Pseudoalteromonas Bacteria Are Capable of Degrading Paralytic Shellfish Toxins

Marine bacterial isolates cultured from the digestive tracts of blue mussels (Mytilus edulis) contaminated with paralytic shellfish toxins (PSTs) were screened for the ability to reduce the toxicity of a PST mixture. Seven isolates reduced the overall toxicity of the algal extract by ≥90% within 3 days. These isolates shared at least 99% 16S rRNA gene sequence similarity with five Pseudoalteromonas spp. Phenotypic tests suggested that all are novel strains of Pseudoalteromonas haloplanktis.Among the marine algal biotoxins identified to date; paralytic shellfish toxins (PSTs) constitute the most serious threat to the safety of the food supply, mainly due to their high acute toxicities and the absence of antidotes or effective medical treatments (8). Paralytic shellfish poisoning is caused by ingestion of one or more of the chemically related PSTs (see Fig. S1 in the supplemental material). PSTs are mainly produced by marine dinoflagellates, including Alexandrium spp., Gymnodinium catenatum, and Pyrodinium bahamense var. compresssum (16). Since bivalve molluscs filter-feed on marine algae, they tend to concentrate PSTs largely, but not exclusively, in their digestive organs (7, 9, 10, 29). Not affected by commercial sterilization (14, 18) or cooking, PSTs present significant risks to the food supply, particularly during periods of toxic algal blooms. Practical methods for PST detoxification of living shellfish do not exist (5).Transformations of PSTs by bacteria have been reported in the literature (23-25, 31, 35, 36, 38); early studies focused on the conversion of hydroxysulfate carbamate derivatives (gonyautoxins 1 and 4) to the more highly toxic saxitoxin (STX) (23-25). In addition, several reports have noted the high capacity of the digestive gland for PST transformation (12, 28, 32, 39), suggesting the presence of toxin-transforming enzymes and/or microorganisms in bivalve molluscs. The partial degradation of gonyautoxins 1 and 4 and C1/C2 by marine bacteria has also been reported (38). In addition, Stewart et al. (37) discovered the bacterial degradation of domoic acid (another marine toxin that causes amnesic shellfish poisoning), collectively suggesting that bacteria might play a role in the elimination of marine toxins from toxic bivalve molluscs. The capacity to catabolize domoic acid is greater in cultures isolated from blue mussels that rapidly eliminate domoic acid than in bacterial isolates from bivalves known to retain the toxin for longer time periods (e.g., scallops), suggesting these bacteria play a role in the elimination of marine toxins.Recently, we reported the kinetics of PST destruction for a group of marine bacteria isolated from toxic blue mussels (11). Here we report the phenotypic and taxonomic characterization of these unique marine bacteria.     

Paralytic Shellfish Toxins in Protogonyaulax tamarensis and Protogonyaulax catenella in Axenic Culture

Paralytic shellfish toxin concentrations were measured and individual toxin profiles were monitored in axenic batch cultures of Protogonyaulax tamarensis and Protogonyaulax catenella. High pressure liquid chromatographic methods were used that allowed the separation of all 12 known paralytic shellfish poisons, including toxins C1, C2, and C3, from a single sample. In isolates of both Protogonyaulax species, total toxin levels were relatively low after inoculation, increased rapidly in early to mid-exponential growth to a value 100 to 300% of that at the initial time point, then decreased by 86 to 95% as the culture aged. Although the concentrations of individual toxins per cell followed the same general pattern as that seen for total moles of toxin per cell, variability in toxin profile with culture age was observed. In P. tamarensis, the mole percent of neosaxitoxin increased substantially from 8 to 44% as total toxin levels per cell decreased. A concomitant decrease in the mole percent of saxitoxin with culture age was noted. Although not as precipitous, changes in the mole percent of specific toxins from P. catenella were also observed. The mole percent of gonyautoxins I and IV increased, while that of gonyautoxins II and III decreased. These data suggest that the toxin profile in isolates of Protogonyaulax can change, sometimes significantly, with changing environmental variables.     

