease in the eating disordered/depressed patients was twice the rate expected (24%) in the general population. Female patients who require psychiatric hospitalization should be routinely evaluated for thyroid function, especially those diagnosed with an eating disorder and depression.

Multiple N-acyl-L-homoserine lactone autoinducers of luminescence in the marine symbiotic bacterium Vibrio fischeri.

In Vibrio fischeri, the synthesis of N-3-oxohexanoyl-L-homoserine lactone, the autoinducer for population density-responsive induction of the luminescence operon (the lux operon, luxICDABEG), is dependent on the autoinducer synthase gene luxI. Gene replacement mutants of V. fischeri defective in luxI, which had been expected to produce no autoinducer, nonetheless exhibited lux operon transcriptional activation. Mutants released into the medium a compound that, like N-3-oxohexanoyl-L-homoserine lactone, activated expression of the lux system in a dose-dependent manner and was both extractable with ethyl acetate and labile to base. The luxI-independent compound, also like N-3-oxohexanoyl-L-homoserine lactone, was produced by V. fischeri cells in a regulated, population density-responsive manner and required the transcriptional activator LuxR for activity in the lux system. The luxI-independent compound was identified as N-octanoyl-L-homoserine lactone by coelution with the synthetic compound in reversed-phase high-pressure liquid chromatography, by derivatization treatment with 2,4-dinitrophenylhydrazine, by mass spectrometry, and by nuclear magnetic resonance spectroscopy. A locus, ain, necessary and sufficient for Escherichia coli to synthesize N-octanoyl-L-homoserine lactone was cloned from the V. fischeri genome and found to be distinct from luxI by restriction mapping and Southern hybridization. N-Octanoyl-L-homoserine lactone and ain constitute a second, novel autoinduction system for population density-responsive signalling and regulation of lux gene expression, and possibly other genes, in V. fischeri. A third V. fischeri autoinducer, N-hexanoyl-L-homoserine lactone, dependent on luxI for its synthesis, was also identified. The presence of multiple chemically and genetically distinct but cross-acting autoinduction systems in V. fischeri indicates unexpected complexity for autoinduction as a regulatory mechanism in this bacterium.

Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri.

Vibrio fischeri is found both as a free-living bacterium in seawater and as the specific, mutualistic light organ symbiont of several fish and squid species. To identify those characteristics of symbiosis-competent strains that are required for successful colonization of the nascent light organ of juvenile Euprymna scolopes squids, we generated a mutant pool by using the transposon Mu dI 1681 and screened this pool for strains that were no longer motile. Eighteen independently isolated nonmotile mutants that were either flagellated or nonflagellated were obtained. In contrast to the parent strain, none of these nonmotile mutants was able to colonize the juvenile squid light organ. The flagellated nonmotile mutant strain NM200 possessed a bundle of sheathed polar flagella indistinguishable from that of the wild-type strain, indicating that the presence of flagella alone is not sufficient for colonization and that it is motility itself that is required for successful light organ colonization. This study identifies motility as the first required symbiotic phenotype of V. fischeri.

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.