Arch Microbiol (2002) 178 : 421–427 DOI 10.1007/s00203-002-0464-9
O R I G I N A L PA P E R
Hui Pan · Lirong Song · Yongding Liu · Thomas Börner
Detection of hepatotoxic Microcystis strains by PCR with intact cells from both culture and environmental samples
Received: 21 September 2001 / Revised: 25 June 2002 / Accepted: 1 July 2002 / Published online: 27 August 2002 © Springer-Verlag 2002
Abstract Microcystins are small hepatotoxic peptides produced by a number of cyanobacteria. They are synthesized non-ribosomally by multifunctional enzyme complex synthetases encoded by the mcy genes. Primers deduced from mcy genes were designed to discriminate between toxic microcystin-producing strains and non-toxic strains. Thus, PCR-mediated detection of mcy genes could be a simple and efficient means to identify potentially harmful genotypes among cyanobacterial populations in bodies of water. We surveyed the distribution of the mcyB gene in different Microcystis strains isolated from Chinese bodies of water and confirmed that PCR can be reliably used to identify toxic strains. By omitting any DNA purification steps, the modified PCR protocol can greatly simplify the process. Cyanobacterial cells enriched from cultures, field samples, or even sediment samples could be used in the PCR assay. This method proved sensitive enough to detect mcyB genes in samples with less than 2,000 Microcystis cells per ml. Its accuracy, specificity and applicability were confirmed by sequencing selected DNA amplicons, as well as by HPLC, ELISA and mouse bioassay as controls for toxin production of every strain used. Keywords Cyanobacteria · Microcystis · Microcystin · Toxin · Peptide synthetase · Whole-cell PCR
H. Pan · L. Song (✉) · Y. Liu Department of Phycology, Institute of Hydrobiology, The Chinese Academy of Sciences, Luojiashan, Wuhan 430072, P. R. China e-mail:
[email protected], Tel.: +86-27-87647715, Fax: +86-27-87647715 T. Börner Institute for Biology, Humboldt University, Chausseestrasse 117, 10115 Berlin, Germany
Introduction In eutrophic water habitats, the proliferation of cyanobacteria results in the formation of water blooms, a worldwide phenomenon that causes the poisoning of animals. Most bloom-forming cyanobacteria are known to synthesize a variety of toxins. Among the cyanotoxins is a family of cyclic heptapeptides called microcystins which are potent hepatotoxins and liver tumor promoters (Carmichael 1996; Sivonen, 1996). The most common producers of microcystins are various Microcystis species (Carmichael 1996; Sivonen, 1996). These species often form a heavy bloom on water surface of lakes or reservoirs. They are also abundant in the sediment of lakes and reservoirs throughout the winter. These overwintering colonies may provide the inoculum for the next bloom (Noriko et al. 1984; Preston and Stewart 1980). Bodies of water that are used for recreational purposes and/or as the source of drinking water should thus be regularly controlled for the presence of toxic cyanobacteria. The majority of Microcystis aeruginosa isolates are toxic and synthesize microcystins. Only toxic isolates contain the genes for the enzymes involved in microcystin biosynthesis (Dittmann et al. 1999; Meissner et al. 1996; Neilan et al. 1999). Microcystins are synthesized nonribosomally by the large modular multifunctional enzyme complexes known as peptide synthetases encoded by the mcy (microcystin synthetase) gene cluster (Dittmann et al. 1999; Tillett et al. 2000; Nishizawa et al. 1999, 2000). Amplification of mcy genes by PCR from DNA isolated from axenic cultures and field samples has proven to be a sensitive means to differentiate between microcystin-producing hepatotoxic and non-hepatotoxic Microcystis strains (Dittmann et al. 1999; Meissner et al. 1996; Neilan et al. 1999). Two pairs of oligonucleotide primers, TOX1P/ TOX1 M and TOX2P/TOX2 M, were designed to detect and characterize microcystin-producing and non-toxic cyanobacteria species by specifically amplifying fragments of the mcyB gene (Dittmann et al. 1999). These PCR-based gene detection procedures established a corre-
422
lation between the existence of mcyB gene and the production of microcystins. Cyanobacteria are gram-negative bacteria with a thick peptidoglycan layer that makes them resistant to routine cell disruption methods. Thus, the available cell disruption protocols may decrease the efficiency of cyanobacterial DNA extraction and inhibit PCR (Howitt 1996; Sidney et al. 1988). Here we describe a method using intact cyanobacteria cells for PCR amplification in order to identify toxic and non-toxic strains of Microcystis. This method is a simple, efficient, and reliable way to identify the toxic cells from axenic strains, isolates, and lyophilized samples as well as from water and sediment samples.
