Production level of tetrodotoxin in Aeromonas is associated with the copy number of a plasmid

Toxicon 101 (2015) 27e34

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Production level of tetrodotoxin in Aeromonas is associated with the copy number of a plasmid Jing Liu a, 1, Fen Wei a, 1, Ying Lu b, Tinglong Ma a, Jing Zhao b, Xiaoling Gong a, Baolong Bao a, * a b

Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, Shanghai 201306, China College of Food Science & Technology, Shanghai Ocean University, Shanghai 201306, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 February 2015 Received in revised form 9 April 2015 Accepted 21 April 2015 Available online 23 April 2015

Tetrodotoxin (TTX) has been identified from taxonomically diverse organisms. Artificial synthesis of TTX has been reported, but the biosynthetic pathway of TTX remains elusive. In this study, we found TTX producing ability was associated with the copy number of plasmid pNe-1 in Aeromonas strain Ne-1 during fermentation, suggesting that at least one gene encoding a TTX-synthesis enzyme is located on this plasmid. Compared with bacterial genomes, plasmids are small and easier to screen for genes associated with TTX biosynthesis. The approximately 100 kb genome of pNe-1 was sequenced. The plasmid contains 60 complete open reading frames (orfs) of which 32 (53.3%) encode hypothetical proteins. Seven genes are related to the type IV secretion system (T4SS) and 2 genes are related to transposons, indicating that the TTX-producing bacterium Aeromonas might have the ability to transfer the TTX biosynthesis gene via the conjugation and contagion of plasmid pNe-1. In addition, we unexpectedly found that Aeromonas Ne-1 contains unknown TTX-degrading materials, indicating there is a homeostatic mechanism to maintain a stable amount of TTX in the bacterium. These results will help us to better understand TTX biosynthesis, the bacterial origin of TTX, and TTX degradation. © 2015 Elsevier Ltd. All rights reserved.

Keywords: TTX-producing bacteria Plasmid copy number TTX biosynthesis TTX degradation Plasmid genome

1. Introduction Tetrodotoxin (TTX), a highly toxic non-protein neurotoxin of low molecular weight, has been identified in taxonomically diverse organisms across 14 different phyla (Chau et al., 2011), including pufferfish from the family Tetraodontidae, some species of goby, newt, frog, starfish, crab, octopus, gastropod and flatworm (Wakely et al., 1966; Noguchi and Hashimoto, 1973; Noguchi et al., 1983; Sheumack et al., 1984; Miyazawa et al., 1985; Mebs and Schmidt, 1989; Hwang et al., 1990; Ritson-Williams et al., 2005; Noguchi et al., 2006; Rodriguez et al., 2008; Wang et al., 2008; McNabb et al., 2010; Itoi et al., 2012; Stokes et al., 2014). The wide distribution of TTX among genetically distant animals makes the origin of TTX controversial. The postulation of exogenous origin of TTX in TTXbearing organisms was first confirmed by Noguchi et al. (Noguchi et al., 1986), who isolated TTX-producing Vibrio from the intestine of xanthid crab. Thereafter, more and more TTX-producing bacterial

* Corresponding author. E-mail address: [email protected] (B. Bao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.toxicon.2015.04.009 0041-0101/© 2015 Elsevier Ltd. All rights reserved.

strains were isolated from numerous organisms and deep-sea sediments. These bacteria included Acinetobacter sp., Aeromonas sp., Alteromonas sp., Bacillus sp., Bacillus horikoshii, Cellulomonas fimi, Kytococcus sedentarius, Lysinibacillus fusiformis, Marinomonas sp., Microbacterium arabinogalactanolyticum, Nocardiopsis dassonvillei, Plesiomonas sp., Pseudoalteromonas haloplanktis tetraodonis, Pseudomonas sp., Raoultella terrigena, Roseobacter sp., Serratia marcescens, Shewanella sp., Tenacibaculum sp., Vibrio alginolyticus and V. fischeri (Noguchi et al., 1987; Narita et al., 1987; Sugita et al., 1987; Hwang et al., 1989; Do et al., 1990; Cheng et al., 1995; Ritchie et al., 2000; Lee et al., 2000; Wu et al., 2005; Wang et al., 2008, 2010; Lu and Yi, 2009; Bragadeeswaran et al., 2010; Yang et al., 2010; Wang and Fan, 2010; Yu et al., 2004, 2011; Pratheepa and Vasconcelos, 2013; Magarlamov et al., 2014). Even now there remains much debate in the literature about whether bacteria are truly the source of TTX in animals (Chau et al., 2011), although the bacterial origin of TTX in TTX-bearing pufferfish has been accepted (Noguchi et al., 2006). Proof of TTX's proposed microbial origin can be achieved by identifying biosynthesis genes involved in its assembly. TTX possesses a unique cage-like structure. The total synthesis of D,L-

