Induction of temperate cyanophage AS-1 by heavy metal – copper

Background It has been reported that some marine cyanophage are temperate and can be induced from a lysogenic phase to a lytic phase by different agents such as heavy metals. However, to date no significant reports have focused on the temperate nature of freshwater cyanophage/cyanobacteria. Previous experiments with cyanophage AS-1 and cyanobacteria Anacystis nidulans have provided some evidence that AS-1 may have a lysogenic life cycle in addition to the characterized lytic cycle. Results In this study, the possible temperate A. nidulans was treated with different concentrations of heavy metal-copper. CuSO4 with concentrations of 3.1 × 10-3 M, 3.1 × 10-4 M, 3.1 × 10-5 M and 3.1 × 10-6 M were used to detect the induction of AS-1 from A. nidulans. The population of the host, unicellular cyanobacteria Anacystis nidulans, was monitored by direct count and turbidity while the amount of virus produced was derived from plaque forming units (PFU) by a direct plating method. The ratio of AS-1 release from A. nidulans was also determined. From these results it appears that AS-1 lysogenic phage can be induced by copper at concentrations from 3.1 × 10-6 M to 3.1 × 10-4 M. Maximal phage induction occurred at 6 hours after addition of copper, with an optimal concentration of 3.1 × 10-6 M. Conclusion Cu2+ is a significant inducer for lysogenic cyanobacterial cells and consequently would be a potential control agent in the cyanobacteria population in fresh water ecosystems.


Background
Anacystis nidulans is a rod-shaped, unicellular prokaryotic cyanobacterium and plays an important role in aquatic ecosystems as a primary producer. It is often used as an indicator for studying the toxic metabolic levels of heavy metals. Many heavy metal studies have been done using A. nidulans as the model system for the reaction of organisms to heavy metal stresses [1][2][3][4][5][6][7][8][9][10]. In freshwater environ-ments, dense algal blooms of cyanobacteria are usually caused by nutrient enrichment (i.e., nitrogen and phosphorus) from sewage, agricultural fertilizers and industrial run-off into waterways [11]. Algal blooms are considered threat to the water system [11,12]. Cyanophage are viruses that infect cyanobacteria and are ubiquitous in both freshwater and marine environments. These phages play important roles in modulating cyanobacterial populations, affecting primary productivity, increasing water quality and may have a profound influence on global biogeochemical cycles [13,14]. Although the interaction between a cyanophage and its host organism is important in maintaining water quality in freshwater systems, little is known about how viruses regulate microbial mortality in natural waters. Recently, it was found that lysogenic infection was common in marine Synechococcus sp. [15]. Cyanophage infecting a single strain of marine Synechococcus sp. can reach 10 3 to 10 5 per ml in seawater [14,[16][17][18]. Suttle and Chan [17] have estimated that between 5-15 % of marine Synechococcus cells were lysed by cyanophage daily. The discovery of a high abundance of viral particles (ca. 10 7 per ml) in natural waters [19,20] initiated the research on the ecological impact of the viral infection and lysis of marine microbes. They also pro-vided evidence that viruses can affect microbial populations by either going through a lytic cycle, causing destruction of the host cell, or maintaining a lysogenic stage, in which the viral genome is inserted and maintained as the prophage in its host cell [18]. There was also evidence to suggest that seasonal changes can cause the prophage to enter a lytic cycle thus leading to the disappearance of algae blooms [21][22][23]. Lysogeny can also be induced to a lytic cycle by pollutants [24].
Although extensive research has been done on the interaction between cyanophage and cyanbacteria in marine systems, there are no significant reports that have focused on freshwater cyanophage/cyanobacteria interactions. The possibility of temperate AS-1 and lysogenic A. nidulans was suggested by Bisen et al [25], but there was no direct The growth curve of AN-T and CuSO 4 treated AN-T evidence provided. It has been reported that UV, mitomycin C and heavy metals such as copper, cadmium can induce the release of cyanophage in marine water [22,26]. In this study, different concentrations of copper sulfate were used to study a possible AS-1 lysogenic life cycle in addition to the previously characterized lytic cycle. Addition of copper sulfate led to a significant increase in phage production, a characteristic of an organism with a lysogenic life cycle. The study of lytic induction from temperate A. nidulans can provide a good model for studying the interaction between cyanophage and cyanobacteria in freshwater ecosystems.