90% methanol. After drying, the eluates were dissolved in 20% methanol, and the fractions were subjected to reversed-phase HPLC column (Shimadzu LC-10A) analysis. ELISA was conducted with monoclonal antibodies kindly provided by Y. Ueno (Yashio Institute of Environmental Sciences). To estimate the total microcystin content, samples were treated twice by freeze-thawing followed by filtration over glass filters (Whatman GF/C, 25 mm in diameter). Microcystin-LR-bovine serum albumin conjugate was coated on a microtiter plate, which was subsequently incubated with standard microcystin-LR or samples and anti-microcystin-LR monoclonal antibody. The amount of antibody bound on the surface of the wells was determined by the reaction of peroxidase-labeled goat anti-mouse IgG (TAGO 4550) with its substrate (0.1 mg 3,3,5,5,tetramethylbenzidine ml–1, 0.005% H2 O2 in 0.1 M acetate buffer, pH 5). Microcystin concentrations were determined from the standard curve of microcystin-LR (also see Ueno et al. 1996). DNA extraction
Materials and methods Cyanobacterial strains, isolates, cultivation and lyophilization Axenic cyanobacteria strains were obtained from the Culture Collections of the Freshwater Algae of the Institute of Hydrobiology (FACHB; Wuhan, China), the National Institute for Environmental Studies (NIES; Tsukuba, Japan), the Culture Collection of Algae at the University of Texas (UTEX; Austin, USA), and the Institute Pasteur (PCC; Paris, France). Cyanobacteria were cultured in liquid MA (Ichimura 1979) medium at 25±2 °C under continuous illumination of 25 µmol photons m–2 s–1. In addition, 16 Microcystis isolates including M. aeruginosa, M. viridis, and Microcystis sp. were sampled from Dianchi Lake (Kunming, China) in 2000 and cultured under conditions as for the axenic strains. The cells of the following axenic Microcystis strains were also investigated after lyophilization and storage at –20 °C: M. aeruginosa 8641, M. wesenbergii 574, M. aeruginosa PCC 7820, M. viridis (Dian-2), M. aeruginosa (Dian-1), and Oscillatoria raciborskii (Daye-1). Environmental samples Cyanobacterial cells from water blooms were collected from Taihu Lake, Wuxi, in December 1999; from East Lake, Wuhan, in September 2000; and from 20 sampling spots of Big Bay and Small Bay of Dianchi Lake, Kunming, monthly from March to December 2000. The top-layer (about 10 cm) sediment samples were taken from Dianchi Lake, Kunming, and from Guanqiao pool, Wuhan, in December 2000. These samples contained single cells and small colonies of Microcystis, mainly M. wesenbergii and M. aeruginosa, as observed with an inverted light microscope (OPTON IM35, Germany). All environmental samples were stored at 4 °C for no more than 1 week. Toxicity measurement and analysis of microcystins To detect microcystins, mouse bioassay, high-pressure liquid chromatography (HPLC), and an indirect competitive enzyme-linked immunosorbent assay (ELISA) were used according to published protocols. In the mouse bioassay, aqueous cell suspensions were injected intraperitonealy, the minimum lethal dose was measured, and hepatic hemorrhage was observed histologically (Song et al. 1999). In HPLC analyses, samples were extracted with 5% acetic acid while being emulsified by ultrasonication After centrifugation(1,250×g, 15 min), the pellets were extracted three times with 100% methanol and the methanolic extracts were dried in a vacuum evaporator. The residues were dissolved in 5% acetic acid and passed through conditioned (10 ml 100% methanol, 50 ml 100% distilled water) Sep-Pak C18 cartridges (Waters). The cartridges were then rinsed with 20% methanol and the samples eluted with
