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Five heterotrophic, aerobic, catalase- and oxidase-positive, non-motile strains from freshwater habitats located in Austria, France, Uganda, P. R. China, and New Zealand were characterized. The strains share 16S rRNA gene similarities ≥ 99.3%. The strains grew on NSY medium over a temperature range of 10–35 °C (two strains also grew at 5 °C, and one strain also grew at 38 °C) and a NaCl tolerance range of 0.0–0.3 % (four strains grew up to 0.5% NaCl). The predominant fatty acids were C16:0, C18:1ω7c, C12:0-3OH, and feature 3 (including C16:1ω7c). The DNA G+C content of the type strain MWH-MoIso2T was 44.9 mol%. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that the five strains formed a monophyletic cluster closely related to Polynucleobacter necessarius (96-97% sequence similarity). This cluster also harbours other isolates as well as environmental sequences, which have been obtained from several habitats. Investigations with taxon-specific FISH probes demonstrated that these bacteria dwell as free-living, planktonic cells in freshwater systems. Based on the revealed phylogeny and pronounced chemotaxonomic differences to P. necessarius (presence of>7 % C12:0-3OH, absence of C12:0 and C12:0-2OH), we propose to establish for the investigated strains the new species Polynucleobacter cosmopolitanus sp. nov. with the type strain MWH-MoIso2T (= DSM 21490T, = CIP 109840T). The proposed species belongs to the minority of described species of free-living bacteria for which both in situ data from their natural environments and culture-based knowledge is available.
K. Heckmann and H.-J. Schmidt described the genus Polynucleobacter for bacterial endosymbionts of freshwater ciliates within the genus Euplotes, and the species Polynucleobacter necessarius for obligate endosymbionts living in the cytoplasm of E. aediculatus (Heckmann and Schmidt, 1987). Phylogenetic analysis of the 16S rRNA gene sequence of an endosymbiotic P. necessarius strain in an E. aediculatus culture revealed the affiliation of the strain to the class Betaproteobacteria (Springer et al., 1996). Recently, it was demonstrated that the obligate endosymbionts representing the species P. necessarius are closely related with obligately free-living strains (Hahn, 2003; Vannini et al., 2007). Therefore, the description of the genus Polynucleobacter and its sole species P. necessarius was emended by addition of descriptions of free-living strains (Hahn et al., in press). Due to the very pronounced differences in lifestyle of the obligately endosymbiotic and obligately free-living strains, the placement of these organisms in the two subspecies P. necessarius subsp. necessarius (for endosymbionts of E. aediculatus and E. harpa) and P. necessarius subsp. asymbioticus (for obligately free-living strains) was proposed (Hahn et al., in press). The obligately free-living P. necessarius strains represent aerobic chemoorganotrophic, non-motile bacteria. Cultivation-independent investigations by fluorescent in situ hybridization (FISH) with P. necessarius-specific, probes demonstrated that the free-living strains possess a planktonic lifestyle and inhabit a large variety of freshwater habitats (Hahn et al., 2005; Wu & Hahn, 2006; Salcher et al., 2008; Alonso et al., in press).
In previous studies, strains were isolated or sequences were obtained by cultivation independent approaches, which were phylogenetically closely related to P. necessarius but did not cluster in trees together with P. necessarius strains (Hahn, 2003; Watanabe et al., 2009; Crump et al., 1999; Zwart et al., 2002). Therefore, these isolates were grouped in separate species-like operational taxonomic units (designated subclusters A, B, C, and D), which were defined by phylogenetic reconstructions (Hahn, 2003). In the present study, we characterize five strains affiliated with the so-called subcluster D (PnecD) of the Polynucleobacter cluster (Hahn, 2003; Wu and Hahn, 2006). These strains share many phenotypic, chemotaxonomic, and ecological traits with strains of P. necessarius subsp. asymbioticus but can be distinguished from those by phylogenetic and chemotaxonomic traits. Based on these differences, we propose to establish for these strains the species P. cosmopolitanus sp. nov. as a second species within the genus Polynucleobacter.
