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Throughout the Baltic Sea redoxcline, virus production and the frequency of lytically-infected prokaryotic cells were estimated from parallel incubations of undiluted seawater and seawater that contained prokaryotes with substantially reduced numbers of viruses (virus dilution approach), effectively preventing viral reinfection during the incubation period. Undiluted seawater incubations resulted in much higher estimates of virus production (6–35×104 mL-1 h-1) and the frequency of infected cells (5–84%) than the virus dilution approach (virus production: 1–3×104 mL-1 h-1; frequency of infected cells: 1–11%). Viral production and the frequency of infected cells from both approaches, however, cannot be directly compared, as data obtained from undiluted incubations were biased by viral reinfection and other uncontrollable processes during the incubation period. High in situ viral abundance (1–2×107 mL-1) together with low virus production rates based on the virus dilution approach resulted in some of the longest viral turnover times (24–84 d) ever reported for the epipelagial. Throughout a wide range of environmental conditions, viral turnover time and burst size were negatively correlated. Given that viral decay estimated in ultra-filtered water was below the detection limit and the burst size was low (1–17), we conclude that prokaryotic viruses in the Baltic Sea redoxcline are investing most of their resources into stress defense (strong capsids) rather than proliferation (high burst size). In summary, the Baltic Sea redoxcline constitutes an environment where low virus production is found in combination with low viral decay, resulting in high viral abundance. [ABSTRACT FROM AUTHOR]

Molecular surveys of marine picoeukaryotes have revealed a large number of sequences unrelated to cultured organisms, such as those forming the marine stramenopile (MAST)-4 clade. Recent FISH (fluorescent in situ hybridization) data have shown that MAST-4 cells are uncultured heterotrophic flagellates of 2–3 μm in size that have a global distribution in non-polar marine waters. However, FISH is time-consuming and hard to apply to the many samples generated during oceanographic cruises, so we developed a real-time quantitative polymerase chain reaction (Q-PCR) protocol to determine rapidly the abundance of this group using environmental DNA. We designed a primer set targeting the 18S rRNA genes (rDNA) of MAST-4 and optimized and calibrated the Q-PCR protocol using a plasmid with the target sequence as insert. The Q-PCR was then applied to quantify MAST-4 rDNA molecules along three marine transects, longitudinal in the Indian Ocean, latitudinal in the Drake Passage and coastal–offshore in the Mediterranean Sea, and to a temporal study in a Mediterranean Sea coastal station. MAST-4 was detected in all samples processed (averaged abundances between 500 and 1000 rDNA molecules ml−1) except in mesopelagic and Antarctic samples, where it was virtually absent. In general, it was more abundant in the coast than offshore and in the deep chlorophyll maximum than at surface. A comparison of Q-PCR and FISH signals in well-controlled microbial incubations indicated that MAST-4 cells have around 30 copies of the rDNA operon. This Q-PCR assay quickly yielded quantitative data of uncultured MAST-4 cells and confirmed their wide distribution and putative ecological importance. [ABSTRACT FROM AUTHOR]

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the “Epsilonproteobacteria” related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 × 10³ to 4.4 × 109 copies ml-1 or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 × 10¹ to 2.2 × 106 copies ml-1 or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml-1. The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment. [ABSTRACT FROM AUTHOR]