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Research article

J. Microbiol. Biotechnol. 2019; 29(1): 59-65

Published online January 28, 2019 https://doi.org/10.4014/jmb.1804.04031

Copyright © The Korean Society for Microbiology and Biotechnology.

The Selective Inhibitory Activity of a Fusaricidin Derivative on a Bloom-Forming Cyanobacterium, Microcystis sp.

So-Ra Ko 1, Young-Ki Lee 2, Ankita Srivastava 1, Seung-Hwan Park 3, Chi-Yong Ahn 1 and Hee-Mock Oh 1*

1Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology, 2IPst Company, 3Infections Disease Research Center, Korea Research Institute of Bioscience and Biotechnology

Correspondence to:

Hee-Mock  Oh
heemock@kribb.re.kr

Received: April 18, 2018; Accepted: October 25, 2018

Abstract

Fusaricidin analogs, produced by Paenibacillus polymyxa, were tested for selective control of a major bloom-forming cyanobacterium, Microcystis sp. Fusaricidin (A and B mixtures) and four analogs were isolated from P. polymyxa E681 and investigated for their inhibition of cyanobacterial cell growth. Among the four fusaricidin analogs, fraction 915 Da (designated as Fus901) showed growth inhibition activity for Microcystis aeruginosa but not for Anabaena variabilis and Scenedesmus acutus. Microcystin concentration decreased up to 70% and its content per cell also decreased over 50% after 3 days. Fusaricidin exhibited growth inhibition against Gram-positive bacteria but Fus901 did not. Molecular weights of fusaricidin A and B were 883 Da and 897 Da, whereas that of Fus901 was 915 Da. Structure analysis by a ringopening method revealed a linear form for Fus901. Expression of the pod gene related to oxidative stress was increased 2.1-fold by Fus901 and that of mcyD decreased up to 40%. These results indicate that Fus901 exerts oxidative stress against M. aeruginosa. Thus, Fus901 can be used as a selective cyanobactericide without disturbing the ecological system and could help in decreasing the microcystin concentration.

Keywords: Bloom control, cyanobacteria, fusaricidin, Microcystis, Paenibacillus polymyxa

Introduction

Freshwater blooms are generally formed by the uncontrolled growth of cyanobacteria, which can produce toxins such as microcystin (MC), nodularin, and anatoxin. MC is encoded by the mcy gene cluster [1]. The mcyABCDEFGHIJ genes are transcribed bidirectionally from a central promoter between mcyA and mcyD. Three peptide synthetases are encoded by mcyABC, mcyD encodes a modular polyketide synthase, mcyE and mcyG encode peptide synthetase and polyketide synthase, respectively, and mcyJ, F and I are putatively involved in tailoring, while mcyH is involved in toxin transport [2,3]. Polymerase chain reaction (PCR) amplification of the mcyA, -B and -C genes has been applied for the detection of potentially toxic Microcystis [4]. Two polyketide synthase modules of mcyD with another two polyketide synthases (mcyE and mcyG) are responsible for the synthesis of the unique Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid) that is used to detect MCs by the protein phosphatase inhibition (PPI) assay [3].

The toxicity of MCs, along with their environmental and economic impact, have triggered the development of methods for the treatment of these toxins [5]. Many chemical and physical methods, such as adsorption, chlorination, oxidation, and ozonation, have been applied to degrade stable MCs. However, until now, only a few biological methods have been reported.

Paenibacillus polymyxa is commonly found in many mineral deposits and the rhizosphere [6]. P. polymyxa strains produce two types of peptide antibiotics. One anti- bacterial group includes polymyxins and polypeptins. The other, which includes fusaricidin A, B, C, and D, is active against fungi and Gram-positive bacteria [7]. Fusaricidins have a ring structure composed of six amino acid residues in addition to 15-guanidino-3-hydroxypentadecanoic acid (GHPD). The general peptide sequence of the fusaricidins was determined to be L-Thr–X1–X2–D-allo-Thr–X3–D-Ala (Fig. 1A). A β-hydroxy fatty acid is attached to the N- terminal L-Thr via an amide linkage and the peptide is cyclized by an ester bond between the C-terminal D-Ala and the β-OH group of the N-terminal L-Thr. The antimicrobial activity of the fusaricidins varies depending on the amino acids at three variable positions (Fig. 1B) [8].

