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Feb 28, 2014 - of paralytic shellfish poisoning are harmful to the aquatic environment and human health (Asakawa et al. 1995;. Higman et al. 2001; Ichimi et al.

Appl Microbiol Biotechnol (2014) 98:4637–4652 DOI 10.1007/s00253-014-5578-x

APPLIED MICROBIAL AND CELL PHYSIOLOGY

Towards molecular, physiological, and biochemical understanding of photosynthetic inhibition and oxidative stress in the toxic Alexandrium tamarense induced by a marine bacterium Yi Li & Hong Zhu & Chengwei Guan & Huajun Zhang & Jiajia Guo & Zhangran Chen & Guanjing Cai & Xueqian Lei & Wei Zheng & Yun Tian & Xiaojing Xiong & Tianling Zheng

Received: 20 November 2013 / Revised: 17 January 2014 / Accepted: 26 January 2014 / Published online: 28 February 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Alexandrium tamarense is a notorious harmful algal bloom species, which is associated with the largest number of paralytic shellfish poisoning cases, causing devastating economic losses and health hazards. The marine bacterium Mangrovimonas yunxiaonensis strain LY01 showed high algicidal effects on A. tamarense. A. tamarense was also susceptible to the supernatant of LY01 as revealed by algicidal activity assay, but washed bacterial cells did not show algicidal activity towards A. tamarense. In this study, we investigated the algicidal effect of the supernatant on growth, photosynthesis and the antioxidative response of A. tamarense. The results indicated that under the algicidal effect of the supernatant, the contents of cellular pigments including chlorophyll a and carotenoids were significantly decreased, and the decline of the maximum quantum yield and relative electron transport rate values suggested that photosynthetic inhibition occurred in the photosystem II system. The content of reactive oxygen species (ROS) increased after 0.5 h exposure, and the surplus ROS induced lipid peroxidation, the destruction of cellular membrane integrity and decreased cellular protein and carbohydrate contents in the algal cells. At the same time, the supernatant also induced the responses of antioxidant enzymes and non-enzymatic antioxidant. The transcription of photosynthesis- and respiration-related genes

Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5578-x) contains supplementary material, which is available to authorized users. Y. Li : H. Zhu : C. Guan : H. Zhang : J. Guo : Z. Chen : G. Cai : X. Lei : W. Zheng : Y. Tian : X. Xiong (*) : T. Zheng (*) State Key Laboratory of Marine Environmental Science and Key Laboratory of MOE for Coast and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361005, China e-mail: [email protected] e-mail: [email protected]

were significantly inhibited during the exposure procedure, which obstructed photosynthetic efficiency and capacity and disturbed the respiratory system, thereby increasing ROS production again. All these results elaborate clearly the entire procedure by which cellular physiological levels respond to the algicidal bacterium and may contribute to a better understanding of the bacterial control of A. tamarense. Keywords Alexandrium tamarense . Mangrovimonas yunxiaonensis LY01 supernatant . ROS . Photosynthetic inhibition . Antioxidative response . Gene transcription inhibition

Introduction Harmful algal blooms (HABs) have broken out frequently all over the world during recent years (McLean and Sinclair 2013; Siswanto et al. 2013), and most coastal countries are threatened by the resulting economic losses and health problems (Ajani et al. 2012; Lewitus et al. 2012). Alexandrium tamarense is a globally distributed toxic dinoflagellate (Franks and Anderson 1992; Hamasaki et al. 2001; Su et al. 2007), which is an important algal bloom causing organism, and the toxins which pass through the food chain after the formation of paralytic shellfish poisoning are harmful to the aquatic environment and human health (Asakawa et al. 1995; Higman et al. 2001; Ichimi et al. 2002). To control HABs caused by A. tamarense, many methods have been implemented (Hathaway et al. 2012; Laanaia et al. 2013). However, the traditional approaches are not only expensive and impractical for application, but also may bring about the secondary damage to the marine environment (Lee et al. 2001). Biological control could be a novel and effective