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J. Liu et al. / Toxicon 101 (2015) 27e34

tetrodotoxin, and the asymmetric total synthesis of tetrodotoxin, have been accomplished (Kishi et al., 1972; Ohyabu et al., 2003; Hinman and Du Bois, 2003; Nishikawa et al., 2004). However, the TTX biosynthetic pathway has yet to be elucidated. A number of TTX biosynthesis pathway proposals have been documented (Woodward and Gougoutas, 1964; Yasumoto et al., 1988; Kotaki et al., 1993; Chau et al., 2011). It was speculated that TTX is assembled by a hybrid polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) enzyme complex which possibly incorporates an amidinotransferase (AMT) (Chau et al., 2011), however, no supporting or experimental evidence has been published for the biosynthesis of TTX. Given that so many phylogenetically unrelated bacteria can produce TTX, it is possible that the TTX-producing bacteria might share the same TTX biosynthetic pathway through horizontal gene transfer (HGT), as for the saxitoxin (STX) pathway (Kellmann et al., 2008a,b). Microbial evolution has been profoundly affected by HGT, even between phyla (Burrus and Waldor, 2004). The ability of bacterial species to acquire new genetic information from other species by HGT is a dominant force of variation in gene content (Koonin and Wolf, 2008). Conjugation, transduction and transformation are the main mechanisms of HGT. One strain of bacteria, Aeromonas sp. Ne-1, isolated from the pufferfish Takifugu obscurus, produces authentic TTX, confirmed by mouse bioassay, enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography coupled with mass spectrometry (LC-MS) (Yang et al., 2010). In the present study, we find that the TTX-producing ability of Aeromonas sp. Ne-1 was lost gradually during fermentation, and this loss was associated with the copy number of a plasmid. We sequenced the plasmid genome and found a series of genes encoding mobile elements such as the type IV secretion system (T4SS) and transposons. 2. Materials and methods 2.1. Bacterial strain and medium TTX-producing Aeromonas sp. strain Ne-1 was isolated from T. obscurus and stored in our lab. The identity of TTX produced by strain Ne-1 had been confirmed (Yang et al., 2010). The strain Ne-1 harbors a plasmid denoted pNe-1. Cells were grown in Ocean Research Institute (ORI) broth, containing 0.2% proteose peptone No. 3 (Difco), 0.2% phytone peptone (BBL), 0.1% yeast extract (Difco), 0.088% ferric citrate, and 3% NaCl. The pH of the medium was adjusted to 8.0.

12000 rpm for 15 min at room temperature. Nucleic acid precipitates were dissolved in 100 mL sterile water and stored at 20
C. The extracted plasmid DNA was subjected to 1% (w/v) agarose gel electrophoresis analysis (Fig. 1A).

2.3. Plasmid sequencing, sequence analysis and annotation An Illumina Genome Analyzer IIx (Illumina, San Diego, CA) was used for plasmid sequencing. Plasmid DNA was first sheared into average 200-300 bp pieces by a Covaris E220 (Covaris, Woburn, MA) instrument, then adapter sequences were added onto the ends of DNA fragments to generate multiplexed paired end sequencing libraries. Detailed sample preparation for sequencing followed the TruSeq™ DNA Sample Preparation Guide (Illumina). Sequencing reads were extracted from the image files generated by the Illumina Genome Analyzer and then processed to produce digital-quality data. Contig assembly used the software package Vector NTI Suite 11 (Invitrogen, Carlsbad, CA) to assemble DNA fragments. Potential open reading frames (orfs) were searched by Vector NTI Suite software, and the predicted protein sequences of orfs were compared with the sequences of proteins in the database using BLASTX (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequence and annotation have been deposited in the GenBank database under accession number KP738729.

2.4. Time-course cell culture and pDNA extraction for real-time QPCR Cells were grown in 500 mL flasks containing 120 mL of medium incubated at 23
C with shaking at 250 rpm. Culture volumes of 100 mL were taken at 12, 18, 21, 24, 31, and 42 h, respectively. Three milliliters of culture were used to determine cell concentration by measuring the turbidity at 600 nm by visible absorbance and the numbers of colony-forming units (CFU) after plating culture aliquots. One hundred milliliters were collected by centrifugation for 15 min at 8000 rpm and then kept at 20
C for the isolation of plasmid pNe-1. The nucleic acid precipitate was dissolved in 50 mL sterile water containing 20 mg/mL RNase A and incubated at 37
C for 30 min. Gel electrophoresis of the plasmid DNA showed no contamination with RNA or protein. The plasmid DNA was stored at 20
C before real-time QPCR.

