Biochemical, morphological, and genetic variations in Microcystis ...

World J Microbiol Biotechnol (2007) 23:663–670 DOI 10.1007/s11274-006-9280-8

ORIGINAL PAPER

Biochemical, morphological, and genetic variations in Microcystis aeruginosa due to colony disaggregation Min Zhang Æ Fanxiang Kong Æ Xiao Tan Æ Zhou Yang Æ Huansheng Cao Æ Peng Xing

Received: 22 May 2006 / Accepted: 12 September 2006 / Published online: 22 March 2007 Springer Science+Business Media B.V. 2007

Abstract Microcystis aeruginosa commonly occurs as large colonial morph under natural conditions, but disaggregates and exists as single cells in laboratory cultures. To demonstrate the adaptive changes, differentiation of carbohydrates, pigments, and protein between colonial and disaggregated M. aeruginosa were examined. Their morphological and ultrastructural characteristics were subsequently observed by scanning electron microscopy and transmission electron microscopy. Results showed that chlorophyll a and phycocyanin in cells, soluble carbohydrate produced in the culture medium, and total carbohydrate in cells and sheath of colonial M. aeruginosa are significantly higher (p < 0.05) compared with those in disaggregated cells. No significant change was found in protein concentration per cell (p > 0.05) between them. Their morphological and ultrastructural characteristics were evidently different, and by morphological criteria they could be separated into two morphotypes. In addition, the genetic diversity of 16S–23S internal transcribed spacer of them were examined and compared with reference strains of M. aeruginosa. The alignment of two sequences revealed that genetic identity level was extremely high M. Zhang F. Kong (&) X. Tan Z. Yang H. Cao P. Xing Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, People’s Republic of China e-mail: [email protected] M. Zhang X. Tan Z. Yang H. Cao P. Xing Graduate School of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China Z. Yang School of Biological Sciences, Nanjing Normal University, Nanjing 210097, People’s Republic of China

(96.94%) and no significant difference was found in the nucleotide diversity (0.014 ± 0.008). This suggested that similar genotypes could present distinct morphotypes in M. aeruginosa. The tree topologies and analysis of molecular variance of the two sequences and reference sequences from GenBank database indicated that the genotypes of M. aeruginosa strains were not always related to their localities and exhibit heterogeneity within a species.

Keywords Carbohydrate Chlorophyll a Microcystis aeruginosa Morphology Phycocyanin Phylogenetics Ultrastructure

Introduction Cyanobacterial blooms, such as Microcystis, pose serious threat to ecological and public health. They dominate the phytoplanktonic assemblage and produce toxins, which affect aquatic organisms or animals, even humans that consume the water, and cause hepatotoxicity and odor problems in lakes and water supplies (Codd et al. 1999; Dinga et al. 1999). Microcystis aeruginosa is the most notoriously reported, among others, bloom-forming cyanobacterium found in both fresh and marine waters (Reynolds and Walsby 1975; Pellegrini et al. 1988; Bucka and Wilk-Wozniak 1999), especially in large shallow lakes or estuaries. M. aeruginosa commonly form large colonial aggregates constrained by an amorphous mucilage or sheath (Holt et al. 1994), which is helpful to vertical migration and defense against predation pressure (Fulton and Paerl 1987). The number of cells per colony can reach tens of thousands in a three-dimensional matrix (Otsuka et al. 2000). More-

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World J Microbiol Biotechnol (2007) 23:663–670

