Identification of a diagnostic marker to detect freshwater cyanophages of filamentous cyanobacteria

Abstract

Cyanophages are viruses that infect the cyanobacteria, globally important photosynthetic microorganisms. Cyanophages are considered significant components of microbial communities, playing major roles in influencing host community diversity and primary productivity, terminating cyanobacterial water blooms, and influencing biogeochemical cycles. Cyanophages are ubiquitous in both marine and freshwater systems; however, the majority of molecular research has been biased toward the study of marine cyanophages. In this study, a diagnostic probe was developed to detect freshwater cyanophages in natural waters. Oligonucleotide PCR-based primers were designed to specifically amplify the major capsid protein gene from previously characterized freshwater cyanomyoviruses that are infectious to the filamentous, nitrogen-fixing cyanobacterial genera Anabaena and Nostoc. The primers were also successful in yielding PCR products from mixed virus communities concentrated from water samples collected from freshwater lakes in the United Kingdom. The probes are thought to provide a useful tool for the investigation of cyanophage diversity in freshwater environments.

FIG. 1.

Partial sequence of the putative AN-15 major capsid protein generated from the PAN15F1 primer. The boldface indicates where the forward primer has annealed, as expected, and also how the degeneracy of the forward primer has enabled it to anneal downstream and also act as the reverse primer.

FIG. 2.

Full nucleotide sequence for the putative MCP of AN-15. The N-terminal sequence match (amplified using the PAN15F1 primer set) is underlined. The positions of the primers MCP5F5 (forward) and MCPR5 (reverse) are underlined and boldface (forward primer position, 564 to 586; reverse primer position, 894 to 918). The primers amplified a 350-bp region of the MCP gene.

FIG. 3.

Analysis of the major capsid protein gene by PCR, using the MCPF5/R5 primer pair. Twenty microliters of the PCR products was run on a 1.2% agarose gel, which was subsequently stained with ethidium bromide. Cyanophage strains: AN-15 (lane 1), A-1 (L) (lane 2), and N-1 (lane 3). Virus concentrates collected from freshwater lakes, with the date of collection (day/month/year) and DNA extraction method (p, phenol; c, CTAB): Priest Pot 260302 (p) (lane 4), Priest Pot 090402 (p) (lane 5), Priest Pot 230402 (p) (lane 6), Priest Pot 080502 (p) (lane 7), Priest Pot 210502 (p) (lane 8), Priest Pot 180602 (p) (lane 9), Priest Pot 160702 (p) (lane 10), Priest Pot 130802 (p) (lane 11), Priest Pot 100904 (p) (lane 12), Priest Pot 081002 (p) (lane 13), Priest Pot 210502 (c) (lane 14), Priest Pot 180602 (c) (lane 15), Esthwaite 160702 (p) (lane 16), Esthwaite 130802 (p) (lane 17), and negative control (lane 18). The positions of the λ Pst markers are labeled on the right-hand side of the gel.