All five investigated strains were isolated by using the filtration-acclimatization method (Hahn, 2003, Hahn et al., 2004) from freshwater habitats located in Austria, France, New Zealand, China, and Uganda, (Table 1). These habitats are located in temperate, subtropical and tropical climatic zones. Four strains were isolated from the surface waters of lakes, but one strain was isolated from a navigable channel fed with water from the Loire River, France. A recent investigation demonstrated that strains closely related to the five strains characterized here, could be directly isolated from agar plates with modified R2A medium (Watanabe et al., 2009).
All investigated strains were routinely grown on NSY medium (Hahn et al., 2004) with strength of 3 g L−1. All strains also grew on R2A medium, and Bacto peptone at least when concentrations were lowered to 3 g L−1 (Hahn, 2003). Growth at different temperatures, and growth under anoxic conditions in an anaerobic chamber were examined on NSY agar. NaCl tolerance was determined using NSY agar supplemented with different NaCl concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1.0, 1.25, 1.5, 1.75, and 2.0%, w/v). The temperature range supporting growth was tested on NSY agar plates exposed to different temperatures (5, 10, 15, 20, 30, 33, 34, 35, 36, and 38 °C). Two of four tested strains grew weakly on an inorganic mineral medium (Hahn et al., 2004) supplemented with acetate as sole carbon and energy source and vitamin B12 (Table 1). However, all three tested strains utilized acetate (Table 2) when this substrate was offered in combination with the complex NSY medium (see below). The investigated strains share the weak growth performance on medium with a sole carbon source with previously investigated P. necessarius subsp. asymbioticus strains (Hahn et al., in press). Due to this weak performance and due to the sake of comparability with the previously investigated Polynucleobacter strains, tests on utilization of various organic compounds were neither performed with sole substrate media, nor by using commercially available test kits. Instead, experiments were performed by applying the method previously used for P. necessarius strains (Hahn et al., in press). Briefly, growth enabled by utilization of a specific substrate was determined by comparison of OD established in liquid one-tenth-strength NSY medium (0.3 gL−1) with and without 0.5 g L−1 test substance. OD differences of <10%, of 10-50%, and of >50% of the OD established on the medium without test substance were scored after 10 days of growth as no utilization (−), weak utilization (w) and good utilization (+), respectively. Sequencing and phylogenetic analyses of 16S rRNA genes was performed as described previously (Hahn, 2003; Hahn et al., 2005). Neighbour-Joining trees were calculated by using the software MEGA4 (Tamura et al., 2007), and Maximum-Likelihood trees were generated by using the RaxML web server (Stamatakis et al., 2008). The morphological characterization of the proposed type strain by electron microscopy was performed as described previously (Yakimov et al., 1998). The G+C content of DNA was determined as described by Tóth et al. (2008). Fatty acid methyl esters (FAMEs) were obtained as described previously (Kämpfer & Kroppenstedt, 1996) and separated by a gas chromatograph (model 6890, Hewlett Packard). Peaks were automatically computed using the Microbial Identification standard software package (Sasser, 1990).
The five investigated strains have with P. necessarius subsp. necessarius in common that they produce only low biomass yields in relatively rich media (Hahn et al., in press). Biomass yields are usually one order of magnitude lower than in other Burkholderiaceae. The results of the chemotaxonomic and phenotypic characterization of the investigated strains are presented in Tables Tables1,1, ,2,2, and and3.3. Experiments on utilization capabilities of three strains resulted only in 68% of the 37 tested substrates in identical results for all three strains (three categories; i.e. no, weak and good utilization) (Table 2). Testing of four P. necessarius subsp. asymbioticus strains with the same substrates even yielded only in 25% of tested substrates identical results (Hahn et al., in press). Despite of the relatively large number of tested substrates no discriminative traits between the three investigated strains and the three P. necessarius subsp. asymbioticus strains were found (Table 2). Therefore, utilization patterns of the remaining two strains, i.e. MWH-TaW3 and MWH-NZ8W13, were not examined. All strains grew in NSY medium as curved rods with small cell sizes (Fig. 1). Doubling times for growth in liquid NSY medium at 20°C were for strains MWH-VicM1, MWH-MoIso2T, and MWH-CaK1 4.5, 5.7, and 5.9 hours, respectively. All three strains grew under these conditions with average cell volumes of < 0.1 μm3 (Hahn, 2003).