over, fluctuations of up to an order of magnitude in mean colony volume can be observed seasonally: for example, M. aeruginosa can have mean colony volumes ranging from 30,000 to 2,500,000 lm3 (Reynolds and Jaworski 1978; Box 1981). However, the aggregating ability of M. aeruginosa observed under natural conditions may be lost (completely or temporarily) during long-term storage and cultivation under laboratory conditions, and then exists mainly as single cells and a few paired cells (Reynolds et al. 1981; Liere and Walsby 1982; Bolch and Blackburn 1996). Colony disaggregation in laboratory cultures suggested that M. aeruginosa colony should have a high phenotypic plasticity, and it may be a kind of adaptive changes to laboratory conditions. To demonstrate the adaptive phenomenon, we investigated the morphological, ultrastructural, carbohydrates, pigments, and protein differentiation between colonial and disaggregated M. aeruginosa isolated from Taihu Lake, China, to determine the changes of biochemical composition and morphology. DNA sequences of 16S–23S internal transcribed spacer (16S–23S ITS) region are known to be more variable and exhibit significant differences in sequences and length (Barry et al. 1991; Navarro et al. 1992). The information obtained from the analysis of the region is useful for species differentiation (Barry et al. 1991). Moreover, the sequences were thought to be effective for clearly demonstrating interpretable variation among closely related species and strains (Otsuka et al. 1999). In this study, the genetic diversity of the 16S–23S ITS sequences from the colonial and disaggregated M. aeruginosa was examined, which will be helpful to illuminate whether genetic variation happens due to colony disaggregation. In addition, the relationship between the studied M. aeruginosa and reference M. aeruginosa strains from France, Spain, Thailand, Japan, and China was analyzed by their 16S–23S ITS sequences. We also sought to compare the morphological, ultrastructural traits and biochemical composition with the sequencing results of the studied M. aeruginosa.

times every day to ensure homogenous mixing of algal cultures.

Materials and methods

Morphological and ultrastructural analyses

Strains and growth conditions

Scanning electron microscopy

Colonial M. aeruginosa were isolated directly from an eutrophic lake, Taihu Lake, in China. Unicellular individuals were obtained from disaggregated colony through long-term laboratory culture, and maintained at 25C and 30 lmol photons m–2 s–1 of cool white fluorescent light under a 12:12-h light:dark photoperiod in flasks containing 150 ml of modified BG–11 medium (Rippka et al. 1979; Huisman et al. 1999). These cultures were shaken three

Fifty milliliters of cultures was fixed in 1% glutaraldehyde at 4C overnight. Then the samples were concentrated by centrifugation at 3000 rpm for 10 min. The pellets were subsequently dehydrated in 25, 50, 70, and 100% ethanol solutions (three times, 10 min for each stage), criticalpoint-dried, mounted on scanning electron micrograph stubs, sputter-coated with gold, and viewed on a JSM 840 (JEOL Ltd., Japan) scanning electron microscope.

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Biochemical composition analysis The colonial and disaggregated M. aeruginosa were activated to the logarithmic phase of growth, and then were inoculated into 150-ml medium in 250-ml flasks by the same initial cell density (2.4 · 104 cells/ml). Culture conditions were the same as stated above. After inoculation, samples (30 ml) were taken at the 10th day with three replicates, and chlorophyll a, phycocyanin, and protein in cells, soluble carbohydrate in the culture medium, and total carbohydrate in cells and sheath were consecutively analyzed. Chlorophyll a was extracted and measured using methods developed by Yan et al. (2004). Samples (5 ml) were extracted with 90% acetone and measured on the spectrofluorophotometer at a scan speed of 60 nm/min with band pass of 5 nm, response time of 2 s, and low PM gain. Phycocyanin measurement (5-ml samples) was extracted using 0.05 M pH 7.0 Tris buffer and determined on the spectrofluorophotometer at an excitation wavelength of 620 nm and an emission wavelength of 647 nm (Abalde et al. 1998; Yan et al. 2004). Carbohydrates were quantified spectrophotometrically by the phenol–sulfuric acid method using glucose as standard (Dubois et al. 1956). Soluble carbohydrate (10-ml samples) was determined in the culture media after cells had been removed by centrifugation. The removed cells were resuspended and used for the determination of total carbohydrate after sonication. This value consequently comprises sheath and intracellular carbohydrates. The interference of the presence of ions in BG-11 cultures was avoided by dialyzing against deionized water (Spectrapor MWCO 3500). Protein (10-ml samples) was measured by the method described in Bradford (1976) using bovine serum albumin as standard. The enumeration of colonial M. aeruginosa was determined by the method of Humphries and Widjaja (1979).


