Bacteria of the genus Limnohabitans, more precisely the R-BT lineage, have a prominent role in freshwater bacterioplankton communities due to their high rates of substrate uptake and growth, growth on algal-derived substrates and high mortality rates from bacterivory. Moreover, due to their generally larger mean cell volume, compared to typical bacterioplankton cells, they contribute over-proportionally to total bacterioplankton biomass. Here we present genetic, morphological and ecophysiological properties of 35 bacterial strains affiliated with the Limnohabitans genus newly isolated from 11 non-acidic European freshwater habitats. The low genetic diversity indicated by the previous studies using the ribosomal SSU gene highly contrasted with the surprisingly rich morphologies and different patterns in substrate utilization of isolated strains. Therefore, the intergenic spacer between 16S and 23S rRNA genes was successfully tested as a fine-scale marker to delineate individual lineages and even genotypes. For further studies, we propose the division of the Limnohabitans genus into five lineages (provisionally named as LimA, LimB, LimC, LimD and LimE) and also additional sublineages within the most diversified lineage LimC. Such a delineation is supported by the morphology of isolated strains which predetermine large differences in their ecology.

, isolated from the meso-eutrophic freshwater Ř ímov reservoir (Czech Republic), were characterized phenotypically, phylogenetically and chemotaxonomically. Both strains were chemo-organotrophic, facultatively anaerobic, non-motile rods, with identical DNA G+C contents of 59.9 mol%. Their major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine and their major fatty acids were C 16 : 1 v7c/C 16 : 1 v6c, C 16 : 0 , C 18 : 1 v7c/C 18 : 1 v6c and C 12 : 0 . Both strains contained Q-8 as the only respiratory quinone component. The 16S rRNA gene sequences of the two strains possessed 99.1 % similarity; however, the level of DNA-DNA reassociation was only 26.7 %. The strains can also be discriminated from each other by several chemotaxonomic and biochemical traits. Phylogenetic analysis of the 16S rRNA gene sequences revealed the affiliation of both strains with the genus Limnohabitans within the family Comamonadaceae. The two investigated strains represent the first isolated members of a narrow phylogenetic cluster (the socalled R-BT065 cluster) formed by a large number of environmental sequences and abundant populations detected in the pelagic zones of various freshwater habitats. We propose to place the two strains in separate novel species within the genus Limnohabitans, Limnohabitans planktonicus sp. nov., with the type strain II-D5 T (5DSM 21594 T 5CIP 109844 T ), and Limnohabitans parvus sp. nov., with the type strain II-B4 T (5DSM 21592 T 5CIP 109845 T ). The description of the genus Limnohabitans is emended accordingly.Only a few, phylogenetically narrow clusters are responsible for the ecological success of betaproteobacteria in freshwater habitats (e.g. Zwart et al., 2002). The so-called R-BT065 cluster (Šimek et al., 2001) represents such a phylogenetically defined group of abundant betaproteobacteria. This cluster was defined based only on environmental sequences retrieved from freshwater habitats, and represents a subgroup of the so-called 'Rhodoferax sp. BAL47' cluster (Zwart et al., 2002). Members of the R-BT065 cluster can be detected and quantified in environmental samples by a specific fluorescent in situ hybridization (FISH) probe (Šimek et al., 2001). Cells targeted by this probe possess a planktonic lifestyle and typically comprise 5-30 % (maximum~50 %) of the total bacterioplankton in non-acidic stagnant freshwater habitats (Šimek et al., 2001, 2005, 2010a Pérez & Sommaruga, 2006;Salcher et al., 2008).Ecological studies on bacteria from the R-BT065 cluster have revealed their ability to respond rapidly to changes in autochthonous (algal-derived) organic substrate supply or Details of the source reservoir, electron micrographs and results of 2D TLC of polar lipids of the novel strains and an extended 16S rRNA gene sequence-based tree including environmental sequences are available as supplementary material with the online version of this paper.