2.5. Real-time QPCR 2.2. Plasmid isolation Aeromonas sp. strain Ne-1 was cultured in ORI medium at 23
C for 21 h. An alkaline lysis method was used for plasmid pNe-1 extraction with minor modifications (Sambrook and Russell, 2001). Cell culture (500 mL) was harvested by centrifugation for 15 min at 8000 rpm. The supernatant was removed. Solution I (0.05 mol/L glucose, 0.025 mol/L Tris-HCl, 0.010 mol/L EDTA, pH 8.0) was added and the cell pellet was resuspended by vortexing after three cycles of 80
C freezer/room temperature. Lysozyme was added to solution I to weaken the cell wall of strain Ne-1. Then, solution II (0.2 mol/L NaOH, 1% (w/v) SDS) was added to the tube and gently mixed by inverting the tube 4e6 times. Immediately, ice-cold solution III (60 mL 5 mol/L KAC, 11.5 mL HAC, 28.5 mL ddH2O) was added to the tube and mixed by inversion. The mixture was centrifuged at 12000 rpm for 20 min at 4
C. The cleared lysate was transferred to a new tube, 0.6 volumes of isopropanol were added and intensively mixed, and the tube was incubated for 10min. The precipitated plasmid nucleic acids were collected by

Quantitative real-time PCR for determination of pDNA was performed by amplification of a 149 bp sequence (position 2252e2400) in the plasmid pNe-1 genome using the forward primer 50 -GACAAGGCTCGGAGACACGCA-30 and the reverse primer 50 -TCTCTTTCATGCTGCGGTTCAGC-30 . PCR reactions were carried out in a CFX96 Touch™ Real-Time PCR detection system using iQ SYBR Green Supermix (Bio-RAD, Hercules, CA), as recommended by the manufacturer. Each 25 mL of final reaction volume contained 12.5 mL of iQ SYBR Green Supermix, 1 mL of each primer (2 mmol/L), 2 mL of sample plasmid, and 8.5 mL PCR grade water. Reactions were incubated at 95
C for 5 min, followed by 30 cycles of 30 s at 95
C, 30 s at 59
C and 30 s at 72
C. Following the final cycle, reactions were kept at 72
C for 10 min and then heat denatured over a temperature gradient from 59 to 95
C over 20 s. CFX Manager Software (Bio-Rad) was used to perform melting curve analysis and determine cycle threshold (Ct) values. The reaction was checked for amplification specificity by 1% agarose gel electrophoresis and sequencing.

J. Liu et al. / Toxicon 101 (2015) 27e34

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Fig. 1. Genetic map of plasmid pNe-1. (A) Gel electrophoresis of plasmid pNe-1 was performed on a 1% agarose gel. (B) Deduced open reading frames (orfs) are shown by open arrows or arrowheads indicating the direction of transcription. The possible functions of the encoded proteins are color coded. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.6. Construction of standard curves for plasmid copy number (PCN) determination Plasmid DNA standards were prepared by growing Aeromonas sp. Ne-1 cells (harboring plasmid pNe-1) in ORI broth at 23
C for 21 h. Plasmid extraction and isolation was performed as above. The concentration of purified pDNA solutions was assayed by Nanodrop 2000 (Thermo Scientific, MA). A 10-fold serial dilution series of pNe-1 was used to construct the standard curves. The concentration of the plasmid was measured using a fluorometer and the corresponding copy number was calculated using the following equation (Eq. (1)) (Whelan et al., 2003)

plasmids 6:02  1023 plasmids 1 mole ¼  bacteria 1 mole single plasmid massðngÞ 

amount of plasmid DNAðngÞ no:of bacteria  1 mL of solution l mL of culture (1)

Ct values in each dilution were measured in triplicate using realtime QPCR as described above to generate the standard curves. The Ct values were plotted against the logarithm of their initial template copy numbers. Each standard curve was generated by a linear regression of the plotted points. 2.7. Time-course cell culture and extraction for TTX measurement Five-hundred milliliter cultures of strain Ne-1 were collected 24, 42, 66, and 96 h after inoculation. Other fermentation conditions were the same as described above. The crude TTX extraction from Aeromonas sp. Ne-1 was as previously described (Yang et al., 2010). Briefly, the cells were harvested by centrifugation at 8000 rpm for 15 min. The pellets were washed three times with distilled water, then suspended in 0.1% acetic acid, ultrasonicated (200 W, 2 s ultrasonication, 3 s interval), and boiled for 15 min. After the cell debris was removed by centrifugation, the cell supernatant together with the culture supernatant was loaded onto an activated charcoal column (Charcoal Factory, Shanghai, China) after the pH had been adjusted to 5.5, and the toxin absorbed in the charcoal was eluted with 1% acetic acid in 20% methanol. The elution was