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Transmission electron microscopy

Statistical and phylogenetic analyses

For transmission electron microscopy (TEM), the cultures were initially washed twice in phosphate buffer (PB), pH 7.2, and concentrated at a low-speed centrifugation (3000 rpm for 5 min), the concentrated cells were embedded in 1% agar solution, and fixed with 3% glutaraldehyde in 0.2 M PB for 2 h. To detect complex polysaccharides, the fixed samples were stained with 0.15% ruthenium red to detect total exopolysaccharides (Colombo and Rascio 1977). Then the samples were post-fixed with 1% osmium tetroxide in 0.2 M PB for 2 h. After a second wash as aforementioned, dehydration was performed with ethanol 30, 50, 70, 80, 90, and 100% before being embedded in Epon resin. Thin sections were made with a Reichert-Ultracut E ultramicrotome and stained by uranyl acetate and lead citrate. They were examined by H-600 (Hitachi Ltd., Japan) transmission electron microscope at 80 kV. Ultramicroscopic observations were conducted on sets of at least three sub-samples.

Statistical analysis was performed with SPSS 10.0 for Windows, using one-way analysis of variance (ANOVA). The sequences, including some from GenBank, were aligned using Clustal X version 1.81 (Jeanmougin et al. 1998). Phylogenetic trees were constructed by the neighbor-joining method on Jukes-Cantor pairwise distance, by maximum parsimony analysis using the MEGA version 3.1 (Kumar et al. 2004) and maximum likelihood analysis using PHYLIP Software Package. Support for tree branch points was estimated by the bootstrap approach (Felsenstein 1993) using 500 replicates of the heuristic search algorithm. Estimations of nucleotide diversity and analysis of molecular variance (AMOVA) was performed using the AMOVA 1.55 (Excoffier 1995).

DNA sequencing DNA for polymerase chain reaction (PCR) was extracted according to the protocol as described in Humbert and Le Berre (2001). Briefly, after centrifugation the pellets were incubated in 1 ml of cell lysis buffer (0.1 M Tris–HCl, 0.1 M NaCl, 50 mM ethylenediaminetetraacetic acid, 1% sodium dodecyl sulfate) at 37C for an hour. Five milligrams of proteinase K per ml was added and the tubes were placed in a 40C water bath and left overnight. After a phenol–chloroform extraction and an ethanol precipitation, DNA was stored at –40C before its utilization. ITS of the 16S–23S rDNA operon was amplified in each DNA extract. The 50-ll PCR mixtures contained 80 ng template DNA, a 90-lM concentration of each of the four dNTPs, 5 ll of PCR reaction buffer (1.5 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl, pH 9), 0.2 lM of each primer (forward primer: 5¢GGGGATGCCTAAGGCAGG GCT3¢; reverse primer: 5¢CCACGTCCTTCTTCGCCTC TG3¢), and 0.5 U of Taq DNA polymerase (TIANGEN Bio-tech). The DNA templates and a negative control were subjected to an initial denaturing step at 95C for 5 min. The following 35 cycles consisted of a 30-s denaturing step at 95C, a 30-s annealing step at 50C, and a 30-s extension step at 72C. A final 10-min extension step was carried out at 72C. Amplifications of the target region were checked by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Positive products were reclaimed, and sequencing was carried out by Shanghai Sangon Biological Engineering Technology & Service Co., Ltd., Shanghai, China.

Results A ANOVA revealed that the biochemical composition, except protein, had a significant difference between colonial and disaggregated M. aeruginosa cells (p < 0.05) (Table 1). Owing to colony disaggregation, chlorophyll a, phycocyanin, soluble carbohydrate, and total carbohydrate obviously decreased from 0.40 ± 0.04, 2.84 ± 0.05, 4.49 ± 0.27, and 31.54 ± 0.68 to 0.19 ± 0.05, 1.39 ± 0.21, 0.88 ± 0.08, and 16.06 ± 0.20 pg/cell, respectively. In particular, the decrease of soluble carbohydrates was the highest and up to 80.4%, and chlorophyll a, phycocyanin, and total carbohydrates decreased 52.5, 51.1, and 49.1%, respectively. The variation of protein was, however, slight and not remarkable (p > 0.05). The colonial and disaggregated M. aeruginosa present evidently different morphologies shown by the scanning electro microscopic images (Fig. 1). Colonial M. aeruginosa present irregular outlines and distinct holes (Fig. 1a), and were wrapped and stuck together by an extracellular layer (Fig. 1b). Smooth surface was observed from the colonial cells (Fig. 1c). However, disaggregated M. aeruginosa existed mainly as dispersed cells, and no

