Virioplankton community structure in Tunisian solar salterns

Abstract

The microbial community inhabiting Sfax solar salterns on the east coast of Tunisia has been studied by means of different molecular and culture-dependent tools that have unveiled the presence of novel microbial groups as well as a community structure different from that of other coastal hypersaline environments. We have focused on the study of the viral assemblages of these salterns and their changes along the salinity gradient and over time. Viruses from three ponds (C4, M1, and TS) encompassing salinities from moderately hypersaline to saturated (around 14, 19, and 35%, respectively) were sampled in May and October 2009 and analyzed by transmission electron microscopy (TEM) and pulsed-field gel electrophoresis (PFGE). Additionally, for all three October samples and the May TS sample, viral metagenomic DNA was cloned in fosmids, end sequenced, and analyzed. Viral concentration, as well as virus-to-cell ratios, increased along the salinity gradient, with around 10(10) virus-like particles (VLPs)/ml in close-to-saturation ponds, which represents the highest viral concentration reported so far for aquatic systems. Four distinct morphologies could be observed with TEM (spherical, tailed, spindled, and filamentous) but with various proportions in the different samples. Metagenomic analyses indicated that every pond harbored a distinct viral assemblage whose G+C content could be roughly correlated with that of the active part of the microbial community that may have constituted the putative hosts. As previously reported for hypersaline metaviromes, most sequences did not have matches in the databases, although some were conserved among the Sfax metaviromes. BLASTx, BLASTp, and dinucleotide frequency analyses indicated that (i) factors additional to salinity could be structuring viral communities and (ii) every metavirome had unique gene contents and dinucleotide frequencies. Comparison with hypersaline metaviromes available in the databases indicated that the viral assemblages present in close-to-saturation environments located thousands of kilometers apart presented some common traits among them in spite of their differences regarding the putative hosts. A small core metavirome for close-to-saturation systems was found that contained 7 sequences of around 100 nucleotides (nt) whose function was not hinted at by in silico search results, although it most likely represents properties essential for hyperhalophilic viruses.

Fig 1

(A) Transmission electron micrographs showing different viral morphotypes found in Sfax salterns. Examples of micrographs used for counting (scale bar in images 1 to 3, 200 nm; viral particles are indicated by arrowheads) are shown, as are details of spherical (scale bar in image 4, 50 nm), spindle-like, and head-tailed (scale bar in image 5, 50 nm) and filamentous (scale bar in image 6, 20 nm) viruses. (B) Distribution of morphotypes (in percentages) detected on every analyzed sample by TEM.

Fig 2

(A) Pulsed-field gel electrophoresis of Sfax saltern viral assemblages. DNA bands are indicated by white triangles. M, lambda PFG marker; sizes of the bands are indicated in kilobases (kb). (B) Southern hybridization between the PFGE viral assemblages and labeled viral DNA from TSOct (the left image corresponds to transferred DNA from the May samples; the right image corresponds to transferred DNA from October samples).

Fig 3

(A) Domain-level classification of the best BLASTx hits produced by the four analyzed metaviromes. (B) Percentages of sequences producing BLASTx matches with halophilic or nonhalophilic Archaea, Bacteria, or viruses.

Fig 4

(A) All 520 ORFs from the distinct Sfax samples were assigned to a distinct numeric locator (from 1 to 520) and compared against themselves by using BLASTp. A diagonal straight line is given by self-matches. (B) Scatter plot of the three principal components based on the dinucleotide usage of Sfax salterns' complete sequence collection. (C) Symbols have been assigned according to a delivery into seven meaningful k-means groups and four distinct source samples.