Cyanophages are viruses that infect cyanobacteria, and some cyanophages reportedly infect different genera of cyanobacteria (Gao E B, et al., 2009; Liu X Y, et al., 2006; Safferman R S, et al. 1963; Yoshida T, et al., 2006). The isolation and cultivation of additional cyanophages that infect different cyanobacteria is needed in order to gain a better understanding of the relationships between cyanophages and their hosts. Although there is evidence that viruses are an important feature of shallow freshwater lake microbial ecosystems and display remarkable morphological diversity, little is known about the precise control mechanisms of freshwater cyanophages that affect the growth and decline of bloom-forming cyanobacteria (Liu Y M, et al., 2006; Yoshida T, et al., 2006; Yoshida M, et al., 2008; Zhang Q Y et al., 2008 and 2012).
M. aeruginosa is one of the most common bloom-forming cyanobacteria found in eutrophic freshwaters (Wood S A, et al., 2012) and usually the dominant cyanobacterial genus in eutrophic lakes, but cultivable cyanophages that infect this genus are scarce (Tan X, et al., 2009; Yoshida T, et al., 2006). In China, Microcystis blooms occur in a large proportion of the aquatic environments, including lakes, reservoirs, and ponds, and often cause serious water management problems (Chen J, et al., 2009; Wu Z X, et al., 2007). Many studies focus on the physical and chemical factors that affect the ecology of Microcystis blooms, but controlling these algal blooms is difficult during warm months (Davis T W, et al., 2009; Oliver R L, et al., 2000; Yoshida T, et al., 2006; Sengco M R, 2009; Yang F, et al., 2013). Some biological factors, such as algicidal bacteria and cyanophages, have been suggested as possible regulators of cyanobacterial blooms (Brussaard C P, 2004; Yang F, et al., 2013; Zhang Q Y, et al., 2009).
The establishment of laboratory phage-host systems would be invaluable to the study of the interactions between cyanophages and their bloom-forming hosts and could help develop methods for controlling Microcystis blooms. In this study, we describe the isolation, cultivation, and partial characterization of a freshwater cyanophage that specifically infects the bloom-forming cyanobacterium M. aeruginosa, which is found in Lake Dianchi, a eutrophic lake in southwestern China. This cyanophage was tentatively named MaMV-DC (Microcystis aeruginosa myovirus from Lake Dianchi) based on its host, morphological features, and location where it can be isolated.
Four cyanobacterial species (21 strains) were used to isolate the MaMV-DC cyanophage and determine its host range (Table 1). Strains were obtained from the Freshwater Algae Culture Bank, Institute of Hydrobiology, Academy of Sciences, Wuhan, China. Each cyanobacterial strain used in this research was clonally grown in BG-11 medium (Gao E B, et al., 2009) at 25 ℃ under a 14-hour light/10-hour dark cycle using 35 μmol photons m-2 s-1 with cool white fluorescent illumination.
Species Strain Susceptibility Microcystic aeruginosa FACHB-524 a + FACHB-526 - FACHB-905 a - FACHB-915 a - FACHB-1179 a - FACHB-1217 - FACHB-1291 a - HAB1801 a - HAB0334 - Anabaena spirioides HAB1211 - FACHB-709 - FACHB-1198 - FACHB-1199 - FACHB-1219 - FACHB-1246 - HAB0502 - HAB0508 - PCC7120 - Anabaena oumiana HAB0984 - Synechococcus sp. FACHB-1061 - Botryococcus sp. FACHB-1108 - aStrains used during the first isolation process. +, sensitive; -, not sensitive.
Table 1. Cyanobacterial strains and their susceptibilities to MaMV-DC.
Freshwater samples were collected from Lake Dianchi, the sixth largest freshwater lake in China, in southwest Kunming City. Centrifugation and filtration were performed at our laboratory to remove zooplankton, phytoplankton, bacteria, and cellular debris from the sample. The sample was centrifuged at 8000 g for 10 minutes at 4 ℃ and filtered through a 0.45-μm pore filter (Millipore, Bedford, MA, USA). Aliquots of the filtrate (200 μL) were added to 24-well plates containing 1 mL of exponentially growing cultures of M. aeruginosa strains; fresh BG-11 medium was used as the negative control. Samples were incubated at 25 ℃ under the previously described lighting conditions. Cultures were visually examined daily for visible changes in cyanobacterial morphology using light microscopy.