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way to solve HAB problems compared with other methods (Zhou et al. 2007). Bacteria play an important role during algal growth, and influence the initiation, growth, maintenance, and possibly determination of HAB populations (Amaro et al. 2005; Wang et al. 2010). It has been reported many times that algicidal bacteria have the potential to control HABs (Kim et al. 2012; Paul and Pohnert 2013; Pokrzywinski et al. 2012), but the algicidal mechanism is still not clear. The action mode of algicidal bacteria on algal cells can generally be divided into two types: One is direct attack, where algicidal bacteria make direct contact with the algal cells and even invade them (Mayali and Azam 2004). The other is indirect attack, where algicidal bacteria produce algicidal substances without contacting the algal cells, and the algicidal substances have an inhibitory effect on algal growth (Pokrzywinski et al. 2012). In the process of growing, algicidal bacteria can release extracellular products into the marine environment, such as carbohydrates, amino acids and peptides, sugars, polyalcohols, vitamins, enzymes, and toxins (Kodani et al. 2002; Mitsutani et al. 2001). These extracellular substances belong to the allelochemicals, which is regarded as a form of interference competition. Allelochemicals from bacteria that inhibit microalgal growth have gained great interest due to their environmental potential as algicides to control HABs (Viktoria et al. 2012). So far, four possible modes of action of allelochemicals on algae are described, including destroying cell structure (Yang et al. 2013), and affecting algal photosynthesis (Yang et al. 2011), respiration (Peñuelas et al. 1996) or enzymatic activities (Churro et al. 2010). Allelochemicals destroy photosynthetic pigment and inhibit the photosynthetic efficiency and capacity of algal cells, simultaneously causing the algal cells to produce numerous reactive oxygen species (ROS) (Apel and Hirt 2004), and the surplus ROS attacks the polyunsaturated fatty acids and destroys cellular protein and carbohydrate. Allelochemicals can also induce the antioxidant system response of algal cells (Gill and Tuteja 2010; Hamilton et al. 2012). The antioxidant system includes enzymatic and nonenzymatic parts. Superoxide dismutase (SOD), catalase (CAT), and peroxidases (PODs) fall into the enzymatic antioxidant system (Shri et al. 2009), whereas glutathione (GSH) and ascorbic acid (AsA) represent the non-enzymatic antioxidant system (Ahmad et al. 2010). Allelochemicals can influence the transcription of photosynthesis-related genes and respiration-related genes (Qian et al. 2010; Yang et al. 2013), however there is little research indicating the change of photosynthesis- and respiration-related gene expressions in A. tamarense. Therefore, we focused on photosynthesisrelated genes (psbA, psbD, and rbcL) and respiration-related genes (cob and cox) to analyze the effect of algicidal bacterium on the photosynthesis and respiration of A. tamarense. Algicidal bacteria can inhibit algal growth and regulate HABs. However, the algicidal mechanism of bacteria on

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A. tamarense has rarely been considered. Our present study demonstrated that Mangrovimonas yunxiaonensis strain LY01 supernatant had a high inhibitory effect on A. tamarense, and our research focused on ROS and the cellular membrane damage, photosynthetic inhibition, antioxidant system response and the relative transcript abundance of photosynthesis- and respiration-related genes. The ultimate goal was to clarify the algicidal mechanism by means of studying ROS production and its effect on growth, photosynthesis, antioxidant system responses of algal cells, and the change of gene expression.

Materials and methods Algal cultures and algicidal bacterium The experimental alga, A. tamarense ATGD98-006, was supplied from the Algal Culture Collection, Institute of Hydrobiology, Jinan University (Guangzhou, China). The algal culture was cultivated in f/2 medium prepared with natural sea water (Guillard 1975) at 20±1 °C under an illumination of 50 μE m−2 s−1 in 12:12 (light/dark). M. yunxiaonensis strain LY01 (GenBank No. JQ937283) was isolated from a surface sediment sample collected in November 2011 at a depth of 20 cm from the Yunxiao Mangrove National Nature Reserve (23° 55′ N 117° 24′ E), Fujian Province of China (Li et al. 2013), and the strain was deposited in BCCM/LMG Bacteria Collection, which accession number is LMG 27142. Bacteria were cultured using 2216E medium (peptone 5 g, yeast extraction 1 g, ferric phosphorous acid 0.1 g, agar 10 g, pH 7.6–7.8, fixed capacity to 1 L using natural sea water) followed by incubation for 24 h at 28 °C with shaking at 120 rpm. Analysis of algicidal activity and algicidal mode In this study, strain LY01 was inoculated into 20 mL 2216E broth and grown to the stationary phase at 25 °C in a shaker at 120 rpm for 24 h, which was the best culturing time for bacterial growth and algicidal activity (Fig. S1). To study the algicidal activity and algicidal mode, different treatments were implemented as follows: (1) adding concentrations of 0.5, 1.0, and 2.0 % bacterial culture into algal cultures; (2) adding concentrations of 0.5, 1.0, and 2.0 % of 0.22-μm Millipore membrane bacterial culture filtrate into algal cultures, to determine if algicidal activity is from extracellular substances; (3) adding concentrations of 0.5, 1.0 and 2.0 % washed bacterial cells resuspended in sterile f/2 after centrifugation at 6,000g for 10 min and washing twice with sterile f/2 medium into algal cultures, to determine if algicidal activity is from the bacterial cells. Control group was normal growth

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algae with adding sterile 2216E or sterile f/2 medium, to avoid the medium influence. The algicidal rate was calculated using the formula: . Algicidal rate ð%Þ ¼ ðN C −N E Þ N C  100