Total genomic DNA was prepared by a commonly used method for cyanobacteria (Sidney and Kaplan 1988). Pretreatments of samples for whole-cell PCR Cyanobacteria for the whole-cell PCR were collected from the environmental water samples by centrifugation. Before resuspension in distilled water to a defined volume, the cells were washed one to three times with distilled water. Sediment sample (about 100 g) was mixed with 500 ml distilled water in a beaker. Cells floating on the surface were collected and treated as described for water samples. The floating cells were also prepared by adding 40 ml sterilized MA medium slowly to a glass tube containing about 10 g sediment sample. Both water and sediment samples were also directly used in PCR without any pretreatment. PCR amplification PCR was performed in a GeneAmp2400 thermocycler (PerkinElmer Cetus, Emeryville, Calif., USA). The thermal cycling protocol included an initial denaturation at 94 °C for 2 min, followed by 35 cycles. Each cycle began with 10 s at 93 °C followed by 20 s at the annealing temperature at 50, 55 or 52 °C for primers MTF2/ MTR2, TOX1P/TOX1 M or TOX2P/TOX2M, respectively, and ended with 1 min at 72 °C (Neilan et al. 1999). When extracted DNA was used, the amplification reactions contained a 10×amplification buffer with 1.5 mm MgCl2, 0.2 mm dNTPs, 20 pmol of each primer and 1 U Taq DNA polymerase, and 3–5 ng purified DNA in a final volume of 50 µl (Dittmann et al. 1999). The PCR amplification with whole cells started with 6 µl of crude sample, pretreated subsample with an approximate cell density of 8× 106 cells/ml, or 0.1 µg lyophilized cyanobacterial cells. The sample was added directly to a 20-µl reaction solution containing bovine serum albumin (0.1 mg/ml) or skim milk (0.1 –100 mg/ml, w/v), and a 10×amplification buffer, which contained 1.5 mM MgCl2, 0.2 mM dNTPs, 20 pmol of each primer, and 0.5 U Taq DNA polymerase (Howitt 1996). The PCR amplifications conditions were identical to those for the samples described above. An extra ramp rate of 3 s/°C between the denaturing and annealing steps was set when a GeneAmp9600 cycler instead of GeneAmp2400 was used for PCR amplification. The dosage for the skim milk ranging from 1 to 100 mg/ml was determined to be appropriate based on the results of PCR. DNA sequencing DNA was sequenced automatically on a PE ABI 377 sequencer (Perkin-Elmer, Foster City, Calif.) using cyclic sequencing with a dye terminator. PCR fragments were sequenced using 300 pmol primers and automated protocols. The similarities between the
423 mcyB genes in GenBank and the PCR products were analyzed with NCBI sequence similarity search tool (BLAST 2.1) and with multiple-sequence cluster alignment software (DNATools 5.1, S.W. Rasmussen, Carlsbad Laboratory, Copenhagen).
Results and discussion Comparison of PCR using extracted DNA and whole cells from axenic strains The primer pair of MTF2/MTR2 was previously used to amplify nonspecifically fragments of all the genes encoding the peptide synthetases in cyanobacteria, allowing the Table 1 Comparison of PCR, HPLC, bioassay and ELISA in determination of toxicity of different cyanobacteria. + Toxic (for HPLC, bioassay and ELISA) or specific band amplified (for PCR); Strains
Microcystis aeruginosa FACHB (Wuda’erchi) Microcystis aeruginosa FACHB (Dianshanhu) Microcystis aeruginosa FACHB (Bao’anhu) Microcystis aeruginosa NIES-101 Microcystis aeruginosa NIES- 90 Microcystis aeruginosa FACHB8641 Microcystis sp. FACHB10 Microcystis aeruginosa FACHB86 Microcystis aeruginosa PCC 7806 Microcystis viridis FACHB (Dianchi-1) Microcystis aeruginosa UTEX2061 Microcystis sp. FACHB20 Microcystis wesenbergii FACHB574 Microcystis aeruginosa FACHB (Dianchi-1) Microcystis aeruginosa FACHB (Dianchi-3) Microcystis viridis FACHB (Dianchi-2) Microcystis aeruginosa FACHB (Wuda’erquan) Microcystis aeruginosa PCC 7820 Microcystis aeruginosa NIES-98 Microcystis sp. FACHB434 Microcystis sp. FACHB573 Microcystis sp. FACHB572 Microcystis sp. FACHB525 Microcystis sp. FACHB526 Microcystis sp. FACHB569 Microcystis sp. FACHB469 Microcystis sp. FACHB502 Microcystis sp. FACHB575 Microcystis elabens NIES-42 Microcystis holsatica NIES-43 Oscillatoria rubescens NIES-610 Oscillatoria agardhii NIES- 595 Oscillatoria raciborskii FACHB (Daye-1) Oscillatoria planctonica UTEX708 Anabaena flos-aquae FACHB1444 Anabaena sp. PCC 7120 Aphanizomeon flos-aquae FACHB 44–1