Figure 1. (A) Primary structure of the fusaricidin-type lipopeptide antibiotics. X1, X2, and X3 indicate three variable positions in fusaricidins. (B) Amino acid substitutions at three variable positions in previously reported fusaricidins and the molecular weights of the fusaricidins.

In this study, we report that one of the fusaricidin analogs has growth inhibition and can decrease intracellular MC content as well. The analog displayed no growth inhibition activity against Gram-positive bacteria. Simultaneously, structural analysis was also performed.

Materials and Methods

Strains and Culture Conditions

Anabaena variabilis NIES 23 and Scenedesmus acutus NIES 94 were obtained from the National Institute for Environmental Studies (NIES), Japan. M. aeruginosa KW was isolated from freshwater in Korea. The strains were grown in BG-11 liquid medium (pH 7.5) under 120 µmol Photons/m2/s1 provided by cool white fluorescent tubes at 27 ± 1°C. Micrococcus luteus (KCTC 1056), Bacillus subtilis (KCTC 1022), Staphylococcus aureus (KCTC 1621), Staphylococcus aureus (KCTC 1916), and Escherichia coli (KCTC 2443) were obtained from Korean Collection for Type Cultures (KCTC), and Erwinia carotovora was provided by Dr. Seung-Hwan Park (KRIBB, Korea). P. polymyxa E681, which was isolated from the roots of winter barley in the Republic of Korea, was cultured in Katznelson and Lochhead medium (KL medium) [9].

Purification of Fusaricidins and Its Analogs

P. polymyxa E681 was grown in 5 L of KL medium at 27°C until they reached the end of the stationary phase (24 h). The liquid culture medium was then centrifuged (12,000 ×g, 30 min), filtered through 0.2-µm filters and heated at 110°C for 10 min. The clarified culture medium was then applied to a CM-Trisacryl column (2 by 10 cm) equilibrated in 25 mM Tris-HCl (pH 8.5). A NaCl gradient of 100 ml (0 to 0.5 M) was used in the same buffer to elute the active components at a flow rate of 1 ml/min. A fraction (1 ml) was collected and tested for antagonistic activities. The active fractions were pooled, diluted three times with Milli-Q water, and applied to another CM-Trisacryl column (2 by 5 cm). Purified materials were eluted with 30 ml of 0.1 M NH4OH at pH 11 and were concentrated and further purified with a C18 Sep-Pack cartridge (Waters, USA) by following the manufacturer’s protocol. Active compounds were eluted from the cartridge in 100% methanol. The methanol was evaporated under reduced pressure and the antagonistic factor was obtained as a white powder that was soluble in methanol.

Mixtures of fusaricidin A and B (denoted as fusaricidin) and its four analogs were isolated from the methanol extract of P. polymyxa E681 by high-pressure liquid chromatography (HPLC). The methanol extract of the cell pellet was analyzed by LC/MS (Shimadzu, Japan) using a mixed solvent of water and acetonitrile containing 0.1% formic acid at a rate of 0.2 ml/min. To make the fusaricidin linearized, it was hydrolyzed by MeOH-H2O-28% aqueous NH3 (4:1:1, pH 9.0) or esterase for 24 h according to the method of Kuroda et al. [8].

Growth Inhibition Activity Assays

The growth inhibition activity of purified fusaricidin and its analogs was measured against Gram-positive and Gram-negative bacteria using the disk diffusion method. LB plates were spread with Gram-positive bacteria (M. luteus KCTC 1056, B. subtilis KCTC 1022, S. aureus KCTC 1621 and S. aureus KCTC 1916) and Gram-negative bacteria (E. coli KCTC 2443 and E. carotovora). Also, for polymyxin E activity, P. polymyxa E681 was cultured in KL medium at 30°C for 24 h. After centrifugation of the culture, the cell pellet was extracted with methanol. The growth inhibition of the mixture of cell extract and supernatant were measured using the disk diffusion method.