Table 1. Differentiation of chlorophyll a (Chl a), phycocyanin (PC), protein, soluble carbohydrate (SC), and total carbohydrate (TC) per cell between colonial and disaggregated M. aeruginosa Single cell

Colony

Chl a (pg/cell)

0.19 ± 0.05

PC (pg/cell)

1.39 ± 0.21

2.84 ± 0.05

11.20 ± 2.52

13.65 ± 1.74

Protein (pg/cell)

0.40 ± 0.04

SC (pg/cell)

0.88 ± 0.08

4.49 ± 0.27

TC (pg/cell)

16.06 ± 0.20

31.54 ± 0.68

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Fig. 1 Electron micrographs of colonial (a–c) and disaggregated (d–f) M. aeruginosa in different zoom multiples

conglutination was found (Fig. 1d and e). The cell surface was comparatively rough and draped (Fig. 1f). The sections of M. aeruginosa showed a typical construction of cyanobacteria cells with thylakoids, vacuoles, granules, lipid droplets, and the plasma membrane (Fig. 2). The photosynthetic apparatus thylakoids ran parallel or perpendicular to the cytoplasmic membrane and surrounded the nucleoplasmic area. Poly-beta-hydroxybutyrate (PBH) and polyhedral granule (PH) were present in the nucleoplasmic area. A scant amount of lipid bodies were mainly found in the cytoplasm of disaggregated cells (Fig. 2a and b). However, visible vacuoles were observed between thylakoids of the colonial cells (Fig. 2c). Ultrastructural cytochemistry using ruthenium red as marker of samples revealed that the cell surfaces of M. aeruginosa

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showed a dense reaction product coat covering the whole cell surface. The polysaccharide envelope of colonial cells surface is thicker than that of disaggregated cells (Fig. 2d). PCR amplification with the primer resulted in the detection of a single band of approximately 360 bp in the two investigated strains. The alignment of two sequences revealed a 96.94% identity. There was no significant difference in the nucleotide diversity (0.014 ± 0.008) of colonial and disaggregated M. aeruginosa. In addition, the distribution of the two sequences and the reference sequences in the phylogenetic tree (Fig. 3) did not show any obvious segregation among different sample sources from China, Japan, Thailand, Spain, and France. In the AMOVA, two groups of populations were defined based on the sample sources: Asia and Europe. For the hierarchical level


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Fig. 2 Transmission electron microscope (TEM) images of disaggregated (a, b) and colonial (c, d) M. aeruginosa. Poly-beta-hydroxybutyrate (PBH), poly granule (PH), lipid droplet (L), thylakoids (T), vacuole (V). (a) A cell showing thylakoids (T) running mainly parallel or perpendicular to the cell wall and the nuleoplasmic area (N) containing PBH, PH. Some lipid droplets (L) are also visible. (b After ruthenium red staining, a thickened envelope is present. (c) A cell showing thylakoids (T) paralleling to the cell wall and visible vacuoles (V). (d) After ruthenium red staining, polysaccharide on the external envelope (arrow) is detectable. Bar = 1 lm

analysis, 1.94% of the variance was due to the group structure. And the fixation index was 0.0004 (not significantly different from 0).

Discussion The quantity analysis of carbohydrates, including soluble extracellular polysaccharide and total polysaccharide, showed that colonial M. aeruginosa cells contained more polysaccharide than disaggregated cells. Moreover, colonial M. aeruginosa had the thicker polysaccharide envelope than unicellular M. aeruginosa by ultrastructural observations. These results suggested that polysaccharide is a

primary content of mucilage or sheath that plays an important role in cyanobacteria colony formation, which is consistent with the description of Bahat-Samet et al. (2004). Dense components and gas vacuoles are mainly contributors which regulate to cyanobacteria buoyancy (Oliver 1994; Walsby 1994). In this study, disaggregated cells contained many lipid droplets, whereas visible vacuoles were evident in colonial cells. The results suggested that colonial M. aeruginosa have better buoyancy ability that contributes to migration than unicellular M. aeruginosa. Furthermore, vertical migration provides a competitive advantage for M. aeruginosa over negatively buoyant and slower migrating species, especially under intermittent mixing conditions (Mitrovic et al. 2001), which results in

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Fig. 3 Distance tree based on the alignment of 16S–23S rDNA ITS sequences of M. aeruginosa strains. Bootstrap values higher than 50% were indicated at nodes. Colonial (colony) and disaggregated (single) sequences were the present of experimental results, and other sequences were obtained from GenBank database (CL1, CL3, CC01 from China; TAC71, TAC86, TAC87, TAC170, NIES44 from Japan; TC6, TC8, TC20-1 from Thailand; UAM253, UAM254 from Spain; others from France).

