Real-Time PCR Detection of Host-Mediated Cyanophage Gene Transcripts during Infection of a Natural Microcystis aeruginosa Population

Abstract

The aim of this study was to develop a quantitative real-time reverse transcription-PCR (real-time RT-PCR) assay to detect and quantify mRNA of cyanophages within infected Microcystis aeruginosa cells in a freshwater pond. Laboratory-based data showed that the relative abundance of the cyanophage g91 mRNA within host cells increased before cyanophage numbers increased in culture. This transcriptional pattern indicated the kinetics of the viral infection suggesting the real-time RT-PCR method to be a potential tool for environmental monitoring of cyanophage infections. In this field survey, the numbers of infected M. aeruginosa cell populations estimated from cyanophage numbers were low at 0.01-2.9 cells mL −1 . The highest relative abundance of phage g91 RNA (10 −2 per rnpB transcript) was at about the same levels of expression as laboratory-based growth data for Ma-LMM01 (estimated density of infected host cells: 10 5 cells mL −1 ); and was observed when cyanophage numbers rapidly increased (as well as a decrease in host cell numbers). Quantification of cyanophage numbers is important to understand ecological relationships between the phage and its hosts. Our data suggest the quantification of phage gene transcripts within a natural host cell population to be a strong tool for investigating the quantitative effects of phage lysis during infection of the host population.Key words: cyanophage, Microcystis aeruginosa, real-time RT-PCR, succession, toxic cyanobacteria Microcystis aeruginosa is a well-known toxic cyanobacterial species that commonly develops blooms in eutrophic freshwater throughout the world. This species includes strains that can produce potent hepatotoxins called microcystins (2). There are several reports of deaths in wild and domestic animals as well as humans due to acute poisoning which causes massive hepatic hemorrhage (1,6,16).Cyanophages are considered to be a significant factor regulating the abundance, clonal diversity, and composition of their cyanobacterial host populations (9,10,12,14,15,19,20,25). The phages also play a major role in nutrient cycling and genetic transfer (21,22,26). In contrast to the vast majority of research having focused on marine cyanophages (12,14,15,19,20,25), there are few studies concerning freshwater cyanophages (9, 10). Reports suggest phages play an important role in regulating the bloom dynamics of M. aeruginosa blooms. Manage et al. (9, 10) observed an increase in cyanophage titers (the numbers of particles forming plaques on an M. aeruginosa lawn) accompanied by a large decrease in the abundance of M. aeruginosa in a natural freshwater environment. Recently, during a field survey of a Japanese freshwater lake, real-time monitoring of M. aeruginosa-cyanophage abundance with quantitative PCR assays showed the seasonal dynamics of the cyanophage community in freshwater that may affect shifts in the clonal composition of diverse M. aeruginosa populations (e.g., microcystin-producing and non-producing populations); rather than having a quantitative impact o...

Rapid Microcystis Cyanophage Gene Diversification Revealed by Long- and Short-Term Genetic Analyses of the Tail Sheath Gene in a Natural Pond

“…To quantify abundances of total M. aeruginosa, a real-time PCR assay was performed using primers 188F and 254R, which are based on sequences of the phycocyanin intergenic spacer (PC-IGS) gene, as described previously (18).…”

“…The majority of the predicted genes in its genome have no detectable homologues in the existing databases, and thus, Ma-LMM01 was assigned as a member of a new lineage in the family Myoviridae (14,15). Since no strain closely related to Ma-LMM01 has been isolated, the degenerate primer set was designed based on environmental sequences of Ma-LMM01 g91 (tail sheath gene), which is phylogenetically distinct from other phage genes and has been used as a genetic marker of this phage (16)(17)(18). Clonal analysis using this primer set reveals high sequence divergence that is derived from single point mutations in natural populations (16).…”

Diurnal Infection Patterns and Impact of Microcystis Cyanophages in a Japanese Pond

“…Hirosawanoike Pond receives high nutrient input in relation to its volume due to its use for raising carp agriculturally. This results in eutrophication and cyanobacterial blooms from early summer to autumn every year (48). Samples of surface water were taken from a boat every 3 h over a period of 24 h on 15 to 16 September and on 21 to 22 October 2009 at a fixed point in the pond.…”

Intricate Interactions between the Bloom-Forming Cyanobacterium Microcystis aeruginosa and Foreign Genetic Elements, Revealed by Diversified Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Signatures

“…Previous studies have shown M. aeruginosa to be genetically highly heterogeneous (44,51); M. aeruginosa populations also undergo temporal changes in genotypic composition (4,52,53). Previously we isolated a phage, Ma-LMM01, infecting M. aeruginosa (57) and observed the potential qualitative impact of the cyanophage and its relatives on natural M. aeruginosa populations (54,56). However, Ma-LMM01 has a narrow host range despite the large genetic diversity of hosts, suggesting that there is a greater diversity of host-cyanophage combinations other than Ma-LMM01 and its host strain in natural M. aeruginosa populations (57).…”