detection of strains synthesizing small peptides nonribosomally (Dittmann et al. 1999; Meissner et al. 1996; Neilan et al. 1999). By using the primer pairs TOX1P/TOX1 M and TOX2P/TOX2 M (Dittmann et al. 1999), it is possible to simply but reliably detect mcyB genes, which are involved in the biosynthesis of microcystins in the Microcystis strains and, to an unknown extent, other cyanobacterial genera . Using one of the three primer pairs mentioned above, both the extracted DNA and whole cells from 38 axenic cyanobacterial strains belonging to different species were analyzed by PCR (Table 1). The same strains were checked with all three of the methods described in Materials and methods for the presence of toxin and toxicity. In most cases, all three methods delivered – non-toxic (for HPLC, bioassay and ELISA) or no specific band amplified (for PCR); / not assayed
Whole-cell PCR Primers MTF/MTR
Primers TOX1P/1M
Primers TOX2P/2M
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + / – – – + – +
+ + + – + + + + + + – – + + / / / + – / / / / / / / / / – – / – – – – – –
+ + + – + + + + + + – – + + + + + + – + – – + – + – – – – – – – – – – – –
HPLC
Bioassay
ELISA
+ + + – + + + + + + – – – + + + + / – / / / / / / / / / / / + + / / / / /
+ + + / + + + + / + – – + + + + + / / / / / / / / / / / / / / / – – – – –
+ + + – + + + + + + – – – + + + + + – + – – + – + – – – – – + + – – – – –
424
Fig. 1 Electrophoresis of the PCR products with primers TOX2P/ TOX2 M from various Microcystis strains cultured axenically. A DNA-template PCR. Lanes: 1 Microcystis sp. FACHB20, 2 Microcystis viridis FACHB (Dianchi-1), 3 Microcystis aeruginosa NIES-90, 4 Microcystis wesenbergii FACHB574, 5 Microcystis aeruginosa FACHB (Dianshanhu), 6 Microcystis aeruginosa UTEX2061, 7 Microcystis aeruginosa FACHB (Wuda’erchi), 8 Microcystisa FACHB8641, 9 Microcystis aeruginosa FACHB86, 10 Microcystis sp. FACHB10, 11 Microcystis aeruginosa FACHB (Dianchi-1), 12 Microcystis aeruginosa FACHB (Bao’anhu). B Whole-cell PCR. Lanes: 1 Microcystis sp. FACHB20, 2 Microcystis aeruginosa UTEX2061, 3 Microcystis aeruginosa NIES-90, 4 Microcystis wesenbergii FACHB574, 5 Microcystisa FACHB (Dianshanhu), 6 Microcystis viridis FACHB (Dianchi-1), 7 Microcystis aeruginosa FACHB (Wuda’erchi), 8 Microcystis aeruginosa FACHB8641, 9 Microcystis aeruginosa FACHB86, 10 Microcystis sp. FACHB10, 11 Microcystis aeruginosa FACHB (Dianchi-1), 12 Microcystis aeruginosa FACHB (Bao’anhu), 13 Genomic DNA of Microcystis aeruginosa PCC 7806 (control), 14 Genomic DNA of Microcystis aeruginosa PCC 7820 (control). A and B M DNA marker. Note that lane 2 and lane 6 in A do not correspond to lane 2 and lane 6 in B
consistent results. In a few cases, however, ELISA was the most sensitive of the three methods (although it occasionally gave false-positive results). The PCR results agreed well with those obtained by HPLC, ELISA and mouse bioassay, except for M. wesenbergii 574, O. rubescens 610 and O. agardhii 595, in which the PCR results differed with those obtained using HPLC and ELISA (Table 1). The MTF2/MTR2-PCR results indicated that all 30 Microcystis strains examined, whether toxic or not, possessed at least one peptide synthetase gene. The prevalence of the peptide synthetase genes among Microcystis strains was also reported in other studies (Christiansen et al. 2001). An expected fragment of about 1,000 bp could be amplified from nontoxic Anabaena flos-aquae 1444 and Aphanizomenon flos-aquae 44–1, but not from the Oscillatoria strains or another Anabaena strain ( Table 1). In this investigation, two pairs of primers, TOX1P/ TOX1 M and TOX2P/TOX2 M, were used to amplify the specific fragments of mcyB. Among the 30 tested Microcystis strains, 18 hepatotoxic Microcystis strains gave positive PCR signals. As illustrated in Fig. 1 and Table 1, a product of 1,300 or 350 bp was measured, depending on which primer pair was used. Conversely, the non-toxic strains yielded no detectable signals. The results with the TOX1P/TOX1 M pair were consistent with the data from the TOX2P/TOX2 M pair in most cases. The PCR data