Major components of whole cell fatty acids were C16:1ω7c, C18:1ω 7c, C16:0 and C12:0-3OH (Table 3). The pattern of P. necessarius was dominated by the same non-hydroxylated acids (Table 3 and Hahn et al., in press). The occurrence of several other fatty acids in minor amounts was shared by P. necessarius and all studied strains of P. cosmopolitanus. These are C14:0, C18:0, and 11-methyl-C18:1ω7c.
On the other hand, the two species could be clearly distinguished by the absence of C12:0 and C12:0-2OH and the presence of high amounts (higher than 7 %) of C12:0-3OH in P. cosmopolitanus. In all P. necessarius strains studied yet, no C12:0-3OH was detected, and the contents of C12:0 and C12:0-3OH were higher than 3 % and 1 %, respectively (Hahn et al., in press). Furthermore, MIDI-feature 2 including C14:0-3OH occurred in P. cosmopolitanus strains in much lower amounts (lower than 4 %) than in P. necessarius strains (higher than 9 %), and the fatty acid C18:ω7c amounted higher than 28 % in P. cosmopolitanus while it amounted less than 21 % in P. necessarius.
The genome size of the five investigated strains was not determined, however, the genome sizes of strains tentatively assigned to the proposed taxon are ranging from 1.9 to 2.1 Mbp (Vannini et al., 2007), which may indicate that P. cosmopolitanus strains possess smaller genome sizes than P. necessarius subsp. asymbioticus strains.
The phylogenetic analyses of 16S rRNA genes of the five strains revealed clustering of the strains in a monophyletic clade representing a sibling taxon of P. necessarius (Fig. 2). The minimal 16S rRNA sequence similarity of the five strains is 99.3 % (sequence stretches ranging from E. coli position 28 to 1542 were analysed). The sequence similarity between 16S rRNA genes of the proposed type strain MWH-MoIso2T and the type strain of P. necessarius subsp. asymbioticus and a sequence representing P. necessarius subsP. necessarius ‘E24’ are 97.3% and 96.8%, respectively. BLAST searches with the 16S rRNA sequences of the investigated strains and subsequent phylogenetic analyses revealed that the five investigated strains form together with 99 cultured and uncultured strains a tight monophyletic cluster (data not shown). This cluster is phylogenetically identical with the previously characterized ‘Polynucleobacter subcluster D’ (Hahn, 2003) for which a minimal within-cluster sequence similarity of 99.1% was reported previously (Wu and Hahn, 2006). Phylogenetic analysis of 16S-23S ITS sequences confirmed the separate clustering of the five investigated strains and previously investigated P. necessarius strains, and demonstrated tight clustering of sequences within both groups, respectively (Fig. 3).
All members of the ‘Polynucleobacter subcluster D’ share the presence of the oligonucleotide sequence 5′-AA(T/G) CCC T(A/T)A GGG GGA AA-3′ within the 16S rRNA gene (E. coli. position 181-197). In almost all cases, the presence of this sequence can be determined by fluorescent in situ hybridization of whole cells by using a mixture of the fluorescently labelled oligonucleotide probes PnecD1-181 and PnecD2-181 (Hahn et al., 2005). The sequences of these two probes differ in the second variable position (E. coli. position 188) of the diagnostic sequence, but not in the first variable position (E. coli. position 183). Analysis of 104 sequences forming the phylogenetic cluster representing the proposed taxon P. cosmopolitanus demonstrated that only one strain differed in the first variable position of the diagnostic sequence from the probe sequences. Besides the FISH probe, a reverse line blot hybridization probe (PolynucD123-144) for detection of the proposed taxon in environmental samples has been developed and successfully applied (Wu et al., 2007).