Chlorophyll-a and Cell Count

Isolated M. aeruginosa KW was cultured in BG-11 liquid medium (pH 7.5) under 120 µmol Photons/m2/s1 provided by cool white fluorescent tubes at 27 ± 1°C. The cell numbers of M. aeruginosa were counted using an optical microscope. Before counting, the cell suspensions of M. aeruginosa were briefly sonicated to disperse any aggregated cell clusters. To determine the chlorophyll-a fraction, samples were filtered using filter paper (Whatman GF/C), then extracted using chloroform-methanol (2:1 (v/v)) [10]. The concentration of chlorophyll-a was then measured by a Turner Quantech fluorometer (Barnstead/Thermolyne, USA) based on a predetermined standard curve programmed into the fluorometer. A blank sample of chloroform was measured and all the readings were readjusted with the blank sample reading.

MC Analysis

To determine the microcystin concentration by PPI assay, 5-ml sample aliquots were filtered through a Whatman GF/C filter. The microcystin in the filter papers was extracted with 5% acetic acid, purified with a Sep-Pak cartridge, and then diluted with 100% methanol. The PPI assay was performed as described by Ward et al. [11]. Briefly, the diluted protein phosphatase 1 (10 µl) was added to 25 µl of sample. After pre-incubation for 1 min, p-nitrophenol (100 µl) was added and measured after 22 min at 405 nm on a microplate reader (Sunrise, Tecan, USA). All enzyme assays were conducted in triplicate. Significant differences (p < 0.05) between the results were determined using a t-test.

RNA Extraction and Real-Time PCR

To study whether Fus901 could affect the expression of toxin-producing gene and antioxidant defense-related genes such as the peroxidase (pod) and superoxide dismutase (sodB) genes, qRT-PCR was carried out to determine the mcyD, pod, and sodB expression levels in Fus901-treated cells (BG11 media, 48 h incubation, final concentration 1 µg/ml) relative to the control cells and normalized with the expression of the reference gene, 16S rRNA, as described by Chini et al. [12].

Total RNA was isolated with the RiboPure-Bacteria Kit (Ambion, USA) according to the manufacturer’s instructions and treated with DNase I at 30°C for 30 min. First-strand cDNA synthesis, using 0.5 µg of RNA as a template, was carried out in a total volume of 20 µl using the iScript cDNA Synthesis Kit (Bio-Rad, USA) according to the manufacturer’s instructions. Any residual DNA was eliminated by incubating RNA preparations for 5 min at 42°C with the gDNA Wipeout Buffer (Qiagen, Germany) and the cDNA synthesis reaction was carried out at 42°C for 15 min in duplicate with or without iScript reverse transcriptase, followed by 3 min incubation at 95°C to inactivate the enzyme. PCR primers for the three genes are listed in Table 1. Real-time PCR was performed in 20 µl of a reaction mixture containing 10 µl of iTaq SYBR Green Supermix with ROX (Bio-Rad, USA), 1 µl (10 pmol/µl) each of forward and reverse primers, 1 µl cDNA and 7 µl distilled water. The amplification reactions were performed by a Chromo4 Four-Color Real-Time PCR Detector (Bio-Rad, USA) under the following conditions: one cycle at 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec, 61°C for 1 min, and 72°C for 20 sec [13].

Table 1 . Primers designed for real-time PCR..

Gene nameForward primer (5’-3’)Reverse primer (5’-3’)
podGCCGTTTTCGATCAAGAGTTGGATGGGATTGGACGTATTG
sodBACACACTTCCCCCTTTACCCGCCGGTTTTGGTAACTTTGA
mcyDaGGTTCGCCTGGTCAAAGTAACCTCGCTAAAGAAGGGTTGA
16S rRNAGGACGGGTGAGTAACGCGTAbCCCATTGCGGAAAATTCCCCc

aKaebernick et al. (2000) [3].

bUrbach et al. (1992) [27].

cNübel et al. (1997) [28].



A threshold cycle (CT) value was determined for each amplification plot. CT values were standardized to 16S rRNA values. The quantitative PCR results were represented as the fold-change in target gene expression. The expression ratio was calculated based on the formula of 2-ΔΔCT [14].

Results

Growth Inhibition of Fusaricidin Analogs

To investigate the growth inhibition, the four analogs and fusaricidin were added to M. aeruginosa KW culture (initial cell density 2.37 ± 0.15 ×107 cells/ml, n =3) at 1μg/ml and incubated at 27°C for 3 days. Fraction 915 among the four analogs showed potent growth inhibition against M. aeruginosa (data not shown). To investigate the specificity of fraction 915, another cyanobacterium, A. variabilis NIES 23 and a green alga, S. acutus NIES 94, were also treated. After 3 days, the growth of A. variabilis NIES 23 and S. acutus NIES 94 was not inhibited, but chlorophyll-a concentration of M. aeruginosa KW decreased from 49.6 μg/l to 18.6 μg/l (Fig. 2). Fraction 915 showing growth inhibition was desig-nated as Fus901. Moreover, the Chlorophyll-a concentration of M. aeruginosa KW was also decreased when Fus901 was added in the middle of the growth phase (Fig. S3).