concentrated into a final volume of 50 mL using a rotary evaporator. After the pH had been adjusted to 6.5e7.4, the sample was filtered using a 0.22 mm membrane (ANPEL, Shanghai, China) for ELISA test. The ELISA test kit for TTX (Zhongwei Inc., Beijing, China) used a monoclonal antibody raised against TTX (MAb-TTX) (Yang et al., 2010). A competitive ELISA method, as described in the manual of the kit, was used to determine TTX. TTX in the sample could compete with the immobilized TTX standard to reduce the signal given in an enzyme-catalyzed reaction. The decreased signal, as indicated by the ratio of absorbance (Ai/Ao, where Ai is the absorbance of the sample, and Ao is the absorbance of the blank with no TTX added), had a linear relationship with the logarithm of the concentration of TTX in the sample within a certain concentration range. A standard curve of TTX was established in the concentration range 10e200 ng/mL. TTX concentration in samples was calculated based on the standard curve. 2.8. In vitro TTX degradation experiment To test the TTX degradation ability of strain Ne-1, 500 mL cultures were collected 66 or 96 h after inoculation. The crude extraction was the same as for the TTX extraction described above. Different amounts of authentic TTX (SigmaeAldrich) were added into the crude extracts to final concentrations of 30, 60, or 90 ng/ mL, respectively. The ORI medium was used as the control group. After incubation at 20
C for 24 h, the TTX amount remaining in each sample was determined using the ELISA method described above. 3. Results 3.1. Sequence and coding regions of plasmid pNe-1 The complete nucleotide sequence of plasmid pNe-1 has 99,401 base pairs (bp). As shown in Fig. 1B, potential complete orfs are distributed around the plasmid. We found 60 complete orfs as shown in Table 1, of which 32 (53.3%) encode proteins similar to hypothetical proteins (Fig. 1B). It is noteworthy that mobile genetic elements were identified in the plasmid. Seven genes are related to the T4SS and two genes are

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J. Liu et al. / Toxicon 101 (2015) 27e34

Table 1 Summary of locations of predicted coding regions in plasmid pNe-1. Orf no.

Position

Nucleic acid length (bp)

Protein length (aa)

Direction

Amino acid identity to informative database match (accession no.)

E value

1 2 3

1e456 993e1322 1289e2431

456 330 1143

151 109 380

sense strand sense strand sense strand

1e-69 8e-50 8e-96

4 5

2358e2564 2561e3817

207 1257

68 418

sense strand sense strand

6 7 8 9 10 11

4157e5122 5153e6631 6648e7535 7867e8607 8604e9947 10784e10909

966 1479 888 741 1344 126

321 492 295 246 447 41

antisense strand antisense strand antisense strand sense strand sense strand sense strand

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

11107e12795 12797e12997 17276e18646 18652e19647 19662e21695 21903e22187 22165e22425 31828e32043 37113e38171 39967e40149 42075e42548 42596e42916 43912e44244 44644e45072 45069e45821 46488e47459 48755e48922 49402e49875

1689 201 1371 996 2034 285 261 216 1059 183 474 321 333 429 753 972 168 474

562 66 456 331 677 94 86 71 352 60 157 106 110 142 250 323 55 157

antisense strand antisense strand antisense strand antisense strand antisense strand sense strand sense strand antisense strand sense strand antisense strand sense strand sense strand sense strand sense strand sense strand sense strand sense strand antisense strand

30 31 32 33 34 35 36

49892e50599 51114e51716 51745e51945 53323e53844 55752e56285 57221e58099 61252e61560

708 603 201 522 534 879 309

235 200 66 173 177 292 102

antisense strand antisense strand antisense strand antisense strand sense strand sense strand sense strand

37 38 39 40 41 42 43 44 45 46 47 48 49

62087e62791 64522e65394 65433e65744 66646e67926 67950e68399 68842e69738 70980e71204 71223e72275 74176e75222 75426e75950 76218e77924 77321e78802 78815e79087

705 873 312 1281 450 897 225 1053 1047 525 1077 1482 273

234 290 103 426 149 298 74 350 348 174 358 493 90

sense strand antisense strand antisense strand sense strand sense strand sense strand sense strand sense strand sense strand sense strand sense strand sense strand sense strand

50 51 52 53 54 55 56

79932e80474 80529e80999 84706e85458 85699e86079 86096e87088 87179e87985 88024e88473

543 471 753 381 993 807 450

180 156 250 126 330 268 149

antisense strand antisense strand sense strand sense strand sense strand sense strand sense strand