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applied at 15 s intervals. rETR could be used as an indicator for the photosynthetic capacity of the algal cells. The Fv/Fm and rETR are the main factors in the photosystem to affect yield and are related to photosynthetic characteristics. ROS and lipid peroxidation assays

where NC represents the number of algal cells in the control group, and NE represents the number of algal cells in the experimental group. The algicidal rate of samples from the algal inoculation system was determined every 12 h. Algal cells were counted after being fixed with Lugol’s iodine reagent. Only intact algal cells were counted. All experiments were repeated in three biological replicates. Lysis process in algal cells To study the lysis process in algal cells, dividing cells were collected and were treated with the 2 % supernatant concentration for 72 h under the microscope (Olympus BX41, Chiyoda-ku, Tokyo, Japan). Photosynthetic pigment assay and chlorophyll a fluorescence measurement Chlorophyll a (chl. a) and carotenoid were measured spectrophotometrically at 665, 645, and 470 nm (Inskeep and Bloom 1985) after 5 mL of algal culture was extracted with 90 % ethanol overnight at 4 °C followed by centrifugation at 4 °C for 10 min at 8,000g. The pigment contents were calculated using the following equations: Chlorophyll aðmg=LÞ ¼ 12:7  A665 −2:69  A645

 Carotenoid ðmg=LÞ ¼ 1; 000  A470 −2:05  C Chlorophyll a =245

Intracellular ROS were detected using a fluorescent probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), based on Choudhury et al. (2013), with slight modifications. The final DCFH-DA concentration in the mixture was 10 μM and this was incubated with the suspended cells at 37 °C in the dark for 30 min and mixed every 5 min during this time. Then, the cells were immediately washed three times with sterile f/2 medium (without silicate) and finally suspended with 500 μL f/2 medium. The fluorescence intensity was monitored using a spectrofluorometer with excitation wavelength at 488 nm and emission wavelength at 525 nm. Lipid peroxidation was measured based on the detection of malondialdehyde, MDA (a by-product of lipid peroxidation). The algal cells in the control and LY01 supernatant treated groups were harvested using centrifugation at 4 °C and 3,000g for 5 min, then washed with PBS (50 mM, pH 7.4). The cells were resuspended in 2 mL PBS and homogenized with an ultrasonic cell pulverizer (NingBo Scientiz Biotechnological Co., Ltd, China) 50 times at 80 W (ultrasonic time: 2 s; rest time: 3 s) at below 4 °C. Then, the homogenate was centrifuged at 10,000g for 10 min at 4 °C. The supernatant was used to analyze the content of MDA. The reaction product of MDA and thiobarbituric acid at high temperature could be measured at 532 nm (Nanjing Jiancheng Bioengineering Institute, China). The analysis method followed the kit’s Operation Manual from Nanjing Jiancheng Bioengineering Institute, China (Zhang et al. 2013). Cellular inclusions assays and antioxidants activities assays of A. tamarense

where A665, A645, and A470 represent absorbance values at wavelengths of 665, 645, and 470 nm. CChlorophyll a represents the content of chl. a. The pulse-amplitude modulation fluorescence measurements were performed using a PAM-CONTROL Fluorometer (Walz, Effeltrich, Germany). Two fluorescence measurements were made: one before (Fo=minimum fluorescence) and one during (Fm=maximal fluorescence) the pulse. The maximum quantum yield (the photosystem II efficiency) was calculated from Fv/Fm (where Fv=Fm−Fo), when the algal cells were dark-adapted for 15 min before measurements under a strong red LED (>3,000 μmol m−2 s−1) (Nymark et al. 2009). The relative electron transport rate (rETR; micromole electrons per square meter per second) were measured followed (Kaplan et al. 2012), eight consecutive light levels of 156, 226, 337, 533, 781, 1,077, 1,593, and 2,130 μmol photons m−2 s−1 were

After the algal cells were treated with LY01 supernatant for 0, 6, 12, 24, 48 and 60 h, they were harvested using centrifugation at 4 °C and 3,000g for 5 min, then washed with PBS and centrifuged twice more. The supernatant, which was prepared in the same manner as that of the MDA extracts, was used to measure the protein and carbohydrate contents and to analyze the enzymatic activities of SOD, CAT, POD, and the nonenzymatic antioxidants activities of GSH and AsA. One milliliter of cellular protein was detected using the “Coomassie brilliant blue protein analysis kit” (Nanjing Jiancheng Bioengineering Institute) with bovine serum albumin as the standard; the rest were stored at −80 °C until they were used to measure the enzymatic and non-enzymatic antioxidant activities. All the analysis methods followed the kit’s Operation Manual from Nanjing Jiancheng Bioengineering Institute,