Figure 2. The chlorophyll-a concentration of A. variabilis NIES 23, S. acutus NIES 94, and M. aeruginosa KW when Fus901 was added. Controls are A. variabilis NIES 23, S. acutus NIES 94, and M. aeruginosa KW without Fus901 treatment.

Decrease in MC Content by Fus901

After 3 days of cultivation, the intracellular MC concentration in Fus901-treated M. aeruginosa KW decreased up to 70%, while extracellular concentration showed no difference, when compared to control (Fig. 3A). These results indicated that Fus901 decreased MC content within M. aeruginosa cells. To confirm this, MC content per cell was determined after treatment with Fus901. The initial cell concentration of M. aeruginosa KW was 2.37 ± 0.15 ×107 cells/ml and the cell concentrations of control and Fus901-treated samples after 72 h were 5.32 × 107 and 2.77 × 107 cells/ml, respectively. The MC content was 41.6 fg/cell in control, whereas that of Fus901-treated cells was 24.0 fg/cell (Fig. 3B).

Figure 3. Effect of Fus901 on MC production by M. aeruginosaKW. (A) Comparison of total MC concentration between control and treatment. (B) Comparison of MC content per cell. Data are means ± SD (n = 3). *p < 0.05 indicate significant differences compared with the corresponding controls.

Growth Inhibition Activity of Fus901

Growth inhibition activity was tested against four Gram-positive bacteria (M. luteus, M. luteus, and Staphylococcus sp. KCTC 1621 and 1916) and two Gram-negative bacteria (E. coli and E. carotovora). Various concentrations (0.625, 1.25, 2.5, 5.0, and 10 mg/ml) of Fus901 were tested to check its growth-inhibiting activity on the M. luteus (Fig. S4). Fusaricidin and Fus901 were tested at 1 mg/ml concentration and crude extract (20 mg/ml) of polymyxin E produced by P. polymyxa E681 was also used. Polymyxin E is known to have strong growth inhibition activity against Gram-negative bacteria [15]. Whereas fusaricidin showed strong growth inhibition activity on the tested Gram-positive bacteria, Fus901 completely lost its growth inhibition activity against the Gram-positive bacteria (Fig. 4A). Similar to fusaricidin, Fus901 showed no growth inhibition activity on E. coli or E. carotovora (Fig. 4B).

Figure 4. Growth inhibition activity of Fus901 compared to the activity of intact fusaricidin. (A) Growth inhibition activity against Gram-positive bacteria, (B) Gram-negative bacteria. a, fusaricidin 1 mg/ml; b, Fus901 1 mg/ml; (A)-c and (B)-d, methanol; (B)-c, crude extract (20 mg/ml) of polymyxin E produced by P. polymyxa E681.

Structure Analysis of Fus901

Fus901 and fusaricidin linearized by a ring-opening method were analyzed by LC/MS/MS. The ring-opening method was carried out by addition of NaOH and esterase. The methanol extract of the Fus901 was analyzed by LC/MS/MS. The (M+H)+ ion peak of Fus901 was 915 at a retention time of 8.23 min (Fig. S1A), while those of fusaricidin were 883 and 897 at retention times of 10.04 min and 10.42 min, respectively (Figs. S1B and S1C). Like Fus901, the (M+H)+ ion peak of ring-opened fusaricidin by treatment of esterase and NaOH was 915 with retention times of 10.20 min and 10.70 min (Figs. S1B and S1C). From the LC/MS/MS results, Fus901 was identified as a linearized form with a broken ester bond (Fig. S2).

Effect of Fus901 on mcyD Transcription and Antioxidant Defenses

A modular polyketide synthase is encoded by mcyD, involved in the synthesis of the β-amino acid Adda that is responsible for the toxicity of the microcystins [2]. Moreover, McyD is essential in microcystin synthesis and the lack of this protein results in the absence of microcystin synthesis [3]. Thus, mcyD was considered for expression studies.