57 58 59 60

88460e90109 91693e91935 97299e97673 98287e98910

1650 243 375 624

549 80 124 207

sense sense sense sense

68% DNA-binding protein Ptr (WP_009385240.1) 70% mobilization protein (WP_026827025.1) 51% relaxase/mobilization nuclease domain-containing protein (WP_012209756.1) hypothetical protein (WP_033679060.1) 38% Type IV secretory pathway, VirD4 component (WP_011668983.1) hypothetical protein (WP_019156870.1) 38% Type II/IV secretion system hypothetical protein (WP_019156872.1) 58% flagellar protein FlgA (WP_000769513.1) hypothetical protein (WP_000761451.1) 46% Single-stranded DNA-binding protein (WP_024465601.1) 49% LtrC (WP_013555054.1) hypothetical protein (WP_016205417.1) 64% TrsK (WP_013555057.1) hypothetical protein (WP_000676587.1) 49% conjugal transfer protein TraA (WP_012552655.1) hypothetical protein (GAJ45310.1) hypothetical protein (WP_000210434.1) hypothetical protein (WP_019156864.1) hypothetical protein (WP_033674956.1) 65% DNA-binding protein (WP_017154134.1) 57% putative DNA repair protein RadC (WP_012301103.1) hypothetical protein (WP_026688364.1) hypothetical protein (WP_033798340.1) hypothetical protein (WP_016205275.1) hypothetical protein (WP_009336127.1) hypothetical protein (WP_006784693.1) 41% putative DNA-binding protein (WP_011255100.1) 56% appr-1-p processing domain-containing protein (EWG08967.1) hypothetical protein (WP_028545839.1) 48% recombinase (KHK53972.1) hypothetical protein (WP_016205417.1) hypothetical protein (WP_001255995.1) hypothetical protein (WP_019156863.1) 59% DNA modification methylase (WP_015010633.1) 34% cell division suppressor protein YneA (WP_016205020.1) hypothetical protein (WP_017473360.1) 99% integrase catalytic subunit (WP_016205490.1) 100% IS426 transposase (WP_016205489.1) hypothetical protein (WP_012301119.1) hypothetical protein (WP_006211781.1) 48% nuclease (WP_002437029.1) hypothetical protein (WP_006211785.1) 62% TrsD (WP_013555119.1) 53% peptidase M23 (WP_011888475.1) hypothetical protein (WP_001043541.1) 53% conjugal transfer protein TraL (WP_014481844.1) hypothetical protein (WP_001202485.1) 47% transition state regulatory protein AbrB (WP_008498882.1) hypothetical protein (WP_012301106.1) hypothetical protein (WP_000389431.1) hypothetical protein (WP_003286251.1) hypothetical protein (WP_019156867.1) 52% peptidase M23 (WP_000502631.1) hypothetical protein (WP_026679216.1) 37% extracellular metalloproteinase, serralysin family (WP_016205130.1) 58% PKD domain-containing protein (WP_016205129.1) 75% transposase (WP_016202602.1) 43% VirD4-like protein (WP_010889632.1) hypothetical protein (WP_000162397.1)

strand strand strand strand

4e-12 2e-68 2e-88 1e-80 8e-97 7e-84 7e-132 7e-04 3e-174 2e-08 3e-99 3e-85 0.0 0.005 3e-21 5e-20 9e-108 5e-14 1e-52 3e-07 6e-11 2e-26 1e-32 0.002 3e-06 8e-47 2e-64 5e-52 2e-08 9e-09 1e-35 6e-79 1e-16 5e-57 0.0 2e-54 2e-130 4e-11 4e-32 3e-11 5e-94 5e-96 5e-20 2e-109 2e-54 8e-13 8e-17 2e-04 1e-69 8e-58 7e-102 4e-18 9e-15 3e-172 8e-27 6e-24 2e-120

