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Heckmann and 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. Pure cultures of the type strain could never be established due to the obligate nature of the symbiotic relationship between the endosymbionts and their hosts. Therefore, P. necessarius is one of a few validly described bacterial species lacking a deposited pure culture. Meanwhile, it was demonstrated that the endosymbiotic P. necessarius type strain is closely related to obligately free-living strains. Similarity values of the 16S rRNA sequence of the type strain and free-living isolates in the range of 99.1 to 99.4% indicate that these organisms belong to the same species. Here, we emend the description of P. necessarius by characterization of free-living strains. The species P. necessarius is characterized by low G+C contents of the DNA (44-46 mol%), small genome sizes (1.5-2.5 Mbp), and a lack of motility. Due to distinct differences in lifestyle and genome size of P. necessarius strains, we propose to establish two subspecies, P. necessarius subsp. necessarius subsp. nov. [with a nameless type strain contained in a deposited E. aediculatus culture (ATCC 30859)], and P. necessarius subsp. asymbioticus subsp. nov. [with the type strain QLW-P1DMWA-1T (=DSM 18221T = YYYYYT)], for the obligate endosymbionts of E. aediculatus and E. harpa and obligately free-living strains, respectively.
The genus Polynucleobacter and the species P. necessarius were proposed by K. Heckmann and H. J. Schmidt in 1987 for bacterial endosymbionts of freshwater ciliates affiliated with the genus Euplotes. The presence of bacterial endosymbionts in Euplotes spp. was reported for the first time by Fauré-Fremiet in 1952. He already described the presence of several nucleoid-like structures in cells of endosymbionts, which could be observed after sufficient staining by light microscopy. This trait, later characterized in detail by ultrastructural investigations (Heckmann, 1975), is described by the genus name. Furthermore, Fauré-Fremiet already suggested that the endosymbionts are essential for the host cells (Fauré-Fremiet, 1952). The bidirectional obligate nature of the symbiotic relationship between the host cells and the intracellular bacterial symbionts was later demonstrated by sophisticated experiments (Heckmann, 1975; Fujishima & Heckmann, 1984; Heckmann et al., 1983). The obligately symbiontic lifestyle of the endosymbionts, indicated by the epithet of the species P. necessarius, seems to be responsible for the failure of all experiments aiming on cultivation of these strains (Heckmann, 1975; Heckmann & Schmidt, 1987; Vannini et al., 2007). These failures resulted in an incomplete phenotypic characterization of the taxon, as well as in the lack of deposition of a pure culture of the P. necessarius type strain proposed by Heckmann and Schmidt. Instead, the type strain is included in a culture of its host E. aediculatus deposited at the American Type Culture Collection. In 1996, the 16S rRNA gene of the P. necessarius type strain was sequenced (Springer et al., 1996), which revealed that the endosymbionts are related to strains currently classified as Ralstonia and Cupriavidus species. Polynucleobacter-like endosymbionts characterized by multiple nucleoid-like structures were reported for eight freshwater Euplotes spp. (Foissner, 1978; Heckmann & Schmidt, 1987), and the brackish-water species E. harpa (Vannini et al., 2005), however, verification of the affiliation with the genus Polynucleobacter by comparative analysis of 16S rRNA genes was only performed for the endosymbionts of three E. harpa strains and a second E. aediculatus strain (Vannini et al., 2005; Vannini et al., 2007).
