Salinity is a major factor controlling the distribution of biota in aquatic systems, and most aquatic multicellular organisms are either adapted to life in saltwater or freshwater conditions. Consequently, the saltwater–freshwater mixing zones in coastal or estuarine areas are characterized by limited faunal and floral diversity. Although changes in diversity and decline in species richness in brackish waters is well documented in aquatic ecology, it is unknown to what extent this applies to bacterial communities. Here, we report a first detailed bacterial inventory from vertical profiles of 60 sampling stations distributed along the salinity gradient of the Baltic Sea, one of world's largest brackish water environments, generated using 454 pyrosequencing of partial (400 bp) 16S rRNA genes. Within the salinity gradient, bacterial community composition altered at broad and finer-scale phylogenetic levels. Analogous to faunal communities within brackish conditions, we identified a bacterial brackish water community comprising a diverse combination of freshwater and marine groups, along with populations unique to this environment. As water residence times in the Baltic Sea exceed 3 years, the observed bacterial community cannot be the result of mixing of fresh water and saltwater, but our study represents the first detailed description of an autochthonous brackish microbiome. In contrast to the decline in the diversity of multicellular organisms, reduced bacterial diversity at brackish conditions could not be established. It is possible that the rapid adaptation rate of bacteria has enabled a variety of lineages to fill what for higher organisms remains a challenging and relatively unoccupied ecological niche.

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Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea

Despite the impressive advances that have been made in assessing the diversity of marine microorganisms, the mechanisms that underlie the participation of microorganisms in marine food webs and biogeochemical cycles are poorly understood. Here, we stress the need to examine the biochemical interactions of microorganisms with ocean systems at the nanometre to millimetre scale--a scale that is relevant to microbial activities. The local impact of microorganisms on biogeochemical cycles must then be scaled up to make useful predictions of how marine ecosystems in the whole ocean might respond to global change. This approach to microbial oceanography is not only helpful, but is in fact indispensable.

Microbial structuring of marine ecosystems

Freshwater bacteria are at the hub of biogeochemical cycles and control water quality in lakes. Despite this, little is known about the identity and ecology of functionally significant lake bacteria. Molecular studies have identified many abundant lake bacteria, but there is a large variation in the taxonomic or phylogenetic breadths among the methods used for this exploration. Because of this, an inconsistent and overlapping naming structure has developed for freshwater bacteria, creating a significant obstacle to identifying coherent ecological traits among these groups. A discourse that unites the field is sorely needed. Here we present a new freshwater lake phylogeny constructed from all published 16S rRNA gene sequences from lake epilimnia and propose a unifying vocabulary to discuss freshwater taxa. With this new vocabulary in place, we review the current information on the ecology, ecophysiology, and distribution of lake bacteria and highlight newly identified phylotypes. In the second part of our review, we conduct meta-analyses on the compiled data, identifying distribution patterns for bacterial phylotypes among biomes and across environmental gradients in lakes. We conclude by emphasizing the role that this review can play in providing a coherent framework for future studies.

A Guide to the Natural History of Freshwater Lake Bacteria

Phytoplankton blooms characterize temperate ocean margin zones in spring. We investigated the bacterioplankton response to a diatom bloom in the North Sea and observed a dynamic succession of populations at genus-level resolution. Taxonomically distinct expressions of carbohydrate-active enzymes (transporters; in particular, TonB-dependent transporters) and phosphate acquisition strategies were found, indicating that distinct populations of Bacteroidetes, Gammaproteobacteria, and Alphaproteobacteria are specialized for successive decomposition of algal-derived organic matter. Our results suggest that algal substrate availability provided a series of ecological niches in which specialized populations could bloom. This reveals how planktonic species, despite their seemingly homogeneous habitat, can evade extinction by direct competition.

http://science.sciencemag.org/content/sci/336/6081/608.full.pdf

Substrate-Controlled Succession of Marine Bacterioplankton Populations Induced by a Phytoplankton Bloom

Marine phytoplankton blooms are annual spring events that are accompanied by a surge in heterotrophic bacteria, primarily roseobacters, flavobacteria and members of the Gammaproteobacteria, which recycle most of the carbon that is fixed by the primary producers. In this Review, Buchan et al. describe the emerging physiological features and functions of these bacterial communities and their interactions with phytoplankton.