J. Liu et al. / Toxicon 101 (2015) 27e34

related to transposons. DNA transfer initiation processing is through the Dtr protein (DNA transfer and replication protein) binding a cognate origin-of-transfer (oriT) sequence (orf 1). Mobilization protein (orf 2), an accessory protein for dsDNA nicking activity, can increase the efficiency of cleavage of DNA relaxases. The DNA relaxase of Dtr protein, which is responsible for site- and strand-specific nicks in double-stranded DNA was identified in pNe-1 (orf 3). Mating-pair formation (mpf) in virulence-associated T4SS depends on the virulence (Vir) factors VirB and VirD4. Several orfs (5, 7, 14, 16, 44, 47 and 59) in plasmid pNe-1 were identified as VirD4-like proteins. The 1775 bp transposon element consisted of terminal IRs (inverted repeats) and two orfs whose functions in transposases were identified (orf 38 and orf 39). Orf38 involves with the process in which a segment of DNA is incorporated into another, usually larger, DNA molecule such as a chromosome. Orf 39 belongs to the IS3 family, which includes various Escherichia coli insertion elements. Putative terminal inverted repeats (underlined in bold) in the left IR were: 50 -TTATTTTAGATTTTTTCTTTTTCTTTTAATAAATAAAAGAAAACTGATTA-30 . In the right terminal of the transposase inverted repeat, 50 -TATATTAATTTTCTTAGTTATTTACTTTTTTAAGAATTAATAGAGACATAG-30 was putatively identified as an IRR. 3.2. Determination of plasmid copy number Amplification specificity was checked by both melting curve analysis and sequencing (Fig. 2A). Absolute quantification of the plasmid pNe-1 DNA sampled from a time-course of cell cultures of Aeromonas sp. Ne-1, ranging from 0 to 1  107 copies/mL, was performed using standard curves (Fig. 2B). Ct values determined were plotted against the logarithm of the known initial copy number (n ¼ 3). A standard curve y ¼ 3.744lgx þ 36 was generated by linear regression in the range tested (R2 ¼ 0.99). The results from the absolute quantification and the calculated plasmid copy numbers are shown in Table 2. One microliter of template DNA extracted from culture in the 18th hour after inoculation contained 884829 plasmid copies, and each clone was estimated to contain 2.51 copies of the plasmid. Based on the number of CFU, the cells were still growing after the 18th hour. However, the estimated PCN per mL of template DNA, and the average PCN of each clone, decreased sharply. By the 42nd hour after inoculation, the plasmid could not be detected in the cell culture. 3.3. Change of TTX amount during TTX-producing bacterial culture A standard curve (y ¼ 0.4232x þ 1.2512) was established to determine TTX levels using a competitive ELISA method (Fig. 3A). The R2 value 0.9935 showed good linearity between the ratio of absorbance and the logarithmic concentration of TTX in the concentration range 10e200 ng/mL. TTX concentration in samples can be calculated with the regression equation derived from the standard curve. The concentration of TTX extracted from cultures of strain Ne-1 at each time-point is shown in Table 3. TTX concentration in 24th h culture was as low as in the medium background (i.e. the negative control). TTX concentration in 42nd h culture was 44.858 ng/mL. Unexpectedly, the TTX concentration at later time points (66 and 96 h) returned to a low level, indicating the presence of an unknown TTX-degrading enzyme in TTX-producing Aeromonas strain Ne-1. In order to determine whether there is such a TTX-degrading enzyme in the strain, an experiment was conducted in which a standard amount of TTX was added to cultures. A standard curve (y ¼ 0.393x þ 1.2543) was established for TTX by the competitive ELISA method (Fig. 3B). The R2 value 0.9612 showed good linearity. The amount of TTX in both 66 and 96 h cultures was significantly

31

lower (P < 0.05) than that in the control group (ORI medium). Compared with the control medium, The 66 h culture degraded 25.59e54.74 % of the TTX, and the 96 h culture degraded 52.61e68.91 % of the TTX from three different initial TTX concentrations (Table 4). 4. Discussion Many plasmids are related to the pathogenicity and virulence of bacteria, such as Escherichia coli enterotoxin and colonization antigen, tetanus toxin, anthrax toxin, Bacillus thuringiensis insecticidal crystal protein, and so on (Makino et al., 1998; Funnell and Phillips, 2004; Zhang et al., 2006). In this study, TTX producing capability is shown to be associated with the copy number of a plasmid in Aeromonas strain Ne-1, suggesting the presence of TTX biosynthesis genes with at least one located in the genome of plasmid pNe-1. In a previous study (Chau et al., 2013), the genes encoding TTX biosynthesis enzymes, along with those associated with toxin regulation and transport, were speculated to be clustered in the genome, in a similar manner to saxitoxin biosynthesis pathways in bacteria (Kellmann et al., 2008a,b). Compared with bacterial genomes, plasmid genomes are relatively small and therefore easier to screen to identify TTX biosynthesis genes. This will be a big help in uncovering the mechanism of TTX biosynthesis, because so far no conclusive evidence has been published on this biosynthesis. TTX biosynthesis genes in plasmid genomes will not of course exclude the possibility that most of the TTX biosynthesis genes are clustered in the bacterial genome. In addition, TTX biosynthesis genes in a plasmid genome indicate the possibility that the TTXproducing ability of Aeromonas strain Ne-1 might have been obtained from other species of bacteria, and/or that Aeromonas can transfer TTX-producing ability to other species via horizontal gene transfer (HGT). Indeed, microbial evolution has been profoundly affected by HGT, even between phyla (Burrus and Waldor, 2004; Gyles and Boerlin, 2014). Complete sequencing reveals that plasmid pNe-1 has a genome of about 100 kb, similar to large conjugation plasmids (Norman et al., 2009). So far, most of the identified conjugative systems are encoded by plasmids and plasmid-derived DNA is the typical substrate for these systems (Wozniak and Waldor, 2010). Conjugative plasmids have enabled HGT over large taxonomic distances (Norman et al., 2009). Several genes encoding intact mobile genetic elements (type IV secretion system and IS426 transposon) were identified in plasmid pNe-1, indicating the TTX-producing bacteria Aeromonas sp. potentially has the ability for conjugation and contagion via this plasmid. The T4SS can translocate DNA or protein substrates across the cell envelope by direct contact with a recipient cell (Cascales and Christie, 2003; Fronzes et al., 2009); this is also used by parasitic bacteria to transfer toxic proteins or other virulence factors into eukaryotic hosts, and it might play roles in the process (Alvarez-Martinez and Christie, 2009). Sequence data including an IS426 insertion sequence are the first biochemical evidence that horizontal gene transfer might exist among TTXproducing bacteria. If a bacterium obtains TTX-producing capability via horizontal gene transfer from other bacteria through plasmid conjugation and contagion, the TTX biosynthesis genes in the plasmid genome of TTX-producing bacteria should encode unique enzymes responsible only for TTX biosynthesis. Genes encoding a hybrid PKS, NRPS enzyme, and AMT responsible for STX biosynthesis, were speculated to be involved in the pathway of TTX biosynthesis (Chau et al., 2011). Those genes are not unique for TTX biosynthesis, mostly locate in bacterial genomes as found in some TTXproducing bacteria (Chau et al., 2013), and were not found in the plasmid genome of pNe-1 in this study. The complete sequence of

