Viruses are now known to be the most abundant biological entities in marine systems as well as important drivers of many ecosystem functions. For example, lytic viral infection has been implicated as a major factor in the seasonal succession of plankton populations (Mühling et al., 2005). Lytic phages infective for marine Synechococcus are easily isolated from seawater, and many of these have been shown to be Myoviridae although some Siphoviridae and Podoviridae have also been isolated.
In addition to working with lytic cyanophages, this lab has been investigating temperate cyanophages and their hosts. Initial experimentation by other researchers demonstrated induction of cyanophage from a cultured marine Synechococcus species isolated from the coastal waters off Kyushu, Japan. Prophage induction occurred in response to UV light, Mitomycin C (Sode et al, 1994), and a heavy metal (Sode et al, 1997). Our work has demonstrated lysogeny in Synechococcus, based on prophage induction in natural populations in both Tampa Bay and the Gulf of Mexico (McDaniel et al, 2002, McDaniel and Paul, 2005). Recent research has indicated that prophage induction events cause changes in bacterial community structure by increasing the diversity and richness of natural microbial communities, highlighting the importance of investigating lysogeny in natural environments (Hewson and Fuhrman, 2007). In addition, cyanophages have been identified as a major factor controlling the seasonal succession of Synechococcus genotypes (Mühling et al, 2005). Because of the seasonal pattern in prophage induction that has been observed in natural populations of Synechococcus in Tampa Bay we hypothesize that Synechococcus prophage may be a reservoir of viruses for this observed seasonal succession.
A research cruise was undertaken to the Gulf of Mexico during July 2005 to further explore environmental factors associated with lysogeny and to directly compare lysogeny in heterotrophic and autotrophic microbial populations. Ambient physical and microbial parameters were measured and prophage induction experiments were performed in contrasting oligotrophic Gulf and eutrophic Mississippi plume areas. Three of eleven prophage induction experiments in heterotrophic bacteria (27%) demonstrated significant induction in response to Mitomycin C. In contrast, there was significant Synechococcus cyanophage induction in six of nine experiments (67%). A strong negative correlation was observed between lysogeny and log-transformed activity measurements for both heterotrophic and autotrophic populations (r = -0.876, p = 0.002 and r = -0.815, p = 0.025, respectively), indicating bacterioplankton with low host growth favor lysogeny. Multivariate statistical analyses indicated that ambient level of viral abundance and productivity were inversely related to heterotrophic prophage induction and both factors combined were most predictive of lysogeny (p = 0.899, p = 0.001). For Synechococcus, ambient cyanophage abundance was most predictive of lysogeny (p = 0.862, p = 0.005). Abundance and productivity of heterotrophic bacteria was strongly inversely correlated with salinity, while Synechococcus was not. This indicated that heterotrophic bacteria were more adaptable to highly variable plume environments, thus providing a possible explanation for differences in lysogeny between the two populations (Long et al. 2007).
We have isolated a series of marine Synechococcus strains from the Gulf of Mexico as well as a group of lytic cyanophages by using Synechococcus WH7803 as a host. In our isolates, eleven of twenty-five strains (46%) produce a statistically significant increase in virus-like particles in response to Mitomycin C treatment. No correlation was found between presence of cyanophage induction and level of resistance to lytic infection.
Investigation of environmental triggers of cyanophage induction has consistently demonstrated that high levels of Synechococcus host, Synechococcus cyanophage and high levels of primary productivity are inversely correlated with cyanophage prophage induction. Work with our putative Synechococcus lysogen has revealed an environmentally relevant trigger of cyanophage induction. Cyanophage induction can be caused by shifting a rapidly growing Synechococcus isolate from normal illumination to high levels of light (McDaniel et al, 2006). These studies indicate that Synechococcus lysogens appear to be prevalent, and that induction of temperate cyanophage can respond to environmental cues.
The putative temperate phage from isolate GM 9914 was induced by continuous high light and isolated for further analysis, including transmission electronic microsocopy and genomic sequencing. TEM analysis of the induced particle revealed that its morphology was non-tailed unlike most lytic cyanophages. This finding may explain the lack of correlation between presence of prophage induction in a Synechococcus isolate and its resistance to lytic infection. Prophages are known to confer immunity to similar viruses and at least in the case of isolate GM 9914 (N), the induced virus appears to be quite dissimilar to typical lytic cyanophages.