Structures of Two Paralytic Shellfish Toxins,Gonyautoxins V and VI,Isolated from a Tropical Dinoflagellate,Pyrodinium bahamense var. compressa

Two paralytic shellfish toxins, gonyautoxin V and gonyautoxin VI, isolated from a tropical dinoflagellate, Pyrodinium bahamense var. compressa, were identified respectively to be derivatives of saxitoxin and neosaxitoxin with a sulfonatocarbamoyl moiety.     

抗麻痹性贝毒素GTX2,3单克隆抗体的制备及特性分析

制备抗麻痹性贝毒GTX2,3单克隆抗体。利用醛化法将GTX2,3与载体牛血清白蛋白(BSA)偶联,制备完全抗原。免疫小鼠,取小鼠脾细胞与Sp2/0细胞融合。GTX2,3与钥孔血蓝蛋白(KLH)偶联作为检测抗原,用间接ELISA法筛选阳性克隆株。将筛选的阳性细胞株制备腹水。获得三株稳定分泌抗GTX2,3单克隆抗体的杂交瘤细胞株F4、F10、G9。间接ELISA法检测F10细胞株腹水抗体效价为1.4×10^-5。半抗原GTX2,3与载体蛋白偶联后,作为免疫原,可制备高滴度的抗GTX2,3抗血清和单克隆抗体。该抗体对于藻毒素具有高特异性和高亲和力,可用于污染海产品的麻痹性贝毒的检测。     

Reevaluation of Production of Paralytic Shellfish Toxin by Bacteria Associated with Dinoflagellates of the Portuguese Coast

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain dinoflagellate and cyanobacterial species. The autonomous production of PSTs by bacteria remains controversial. In this study, PST production by two bacterial strains, isolated previously from toxic dinoflagellates, was evaluated using biological and analytical methods. Analyses were performed under conditions determined previously to be optimal for toxin production and detection. Our data are inconsistent with autonomous bacterial PST production under these conditions, thereby challenging previous findings for the same strains.     

In Silico Analysis of Putative Paralytic Shellfish Poisoning Toxins Export Proteins in Cyanobacteria

Paralytic shellfish poisoning toxins (PSTs) are a family of more than 30 natural alkaloids synthesized by dinoflagellates and cyanobacteria whose toxicity in animals is mediated by voltage-gated Na⁺ channel blocking. The export of PST analogues may be through SxtF and SxtM, two putative MATE (multidrug and toxic compound extrusion) family transporters encoded in PSTs biosynthetic gene cluster (sxt). sxtM is present in every sxt cluster analyzed; however, sxtF is only present in the Cylindrospermopsis-Raphidiopsis clade. These transporters are energetically coupled with an electrochemical gradient of proton (H⁺) or sodium (Na⁺) ions across membranes. Because the functional role of PSTs remains unknown and methods for genetic manipulation in PST-producing organisms have not yet been developed, protein structure analyses will allow us to understand their function. By analyzing the sxt cluster of eight PST-producing cyanobacteria, we found no correlation between the presence of sxtF or sxtM and a specific PSTs profile. Phylogenetic analyses of SxtF/M showed a high conservation of SxtF in the Cylindrospermopsis-Raphidiopsis clade, suggesting conserved substrate affinity. Two domains involved in Na⁺ and drug recognition from NorM proteins (MATE family) of Vibrio parahaemolyticus and V. cholerae are present in SxtF/M. The Na⁺ recognition domain was conserved in both SxtF/M, indicating that Na⁺ can maintain the role as a cation anti-transporter. Consensus motifs for toxin binding differed between SxtF and SxtM implying differential substrate binding. Through protein modeling and docking analysis, we found that there is no marked affinity between the recognition domain and a specific PST analogue. This agrees with our previous results of PST export in R. brookii D9, where we observed that the response to Na⁺ incubation was similar to different analogues. These results reassert the hypothesis regarding the involvement of Na⁺ in toxin export, as well as the motifs L³⁹⁸XGLQD⁴⁰³ (SxtM) and L³⁹⁰VGLRD³⁹⁵ (SxtF) in toxin recognition.     