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J. Liu et al. / Toxicon 101 (2015) 27e34

Fig. 2. Confirmation of PCR amplification specificity and construction of standard curves. (A) The melting temperature and the amplicon size were 82
C and 149 bp, respectively, for the target fragment (position 2252e2400). Gel electrophoresis of the PCR products was performed on a 1% agarose gel. The identity of the amplified products was confirmed by DNA sequencing analysis. (B) Standard curves were constructed with serial 10-fold dilutions of the pNe-1 plasmid, ranging from 0 to 1  107 copies/mL. Each standard dilution was amplified by real-time QPCR in triplicate. M. 100 bp DNA Ladder; 1. the standard sample; 2. the 12, 18, 21, 24, 31, and 42 h sample; 3. negative control.

Table 2 Estimated PCN by absolute quantification. Time-course sample 12 18 21 24 31 42

h h h h h h

CFU/mL culture 7.00 3.52 8.52 3.84 7.86 9.60

     

7

10 108 108 109 109 109

Ct (Avg. ± S.D.) 24.11 13.76 15.94 19.85 19.89 30.50

± ± ± ± ± ±

0.24 0.29 0.08 0.04 0.22 0.427

PCN/mL template DNA (Avg. ± S.D.) 1515 884829 228219 20546 20190 30

± ± ± ± ± ±

266 184378 14383 695 3290 9

Average PCN of each clone 0.022 2.51 0.27 0.005 0.003 0

± ± ± ± ± ±

0.0035 0.525 0.017 0.00058 0.00058 0

Fig. 3. Standard curve established with the competitive ELISA method for detection of TTX. (A) The standard curve for TTX detection in a cell culture time-course. (B) The standard curve for the TTX degradation experiment.

J. Liu et al. / Toxicon 101 (2015) 27e34

33

Table 3 TTX amount at different time-points during culture of Aeromonas strain Ne-1. Time-course culture

OD450 (Avg. ± S.D.)

Medium (control) Ne-1 e 24 h Ne-1 e 42 h Ne-1 e 66 h Ne-1 e 96 h

1.896 1.984 1.262 1.960 2.126

± ± ± ± ±

0.124 0.064 0.073 0.074 0.224

TTX concentration (ng/mL) 10.358 8.249 44.858 8.748 6.370

± ± ± ± ±

3.061 1.291 7.526 1.575 2.746

CFU/mL culture

TTX amount for each 1010 clone (ng)

d 2.59 2.79 3.01 2.88

d 0.317 1.602 0.292 0.220

   

1011 1011 1011 1011

± ± ± ±

0.050 0.269 0.052 0.095

Table 4 In vitro TTX elimination by cultures of Aeromonas strain Ne-1 grown for different time periods. Initial concentration of TTX (ng/mL)

OD450 of ORI (control) (Avg. ± S.D.)

TTX concentration in ORI (control) (ng/mL)

OD450 of 66 h culture of Ne-1 (Avg. ± S.D.)

TTX concentration in 66 h culture of Ne-1 (ng/mL)

TTX elimination by 66 h culture versus ORI (%)

OD450 of 96 h culture of Ne-1 (Avg. ± S.D.)

