Actinorhodopsin genes discovered diverse in freshwater habitats and among cultivated Actinobacteria

Microbial rhodopsins are membrane proteins that utilize a retinal chromophore to harvest sunlight for energetic and photosensory functions. Recently, a group of novel rhodopsin sequences named ‘actinorhodopsins’ (ActRs) was hypothesized to exist among uncultured planktonic Actinobacteria. ActRs were discovered by mining metagenomic data obtained during the Venter Institute’s Global Ocean Sampling expedition, from a hypersaline lagoon, two estuaries and a freshwater lake. On the basis of these findings, and many studies that show Actinobacteria are common inhabitants of lakes, we predicted that ActR genes would likely be present in other freshwater habitats and among the genomes of cultivated Actinobacteria. Using degenerate polymerase chain reaction primers, we discovered an ActR gene present in an actinobacterial isolate of the family Microbacteriaceae. Isolate MWH-Uga1 was cultivated prior to this study from a freshwater pond in Uganda and belongs to a group of Actinobacteria previously identified in freshwater ecosystems. ActR genes were also discovered present in numerous mixed cultures containing freshwater Actinobacteria and among environmental DNA samples obtained from three freshwater sources; a small woodland pond and the Laurentian Great Lakes Superior and Erie. An analysis of small subunit ribosomal RNA genes from metagenomic DNA samples harboring ActR genes suggests that organisms belonging to the acI lineage, an uncultured group of Actinobacteria commonly present in fresh waters, may utilize rhodopsins. The co-occurrence of an acI organism with a specific ActR variant in a mixed culture supports our hypothesis

The Passive Yet Successful Way of Planktonic Life: Genomic and Experimental Analysis of the Ecology of a Free-Living Polynucleobacter Population

Background: The bacterial taxon Polynucleobacter necessarius subspecies asymbioticus represents a group of planktonic freshwater bacteria with cosmopolitan and ubiquitous distribution in standing freshwater habitats. These bacteria comprise,1 % to 70 % (on average about 20%) of total bacterioplankton cells in various freshwater habitats. The ubiquity of this taxon was recently explained by intra-taxon ecological diversification, i.e. specialization of lineages to specific environmental conditions; however, details on specific adaptations are not known. Here we investigated by means of genomic and experimental analyses the ecological adaptation of a persistent population dwelling in a small acidic pond. Findings: The investigated population (F10 lineage) contributed on average 11 % to total bacterioplankton in the pond during the vegetation periods (ice-free period, usually May to November). Only a low degree of genetic diversification of the population could be revealed. These bacteria are characterized by a small genome size (2.1 Mb), a relatively small number of genes involved in transduction of environmental signals, and the lack of motility and quorum sensing. Experiments indicated that these bacteria live as chemoorganotrophs by mainly utilizing low-molecular-weight substrates derived from photooxidation of humic substances. Conclusions: Evolutionary genome streamlining resulted in a highly passive lifestyle so far only known among free-living bacteria from pelagic marine taxa dwelling in environmentally stable nutrient-poor off-shore systems. Surprisingly, such a lifestyle is also successful in a highly dynamic and nutrient-richer environment such as the water column of the investigate

Metagenomic and Metabolic Profiling of Nonlithifying and Lithifying Stromatolitic Mats of Highborne Cay, The Bahamas

BACKGROUND: Stromatolites are laminated carbonate build-ups formed by the metabolic activity of microbial mats and represent one of the oldest known ecosystems on Earth. In this study, we examined a living stromatolite located within the Exuma Sound, The Bahamas and profiled the metagenome and metabolic potential underlying these complex microbial communities. METHODOLOGY/PRINCIPAL FINDINGS: The metagenomes of the two dominant stromatolitic mat types, a nonlithifying (Type 1) and lithifying (Type 3) microbial mat, were partially sequenced and compared. This deep-sequencing approach was complemented by profiling the substrate utilization patterns of the mats using metabolic microarrays. Taxonomic assessment of the protein-encoding genes confirmed previous SSU rRNA analyses that bacteria dominate the metagenome of both mat types. Eukaryotes comprised less than 13% of the metagenomes and were rich in sequences associated with nematodes and heterotrophic protists. Comparative genomic analyses of the functional genes revealed extensive similarities in most of the subsystems between the nonlithifying and lithifying mat types. The one exception was an increase in the relative abundance of certain genes associated with carbohydrate metabolism in the lithifying Type 3 mats. Specifically, genes associated with the degradation of carbohydrates commonly found in exopolymeric substances, such as hexoses, deoxy- and acidic sugars were found. The genetic differences in carbohydrate metabolisms between the two mat types were confirmed using metabolic microarrays. Lithifying mats had a significant increase in diversity and utilization of carbon, nitrogen, phosphorus and sulfur substrates. CONCLUSION/SIGNIFICANCE: The two stromatolitic mat types retained similar microbial communities, functional diversity and many genetic components within their metagenomes. However, there were major differences detected in the activity and genetic pathways of organic carbon utilization. These differences provide a strong link between the metagenome and the physiology of the mats, as well as new insights into the biological processes associated with carbonate precipitation in modern marine stromatolites