Relative transcriptional change by Fus901 based on the housekeeping gene, 16S rRNA, is shown in Fig. 5. The level of pod transcript increased 2.1-fold when M. aeruginosa KW was grown under Fus901. Meanwhile, sodB transcript level hardly changed. The level of mcyD transcript slightly decreased (up to 40%) and this result corresponded with the decrease of MC content.

Figure 5. Level of mcyD, pod, and sodB mRNA expression in M. aeruginosa KW as a response to Fus901 by real-time PCR. The error bar is the mean ± SD (*p < 0.05).

Discussion

Several control techniques for algal bloom have been developed such as the use of yellow loess [16] and clay [17]. Even though effective, yellow loess and clay can cause secondary effects on other aquatic organisms. The application of chemical cyanobactericidal agents such as copper sulfate [18] and hydrogen peroxide [19] are also effective in controlling cyanobacterial blooms within a short time, but these chemicals can inhibit the entire phytoplankton community and cause water quality deterioration [20]. Recent studies have focused on the identification of bacteria capable of inhibiting or degrading algal blooms in marine and freshwater environments [21]. Application of biological agents, such as bacteria [22], viruses [23] and planktonic ciliates [24], in aquatic systems, faces difficulty posed by the demands of high bacterial cell density. Five strains (HYY0510-SK04, HYY0511-SK09, HYK0512-SK12, HYK0512-PK04 and HYY0512-PK05) isolated by Kang et al. [22] degraded Stephanodiscus hantzschii cells when those bacteria were inoculated at a concentration of ≥107 cells/ml. Moreover, these bacteria showed growth inhibition activity against several algae and cyanobacteria. Mayali and Doucette [24] isolated Cytophaga strain 41-DBG2 from Gulf of Mexico waters which showed algicidal activity against Karenia brevis (Dinophyceae), when added at ≥ 106 cells/ml.

Several aquatic plants produce metabolites inhibiting the growth of algae and cyanobacteria. These substances are known as allelochemicals. Many allelochemicals have been isolated and identified. Nakai et al. [25] reported that macrophyte Myriophyllum spicatumreleased allelopathic polyphenols, which inhibited the growth of M. aeruginosa at 1.26 mg/l. However, none of these methods can specifically control algal blooms without causing problems to other organisms in aquatic environments.

Tandem mass spectrophotometry (MS/MS) has been used for elucidating the primary structures of peptides or proteins. In this study, to investigate the structural differences in the four analogs derived from fusaricidin, we purified them by HPLC. The (M+H)+ ion peaks of fusaricidin A, B, C, and D were 883, 897, 947, and 961, respectively. But, those of the four analogs were 915, 929, 943, and 957, respectively (data not shown). Because fusaricidin A and B comprise a large proportion of the fusaricidin produced by P. polymyxa E681, we made a ring-opened fusaricidin (A and B mixture) by NaOH treatment and compared those structures with Fus901 by MS/MS analysis. The (M+H)+ ion peak of ring-opened fusaricidin was 915 and this molecular weight of m/z 915 corresponded with that of Fus901. Ring-opened fusaricidin was probably made by the ring-opening process between the β-carbon atom and the oxygen atom at the β-position of threonine (Fig. S2A asterisk).

Fusaricidin B showed strong growth inhibition activity against a wide variety of fungi and also had low minimum inhibitory concentration values against Gram-positive bacteria (S. aureus and M. luteus) with 1.56 μg/ml. On the other hand, it showed relatively low sensitivity against Gram-negative bacteria (E. coli and P. aeruginosa) [26]. However, Fus901 did not show growth inhibition activity against Gram-positive bacteria or Gram-negative bacteria at 1 μg/ml. This indicated that Fus901 is an eco-friendly cyanobactericide that does not disturb other aquatic microorganisms.

A few studies have focused specifically on the MC biosynthesis genes and have shown the effects of single or combined factors on MC synthesis [3]. The current study selected mcyD as representative of decreased MC production and used real-time PCR to analyze the transcript levels of this gene under Fus901 treatment. Expression of mcyD transcript level was slightly decreased (up to 40%) by Fus901. A decrease in mcyD transcript level resulted in the reduction of MC synthesis. In photosynthetic organisms, environmental stress can create oxidative stress through overproduction of reactive oxygen species, which induce the expression of the superoxide dismutase (SOD) and peroxidase (POD) genes. Under oxidative stress, plants and microalgae respond by increasing antioxidant defenses such as SOD and POD enzymes. Fus901 increased pod gene transcription in M. aeruginosa, whereas sodB transcript level did not change. These results indicated that Fus901 creates oxidative stress in M. aeruginosa and consequently inhibits the growth of M. aeruginosa while also reducing the MC production.