TTX concentration in 96 h culture of Ne-1 (ng/mL)

TTX elimination by 96 h culture versus ORI (%)

30 60 90

2.43. ± 0.11 2.34 ± 0.09 2.08 ± 0.16

8.75 ± 0.39 10.34 ± 0.40 16.41 ± 0.11

2.88 ± 0.17 2.51 ± 0.06 2.24 ± 0.09

3.96 ± 0.26 7.61 ± 0.18 12.21 ± 0.48

54.74 26.4 25.59

3.09 ± 0.05 2.76 ± 0.06 2.66 ± 0.14

2.72 ± 0.05 4.9 ± 0.12 5.77 ± 0.25

68.91 52.61 64.84

the pNe-1 genome containing 60 complete orfs provides an opportunity to identify unique genes for TTX biosynthesis in the future. Plasmid pNe-1 has a low copy number, estimated at about 2.5 copies per cell in the 18th hour of culture in this study. This is similar to other conjugative plasmids typically found in low copy numbers (<10 copies/cell) (Paulsson, 2002). Low copy number plasmids have to rely on active mechanisms rather than on random segregation during bacterial cell division to be stably maintained in the population (Norman et al., 2009; Dmowski and Jagura-Burdzy, 2013). However, we found plasmid pNe-1 in Aeromonas sp. was becoming unstable after 18 h of culture in ORI medium. Plasmid stability might be influenced by the nature of the host cell, the type of plasmid and the environmental conditions (McLoughlin, 1994). What causes the culture population of Aeromonas to lose the plasmid pNe-1? TTX-producing Aeromonas sp. was isolated from the ovary of pufferfish T. obscures as a symbiotic microbe in our previous study (Yang et al., 2010). Plasmid pNe-1 encoding TTX biosynthesis enzymes to produce TTX may confer a selective advantage on the host Aeromonas in colonizing pufferfish, because TTX is the major defense strategy the pufferfish appears to use against predators and it also used as a male-attracting pheromone during spawning (Fuhrman, 1986; Itoi et al., 2014; Matsumura, 1995). However, in in vitro culture of Aeromonas, cells containing plasmid pNe-1 will not own a selective advantage; on the contrary, the plasmid will be an energy drain due to production of TTX. Loss of plasmid pNe-1 after 18 h of culture causing a decrease in TTX-producing ability led us to discover the presence of an unexpected, unknown TTX-degrading enzyme in strain Ne-1. This conclusion was supported by in vitro TTX degradation experiments, indicating there is a homeostatic mechanism to maintain a stable amount of TTX in the bacteria. These findings further indicate that the genes encoding the unknown enzymes are not located in plasmid pNe-1. It is noteworthy that the unknown TTX-degrading materials were extracted using the procedure for TTX extraction in this study; that will help us to purify and identify the unknown enzymes in the future to understand the TTX degradation pathway in TTX-producing bacteria. 5. Conclusions In this study, TTX producing capability is shown to be associated with the copy number of a plasmid in Aeromonas strain Ne-1. Complete sequencing reveals that plasmid pNe-1 has a genome of about 100 kb with 60 complete orfs, including several genes

encoding intact mobile genetic elements (of the type IV secretion system and IS426 transposon). Final, we found, for the first time, that a TTX-producing bacterium Aeromonas strain Ne-1 also has TTX-degrading ability. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the National Science Founding of China (grant number 41176108), the Shanghai Municipal Education Commission Grant (grant number 14ZZ145), the Pujiang Foundation from Science and Technology Commission of Shanghai Municipality (grant number 11PJ1404400), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning, Zhejiang Provincial Natural Science Foundation of China (grant number Y3110477), and the Shanghai Universities First-class Disciplines Project of Fisheries from Shanghai Municipal Education Commission. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2015.04.009. References Alvarez-Martinez, C.E., Christie, P.J., 2009. Biological diversity of prokaryotic type IV secretion systems. Microbiol. Mol. Biol. Rev. 73, 775e808. Bragadeeswaran, S., Therasa, D., Prabhu, K., Kathiresan, K., 2010. Biomedical and pharmacological potential of tetrodotoxin-producing bacteria isolated from marine pufferfish Arothron hispidus (Muller, 1841). J. Venom. Anim. Toxins Incl. Trop. Dis. 16, 421e431. Burrus, V., Waldor, M.K., 2004. Shaping bacterial genomes with integrative and conjugative elements. Res. Microbiol. 155, 376e386. Cascales, E., Christie, P.J., 2003. The versatile bacterial type IV secretion systems. Nat. Rev. Microbiol. 1, 137e149. Chau, R., Kalaitzis, J.A., Neilan, B.A., 2011. On the origins and biosynthesis of tetrodotoxin. Aquat. Toxicol. 104, 61e72. Chau, R., Kalaitzis, J.A., Wood, S.A., Neilan, B.A., 2013. Diversity and biosynthetic potential of culturable microbes associated with toxic marine animals. Mar. Drugs 11, 2695e2712. Cheng, C.A., Hwang, D.F., Tsai, Y.H., Chen, H.C., Jeng, S.S., Noguchi, T., Ohwada, K., Hasimoto, K., 1995. Microflora and tetrodotoxin-producing bacteria in a gastropod, Niotha clathrata. Food Chem. Toxicol. 33, 929e934. Dmowski, M., Jagura-Burdzy, G., 2013. Active stable maintenance functions in low copy-number plasmids of Gram-positive bacteria I. Partition systems. Pol. J. Microbiol. 62, 3e16.

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