Master recyclers: features and functions of bacteria associated with phytoplankton blooms

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes In particular, the abundances of aquatic Actinobacteria were significantly underestimated We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss Incubations with high concentrations of lysozyme (10 mg ml 1 ) followed by achromopeptidase (60 U ml 1 ) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%) Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%) Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton The taxonomic composition of picoplankton communities profoundly differs in freshwater and marine systems Cultivation-independent molecular approaches have demonstrated that bacteria from large phylogenetic lineages of typical freshwater microbes, eg, the -subdivision of the proteobacteria, are virtually absent in the marine picoplankton (15, 23) For other bacterial lineages the evidence is less conclusive Comparative analysis of 16S rDNA genes indicate that uncultured members of the class Actinobacteria are ubiquitous in lakes of various trophic state, size or geographic location (16, 36), but actinobacterial sequence types are also known from marine systems (31)

An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization.

We analysed the phylogenetic relatedness of 16S rRNA genes from freshwater bacteria affiliated with the class Actinobacteria. A polymerase chain reaction assay was developed to identify reliably rare Actinobacteria-related inserts within 16S rRNA gene clone libraries. In 18 libraries constructed from seven freshwater systems, altogether 63 actinobacterial sequence types were collected from a total of > 1800 clones. Sixty of the newly obtained sequences grouped within four distinct phylogenetic lineages. They constitute approximately 75% of the nearly complete sequences within these clusters that are presently available. A comparison with > 300 sequences from various soil habitats revealed that two of these monophyletic actinobacterial clades (acI and acII) almost exclusively harbour 16S rRNA sequence types from freshwaters and estuaries. This may indicate that such bacteria are not inoculated to freshwaters from terrestrial sources, but are autochthonous components of freshwater microbial assemblages. In contrast, sequence types from freshwaters, marine sediments and soils were clearly mixed in another of the actinobacterial lineages (acIV). Sequence divergence within acIV was the highest of all four lineages (88% minimum similarity), which potentially reflects its radiation across several habitat types. Within the freshwater lineages, groups of essentially identical sequence types were retrieved from geographically distant aquatic systems with strikingly different hydrological and limnological characteristics. This points to the necessity to investigate genotypic variability, in situ abundances and activities of these Actinobacteria in freshwater plankton in greater detail by cultivation-independent techniques.

Actinobacterial 16S rRNA genes from freshwater habitats cluster in four distinct lineages

Members of the class Actinobacteria (44) are regarded as typical inhabitants of soil environments (14). Evidence is accumulating that these bacteria are also present in a variety of freshwater habitats (7, 10, 17, 48, 49). Bacteria from this group may in fact constitute a large fraction of the bacterioplankton in oligo- to mesotrophic lakes or in humic lakes (8, 13, 36). In order to understand the role of Actinobacteria in different freshwater habitats, it is important to distinguish between the various phylogenetic groups within this lineage (13, 47, 48) and to determine their population sizes in situ. This can be achieved by fluorescence in situ hybridization (FISH) with specific rRNA-targeted oligonucleotide probes (36).

However, the occurrence of particular phylogenetic groups of Actinobacteria in the plankton of lakes does not necessarily imply that these bacteria are growing in this environment. Many cultivated Actinobacteria perform a life cycle which may involve a vegetative and a resting stage (i.e., spores) (11). Spores are highly resistant to unfavorable environmental conditions and can survive for extended periods of time. This life strategy promotes the survival and dispersal of Actinobacteria in soils. Thus, actinobacterial phylotypes that are found in freshwater might potentially be resting stages of predominently soil-dwelling allochthonous microorganisms. In order to prove that this phylogenetic group is an indigenous component of freshwater planktonic microbial assemblages, it is necessary to demonstrate cell growth in situ, e.g., by visualization of DNA de novo synthesis at the single-cell level (31).