Four fusaricidin analogs were isolated and purified from P. polymyxa E681. One analog, designated Fus901, exhibited highly species-specific growth inhibition on M. aeruginosa. The growth of M. aeruginosa was inhibited and the MC concentration also decreased up to 70% when M. aeruginosa was grown in 1 μg/ml of Fus901. MC content also decreased from 41.6 fg/cell to 24.0 fg/cell. Real-time PCR analyses of the mcyD gene under Fus901 revealed that the level of mcyD transcript slightly decreased (0.4-fold) as compared to the control. The level of pod transcript increased in response to Fus901. The data indicated that Fus901 induced oxidative stress in M. aeruginosa. Fus901, unlike fusaricidin, showed no growth inhibition against Gram-positive bacteria. Therefore, Fus901 appears to be a promising cyanobactericide that does not disturb the aquatic ecosystem.

Supplemental Materials

Acknowledgments

This research was supported by the Basic Core Technology Development Program for the Oceans and the Polar Regions of the National Research Foundation (NRF) funded by the Ministry of Science and ICT (2016M1A5A1027453) and KRIBB Research Initiative Program.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.(A) Primary structure of the fusaricidin-type lipopeptide antibiotics. X1, X2, and X3 indicate three variable positions in fusaricidins. (B) Amino acid substitutions at three variable positions in previously reported fusaricidins and the molecular weights of the fusaricidins.
Journal of Microbiology and Biotechnology 2019; 29: 59-65https://doi.org/10.4014/jmb.1804.04031

Fig 2.

Figure 2.The chlorophyll-a concentration of A. variabilis NIES 23, S. acutus NIES 94, and M. aeruginosa KW when Fus901 was added. Controls are A. variabilis NIES 23, S. acutus NIES 94, and M. aeruginosa KW without Fus901 treatment.
Journal of Microbiology and Biotechnology 2019; 29: 59-65https://doi.org/10.4014/jmb.1804.04031

Fig 3.

Figure 3.Effect of Fus901 on MC production by M. aeruginosaKW. (A) Comparison of total MC concentration between control and treatment. (B) Comparison of MC content per cell. Data are means ± SD (n = 3). *p < 0.05 indicate significant differences compared with the corresponding controls.
Journal of Microbiology and Biotechnology 2019; 29: 59-65https://doi.org/10.4014/jmb.1804.04031

Fig 4.

Figure 4.Growth inhibition activity of Fus901 compared to the activity of intact fusaricidin. (A) Growth inhibition activity against Gram-positive bacteria, (B) Gram-negative bacteria. a, fusaricidin 1 mg/ml; b, Fus901 1 mg/ml; (A)-c and (B)-d, methanol; (B)-c, crude extract (20 mg/ml) of polymyxin E produced by P. polymyxa E681.
Journal of Microbiology and Biotechnology 2019; 29: 59-65https://doi.org/10.4014/jmb.1804.04031

Fig 5.

Figure 5.Level of mcyD, pod, and sodB mRNA expression in M. aeruginosa KW as a response to Fus901 by real-time PCR. The error bar is the mean ± SD (*p < 0.05).
Journal of Microbiology and Biotechnology 2019; 29: 59-65https://doi.org/10.4014/jmb.1804.04031

Table 1 . Primers designed for real-time PCR..

Gene nameForward primer (5’-3’)Reverse primer (5’-3’)
podGCCGTTTTCGATCAAGAGTTGGATGGGATTGGACGTATTG
sodBACACACTTCCCCCTTTACCCGCCGGTTTTGGTAACTTTGA
mcyDaGGTTCGCCTGGTCAAAGTAACCTCGCTAAAGAAGGGTTGA
16S rRNAGGACGGGTGAGTAACGCGTAbCCCATTGCGGAAAATTCCCCc

aKaebernick et al. (2000) [3].

bUrbach et al. (1992) [27].

cNübel et al. (1997) [28].


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