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China. The content of carbohydrate was determined following the phenol–sulfuric acid colorimetric method, using glucose as the standard (Masuko et al. 2005). RNA extraction, reverse transcription, and real-time analysis Forty milliliters of algal culture was centrifuged at 3,000g for 5 min at 4 °C after treated with LY01 supernatant for 6 and 24 h. RNA was extracted as soon as possible using the RNAiso kit (TaKaRa Company, Dalian, China) following the manufacturer’s instructions. For reverse transcription, 1 μg of total RNA was mixed with RT Primer Mix and reverse transcriptase following the instructions of the PrimeScript RT reagent kit (TaKaRa Company). Real-time PCR was carried out using an “SYBR Premix EX TaqTM II” (TaKaRa Company). The primer pairs for three target photosynthesis-related genes (psbA, psbD, and rbcL) and two target respirationrelated genes (cob and cox) are listed in Table 1. The PCR program was: one denaturation step at 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 30 s, then increasing from 60 °C to 95 °C by 0.5 °C each 5 s. 18S rRNA was used as a housekeeping gene to normalize the expression changes. The relative gene expression among the treatment groups was quantified using the 2 −ΔΔCt method (Livak and Schmittgen 2001).

Results Algicidal activity and mode of algicidal action Algicidal activity was studied by adding different concentrations (0.5, 1.0 and 2.0 %) of bacterial culture into the algal cultures. After treatment for 12 h with the 1.0 and 2.0 % LY01 cultures, the algicidal rate reached 58.7 % (Fig. 1a) and Table 1 Sequences of primer pairs used with A. tamarense for real-time PCR Primer

Sequence (5'–3')

18S rRNA

F: 5′-TGCATGGCCGTTCTAGTTGGTGG-3′ R: 5′-CACCTACGGAAACCTTGTTACGAC-3′ F: 5′-CGAGATCAGCCTAGCTATTT-3′ R: 5′-TTGGTGGCGTACTGTCATTG-3′ F: 5′-ATCAACACCAGCAAACAG-3′ R: 5′-GGACAGCCATACCAAAGAC-3′ F: 5′-CCAAGCTTCTCCTTCCAKCCHGGRTADAT-3′ R: 5′-CTGTTCGACCGCAACMTCACCGATGG-3′ F: 5′-AGCATTTATGGGTTATGTNTTACCTTT-3′ R: 5′-AGCTTCTANDGMATTATCTGGATG-3′ F: 5′-GAGGTGGAACAGGATGGA-3′ R: 5′-GTGTAACAATGGCGGATT-3′

psbA psbD rbcL cob cox

Fig. 1 Algicidal activity of bacterial strain LY01 on A. tamarense cultures: a bacterial culture added; (b) supernatant added; (c) washed bacterial cells added; all error bars indicate the SE of the three biological replicates

A. tamarense cells appeared to be plasmolysed. The cell wall and membrane began to separate, and then one side of the cell wall started to break (Fig. 2b, c). With increased exposure time, the cell wall lost its integrity, the cells were disrupted, the cellular membrane broken, and part of the cellular substances were decomposed and released (Fig. 2d, e). After 72 h of treatment, the algicidal rate was up to 93.9 % (Fig. 1a), and the A. tamarense cells were lysed completely (Fig. 2f). However, almost no algicidal effect was observed in the treatment with the 0.5 % concentration of bacterial culture. The

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Fig. 2 Lysis process of A. tamarense by strain LY01. a control cells presenting normal morphology; b–f cells exposed to bacterial supernatant for 6, 12, 24, 48, and 72 h. Scale bar 10 μm

concentration of the bacterial culture thus had a great impact on the algicidal effect. There was a different algicidal effect between adding the supernatant or washed bacterial cells (Fig. 1b, c). When we treated the algal culture using different concentrations of supernatant, the algicidal activity was also obvious, and the result did not show any clear difference from adding various concentrations of bacterial culture to the algal cultures. The algicidal rate reached 48.7 % when treated for 12 h with supernatant concentrations of 1.0 and 2.0 % and, after 60 h, the algicidal rate reached 86.4 %, and most algal cells were lysed. The 0.5 % concentration of supernatant did not show any effect on the growth of A. tamarense. At the same time, the addition of washed bacterial cells did not show any obvious effect on the growth of A. tamarense regardless of the bacterial concentration. Throughout the results, the algicidal mode of LY01 was indirect attack, and it lysed algal cells by releasing algicidal substances without making contact with the cell surfaces. Effects of LY01 supernatant on cellular pigments and chl. a fluorescence measurements In the process of algal growth, the cellular pigments (chl. a and carotenoid) contents in the algal cells were significantly decreased under the function of supernatant at concentrations of 1.0 and 2.0 % (Fig. 3a), compared with the control and a concentration of 0.5 %. After 6 h exposure to the LY01 supernatant, the chl. a content was significantly (p

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