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Comparative Study
. 1998 Jun;64(6):2051-60.
doi: 10.1128/AEM.64.6.2051-2060.1998.

Occurrence of a sequence in marine cyanophages similar to that of T4 g20 and its application to PCR-based detection and quantification techniques

N J Fuller  1 , W H Wilson, I R Joint, N H Mann
Affiliations
Comparative Study

Occurrence of a sequence in marine cyanophages similar to that of T4 g20 and its application to PCR-based detection and quantification techniques

N J Fuller et al. Appl Environ Microbiol. 1998 Jun.

Abstract

Viruses are ubiquitous components of marine ecosystems and are known to infect unicellular phycoerythrin-containing cyanobacteria belonging to the genus Synechococcus. A conserved region from the cyanophage genome was identified in three genetically distinct cyanomyoviruses, and a sequence analysis revealed that this region exhibited significant similarity to a gene encoding a capsid assembly protein (gp20) from the enteric coliphage T4. The results of a comparison of gene 20 sequences from three cyanomyoviruses and T4 allowed us to design two degenerate PCR primers, CPS1 and CPS2, which specifically amplified a 165-bp region from the majority of cyanomyoviruses tested. A competitive PCR (cPCR) analysis revealed that cyanomyovirus strains could be accurately enumerated, and it was demonstrated that quantification was log-linear over ca. 3 orders of magnitude. Different calibration curves were obtained for each of the three cyanomyovirus strains tested; consequently, cPCR performed with primers CPS1 and CPS2 could lead to substantial inaccuracies in estimates of phage abundance in natural assemblages. Further sequence analysis of cyanomyovirus gene 20 homologs would be necessary in order to design primers which do not exhibit phage-to-phage variability in priming efficiency. It was demonstrated that PCR products of the correct size could be amplified from seawater samples following 100x concentration and even directly without any prior concentration. Hence, the use of degenerate primers in PCR analyses of cyanophage populations should provide valuable data on the diversity of cyanophages in natural assemblages. Further optimization of procedures may ultimately lead to a sensitive assay which can be used to analyze natural cyanophage populations both quantitatively (by cPCR) and qualitatively following phylogenetic analysis of amplified products.