From 1996 on, many investigations employing cultivation-independent techniques for revealing the diversity of freshwater bacterioplankton retrieved 16S rRNA sequences closely related to the sequence of the endosymbiotic P. necessarius type strain (e.g., Bahr et al., 1996; Hiorns et al., 1997; Crump et al., 1999). Thus, P. necessarius-like bacteria were detected in the water column of lakes and rivers, which is an environment usually not inhabited by the benthos-dwelling Euplotes spp. hosting Polynucleobacter endosymbionts, therefore, it was uncertain if these environmental sequences represent endosymbionts of ciliates. Recently, the first strains matching environmental sequences have been brought into pure cultures in the laboratory (Hahn, 2003). Phylogenetic analyses with 16S rRNA gene sequences of the endosymbiont, the cultivated strains, and environmental sequences revealed one monophyletic cluster (operationally designated as ‘Polynucleobacter cluster’) subdivided in four narrow subclusters (designated as PnecA, PnecB, PnecC, and PnecD, respectively), each characterized by internal minimal 16S rRNA similarity values >98.2% (Hahn, 2003; Wu & Hahn, 2006a). All available sequences of endosymbionts but also several sequences of free-living strains clustered within subcluster PnecC (Hahn, 2003; Vannini et al. 2005), which is characterized by a minimal 16S rRNA similarity value of 98.5%, and thus resembles a species-like taxon (Stackebrandt & Ebers, 2006). Development and application of a FISH probe specific for the entire subcluster PnecC demonstrated the presence of free-living bacteria affiliated with this subcluster in the water column of freshwater systems (Hahn et al., 2005, Wu & Hahn, 2006a), thus, this species–like subcluster contains both free-living and endosymbiotic strains. Recently, it was demonstrated that the endosymbiotic P. necessarius and the cultivated strains posses the closest phylogenetic relationship ever revealed between obligate endosymbionts and obligately free-living bacteria (Vannini et al., 2007).
Free-living strains affiliated with the ‘Polynucleobacter cluster’ posses a wide distribution in freshwater habitats. Environmental sequences and cultivated strains were obtained from an ecologically broad variety of habitats located in all climatic zones (Hahn, 2003) and characterized by various chemical conditions. Bacteria affiliated with subcluster PnecC were even detected in lakes located at altitudes of 5000 meters above sea level (Wu et al., 2006). Furthermore, investigations employing FISH probes specific for the phylogenetically characterized subclusters revealed relative abundances in the range of <1% to 60% of total bacterial numbers in the water column of various freshwater habitats (Hahn et al., 2005; Wu & Hahn, 2006a; Wu & Hahn, 2006b), thus, this group of bacteria comprises an important part of the freshwater bacterioplankton.
Here, we present an emended description of the genus Polynucleobacter and its sole species P. necessarius, which considers both endosymbiotic and free-living strains. This description is based on the phenotypic and chemotaxonomic characterization of four free-living strains affiliated with subcluster PnecC.
The investigated strains were isolated from freshwater habitats (Supplemental Material Table S1) by using the filtration-acclimatization method (Hahn et al., 2004) or the dilution-acclimatization method as reported previously (Hahn, 2003; Hahn et al., 2005). Strains closely related to the strains presented here, could also be isolated from NSY agar plates directly inoculated with freshwater samples (Hahn, unpublished data). Strains were routinely grown on NSY medium (Hahn et al., 2004) with strength of 3 g L−1 (Supplemental Material Fig. S1). All strains also grew on R2A, peptone and Luria-Bertani agar (Hahn, 2003). Growth at different temperatures (5–38 °C), 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, and 36 °C). Three of the four investigated strains could grow on a mineral medium (Hahn et al., 2004) supplemented with vitamin B12 and acetate as sole carbon and energy source, however, in some experiments the inoculated cultures did not grew on this minimal medium but repetition of the experiment clearly resulted in growth. By contrast, experiments with three Cupriavidus spp. and the Burkholderia gladioli type strain always resulted in strong and reliable growth. Due to the unreliability of growth and due to the establishment of only relatively low cell concentrations, tests on assimilation of various organic compounds were neither performed with media containing only a sole substrate source, nor by utilization of commercially available using test kits. Instead, experiments were performed 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 (pH 7.2). OD differences of <10%, of 10-50%, and of >50% of the OD established on the medium without test substance were scored as no assimilation, weak assimilation and assimilation, respectively. Sequencing and phylogenetic analyses of 16S rRNA genes was mainly preformed 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 constructed by using the software RAxML version 7.0.4 (Stamatakis et al., 2008). Both, the G+C content of DNA, and the composition of cellular fatty acids were determined as described by Tóth et al. (2008). Genome sizes were examined by pulsed field gel electrophoresis (PFGE) as described previously (Vannini et al., 2007), and for two strains genome sizes were determined in a genome-sequencing project.