97 CL1 CL3 TAC86

57

96 TAC87 TAC170 CC01 SC83 97

SG75

TC20-1 SINGLE

81 100

COLONY

SC113 SG76 TC8 CC921

99

CC922 71

CC14 SC93 TC6 NIES44 UAM253

100 UAM254

73 CC13 55

CG211 SC52 99 SG72 TAC71

0.01

that M. aeruginosa has more chances to use light energy. In the case, pigment concentration per cell of colonial M. aeruginosa was higher than that of unicellular individuals, which confirmed that colonial M. aeruginosa owns better potential ability of photosynthesis than unicellular M. aeruginosa. In this study, colonial and disaggregated M. aeruginosa could be separated into two morphotypes by morphological criteria. However, the two distinct morphotypes had highly similar 16S–23S ITS sequences. This suggested that no significant genetic variation was found during M. aeruginosa colony disaggregation. Palinska et al. (1996) showed that the great morphological diversity observed in nature and (partially) in culture does not necessarily reflect genetic diversity, and mentioned the likelihood that more ecophenic and/or phenotype forms have been described than genotypic species. The lack of genetic differentiation between colonial and disaggregated M. aeruginosa suggested that there was no genetically differentiated phenotype. Thus, M. aeruginosa was able to exist in different

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morphologies, unicellular and colonial formation, and adapt to different environment conditions. In addition, early studies about M. aeruginosa taxonomy showed that M. aeruginosa morphological characteristics were not consistent with phylogenetic analysis. Furthermore, that morphology does not correlate with genotype in M. aeruginosa, or Microcystis generally, and that one genotype could present more than one morphotype (Otsuka et al. 1999). The present result supported the conclusion and was in line with the studies using the phycocyanin intergenic spacer of Brazilian M. aeruginosa strains (BittencourtOliveira et al. 2001). And it did not support the taxonomy based on morphological characteristics. In the constructed phylogenetic tree, all sequences were 16S–23S ITS of M. aeruginosa. Therefore, the identity of sequences was still high (83.4–100%) after excluding positions with gaps which was the reason for the low bootstrap probability obtained for the tree. In the tree, all sequences from different lakes or regions separated by geographic distance were distributed in different clusters.


There was no cluster that only contained the sequences from the same lake or region. Especially, TAC71 from Japan has identical sequence with the one from France. The AMOVA also showed that there is no significant separation between ITSs from Asia and Europe. Therefore, the genotypes of M. aeruginosa strains were not always related to their localities and exhibit heterogeneity within a species, which agrees with Nishihara et al. (1997). The same conclusion was also obtained by assessing the genetic diversity of geographically unrelated M. aeruginosa strains using amplified fragment length polymorphisms (Oberholster et al. 2005). In conclusion, pigment and carbohydrate concentration per cell significantly decreased after M. aeruginosa colony disaggregation. Colonial and disaggregated M. aeruginosa are evidently different in morphology and ultrastructure, whereas 16S–23S ITS sequences have a high similarity. The taxonomy of M. aeruginosa based on morphological criteria was not supported by phylogenetic analysis based on 16S–23S ITS sequences. Highly similar genotypes could present distinct morphotypes in M. aeruginosa. Moreover, the genotypes of M. aeruginosa strains were not always related to their localities and exhibit heterogeneity within a species. Acknowledgments We thank Dr. Guang Gao and Dr. Feizhou Chen for kindly providing some necessary apparatus. The work was supported by the project of the State Key Fundamental Research and Development Program (2002CB412300), National Natural Science Foundation of China (40471045, 30670404), and the 100-Researcher Program of CAS.

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