with the latter primer pair detected more toxic strains and were consistently in agreement with results from the determination of toxin and toxicity (Table 1). Therefore, the TOX2P/TOX2 M primer pair appears to be more reliable and was thus used in subsequent experiments. Among the eight filamentous cyanobacteria, no PCRamplified product was measured from the non-hepatotoxic strains, which included A. flos-aquae 1444, Anabaena sp. 7120, Ap. flos-aquae 44–1, and O. planctonica 708. Table 1 also shows that neither O. agardhii NIES-595 nor O. rubescens NIES-610 gave a detectable signal when the primer pair TOX2P/TOX2 M was used. In the context of the present study, the most important observation is that the PCR with extracted DNA and with whole cells without pretreatment gave identical results (Table 1, Fig. 1). Therefore, for quick identification of microcystin-producing strains, whole cells may be used directly. Whole-cell PCR used in samples other than axenic cultures With the positive results from axenically cultivated cells, the same simple protocol was applied to lyophilized
Fig. 2 Discrimination of the hepatotoxic Microcystis by wholecell PCR. A Water samples, B lyophilized cells. Lanes 1–4: Water samples from Taihu Lake, December, 1999. 5 Genomic DNA of Microcystis aeruginosa PCC 7806 (control), 6 Microcystis aeruginosa FACHB8641, 7 Microcystis wesenbergii FACHB574, 8 Microcystis aeruginosa PCC 7820, 9 Microcystis viridis FACHB (Dianchi-2), 10 Microcystis aeruginosa FACHB (Dianchi-1), 11 Oscillatoria raciborskii FACHB (Daye-1). M DNA marker
425 Table 2 Identification of toxic Microcystis strains from sediment samples by whole-cell PCR
Samples
Sampling spots
Results of TOX-PCR TOX1P/1F TOX2P/2F
Sediment samples
Haigeng No. 13 No. 17
+ + +
+ + +
Floating cells collected from sediment samples after being stirred and being suspended in distilled water
Haigeng No. 13 No. 17
+ + +
+ + +
Cells collected from and cultured in medium MA for 1 week
Haigeng No. 13 No. 17
+ + +
+ + +
Cells collected from sediment samples and cultured in MA media for 1 week
Haigeng No. 13 No. 17
+ + +
+ + +
Corresponding water samples above sediment samples
Haigeng No. 13 No. 17
+ + +
+ + +
Microsystis isolates from sediment samples of Dian Chi Lake
No. 1 No. 2 No. 3 No. 4 No. 5
+ + + + +
+ + + + +
Microcystis.aeruginosa PCC 7806 Microcystis.aeruginosa UTEX2061 Microcystis.aeruginosa FACHB -(Dianchi-4) Microcystis.aeruginosa FACHB -(Bao’anhu)
+
+
–
–
+
+
+
+
Sterilized sediment samples Microcystis strains inoculated in sterilized sediments
cyanobacterial cells, uncultured environmental samples, such as Microcystist bloom samples, and sediments with a mixed background of other cyanobacterial species and bacteria besides Microcystis. Six samples of the lyophilized cyanobacteria cells were directly examined by PCR without any pretreatment or DNA extraction. M. aeruginosa 8641, M. wesenbergii 574, M. aeruginosa PCC 7820, M. viridis (Dian-2) and M. aeruginos (Dian-1) were proven to produce toxins by HPLC, ELISA and mouse bioassay. As a negative control, lyophilized O. raciborskii (Daye-1) cells generated no detectable PCR signals (Fig. 2B). More than 200 water samples collected from the blooms in Dianchi Lake, East Lake, and Taihu Lake were also checked . Whole-cell PCR amplified the mcyB fragments in all 200 samples that were also found positive by ELISA. More than 95% of the samples were positive in microcystin assays by ELISA. Every month from March to December, 2000, the mixed water samples were tested by HPLC, and the results were consistent with those of whole-cell PCR (Fig. 2A.). A similar consistency was also observed in the tests of the sediment samples from