Implication of Catecholamines During Spawning in Marine Bivalve Molluscs

Summary

During the tail-bud stage of Ascidiella aspersa embryogenesis, the test cells or innermost cells of the egg envelope manifest locomotive activities. Light microscopy further reveals that the adhesive behaviour of test cells changes in the course of embryogenesis. Mechanical dechorionation experiments performed on 846 embryos demonstrate that up to the tail-bud stage all test cells are attached to the inner surface of the chorion. The embryo is completely devoid of test cells. At the onset of larval tunic secretion, increasing numbers of test cells settle on the embryo until all test cells adhere to it. This switch in adhesive properties is completed within 65 min. The hatched larva carries the entire complement of test cells until the onset of metamorphosis. SEM and observations show that test cells do not establish direct cell-to-cell contacts to ectodermal cells but attach to the larval tunic.     

Biotransformations of 2-Methylisoborneol by Camphor-Degrading Bacteria

Many camphor-degrading bacteria that are able to transform 2-methylisoborneol (2-MIB) have been identified. Three of these strains have been examined in detail. Rhodococcus ruber T1 metabolizes camphor through 6-hydroxycamphor but converts 2-MIB to 3-hydroxy-2-MIB. Pseudomonas putida G1, which metabolizes camphor through 5-hydroxycamphor, converts MIB primarily to 6-hydroxy-2-MIB. Rhodococcus wratislaviensis DLC-cam converts 2-MIB through 5-hydroxy-2-MIB to 5-keto-2-MIB. Together, these three strains produce metabolites resulting from hydroxylation at all of the three available secondary carbons on the six-member ring of 2-MIB.     

Ultrastructural Characteristics of Interneuronal Connections of the Central Nervous System of Bivalve Molluscs

Ultrastructure and peculiarities of interneuronal contacts are studied in visceral ganglia of two species of bivalve molluscs, Anadara broughtoni and Mactra sulcatoria. Gap, desmosome-like, symmetrical, and classic synaptic junctions between neuronal bodies and their main processes are described. The major part of interneuronal junctions in the ganglia of the molluscs studied are symmetrical. Complex synaptic complexes formed by specialized and non-specialized junctions are observed.     

微小亚历山大藻麻痹性贝类毒素小鼠生物检测

应用小鼠生物检测法对培养的微小亚历山大藻（LJX02株系）麻痹性贝类毒素进行了检测。首先建立微小．[X02亚历山大藻培养技术,利用对数期藻细胞接种,可使藻细胞处于快速生长,并在7d内藻细胞密度最高达到30000ml^-1。从培养株系中提取的藻毒素,经液相质谱分析主要为GTX1／4和GTX2／3,其含量分别为12．23μg／ml和9．742μg／ml,12L藻体培养液中可获得18．35μgGTX1／4和14．61μgGTX2／3毒素量。采用小鼠生物检测法建立了藻毒素剂量-小鼠死亡时间曲线,其曲线方程为Y=74．017X^-1.5896,将死亡时间t变为时间倒数1／t,GTX毒性MU取对数为logMU,得直线回归方程为Y=3．7009X-0．0858（R^2=0．9824）,求得了昆明小鼠一个鼠单位所代表的GTX1／4和GTX2／3毒素剂量分别为0．026μg和0．021μg。     

Feeding and Digestion in Suspension-Feeding Bivalve Molluscs: The Relevance of Physiological Compensations

SYNOPSIS. Intertidal suspension-feeding bivalves face reductionsin the time available for feeding proportional to the durationof aerial exposure. Such individuals do not compensate by increasingtheir rates of feeding during immersion. Simulations of feedingbehaviour, in terms of the energetic balance between the costsand the gains of changes in feeding rate, suggest that variationfrom the rates characteristic of the sublittoral condition wouldnot enhance net energy gain for intertidal individuals. Thesefindings are consistent with evidence of potential food limitationin the natural sublittoral habitat. Nevertheless, hydrodynamicfactors may enhance the supply of food on the shore, so amelioratingthe disadvantages of reduced feeding time. In addition, potentialphysiological compensations for reduced dietary quality includechanges in gut residence time, the volume of gut occupied fordigestion and (possibly) also in the provision of appropriatedigestive enzymes. However, these compensations require daysor weeks and are inappropriate on tidal time scales.     