Strains affiliated to the phylogenetic cluster represented by the proposed species were isolated from freshwater habitats located in Eurasia, South America, Africa, and Oceania (Hahn, 2003; Hahn et al., unpublished data). Even from a freshwater habitat located on the Hawaiian Archipelago a strain could be isolated (strain MWH-Haw2W10a, accession number AM110098). Recently, many strains were isolated from various lakes located in Japan (Watanabe et al., 2009). Furthermore, cultivation-independent investigations indicated the presence of this taxon at various places including Northern America (Crump et al., 1999; Zwart et al., 2002; Simpson et al., 2004; Crump & Hobbie, 2005), a river estuary of northern Taiwan (Liao et al., 2007), and a running water in Germany (Beier et al., 2008). Even in a pond located at the Tibetan Plateau at an altitude of 3210 m.a.s.l the phylogenetic group representing the proposed species was detected by cultivation-independent methods (Wu et al., 2006). The geographic distribution of habitats colonized by strains phylogenetically belonging to the proposed new species clearly indicates a cosmopolitan distribution of the new taxon over all continents and all climatic zones.
Members of the phylogenetic cluster represented by the proposed species were isolated and detected in the water columns of running and stagnant freshwater habitats like ponds, lakes and streams. A recent investigation compared the salinity tolerance determined by ecophysiological experiments with six pure culture strains of the proposed taxon with the occurrence of the taxon along a salinity gradient of sixteen lakes (Wu et al., 2006). This gradient ranged from 0.2 g L−1 (freshwater) to 222 g L−1 salinity (hypersaline). While the tested strains grew in the laboratory up to maximal salinities of 3.6 to 5.6 g L−1 (note that the salinity tolerance considers the total amount of dissolved salt, while the NaCl tolerance mentioned above considers exclusively the NaCl added to the medium but not the other salt contained in the medium), the taxon was in situ detected by cultivation-independent methods exclusively in habitats with salinities < 1 g L−1. On the other hand, the new taxon was detected in a metagenomic study in two estuaries, and for one of these estuaries (Chesapeake Bay, MD) a salinity of 3.5 g L−1 (oligosaline) was reported (Shaw et al., 2008). The detection at terrestrial or offshore marine sites was never reported. All these observations indicate that the new taxon mainly inhabits the water column of freshwater habitats but may also occur in oligosaline habitats, and is absent in eusaline (oceans) and hypersaline environments.
The new taxon could be detected by the FISH probes PnecD1-181/PnecD2-181 in several habitats (Wu and Hahn, 2006, Wu and Hahn, unpublished data). In a survey of 60 freshwater lakes, the proposed taxon was detected by FISH in 15 habitats. Usually, detections ranged from 0 % (below detection limit by FISH) to 0.9 % of total prokaryotic numbers. The highest recorded relative abundance of 8.1 % of total prokaryotic cells was observed in a freshwater pond located at the Tibetan Plateau (see above). All cells detected by the probes in the various investigated habitats possessed morphologies of curved rods. In a seasonal study on shallow, eutrophic Taihu Lake located in subtropical China, two population peaks both with 0.3 % relative abundance (equalling 1 × 104 cells mL−1) in winter and late spring could be observed (Wu and Hahn, 2006).
In a study focusing on the vulnerability of strains affiliated with the new taxon to predation by bacterivorous flagellates a relatively low vulnerability was observed (Boenigk et al., 2004). Predation by such flagellates represent in general a major mortality factor of planktonic freshwater bacteria (Hahn & Höfle, 2001). The revealed low vulnerability was explained by the small cell sizes of the strains (< 0.1 μm3, ultramicrobacteria), however, a complete resistance to predation was not observed. Thus, factors determining the occurrence and relative abundance of the new taxon in various freshwater habitats remain to be revealed.