Small bacterivorous eukaryotes play a cardinal role in aquatic food webs and their taxonomic classification is currently a hot topic in aquatic microbial ecology. Despite increasing interest in their diversity, core questions regarding predator-prey specificity remain largely unanswered, e.g., which heterotrophic nanoflagellates (HNFs) are the main bacterivores in freshwaters and which prokaryotes support the growth of small HNFs. To answer these questions, we fed natural communities of HNFs from Římov reservoir (Czech Republic) with five different bacterial strains of the ubiquitous betaproteobacterial genera Polynucleobacter and Limnohabitans. We combined amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting eukaryotic 18 S rRNA genes to track specific responses of the natural HNF community to prey amendments. While amplicon sequencing provided valuable qualitative data and a basis for designing specific probes, the number of reads was insufficient to accurately quantify certain eukaryotic groups. We also applied a double-hybridization technique that allows simultaneous phylogenetic identification of both predator and prey. Our results show that community composition of HNFs is strongly dependent upon prey type. Surprisingly, Cryptophyta were the most abundant bacterivores, although this phylum has been so far assumed to be mainly autotrophic. Moreover, the growth of a small lineage of Cryptophyta (CRY1 clade) was strongly stimulated by one Limnohabitans strain in our experiment. Thus, our study is the first report that colorless Cryptophyta are major bacterivores in summer plankton samples and can play a key role in the carbon transfer from prokaryotes to higher trophic levels.

The distribution of the phylogenetically narrow R-BT065 cluster (Betaproteobacteria) in 102 freshwater lakes, reservoirs, and various ponds located in central Europe (a total of 122 samples) was examined by using a cluster-specific fluorescence in situ hybridization probe. These habitats differ markedly in pH, conductivity, trophic status, surface area, altitude, bedrock type, and other limnological characteristics. Despite the broad ecological diversity of the habitats investigated, the cluster was detected in 96.7% of the systems, and its occurrence was not restricted to a certain habitat type. However, the relative proportions of the cluster in the total bacterioplankton were significantly lower in humic and acidified lakes than in pH-neutral or alkaline habitats. On average, the cluster accounted for 9.4% of the total bacterioplankton (range, 0 to 29%). The relative abundance and absolute abundance of these bacteria were significantly and positively related to higher pH, conductivity, and the proportion of low-molecular-weight compounds in dissolved organic carbon (DOC) and negatively related to the total DOC and dissolved aromatic carbon contents. Together, these parameters explained 55.3% of the variability in the occurrence of the cluster. Surprisingly, no clear relationship of the R-BT065 bacteria to factors indicating the trophic status of habitats (i.e., different forms of phosphorus and chlorophyll a content) was found. Based on our results and previously published data, we concluded that the R-BT065 cluster represents a ubiquitous, highly active segment of bacterioplankton in nonacidic lakes and ponds and that alga-derived substrates likely form the main pool of substrates responsible for its high growth potential and broad distribution in freshwater habitats.Heterotrophic bacterioplankton assemblages found in a broad variety of freshwater ecosystems are frequently dominated by representatives of a few phylogenetic clusters of Betaproteobacteria and Actinobacteria (1,7,9,17,21,23,34,40). One of these clusters is the phylogenetically defined R-BT065 group (34), which is currently represented by Ͼ700 environmental ribosomal sequences deposited in the GenBank database. These sequences were obtained in several nonquantitative diversity studies of many European and North American freshwater habitats (5,10,18,27,30,42). The R-BT065 cluster is characterized by a minimum level of intragroup 16S rRNA sequence similarity of 97.3%, and investigations of recently cultivated strains demonstrated that members of the group are heterotrophic bacteria (V. Kasalický, J. Jezbera, K. Šimek, and M. W. Hahn, submitted for publication). This cluster forms a phylogenetically distinct subgroup of the so-called "Rhodoferax sp. BAL47" cluster (42). The new genus Limnohabitans was recently established for some strains affiliated with the "Rhodoferax sp. BAL47" cluster (11), and it has to be assumed that bacteria belonging to the R-BT065 cluster are also affiliated with this genus (Kasalický and coworkers, submitted for publicat...