The highest abundance obtained by induction to date is approximately 5.5 x 108 VLP’s ml-1 in comparison to lytic cyanophages which usually display at least an order of magnitude higher abundance. The genome appears to be approximately 7.8 kb in size. Lane 1 contains marker. Lanes 2 and 3 contain phi X174 (5.4 kb) and Lane 4 contains induced cyanophage N CsCl purified genomic DNA. The gel was stained with SYBR Gold instead of Ethidium Bromide to allow detection of small quantities of nucleic acid. Several single clone sequences obtained to date contain gene sequences highly similar to known Synechococcus or Prochlorococcus genes, providing support for the contention that the viral particle derives from the cyanobacteria rather than a heterotrophic contaminant.
Differential nucleic acid digestion experiments revealed the nucleic acid content of the inducible particle was single-stranded (ssDNA) rather than dsDNA. This was also confirmed by the lack of visibility of DAPI-stained N-induced lysates compared to easily visible SYBR-stained particles by epifluorescence microscopy. Most known cyanophages contain dsDNA, however, according to the International Committee on Taxonomy of Viruses (ICTV) the non-tailed morphology and content of ssDNA makes this viral particle most similar to the viral group Microviridae. Phi X174, which is a bacteriophage infecting Escherichia coli is the type strain of this small viral family. Such a prophage-like particle is unlike any prophage described to date, implying that the process of lysogeny is unique in certain marine Synechococcus strains.
Recently, a novel approach has been developed for investigating lysogenic gene expression. To obtain sequence information on natural populations of temperate phage, a large sample of the ambient microbial population was concentrated by tangential flow filtration and then induced with Mitomycin C. The treatment sample demonstrated a low but statistically significant increase in viruses in comparison to the untreated sample. These induced viruses were collected by centrifugation and 0.2 µm filtration of the concentrated, induced sample. The induced viruses were then purified by CsCl banding and sequenced by the 454/Pyrosequencing method.
This sequencing yielded 294,068 reads, of which 19,536 are “known” (6.6%). Preliminary analysis of the metagenome revealed many phage hits-7629 top BLAST hits were to phage-like sequences and 5279 were to cyanophage. There were 103 Integrase hits in the unassembled reads, yet only 4 strongly significant ones. Primers and probes for real-time PCR were designed for each of four integrases and were tested utilizing DNA extracted from the microbial fraction of Tampa Bay. Assays for Vibrio-like integrase and Oceanicola-like integrase revealed 1.1 x 10E5 (0.005% of ambient population), and 300 gene copies per liter respectively. The other two integrases were not detected. The integrase assay was then tested on the microbial RNA fraction extracted from 200 ml of Tampa Bay water over an annual cycle. The Vibrio-like integrase gene in three of the samples, with an estimated copy number range from 2.4-1280 gene copies per liter. The Clostridium-like integrase gene expression was detected in 6 samples, with an estimated copy number ranging from 37 to 265 copies per liter. In all cases, detection of integrase gene expression corresponded to statistically significant prophage induction events in the natural microbial populations.
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Long, A., McDaniel, L., Mobberley, and Paul, J.H., 2007 Comparison of Lysogeny (Prophage Induction) in Heterotrophic and Autotrophic Microbial Populations in the Gulf of Mexico. Journal of the International Society for Microbial Ecology, submitted.
McDaniel, L., delaRosa, M. & Paul, J.H., 2006. Temperate and lytic cyanophages from the Gulf of Mexico. Journal of the Marine Biological Association of the U.K., 86, 517-527.
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McDaniel, L. & Paul, J.H., 2005. Effect of nutrient addition and environmental factors on prophage induction in natural populations of marine Synechococcus species. Applied and Environmental Microbiology, 71, 842-850.
Mühling, M., Fuller, N.J., Millard, A., Somerfield, P.J., Marie, D., Wilson, W.H., Scanlan, D.J., Post, A.F., Joint, I. & Mann, N.H., 2005. Genetic diversity of marine Synechococcus and co-occuring cyanophage communities: evidence for viral control of phytoplankton. Environmental Microbiology, 7(4), 499-508.
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Sode, K., Oozeki, M., Asakawa, K., Burgess, J.G. & Matsunaga, T., 1994. Isolation of a marine cyanophage infecting the marine unicellular cyanobacterium, Synechoccus sp. NKBG 042902. Journal of Marine Biotechnology, 1, 189-192.