Results and discussion
In order to determine if heavy metals could cause induction of AS-1 from temperate A. nidulans (AN-T), CuSO 4 was added at concentrations of 3.1 × 10 -3 M, 3.1 × 10 -4 M, 3.1 × 10 -5 M and 3.1 × 10 -6 M at day 4 post innoculation, the exponential growth stage of the culture. Growth of AN-T was severely inhibited at concentrations of 3.1 × 10 -3 M and 3.1 × 10 -4 M. Growth was affected to a certain extent in 3.1 × 10 -5 M of CuSO 4 ; growth rate in 3.1 × 10 -6 M CuSO 4 was very similar to the control ( Figure 1).
While there is no clear evidence to explain why induction decreases over time, it is possible that either the phage/ host interaction stabilizes after the initial stress or the toxic effect of heavy metal on the host causes a disruption in phage production.
Although the heavy metal induction rates varied depending on the concentrations of the heavy metal, the overall induction of copper compared to control is clear. The results suggest that Cu 2+ is a significant inducer for temperate AS-1 released from AN-T. The results correlated well with the study of induction for marine cyanobacterial lysogen although AS-1 release rate and induction rate by copper were much lower than the marine cyanophage/ cyanobacterial lysogen studies [14,21,26]. Further study with other reported inducers mitomycin C and UV was also carried out to compare the PIR of both mitomycin C and UV with copper studies.

Conclusion
These results suggest that AS-1 lysogenic phage can be induced by copper with a concentration range from 3.1 × 10 -6 M to 3.1 × 10 -4 M. The best condition for phage induction occurred at 6 hours after addition of all these concentrations. Copper concentrations of 3.1 × 10 -6 M showed the highest level of viral induction. Cu 2+ is an important inducer for lysogenic cyanobacterial cells and consequently could be a potential trigger in the cyanobacteria population in freshwater aquatic environments.

I. Maintenance of cultures of anacystis nidulans and AS-1 1. Culture and maintenance of anacystis nidulans
Anacystis nidulans was obtained from Dr. R. McGowan, Brooklyn College, N.Y. The culture was inoculated aseptically in a 250 ml Erlenmeyer flask with 100 ml Mauro's Modified Medium (3 M medium) at pH 7.9 [27]. The culture was grown in ambient temperature, with constant fluorescent light and continuous agitation at 100 rpm. Cell growth was monitored by direct cell count using a hemacytometer and turbidity studied using a Baush & Lomb Spectronic 20 at OD 750 nm [13]. The cultures of A. nidulans were checked periodically for bacteria contamination by plating 100 µl of the culture on nutrient agar plates and observing after a 2 to 3 day incubation period. The stock cultures were maintained on 3 M agar plates and slants that were made with 3 M medium containing 2 % agar. itored by checking the lysis of the host cell. Host cell lysis was determined by turbidity studies using a Baush & Lomb Spectronic 20 at OD 750 nm [28]. The lysis curve was generated by determining a decrease in the turbidity of the infected culture as well as by direct cell count using a hemacytometer as previously described.
The population of AS-1 was also monitored by plaque forming units (PFU). Pure non-viral infected A. nidulans (10 ml; AN-P) culture was centrifuged at 5,000 rpm for 10 minutes and the cell pellet was collected. At different time intervals, 2.5 ml were removed from cultures of temperate A. nidulans (AN-T) treated with different concentrations of copper and added into the cell pellet and mixed well. Melted 1 % 3 M soft agar (1 ml) was added to the mixture and vortexed. The mixture was then poured onto prewarmed 2 % 3 M agar plates. After the soft agar solidified, the plate was placed under continuous "cool-white" fluorescent light for 5-7 days until the plaques (clear zone) were formed, and counted.

II. Copper induction
Five ml of AN-T were inoculated respectively into 5 flasks containing 95 ml of 3 M medium to achieve a concentration of 1.0 × 10 7 cells/ ml. The cultures were grown for 4 days to reach exponential growth stage. Copper was then added to the cultures respectively using the following concentrations: CuSO 4 3.1 × 10 -3 M, 3.1 × 10 -4 M, 3.1 × 10 -5 M, and 3.1 × 10 -6 M; a culture with no heavy metal was used as a control. The growth of AN-T was monitored by direct cell count using a hemacytometer and turbidity measured using a Baush & Lomb Spectronic 20 at OD 750 nm for an 11 day period. The released AS-1 in the culture was monitored by the plaque plating method at 6 hours, 24 hours and 48 hours after addition of copper.