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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]

Profiles microbiologist Peter Hirsch who made contributions in the field of microbial ecology through various research and publication of papers and reviews, in line with his 75th birthday. Branch of science that are of interest to Hirscht; Educational background; Career highlights as a microbiologist; List of original papers and reviews that have been published by Hirsch.

In the Baltic Sea redoxcline, lysogenic viruses infecting prokaryotes have rarely been detected using the commonly used inducing agent mitomycin C. However, it is well known that not all viruses are induceable by mitomycin C and growing evidence suggests that changes in trophic conditions may trigger the induction of lysogenic viruses. We hypothesized that using antibiotics to simulate a strong change in trophic conditions for antibiotica-resistant cells due to reduced competition for resources might lead to the induction of lysogenic viruses into the lytic cycle within these cells. This hypothesis was tested by incubating prokaryotes obtained throughout the Baltic Sea redoxcline in seawater with substantially reduced numbers of viruses. We used a mixture of the protein synthesis-inhibiting antibiotics streptomycin and erythromycin to induce the desired changes in trophic conditions for resistant cells and at the same time ensuring that no progeny viruses were formed in sensitive cells. No inducible lysogenic viruses could be detected in incubations amended with mitomycin C. Yet, the presence of streptomycin and erythromycin increased virus-induced mortality of prokaryotes by 56–930% compared to controls, resulting in the induction of lysogenic viruses equivalent to 2–14% of in situ prokaryotic abundance. The results indicate the existence of a previously unrecognized induction mechanism for lysogenic viruses in the Baltic Sea redoxcline, as the mode of action distinctly differs between the used antibiotics (no virus production within affected cells) and mitomycin C (lysogenic viruses are produced within affected cells). Obtaining accurate experimental data on levels of lysogeny in prokaryotic host cells remains challenging, as relying on mitomycin C alone may severely underestimate lysogeny. [ABSTRACT FROM AUTHOR]

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]

The biodegradability of terrigenous dissolved organic matter (tDOM) exported to the sea has a major impact on the global carbon cycle, but our understanding of tDOM bioavailability is fragmentary. In this study, the effects of preparative tDOM isolation on microbial decomposition were investigated in incubation experiments consisting of mesocosms containing mesohaline water from the Baltic Sea. Dissolved organic carbon (DOC) consumption, molecular DOM composition, bacterial activities, and shifts in bacterial community structure were compared between mesocosms supplemented with riverine tDOM, either as filtered, particle-free river water or as a concentrate obtained by lyophilization/tangential ultrafiltration, and those containing only Baltic Sea water or river water. As shown using ultra-high-resolution mass spectrometry (15 Tesla Fourier-transform ion cyclotron resonance mass spectrometry, FT-ICR-MS) covering approximately 4600 different DOM compounds, the three DOM preparation protocols resulted in distinct patterns of molecular DOM composition. However, despite DOC losses of 4–16% and considerable bacterial production, there was no significant change in DOM composition during the 28-day experiment. Moreover, tDOM addition affected neither DOC degradation nor bacterial dynamics significantly, regardless of the tDOM preparation. This result suggested that the introduced tDOM was largely not bioavailable, at least on the temporal scale of our experiment, and that the observed bacterial activity and DOC decomposition mainly reflected the degradation of unknown, labile, colloidal and low-molecular weight DOM, both of which escape the analytical window of FT-ICR-MS. In contrast to the different tDOM preparations, the initial bacterial inoculum and batch culture conditions determined bacterial community succession and superseded the effects of tDOM addition. The uncoupling of tDOM and bacterial dynamics suggests that mesohaline bacterial communities cannot efficiently utilize tDOM and that in subarctic estuaries other factors are responsible for the removal of imported tDOM. [ABSTRACT FROM AUTHOR]

Marine pelagic redoxclines are zones of high dark CO2 fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO2 fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO2 incorporation in 13C-bicarbonate pulse experiments. The incorporation of 13C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in 13C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO2-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the 13C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO2-fixing Proteobacteria within this habitat. [ABSTRACT FROM AUTHOR]