The phenotypic characterization of the investigated free-living strains was complicated by their relatively weak growth on complex media (Supplemental Material Fig. S2), and by their weak and unreliable growth performance on media with only a single carbon source. The results of the phenotypic and chemotaxonomic characterization of the investigated free-living strains is presented in Tables Tables11 and and22.
The phylogenetic analyses of 16S rRNA genes of free-living strains and endosymbiotic Polynucleobacter strains revealed the affiliation with the family Burkholderiaceae (Fig. 1). The cluster formed by the Polynucleobacter taxa is closest related to the Ralstonia spp./Cupriavidus spp. lineage (94% maximal sequence similarity). The minimal 16S rRNA sequence similarity of the four free-living strains and the originally proposed endosymbiotic type strain of Polynucleobacter necessarius is 99.1% (1500bp sequence stretches analysed), and the minimal sequence similarity among the four free-living strains is 99.6%. A very close phylogenetic relationship between the obligate endosymbionts of E. aediculatus and E. harpa, and the four obligately free-living strains was also demonstrated by the analysis of 16S-23S ITS sequences (Vannini et al., 2007). Furthermore, the endosymbionts and the free-living strains share low and almost identical G+C contents of their DNA, small genome sizes, and the lack of motility. All these characteristics are absent or exceptional among other Burkholderiaceae (Table 3). Almost all observed differences between free-living and endosymbiotic strains (Table 4) are likely a result of the evolutionary adaptation of endosymbionts to an obligate dependence on their host cells (Heckmann, 1975; Vannini et al., 2007). A lack of dependence on self-synthesis of essential compounds due to delivery of such compounds by the host could have made possible the deletion of involved genes, which could have resulted in a decrease of genome size. This could have also resulted in more complex nutritional demands of the obligate endosymbionts causing the lack of cultivability. An increase in size is usually a disadvantage for free-living planktonic bacteria with small cell sizes, because of a resulting increase of predation by bacterivorous protists (Hahn & Höfle, 2001), however, the obligate endosymbionts are completely protected by their hosts against direct predation.
Based on the close phylogenetic relationship between the free-living and the endosymbiotic strains, we propose to consider these organisms differing so strongly in lifestyle as members of the species Polynucleobacter necessarius. Unfortunately, this proposal cannot be verified by DNA-DNA reassociation experiments due to the lack of sufficient amounts of pure DNA extracted from endosymbiotic strains. Strains affiliated with P. necessarius can be clearly differentiated from other valid taxa within the family Burkholderiaceae by means of FISH with the P. necessarius-specific probe PnecC-16S-445 (Hahn et al., 2005), by the G+C content of their DNA, and by the lack of motility (Table 3). Members of Polynucleobacter necessarius may be differentiated from most Ralstonia and Cupriavidus species by the lack of 2-hydroxylated fatty acids others than 2-OH-C12:0 (Table 2). The mentioned FISH probe also enables the differentiation of P. necessarius from so far undescribed taxa putatively affiliated with the genus Polynucleobacter.
Based on distinct differences in lifestyle and genome size, we propose to place obligately endosymbiotic, and obligately free-living P. necessarius separately in two new subspecies.
Polynucleobacter [Pol.y.nuc'leo.bac.ter. Gr. adj. polys numerous; L. masc. n. nucleus nut, kernel; masc. bacter the equivalent of Gr. neut. n. bactrum a rod; N.L. masc. Polynucleobacter rod with many nucleoids].
The monotypic genus Polynucleobacter belongs to the family Burkholderiaceae, and harbours endosymbiotic strains of several Euplotes species and free-living strains dwelling in the water column of freshwater lakes, ponds and streams. According to the description by Heckmann and Schmidt (1987), endosymbionts of the freshwater species E. aediculatus, E. eurystomus, E. plumipes, E. daidaleos, E. octocarinatus, E. patella, and E. woodruffi, which are penicillin-sensitive and posses multiple nuclei-like structures belong to this genus. Furthermore, endosymbionts of Euplotes moebiusi f. quadricirratus (Foissner, 1978) posses the mophological characteristics described for Polynucleobacter spp. However, for most of these endosymbionts the examination of their phylogenetic affiliation with the genus Polynucleobacter is lacking. Vannini and colleagues (2005) demonstrated by phylogenetic and ultrastructural investigations that endosymbionts of the brackish-water species E. harpa also belong to this genus. Type species is P. necessarius.