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Figures

FIG. 1
FIG. 1
Alignment of the nucleic acid sequences of ORFs of three marine cyanomyovirus strains (S-PM2, S-WHM1, and S-BnM1; GenBank accession no. AFO16384, AFO16385, and AFO16386, respectively) with the sequence of coliphage T4 gene 20, which encodes a capsid assembly protein (34), and with partial sequence data for the same region of coliphage RB49 (37). Conserved bases are enclosed in boxes. The 25-mer forward primer CPS1 (5′-GTAG[T/A]ATTTTCTACATTGA[C/T]GTTGG-3′) and the 23-mer reverse primer CPS2 (5′-GGTA[G/A]CCAGAAATC[C/T]TC[C/A]AGCAT-3′) cyanophage-specific sequences are indicated by arrows.
FIG. 1
FIG. 1
Alignment of the nucleic acid sequences of ORFs of three marine cyanomyovirus strains (S-PM2, S-WHM1, and S-BnM1; GenBank accession no. AFO16384, AFO16385, and AFO16386, respectively) with the sequence of coliphage T4 gene 20, which encodes a capsid assembly protein (34), and with partial sequence data for the same region of coliphage RB49 (37). Conserved bases are enclosed in boxes. The 25-mer forward primer CPS1 (5′-GTAG[T/A]ATTTTCTACATTGA[C/T]GTTGG-3′) and the 23-mer reverse primer CPS2 (5′-GGTA[G/A]CCAGAAATC[C/T]TC[C/A]AGCAT-3′) cyanophage-specific sequences are indicated by arrows.
FIG. 2
FIG. 2
Design of the internal standard for cPCR in which the cyanophage-specific primers CPS1 and CPS2 are used. A 1.5-kb PstI-EcoRI fragment, isolated from cyanophage S-BnM1, was cloned into pUC19 (pWHW04). A 193-bp SphI-PvuII fragment from pUC19 was subsequently cloned into an EcoNI site in pWHW04 to enlarge the region amplified by the primers.
FIG. 3
FIG. 3
Determination of the sensitivity of cyanophage-specific PCR primers CPS1 and CPS2. Serial dilutions of a filtered (pore size, 0.2 μm) cyanophage strain S-BnM1 lysate were enumerated by TOTO-1 iodide staining and were amplified in the absence of competitor DNA. Lanes 1 to 6, preparations containing ca. 1.9 × 106, 1.9 × 105, 1.9 × 104, 1.9 × 103, 190, and 19 cyanophage strain S-BnM1 particles per reaction mixture, respectively. A 15-μl aliquot of each PCR mixture was electrophoresed on a 1.2% agarose gel in 1× TBE buffer.
FIG. 4
FIG. 4
cPCR of decimal dilutions of cyanophage strain S-BnM1 lysate, each coamplified with 2 pg of competitor DNA (Fig. 2) by using cyanophage-specific primers CPS1 and CPS2. Lanes 1 to 5, preparations containing ca. 190, 1.9 × 103, 1.9 × 104, 1.9 × 105, and 1.9 × 106 cyanophage strain S-BnM1 particles per reaction mixture, respectively. Cyanophage counts were determined by epifluorescence microscopy of lysate stained with the nucleic acid dye TOTO-1. A 15-μl aliquot of each PCR mixture was electrophoresed on a 1.2% agarose gel in 1× TBE buffer.
FIG. 5
FIG. 5
cPCR calibration curves for target cyanophage strains S-BnM1 (▪), S-MM5 (•), and S-BM3 (▴), obtained by using DNase-treated lysates. The data are plotted as log10 target product/competitor product relative intensities (estimated with a computer densitometer) (y axis) against log10 total number cyanophage particles enumerated by fluorescence microscopy with TOTO-1 iodide staining (x axis). Portions (2 pg) of competitor DNA (Fig. 2) were added to decimal dilutions of each cyanophage, and the DNAs were coamplified by using cyanophage-specific primers CPS1 and CPS2. Standard deviations of the means (n = 3) are indicated by error bars.
FIG. 6
FIG. 6
Analysis of fragments amplified by PCR from natural cyanophage communities collected from coastal seawater off Trinidad and Barbados by using primers CPS1 and CPS2. Lanes 2 through 5 contained amplified products from either 1-μl (lanes 2 and 4) or 10-μl (lanes 3 and 5) aliquots of unconcentrated Trinidad seawater added to PCR mixtures; seawater was both filtered (pore size, 0.2 μm) (lanes 2 and 3) and not filtered (lanes 4 and 5) prior to amplification. Lanes 6 through 9 contained amplified products from either 1-μl (lanes 6 and 8) or 10-μl (lanes 7 and 9) aliquots of unconcentrated Barbados seawater added to PCR mixtures; seawater was both filtered (pore size, 0.2 μm) (lanes 6 and 7) and not filtered (lanes 8 and 9) prior to amplification. Lanes 10 and 11 contained amplified products from 1- and 10-μl aliquots, respectively, of filtered (pore size, 0.2 μm) Barbados seawater concentrated 100× by ultracentrifugation. Lane 1 was a positive control lane containing amplified cyanophage strain S-BnM1 lysate added to filtered (pore size, 0.2 μm) Barbados water. A 15-μl aliquot of each PCR mixture was electrophoresed on a 1.2% agarose gel in 1× TBE buffer.
FIG. 7
FIG. 7
Alignment of the predicted amino acid sequences encoded by ORFs of marine cyanomyovirus strains S-PM2, S-WHM1, and S-BnM1 (GenBank accession no. AFO16384, AFO16385, and AFO16386, respectively) with the sequence of the product of coliphage T4 gene 20, which encodes a capsid assembly protein (34), and with the predicted sequence of amino acids translated from partial sequence data for the same region of coliphage RB49 (37). Conserved residues are enclosed in boxes.
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