Enrichment of Omnivorous Cercozoan Nanoflagellates from Coastal Baltic Sea Waters

Free-living nano-sized flagellates are important bacterivores in aquatic habitats. However, some slightly larger forms can also be omnivorous, i.e., forage upon both bacterial and eukaryotic resources. This hitherto largely ignored feeding mode may have pronounced implications for the interpretation of experiments about protistan bacterivory. We followed the response of an uncultured group of omnivorous cercozoan nanoflagellates from the Novel Clade 2 (Cerc_BAL02) to experimental food web manipulation in samples from the Gulf of Gdańsk (Southern Baltic Sea). Seawater was either prefiltered through 5 µm filters to exclude larger predators of nanoflagellates (F-treatment), or prefiltered and subsequently 1∶10 diluted with sterile seawater (F+D-treatment) to stimulate the growth of both, flagellates and bacteria. Initially, Cerc_BAL02 were rapidly enriched under both conditions. They foraged on both, eukaryotic prey and bacteria, and were highly competitive at low concentrations of food. However, these omnivores were later only successful in the F+D treatment, where they eventually represented almost one fifth of all aplastidic nanoflagellates. By contrast, their numbers stagnated in the F-treatment, possibly due to top-down control by a concomitant bloom of other, unidentified flagellates. In analogy with observations about the enrichment of opportunistically growing bacteria in comparable experimental setups we suggest that the low numbers of omnivorous Cerc_Bal02 flagellates in waters of the Gulf of Gdańsk might also be related to their vulnerability to grazing pressure

Top-down and bottom-up induced shifts in bacterial abundance, production and community composition in an experimentally divided humic lake

We examined in situ abundance and activities of the major bacterial groups in the two most distinct compartments of experimentally divided Lake Grosse Fuchskuhle (Germany). The selected south‐west (SW) and north‐east basin (NE) differ substantially in their major chemical and biological parameters that potentially influence the dynamics and composition of microbial communities. Water from the basins were incubated in dialysis bags, which allowed for a relatively free exchange of nutrients, limiting solutes and low molecular organic matter but fully prevented exchange of organisms. To investigate the effect of top‐down and bottom‐up manipulations three size fractions of water samples were produced: (i) unfiltered, (ii) pre‐filtered through 5.0 μm pore size membranes to remove large particles, as well as grazers and (iii) pre‐filtered through 0.8 μm filters to remove all potential bacterivores. One set of dialysis bags was either incubated in acidic SW (rich in humic matter) or in almost neutral NE basin whereas a second set was transferred from the SW to the NE basin and vice versa. Our study revealed pronounced differences in growth rates among the major bacterial groups in relation to the treatments. Members of the Betaproteobacteria, in particular of the subgroup targeted by the BETA2‐870 probe, were highly abundant in both basins, and most of them belonged to the Polynucleobacter necessesarius subcluster PnecC. Their specific growth rates surprisingly increased in all treatments when being transplanted into the acidic SW basin, indicating that pH and humic substances greatly affected growth of this particular group in the lake. In contrast, members of the Sphingobacteria/Flavobacteria group of the Bacteroidetes (both basins) as well as Actinobacteria (SW basin) were less abundant, especially in the presence of flagellates (< 5.0 μm treatments). However, because of their extremely low initial numbers, grazing of heterotrophic nanoflagellates mostly controlled only a small part of the bacterial production (≤ 12%)

Shifts in cell size and community composition of bacterioplankton due to grazing by heterotrophic flagellates: evidence from a marine system

The effect of flagellate grazing on bacterioplankton community composition, e.g. cell size and diversity, has been well studied in fresh waters, but much less is known for marine systems. We conducted experiments with communities from an oligotrophic bay in the NW Mediterranean Sea; size fractionation was used to alter grazing by flagellates, and incubation in dialysis bags was used to retain otherwise in situ conditions. Grazing reduced bacterial abundance and growth. In contrast to a current model of the effects of grazing, cell size distribution was unimodal and not bimodal. Significant shifts towards larger cell size classes were observed. This is in accordance with the idea that increasing the cell length is a bacterial strategy to reduce susceptibility to grazing by small flagellates. Only weak evidence was found for the complementary strategy of reducing cell size. In all experiments, bacterial community structure as assessed by PCR-based 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) showed significant differences associated with the presence of grazers. Sequence analyses of DGGE bands were used to select probes for catalyzed reporter deposition-fluorescence in situ hybridization analysis for specific taxa. In our experiments, Polaribacter showed patterns of defense against grazing, while Roseobacter was highly susceptible to grazing. Although the effect of grazing may be stronger in freshwater than in oligotrophic marine systems, our data support the hypotheses that (1) increasing the cell size is a bacterial defense strategy against grazing by small flagellates typical in marine systems and (2) grazing influences bacterial community composition