Our knowledge on the environmental conditions that favor the occurrence of Actinobacteria in lakes is scarce. The first sequence types from this group originate from acidified lakes in the Adirondack Mountains (17). Actinobacteria were conspicuously abundant in the water column of an alpine lake that is characterized by high levels of incident solar UV radiation and by high water transparency (13, 42). Solar UV radiation is harmful to various aquatic microorganisms, including viruses, bacteria, and unicellular eukaryotes (19, 40, 45). Furthermore, UV radiation is known to inhibit bacteria-mediated processes (41), and picoplankton organisms from clear-water ecosystems accumulate significant UV-induced DNA damage (6). DNA rich in A+T content can be particularly susceptible due to the higher probability of production of cyclobutane thymine dimers. One typical feature of the Actinobacteria is their high genomic G+C content (5). It is thus conceivable that such bacteria might be favored in the bacterioplankton assemblages of UV-transparent lakes.

In order to test this hypothesis, the relative abundances of Actinobacteria were determined in the bacterial assemblages of 10 mountain lakes. These lakes are located along an altitude gradient, and they cover a wide range of water column UV transparencies. The composition of the microbial assemblages in these lakes was analyzed using a set of available and newly designed FISH probes. Specifically, we attempted to identify the numerically important taxa of freshwater Actinobacteria. In addition, the proportions of growing Actinobacteria were determined by pulse-labeling with bromodeoxyuridine (31).

Abundances, Identity, and Growth State of Actinobacteria in Mountain Lakes of Different UV Transparency

Humic lakes are systems often characterized by irregular high input of dissolved organic carbon (DOC) from the catchment. We hypothesized that specific bacterial groups which rapidly respond to changes in DOC availability might form large populations in such habitats. Seasonal changes of microbial community composition were studied in two compartments of an artificially divided bog lake with contrasting DOC inputs. These changes were compared to community shifts induced during short-term enrichment experiments. Inocula from the two compartments were diluted 1:10 into water from the more DOC-rich compartment, and inorganic nutrients were added to avoid microbial N and P limitation. The dilutions were incubated for a period of 2 weeks. The microbial assemblages were analyzed by cloning and sequencing of 16S rRNA genes and by fluorescence in situ hybridization with specific oligonucleotide probes. β-Proteobacteria from a cosmopolitan freshwater lineage related to Polynucleobacter necessarius (beta II) were rapidly enriched in all treatments. In contrast, members of the class Actinobacteria did not respond to the enhanced availability of DOC by an immediate increase in growth rate, and their relative abundances declined during the incubations. In lake water members of the beta II clade seasonally constituted up to 50% of all microbes in the water column. Bacteria from this lineage annually formed a significantly higher fraction of the microbial community in the lake compartment with a higher allochthonous influx than in the other compartment. Actinobacteria represented a second numerically important bacterioplankton group, but without clear differences between the compartments. We suggest that the pelagic microbial community of the studied system harbors two major components with fundamentally different growth strategies.

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Members of a Readily Enriched β-Proteobacterial Clade Are Common in Surface Waters of a Humic Lake

Summary Members of the phylum Bacteroidetes are among the most abundant microbes in coastal marine waters, but it is unclear to which extent the diversity within this phylum is covered by currently available 16S rRNA gene sequence information. We, thus, obtained a comprehensive collection of sequence types affiliated with Bacteroidetes in coastal North Sea surface waters and we compared this local diversity with the available sequences of marine planktonic and other aquatic Bacteroidetes. Approximately 15% of > 600 clones from two libraries (August 2000, June 2001) were related to Bacteroidetes, specifically to the Flavobacteria. Local diversity appeared to be almost exhaustively sampled. However, the diversity of the two libraries virtually did not overlap, indicating a pronounced temporal variability of the planktonic Flavobacteria assemblage. The majority of sequence types represented novel phylogenetic lineages, adding 6‐7% to the currently known genera and species of Bacteroidetes in marine waters. Different diversity estimators suggested that so far only approximately half of the global diversity of planktonic marine Bacteroidetes has been described. The data set moreover indicated that cultivationindependent techniques and isolation approaches have recovered almost equally sized and virtually non-overlapping fractions of the currently known diversity within this phylum. Interestingly, only 15% of genera of Bacteroidetes from various aquatic environments appear to occur in more than one habitat type.

Falk Warnecke

Papers

An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization.

Actinobacterial 16S rRNA genes from freshwater habitats cluster in four distinct lineages

Abundances, Identity, and Growth State of Actinobacteria in Mountain Lakes of Different UV Transparency

Members of a Readily Enriched β-Proteobacterial Clade Are Common in Surface Waters of a Humic Lake

High local and global diversity of Flavobacteria in marine plankton