Polynucleobacter necessarius (nec.es.sar'i.us. L. adj. necessarius indispensable, necessary). The species contains obligate endosymbionts living in ciliates of the species E. aediculatus and E. harpa, as well as obligately free-living strains inhabiting freshwater systems. The species possesses the following characteristics. The DNA G+C content is 44-46 mol%, and genome sizes range from 1.5 to 2.5 Mbp. All strains are non-motile. Members of the species can be distinguished from other taxa by the presence of the oligonucleotide sequence 5′-GAG CCG GTG TTT CTT CCC-3′ (E. coli. position 445-463) within the 16S rRNA gene. The presence of this sequence can be determined by fluorescent in situ hybridization of whole cells by using the fluorescently labelled oligonucleotide probe PnecC-16S-445 (Hahn et al., 2005).
Polynucleobacter necessarius subspecies necessarius (nec.es.sar'i.us. L. adj. necessarius indispensable, necessary).
The subspecies possesses the characteristics previously described by Heckmann and Schmidt (1987) for the species P. necessarius. This subspecies contains P. necessarius strains obligately living as endosymbionts in cells of the ciliates E.aediculatus and E. harpa. This includes the P. necessarius type strain contained in the E. aediculatus E24 culture (= ATCC 30859, = clone 15 (Heckmann and Schmidt, 1987)) and the endosymbiontic strain STIR1 contained in the E. aediculatus STIR1 culture (Petroni et al., 2002; Vannini et al., 2007), as well as three strains detected in three cultures of the brackish water ciliate E. harpa (Vannini et al., 2005, Vannini et al., 2007). Strains usually have elongated cells with cell lengths > 2.5 μm. Genome size is ranging from 1.5 to 1.8 Mbp. All attempts to cultivate the obligate endosymbionts outside of their host cells failed so far, therefore the subspecies is lacking a type strain cultivated as pure culture.
Polynucleobacter necessarius subspecies asymbioticus (a.sym.bio'ti.cus. Gr. pref a not; M.L. symbioticus, -a, -um living together; M.L. masc. adj. asymbioticus not symbiotic).
This subspecies exclusively contains free-living P. necessarius strains. Strains have cell morphologies of straight or curved rods, 0.5–3.0 μm in length and 0.3–0.5 μm in width. Strains forming short curved rods grow in NSY medium with relatively even cell lengths, but strains with larger cells usually show uneven lengths distributions, and even a few elongated cells of up to 15 μm were observed in some of the cultures, however, the majority of cells posses cell lengths < 2 μm. Chemoorganotroph, aerobic, at least some of the strains facultatively anaerobic. Can be cultivated on NSY, R2A, Luria-Bertani, and peptone medium. Colonies grown on NSY agar are unpigmented, circular and convex with smooth surface. Growth occurs at 5–30 °C, some strains also grow at 34 and 35 °C. Growth occurs with 0–0.3 % (w/v) NaCl; some strains also grow at 0.4 and 0.5 % (w/v) NaCl. Growth does not occur at 0.6 % (w/v) NaCl or higher. Assimilates acetate, pyruvate, malate, succinate, fumarate, D-galacturonic acid and L-cysteine. None of the strains assimilates glycolate, oxalate, L-serine, and citrate. Major cellular fatty acids are C16:1w7c, C16:0, C18:1 w7c and summed feature 2 (including 3-OH-C12:0). Sole 2-hydroxylated compound is 2-OH-C12:0. Genome size is ranging from 2.1 to 2.5 Mbp.
The type strain is QLW-P1DMWA-1T (= DSM 18221T = yyyy), isolated from a small acidic freshwater pond located in the Austrian Alps at an altitude of 1300 meters above sea level (Hahn et al., 2005).
We are grateful to E. Stackebrandt and J. P. Euzéby for taxonomic advice, R. M. Kroppensted for performing the fatty acid analyses, to P. Schumann for determining the G+C content, and to M. Kopitz 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 QLW-P1DMWA-1T, MWH-JaK3, MWH-MoK4, and MWH-HuW1 are AJ879783, AJ550657, AJ550654, and AJ550666, respectively.