Degradation of Parathion by Bacteria Isolated from Flooded Soil

Two bacteria, Bacillus sp. and Pseudomonas sp., were isolated from parathionamended flooded alluvial soil which exhibited parathion-hydrolyzing ability. Bacillus sp. readily liberated nitrite from the hydrolysis product, p-nitrophenol, but not from intact parathion. Pseudomonas sp. hydrolyzed parathion and then released nitrite from p-nitrophenol. These studies establish bacterial degradation of parathion past the p-nitrophenol stage to the end product, nitrite.     

Degradation of Methylmercury by Bacteria Isolated from Environmental Samples

A total of 207 bacterial cultures, isolated from environmental samples, was screened for ability to degrade methylmercury. Of these, 30 were found positive for aerobic demethylation. Twenty-two of these were shown to be facultative anaerobes and 21 of these degraded methylmercury anaerobically. All positive species volatilized methylmercury aerobically, and methane was produced as a degradation product. Although methylmercury degradation was complete in most cases, material balances indicated some of the inorganic mercury formed was not volatilized and is presumed bound to the cells. All positive isolates were tolerant to at least 0.5 mug of methylmercury per ml, and the extent of volatilization of mercury increased with concentration to the threshold value. The results indicate that demethylating species are prevalent in the environment and may be important in suppressing the methylmercury content of sediments.     

Phylogeography of Cylindrospermopsin and Paralytic Shellfish Toxin-Producing Nostocales Cyanobacteria from Mediterranean Europe (Spain)

Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanotoxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanotoxin production, and the presence of cyanotoxin biosynthesis genes. The potent cytotoxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 μg CYN mg⁻¹ [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic shellfish poisoning (PSP) neurotoxins (saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.     

High Specificity of a Quantitative PCR Assay Targeting a Saxitoxin Gene for Monitoring Toxic Algae Associated with Paralytic Shellfish Toxins in the Yellow Sea

The identification of core genes involved in the biosynthesis of saxitoxin (STX) offers a great opportunity to detect toxic algae associated with paralytic shellfish toxins (PST). In the Yellow Sea (YS) in China, both toxic and nontoxic Alexandrium species are present, which makes it a difficult issue to specifically monitor PST-producing toxic algae. In this study, a quantitative PCR (qPCR) assay targeting sxtA4, a domain in the sxt gene cluster that encodes a unique enzyme involved in STX biosynthesis, was applied to analyze samples collected from the YS in spring of 2012. The abundance of two toxic species within the Alexandrium tamarense species complex, i.e., A. fundyense and A. pacificum, was also determined with TaqMan-based qPCR assays, and PSTs in net-concentrated phytoplankton samples were analyzed with high-performance liquid chromatography coupled with a fluorescence detector. It was found that the distribution of the sxtA4 gene in the YS was consistent with the toxic algae and PSTs, and the quantitation results of sxtA4 correlated well with the abundance of the two toxic species (r = 0.857). These results suggested that the two toxic species were major PST producers during the sampling season and that sxtA-based qPCR is a promising method to detect toxic algae associated with PSTs in the YS. The correlation between PST levels and sxtA-based qPCR results, however, was less significant (r = 0.552), implying that sxtA-based qPCR is not accurate enough to reflect the toxicity of PST-producing toxic algae. The combination of an sxtA-based qPCR assay and chemical means might be a promising method for monitoring toxic algal blooms.     