Different bacterial strains can have different value as food for heterotrophic nanoflagellates (HNF), thus modulating HNF growth and community composition. We examined the influence of prey food quality using four Limnohabitans strains, one Polynucleobacter strain and one freshwater actinobacterial strain on growth (growth rate, length of lag phase and growth efficiency) and community composition of a natural HNF community from a freshwater reservoir. Pyrosequencing of eukaryotic small subunit rRNA amplicons was used to assess time-course changes in HNF community composition. All four Limnohabitans strains and the Polynucleobacter strain yielded significant HNF community growth while the actinobacterial strain did not although it was detected in HNF food vacuoles. Notably, even within the Limnohabitans strains we found significant preyrelated differences in HNF growth parameters, which could not be related only to size of the bacterial prey. Sequence data characterizing the HNF communities showed also that different bacterial prey items induced highly significant differences in community composition of flagellates. Generally, Stramenopiles dominated the communities and phylotypes closely related to Pedospumella (Chrysophyceae) were most abundant bacterivorous flagellates rapidly reacting to addition of the bacterial prey of high food quality.

Limnohabitans curvus gen. nov., sp. nov., a planktonic bacterium isolated from a freshwater lake A chemo-organotrophic, aerobic, facultatively anaerobic, non-motile strain, MWH-C5 T , isolated from the water column of the oligomesotrophic Lake Mondsee (Austria), was characterized phenotypically, phylogenetically and chemotaxonomically. The predominant fatty acids of the strain were C 16 : 1 v7c/v6c, C 16 : 0 , C 12 : 1 and C 8 : 0 -3OH, the major quinone was ubiquinone Q-8 and the G+C content of the DNA of the strain was 55.5 mol%. 16S rRNA gene similarity to the closest related type strains was 96.6 % (Curvibacter delicatus LMG 4328 T ) and 95.7 % (Rhodoferax fermentans FR3 T ). Phylogenetic analysis of 16S rRNA gene sequences revealed the affiliation of the strain with the family Comamonadaceae (Betaproteobacteria); however, the phylogenetic position of the strain did not support an affiliation to any previously described genus within this family. A family-wide comparison of traits revealed that the strain possesses a unique combination of DNA G+C content, major fatty acids and major 3-hydroxy fatty acid. Furthermore, the strain differs in several traits from the closest related genera. Based on the phylogeny of the strain and differences from closely related genera, we propose to establish the new genus and species Limnohabitans curvus gen. nov., sp. nov. to accommodate this strain. The type strain of Limnohabitans curvus is MWH-C5 T (5DSM 21645 T 5CCUG 56720 T ). The type strain is closely related to a large number of uncultured bacteria detected by cultivation-independent methods in various freshwater systems.The bacterioplankton of freshwater habitats is composed mainly of phylogenetic groups absent from marine bacterioplankton and terrestrial habitats (Hahn, 2006; Zwart et al., 2002). Investigations with cultivationindependent methods have demonstrated that the majority of taxa dwelling in the water column of freshwater lakes and ponds represent uncultured and undescribed taxa (Crump et al., 1999;Eiler & Bertilsson, 2004; Zwart et al., 2002). In this paper, we characterize a strain isolated from the pelagic zone of a freshwater lake that is closely related to uncultured bacteria numerously detected in freshwater samples and propose to establish a new genus and species within the family Comamonadaceae (Betaproteobacteria) to accommodate this strain.Strain MWH-C5 T was isolated from deep, oligomesotrophic Lake Mondsee (47 u 509 2.920 N 13 u 229 25.980 E) located in Austria. The strain was obtained by using the dilution-acclimatization method (DAM) and nutrient broth soytone yeast extract (NSY) medium (Hahn et al., 2004(Hahn et al., , 2005. The isolated bacterium grows on a variety of solidified complex media, e.g. Luria-Bertani agar (Difco BD), casitone agar (Difco BD), R2A agar (Remel) and NSY agar (Hahn et al., 2004), forming unpigmented, smooth, convex colonies.Tests of growth of the strain on single carbon sources resulted only in weak growth. In order to avoid falsenegative results in such tests caus...

