Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

Recently developed techniques for estimating bacterial biomass and productivity indicate that bacterial biomass in the sea is related to phytoplankton concentration and that bacteria utilise 10 to 50 % of carbon fixed by photosynthesis. Evidence is presented to suggest that numbers of free bacteria are controlled by nanoplankton~c heterotrophic flagellates which are ubiquitous in the marine water column. The flagellates in turn are preyed upon by microzooplankton. Heterotrophic flagellates and microzooplankton cover the same size range as the phytoplankton, thus providing the means for returning some energy from the 'microbial loop' to the conventional planktonic food chain.

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The Ecological Role of Water-Column Microbes in the Sea*

We review the biogeography of microorganisms in light of the biogeography of macroorganisms A large body of research supports the idea that free-living microbial taxa exhibit biogeographic patterns Current evidence confirms that, as proposed by the Baas-Becking hypothesis, 'the environment selects' and is, in part, responsible for spatial variation in microbial diversity However, recent studies also dispute the idea that 'everything is everywhere' We also consider how the processes that generate and maintain biogeographic patterns in macroorganisms could operate in the microbial world

/pdf/microbial-biogeography-putting-microorganisms-on-the-map-1k7e5do5pn.pdf

Microbial biogeography : putting microorganisms on the map

If stretched end to end, the estimated 1030viruses in the oceans would span farther than the nearest 60 galaxies. This reservoir of genetic and biological diversity continues to yield exciting discoveries and, in this Review, Curtis A. Suttle highlights the areas that are likely to be of greatest interest in the next few years. Viruses are by far the most abundant 'lifeforms' in the oceans and are the reservoir of most of the genetic diversity in the sea. The estimated 1030 viruses in the ocean, if stretched end to end, would span farther than the nearest 60 galaxies. Every second, approximately 1023 viral infections occur in the ocean. These infections are a major source of mortality, and cause disease in a range of organisms, from shrimp to whales. As a result, viruses influence the composition of marine communities and are a major force behind biogeochemical cycles. Each infection has the potential to introduce new genetic information into an organism or progeny virus, thereby driving the evolution of both host and viral assemblages. Probing this vast reservoir of genetic and biological diversity continues to yield exciting discoveries.

Marine viruses — major players in the global ecosystem

Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na+(Li+)/H+ antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies.

Metagenomics: Application of Genomics to Uncultured Microorganisms

Total bacterial abundances estimated with different epifluorescence microscopy methods (4′,6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, “live” bacteria (determined as such with the Molecular Probes Live/Dead BacLight bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and “dead” bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of “live” cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.

https://aem.asm.org/content/aem/65/10/4475.full.pdf

Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria.

Because of their small size, great abundance and easy dispersal, it is often assumed that marine planktonic microorganisms have a ubiquitous distribution that prevents any structured assembly into local communities. To challenge this view, marine bacterioplankton communities from coastal waters at nine locations distributed world-wide were examined through the use of comprehensive clone libraries of 16S ribosomal RNA genes, used as operational taxonomic units (OTU). Our survey and analyses show that there were marked differences in the composition and richness of OTUs between locations. Remarkably, the global marine bacterioplankton community showed a high degree of endemism, and conversely included few cosmopolitan OTUs. Our data were consistent with a latitudinal gradient of OTU richness. We observed a positive relationship between the relative OTU abundances and their range of occupation, i.e. cosmopolitans had the largest population sizes. Although OTU richness differed among locations, the distributions of the major taxonomic groups represented in the communities were analogous, and all local communities were similarly structured and dominated by a few OTUs showing variable taxonomic affiliations. The observed patterns of OTU richness indicate that similar evolutionary and ecological processes structured the communities. We conclude that marine bacterioplankton share many of the biogeographical and macroecological features of macroscopic organisms. The general processes behind those patterns are likely to be comparable across taxa and major global biomes.

Global patterns of diversity and community structure in marine bacterioplankton

The distribution of phytoplankton primary production into four size fractions (>10 μm, 10-3 μm, 3-0.2 μm and <0.2 μm), the utilization of algal exudates by bacteria and the bacterial production were studied in a eutrophication gradient in the northern Baltic proper. The polluted area exhibits substantially increased nutrient, especially nitrogen, levels while only minor differences occur in salinity and temperature regimes. Total primary production was 160 g C · m-2 · yr-1 at the control station and about 275 g C · m-2 · yr-1 at the eutrophicated stations. The estimated total exudate release was 16% of the totally fixed 14CO2 in the control area and 12% in the eutrophicated area (including the estimated bacterial uptake of exudates). The difference in14CO2 uptake rates between incubation of previously filtered water (<3, <2, <1 μm) and unfiltered water was used to estimate bacterial uptake of phytoplankton exudates which were found to contribute about half of the estimated bacterial carbon requirement in both areas. Bacterial production was estimated by the frequency of dividing cells (FDC) method as being 38 g C · m-2 · yr-1 at the control station and 50 g C · m-2 · yr-1 at the eutrophicated stations. To estimate the mean in situ bacterial cell volume a correlation between FDC and cell volume was used. The increased annual primary production in the eutrophicated area was due mainly to higher production during spring and autumn, largely by phytoplankton cells (mainly diatoms) retained by a 10 μm filter. Primary production duringsummer was similarin the two areas, as was the distribution on different size fractions. This could possibly explain the similar bacterial production in the trophic layers at all stations since the bulk of bacterial production occurs during summer. It was demonstrated that selective filtration does not quantitatively separate photoautotrophs and bacteria. A substantial fraction of the primary production occurs in the size fraction <3 μm. The primary production encountered in the 3-0.2 μm fraction was due to abundant picoplankton (0.5 to 8 · 107 ind · l-1), easily passing a 3 μm filter. The picoplankton was estimated to constitute up to 25% of the total phytoplankton biomass in the control area and up to 10% in the eutrophicated area.

Fractionated phytoplankton primary production, exudate release and bacterial production in a Baltic eutrophication gradient

Counts of heterotrophic bacteria in marine waters are usually in the order of 5 x 10(sup5) to 3 x 10(sup6) bacteria ml(sup-1). These numbers are derived from unspecific fluorescent staining techniques (J. E. Hobbie, R. J. Daley, and S. Jasper, Appl. Environ. Microbiol. 33:1225-1228, 1977; K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980) and are subsequently defined as total counts of bacteria. In samples from the Baltic Sea, the North Sea (Skagerrak), and the northeastern Mediterranean Sea, we found that only a minor fraction (2 to 32%) of total counts can be scored as bacteria with nucleoids. Lack of DNA no doubt means inactive cells; therefore, a much lower number of bacteria that grow at rates higher than those previously estimated must be responsible for the measured bacterial production in these seas. The remaining bacterium-sized and/or -shaped particles included in total counts may be cell residues of virus-lysed bacteria (ghosts) or remains of protozoan grazing.

Total counts of marine bacteria include a large fraction of non-nucleoid-containing bacteria (ghosts).

Consumption of dissolved organic carbon by marine bacteria and demand for inorganic nutrients.

/pdf/consumption-of-dissolved-organic-carbon-by-marine-bacteria-2db54m1gaw.pdf

Åke Hagström

Papers

Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria.

Global patterns of diversity and community structure in marine bacterioplankton

Fractionated phytoplankton primary production, exudate release and bacterial production in a Baltic eutrophication gradient

Total counts of marine bacteria include a large fraction of non-nucleoid-containing bacteria (ghosts).

Consumption of dissolved organic carbon by marine bacteria and demand for inorganic nutrients