Results from the phylogenetic analysis and chemotaxonomic investigations demonstrated pronounced differences between the five investigated strains and strains of the species P. necessarius (Figs (Figs22 and and3,3, Table 4). Furthermore, the determined 16S rRNA gene sequence similarities between the type strain of the proposed new species and strains representing the two P. necessarius subspecies of ≤ 97.3 % leads us to the conclusion that strain MWH-MoIso2T represents a new species (Stackebrandt & Ebers, 2006). Coherence of the taxon represented by the five investigated strains could not be checked by DNA-DNA hybridization experiments, because of limitations in production of DNA amounts sufficient for such investigations. However, the phylogenetic analysis of 16S rRNA gene sequences and of the much less conserved 16S-23S ITS sequences of Polynucleobacter spp. strains (Fig. 3) strongly indicates that the five investigated strains represent a coherent taxon. The latter phylogenetic marker is much less conserved than 16S rRNA gene sequences, thus significant intra-taxon heterogeneity would be expected to be indicated by this marker. We propose to establish for strain MWH-MoIso2T the new species Polynucleobacter cosmopolitanus sp. nov., and to assign tentatively the other four investigated strains to this new species.
Polynucleobacter cosmopolitanus (cos.mo.po.li’ta.nus. Gr. adj. kosmopolites, an inhabitant of the world, a cosmopolitan; N.L. masc. adj. cosmopolitanus cosmopolitan). Curved, non-motile rods, 0.5–1.4 μm in length and 0.3–0.5 μm in width. Chemoorganotrophic, aerobic strains, at least some of the strains are able to grow anaerobically. Can be cultivated on NSY, R2A, and Bactopeptone medium, and some other complex media with concentrations of ≤ 3g L−1. Colonies grown on NSY agar are unpigmented, circular and convex with smooth surface and reach a diameter of 1 mm after 5-10 days at 20-24° C. Growth occurs at 10–35 °C, four strains also grow at 5 °C, and two strain also grow at 38 °C. Grows without NaCl. Maximum NaCl concentration tolerated is strain dependant 0.3 - 0.5 % (w/v). Utilizes acetate, pyruvate, propionate, oxaloacetate, malate, succinate, fumarate, D-galacturonic acid, D-galactose, L-cysteine, and L-alanine when these substrates are provided in a medium containing low amounts of NSY. None of the strains utilizes formiate, glycolate, oxalate, D-lyxose, D-fructose, D-fucose, D-sorbitole, L-aspartate, L-serine, L-asparagine, and betaine. Major cellular fatty acids are C18:1ω7c, C16:0 and C12:0-3OH. No 2-hydroxylated compounds detected. The DNA G+C content of the type strain is 44.9 %. Fractions of natural populations pass through 0.2μm filters. Can be distinguished from P. necessarius by using the P. necessarius-specific FISH probe PnecC-16S-445 (Hahn et al., in press), as well as by differences in their fatty acids profiles (Table 3). In contrast to P. necessarius, exclusively free-living strains and no endosymbiotic strains were reported, however, the existence of endosymbiotic strains affiliated to P. cosmopolitanus sp. nov. cannot be completely excluded at present. Tentatively, all strains possessing the oligonucleotide sequence 5′-AA(T/G) CCC T(A/T)A GGG GGA AA-3′ (E. coli. position 181-197) within the 16S rRNA gene shall be assigned to the proposed species. In almost all cases (one known exception), the presence of the diagnostic sequence can be determined by FISH with a mixture of the two probes PnecD1-181 and PnecD2-181 (Hahn et al., 2005). The type strain is MWH-MoIso2T (= DSM 21490T = CIP 109840T), isolated from the water column (pelagic zone) of Lake Mondsee located in the Salzkammergut Lake District in Austria (Hahn, 2003).
We are grateful to R. M. Kroppenstedt for performing the fatty acid analyses, to P. Schumann for determining the G+C content, and to M. Kopitz and Matthias Pöckl for technical assistance. This study was supported by the Austrian Science Fund (Projects P15655 and P19853 granted to MWH).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains MWH-MoIso2T, MWH-CaK1, and MWH-VicM1, MWH-TaW3, and MWH-NZ8W13 are AJ550672, AJ550667, and AJ550651, AJ550661, and AM110084, respectively.