Conversion of Unsaturated Fatty Acids by Bacteria Isolated from Compost

A compost mixture amended with soybean oil was enriched in microorganisms that transformed unsaturated fatty acids (UFAs). When oleic acid or 10-ketostearic acid was the selective fatty acid, Sphingobacterium thalpophilum (NRRL B-23206, NRRL B-23208, NRRL B-23209, NRRL B-23210, NRRL B-23211, NRRL B-23212), Acinetobacter spp. (NRRL B-23207, NRRL B-23213), and Enterobacter cloacae (NRRL B-23264, NRRL B-23265, NRRL B-23266) represented isolates that produced either hydroxystearic acid, ketostearic acid, or incomplete decarboxylations. When ricinoleic (12-hydroxy-9-octadecenoic) acid was the selective UFA, Enterobacter cloacae (NRRL B-23257, NRRL B-23267) and Escherichia sp. (NRRL B-23259) produced 12-C and 14-C homologous compounds, and Pseudomonas aeruginosa (NRRL B-23256, NRRL B-23260) converted ricinoleate to a trihydroxyoctadecenoate product. Also, various Enterobacter, Pseudomonas, and Serratia spp. appeared to decarboxylate linoleate substrate incompletely. These saprophytic, compost bacteria were aerobic or facultative anaerobic Gram-negative and decomposed UFAs through decarboxylation, hydroxylation, and hydroperoxidation mechanisms. Received: 3 November 1998 / Accepted: 30 November 1998     

Utilization of Ammonia Nitrogen by Intestinal Bacteria Isolated from Pigs

In a medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, 17 of 24 Bacteroidaceae strains, 3 of Selenomonas strains, 1 of 7 curved rods, 3 of 7 Spirochaetaceae strains, 8 of 20 Eubacterium strains, 8 of 13 Peptococcaceae strains, 3 of 4 Clostridium strains, 19 of 20 Enterobacteriaceae strains, and 1 of 8 Streptococcus strains utilized ammonia nitrogen preferentially to proteose peptone nitrogen. To determine the ability of intestinal microbes to synthesize amino acids from ammonia, ammonia utilization by Bacteroides ruminicola strain 9 was studied in defined media containing ammonia and other nitrogen sources. In another medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, ammonia was preferentially utilized even when the proteose peptone nitrogen content was eight times greater than that of ammonia nitrogen. In a medium containing ammonia, an amino acid, and cysteine, the lowest uptake of ammonia nitrogen was observed when the medium contained aspartic acid, glutamic acid, threonine, or alanine; but ammonia was utilized more effectively than any of the amino acids. Incorporation of ¹⁵N from [¹⁵N]ammonia into bacterial amino acids was studied. ¹⁵N was incorporated into every amino acid of B. ruminicola strain 9, and the highest uptake was observed in aspartic acid and alanine.     

Properties of Bacteria Isolated from Deep-Sea Sediments

Thirty-eight isolates were subjected to taxonomic analysis by computer. Of the 38 isolates, 31 were from sediment samples collected at depths from 9,400 to 10,400 meters in the Philippine and Marianas Trenches of the Pacific Ocean, and 7 cultures were from seawater samples collected at various depths from surface to 4,000 meters and from several locations in the Pacific Ocean. A total of 116 characteristics were determined for each isolate, coded, and transferred to punch cards. Similarity values were obtained by computer analysis, with the use of two recently developed computer programs. Five distinct phenetic clusters were observed from the numerical analyses. Four of the clusters were identified as species of the genus Pseudomonas, and one, as an aerogenic species of Aeromonas. Group IV was identified as pigmented Pseudomonas fluorescens, and the major cluster, consisting of groups I and II, which merged at a species level of similarity, was treated as a new species of Pseudomonas. The 38 strain data were compared with data for 132 marine and nonmarine strains previously subjected to computer taxonomic analysis. The barotolerant deep-sea strains, with the exception of the deep-sea P. fluorescens isolates, clustered separately from all other marine strains.