Dianchi Lake and Guanqiao pool. As shown in Table 2, positive results of these samples were confirmed by the expected amplification products in the corresponding water samples. However, the crude sediment samples without any pretreatments yielded no whole-cell PCR products. Several reports have suggested that humic acids or other materials in sediments might remain associated with the cyanobacteria cells, inhibiting DNA polymerases and thus preventing PCR (Carol 1996; Higuchi 1989; Picard et al. 1992; Tsai and Olson 1992a, b). After extensive treatment steps, such as adding skim milk, bovine serum albumin, or phage-T4 gene 32 protein to the PCR reaction mixture (Carol 1996), this PCR inhibition could be alleviated. The addition of BSA or skim milk was found to be essential for the whole-cell PCR; without this step, no DNA fragment could be amplified even from the whole cyanobacterial cells. The whole-cell PCR protocol can be optimized by rinsing the cyanobacterial cells one to three times with distilled water and by adjusting the density of the primers
426
Fig. 3 Correlation between the cell concentration and the intensity of the PCR signals in toxic strains of Microcystis. Lanes: 1 Genomic DNA of Microcystis aeruginosa PCC 7806 (control), 2 crude water sample, 3 cells from the water sample rinsed with distilled water three times, 4–10 dilutions of cell suspensions of Microcystis aeruginosa PCC 7806 (5-, 10-, 50-,100-, 500-, 1,000-, and 2,000-fold dilutions). The starting cell concentration was 4×106 cells/m l. M DNA marker
and cells in the reactions. Following such steps, amplification of non-specific products could be minimized. Hence, we suggest that the non-specific products do not come from cyanobacteria themselves, but perhaps from other bacteria associated with cyanobacteria.
high level of identity, ranging from 97 to 99% (data not shown). The present study presents a simple and rapid PCR protocol which, by virtue of using intact cells, permits the assessment of toxicity in large number of samples simultaneously without the tedious DNA extraction and purification steps. This method could be applied to routinely monitor the temporal and spatial changes of the toxic Microcystis populations in bodies of water, thus, supporting their long-term evaluation. Whole-cell PCR was demonstrated to be applicable not only to the Microcystis strains but also to other cyanobacteria, such as An. flos-aquae 1444 or Ap. flos-aquae 44–1. In order to develop a reliable detection of all the mcy genes in Microcystis as well as other microcystin-producing genera, further efforts are essential to select more optimum primers and PCR conditions.. Acknowledgements The authors would like to thank two anonymous reviewers for their critical comments to the paper. Thanks are also due to Lian Min and Shen Qiang for their kind assistance in the ELISA and HPLC analysis in this study. This research was supported by the National 863 Program (no. 2001AA641030) and the Chinese Academy of Sciences Project (STZ-01–31) to L. Song, and the National Key Project on “Dianchi Lake Bloom Control” (K99–05–35–01) to Y. Liu. This research was also financially supported by the Frontier Science Projects Programme of the Institute of Hydrobiology, CAS
References Sensitivity of the whole-cell PCR The sensitivity of the whole-cell PCR was investigated with axenically cultured toxin-producing Microcystis cells. A concentration as low as about 2,000 cells/ml was found sufficient to give the expected PCR product (Fig. 3). For lyophilized cyanobacterial cells, 1×10–4mg (dry wt) toxic cells were necessary to amplify the mcyB fragments. By this means, the toxic Microcystis cells could be roughly estimated by the intensity of the PCR signals. Sequences and comparisons To confirm that the amplified DNA fragments were indeed parts of the mcyB genes, 11 PCR products were selected for sequencing. These fragments were derived either from DNA templates or from the whole-cell PCR. All 11 sequenced PCR products exhibited 98–99% identity with the corresponding mcyB from M. aeruginosa (GenBank accession no. U97078). For the axenic strain M. aeruginosa PCC 7806 and a fresh water sample collected from Dianchi Lake, the products of both types of PCR were sequenced; no difference was found between the two methods. Among seven other strains, including M. aeruginosa PCC 7820, M. aeruginosa 90, M. aeruginosa (Wuda’erchi), M. aeruginosa (Dianchi-3), M. viridis (Dianchi-1), M. viridis (Dianchi-2) and M. wesenbergii 574, the alignments revealed that their sequences had a
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