We examined the proportions of major Betaproteobacteria subgroups within bacterial communities in diverse nonaxenic, monospecific cultures of algae or cyanobacteria: four species of cryptophyta (genera Cryptomonas and Rhodomonas), four species of chlorophyta (genera Pediastrum, Staurastrum, and Chlamydomonas), and two species of cyanobacteria (genera Dolichospermum and Aphanizomenon). In the cryptophyta cultures, Betaproteobacteria represented 48 to 71% of total bacteria, the genus Limnohabitans represented 18 to 26%, and the Polynucleobacter B subcluster represented 5 to 16%. In the taxonomically diverse chlorophyta group, the genus Limnohabitans accounted for 7 to 45% of total bacteria. In contrast, cyanobacterial cultures contained significantly lower proportions of the Limnohabitans bacteria (1 to 3% of the total) than the cryptophyta and chlorophyta cultures. Notably, largely absent in all of the cultures was Polynucleobacter necessarius (Polynucleobacter C subcluster). Subsequently, we examined the growth of Limnohabitans strains in the presence of different algae or their extracellular products (EPP). Two strains, affiliated with Limnohabitans planktonicus and Limnohabitans parvus, were separately inoculated into axenic cultures of three algal species growing in an inorganic medium: Cryptomonas sp., Chlamydomonas noctigama, and Pediastrum boryanum. The Limnohabitans strains cocultured with these algae or inoculated into their EPP consistently showed (i) pronounced population growth compared to the control without the algae or EPP and (ii) stronger growth stimulation of L. planktonicus than of L. parvus. Overall, growth responses of the Limnohabitans strains cultured with algae were highly species specific, which suggests a pronounced niche separation between two closely related Limnohabitans species likely mediated by different abilities to utilize the substrates produced by different algal species.There is compelling evidence that phytoplankton community dynamics have a significant impact on the composition of bacterioplankton communities (for example, see references 6, 19, and 21). The apparent driving force of such alga-bacterium interactions is likely the nature and quantity of alga-derived substrates available in the form of extracellular phytoplankton products (EPP) or decaying algal biomass. Although it is usually not known which algal species are the major EPP producers in situ, tight species-specific alga-bacterium relationships have been suggested as characterizing bacterium-alga consortia (e.g., 6, 21, 33). Interactions of phytoplankton and bacteria range from symbiotic to parasitic relationships (3). It is not surprising, then, that specific bacterial assemblages associated with different algae can also stimulate or even inhibit algal growth, as documented for cultures of marine diatoms (6). The latter study also demonstrated that free-living and phytoplankton-associated (i.e., attached to algal surfaces) bacteria are significantly different from each other and are dominated by distinct phylog...

How symbioses between bacteria and aquatic animals influence food webs in freshwater ecosystems is a fundamental question in ecology. We investigated symbiosis between a crustacean zooplankton Daphnia magna and its dominant bacterial symbiont Limnohabitans, an abundant and globally distributed freshwater Betaproteobacteria. Aposymbiotic juvenile Daphnia were prepared and exposed to any of four Limnohabitans sp. - Limnohabitans strains DM1, 2KL-3, 2KL-7 and Limnohabitans planktonicus strain II-D5, all previously found in D. magna digestive tract or culture. Re-infected Daphnia were cultured until they produced the first clutch of juveniles. Limnohabitans strain DM1 and L. planktonicus strain II-D5 successfully re-infected Daphnia through single exposure at the first instar juvenile stage. In contrast to aposymbiotic Daphnia that produced non-viable juveniles, re-infected Daphnia produced viable juveniles and increased fecundity to levels of that of symbiotic Daphnia. Re-infected Daphnia did not increase their number of eggs nor growth rates. Limnohabitans strains 2KL-7 and 2KL-3 could not recover fecundity even in multiple exposures during culture. This study shows the functional evidence demonstrating that a single bacterium Limnohabitans regulates fecundity of the consumer Daphnia through symbiosis. Our results indicated that symbiotic relationship between major bacterioplankton and zooplankton is important for maintaining the population of zooplankton in freshwater ecosystems.