The cultured M. aeruginosa FACHB-524 cells were treated with successive plaque purification cycles in order to develop the clonal and lytic MaMV-DC cyanophage. Briefly, the supernatant of the lysed cells was serially diluted with BG-11 medium and each 20 μL dilution of supernatant was mixed with 0.5 mL of the exponentially growing M. aeruginosa FACHB-524 cultures (approximately 9.5×108 cells mL-1). Following incubation at 25 ℃ for 1 hour, 2.5 mL of top agar (0.4% molten BG-11 agar medium) containing the supernatant dilution and M. aeruginosa cells was spread onto 1% BG-11 agar plates. M. aeruginosa FACHB-524 cultures without the cyanophage suspension served as the negative control. All plates were incubated under the previously described conditions and monitored daily. After plaque formation, isolated plaques were selected and used to infect fresh cyanobacterial cultures. A new round of cyanophage infection and plaque isolation was conducted, as described above, until homogeneous plaques appeared.
The host range of MaMV-DC was determined by adding 0.2 mL aliquots of the MaMV-DC suspension to 0.8 mL of exponentially growing cyanobacterial cultures (Table 1). Cyanobacterial cultures without the MaMV-DC suspension served as the negative control. All cultures were monitored daily for host cells lysis using light microscopy. Cyanobacterial strains that did not lyse after 14 days of incubation were defined as insusceptible hosts for MaMV-DC.
One-step growth experiments were performed to evaluate the development and burst size of MaMV-DC. Ten mL of the MaMV-DC suspension (cyanophage titer 6×108 infectious units) was added to 100 mL of exponentially growing M. aeruginosa FACHB-524 cultures (approximately 107 cell mL-1). Cultures were incubated for 6 days in an illuminating incubator. After inoculation, the density of the cyanobacterial cells was determined daily by direct counting using a hemocytometer (Shanghai Medical Optical Instrument Plant, Shanghai, China), and the MaMV-DC titer was estimated using the most-probable-number technique, as previously described for the PaV-LD cyanophage (Gao E B, et al., 2009).
The lysis curves for MaMV-DC were established as described above. M. aeruginosa FACHB-524 cultures were monitored daily by measuring the optical density of the wavelength at 680 nm (OD680) after inoculation with the MaMV-DC suspension or an autoclaved MaMV-DC suspension.
Cyanophage particles from M. aeruginosa FACHB-524 cultures were purified by sucrose density gradient centrifugation (20% -60%). The cyanophage lysates were treated with chloroform and centrifuged to remove cell debris, and the cyanophage particles were concentrated using polyethylene glycol 8000 (9.3% [wt/vol]). The concentrates were centrifuged on a discontinuous sucrose density gradient at 24, 000 rpm for 40 minutes (Beckman Optima L-90 K, SW 41 swing-out rotor; Beckman, Fullerton, CA). The resulting bands in the 30% -60% range were separately collected and washed twice with sterile phosphate-buffered saline (PBS). The purified cyanophage particles were resuspended in sterile PBS and transferred to an Eppendorf tube for negative stain electron microscopy and genomic DNA extraction.
The purified cyanophage particles were negatively stained with 2% uranyl acetate (w/vol) and observed at 80 kV using a JEM-1230 transmission electron microscope (JEOL, Tokyo, Japan). Cyanophage DNA was prepared for restriction endonuclease cleavage analysis according to the method described by Gao et al (2012). Briefly, the purified cyanophage particles were treated with sodium dodecyl sulfate-proteinase K digestion, and genomic DNA was subjected to phenol-chloroform extraction and ammonium acetate-isopropanol precipitation.
To assess the sensitivity of the MaMV-DC nucleic acids to restriction enzymes, the extracted genomic DNA was prepared for restriction enzyme treatment. The nucleic acid was incubated overnight with two restriction enzymes—Pst Ⅰ (12 U/μL; TaKaRa, Kyoto, Japan) and Sac Ⅰ (12 U/μL; TaKaRa)—at 37 ℃ according to the manufacturer's recommendations. The restriction enzyme-digested products were electrophoresed in 1% agarose (wt/vol) and visualized using a gel analysis system (Syngene G: Box; Cambridge, UK) after staining with ethidium bromide (Yoshida T, et al., 2006).
The purified MaMV-DC particles were mixed with an equal volume of 2×SDS-PAGE loading buffer (100 mmol/L Tris-HCl [pH 6.8], 4% SDS, 0.2% bromphenol blue, 2% 2-mercaptoethanol, 20% glycerol), boiled for 5 minutes, and loaded onto 15% SDS-polyacrylamide gels. The proteins were separated at a constant voltage (120 V) and visualized using the Coomassie brilliant blue staining method.