Limnohabitans planktonicus sp. nov. and Limnohabitans parvus sp. nov., planktonic betaproteobacteria isolated from a freshwater reservoir, and emended description of the genus Limnohabitans

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 shifts in predation pressure by bacterivorous protists (Jezbera et al., 2006; Šimek et al., 2006, 2008, Horňák et al., 2008). Moreover, the R-BT065 cluster usually represents the fastest growing segment of bacterioplankton in lakes (e.g. Šimek et al., 2006; Salcher et al., 2008). So far, the R-BT065 cluster contains no species with validly published names. Here, we describe two strains affiliated with this cluster that represent two novel species in the genus Limnohabitans (Hahn et al., 2010).

The two bacterial strains, II-B4^T and II-D5^T, were isolated from the water column of the Římov reservoir, Czech Republic, using the filtration–acclimatization method (Hahn et al., 2004); for details of the isolation source, see Supplementary Table S1, available in IJSEM Online. Briefly, a whole water sample was filtered through a 0.8 μm polycarbonate membrane filter (Millipore) and subsequently diluted with sterile medium in order to obtain cell concentrations suitable for inoculation of 24-well microplates with approximately 0.5 cells per well. Established cultures were acclimatized to growth in NSY medium by stepwise addition of increasing doses of NSY medium (Hahn et al., 2004).

The isolated strains were routinely grown in NSY medium with strength of 3 g l⁻¹, either liquid or solidified with 1.5 % (w/v) agar. Both strains were tested for growth on commercially available complex media. The strength of complex medium and agar concentrations were adjusted to 3 g l⁻¹ and 1.5 %, respectively. Due to inefficient growth of the investigated strains on media containing only a single carbon source, assimilation experiments were performed by comparison of OD₅₇₅ established in liquid one-tenth-strength NSY medium (0.3 g l⁻¹) with and without 0.5 g test substance l⁻¹ (pH 7.2), as described previously (Hahn et al., 2009). Differences in OD₅₇₅ of <10 %, 10–50 % and >50 % of the OD₅₇₅ established on medium without test substance were scored as no assimilation, weak assimilation and assimilation, respectively. Growth at 4, 6, 8, 10, 12, 15, 21, 34 and 36 °C and growth under anoxic conditions in an anaerobic chamber were examined on NSY medium amended with 1.5 % agar. NaCl tolerance was determined using NSY agar supplemented with 0, 0.05, 0.075, 0.1, 0.15, 0.2, 0.3, 0.5, 1.0 and 1.5 % (w/v) NaCl.

Gram staining was performed as described by Hucker & Conn (1927). Catalase activity was tested by bubble formation in a 3 % (v/v) H₂O₂ solution. Oxidase activity was determined by oxidation of 1 % p-aminodimethylaniline oxalate. Cell morphologies of DAPI-stained cells from cultures grown for 3 days in 3 g NSY medium l⁻¹ at room temperature were inspected with an Olympus BX 60 microscope using the semiautomatic image analysis system LUCIA D (Lucia 3.52; Laboratory Imaging, Prague, Czech Republic).

The 16S rRNA genes of the two strains were sequenced and analysed as described previously (Hahn et al., 2009). 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). Comparative analysis of the almost full-length 16S rRNA gene sequences (>1400 bp) of strains II-B4^T and II-D5^T and Limnohabitans curvus MWH-C5^T were performed by using the pairwise alignment menu of the EzTaxon server (Chun et al., 2007).

Cellular fatty acid contents of the strains were characterized by using the MIS Sherlock automatic identification system (MIDI, Inc.) and the Sherlock aerobic bacterial database (TSBA60) as described by Greenblatt et al. (1999). For this analysis, biomass of replicate cultures of each strain grown in NSY medium (3 g l⁻¹) for 2 days at 21 °C was analysed.

Determination of the DNA G+C content and respiratory lipoquinones, polar lipid analyses and DNA–DNA hybridizations were carried out by the Identification Service of the DSMZ and Dr Brian J. Tindall (DSMZ, Braunschweig, Germany). For DNA–DNA reassociation experiments, DNA was isolated using a French pressure cell (Thermo Spectronic) and purified by chromatography on hydroxyapatite, as described by Cashion et al. (1977). DNA–DNA hybridization was carried out under optimal conditions for DNA–DNA reassociation as described by De Ley et al. (1970), with the modifications described by Huß et al. (1983), using a Cary 100 Bio UV/Vis spectrophotometer equipped with a Peltier-thermostatted 6×6 multicell changer and a temperature controller with an in situ temperature probe (Varian). The base composition of the DNA of the strains was determined as described by Tóth et al. (2008).

Strains II-B4^T and II-D5^T were Gram-negative, rod-shaped, non-motile bacteria (Supplementary Fig. S1). Both grew well on NSY medium, R2A agar (Remel), standard methods agar (Remel), peptone agar, Casitone agar, solidified Luria–Bertani (LB) broth, solidified brain heart infusion (BHI) and solidified tryptic soy broth (TSB) (all from BD-Difco), whereas no growth occurred on Löwenstein medium base (BD-Difco). Growth of both strains was observed in the temperature range 4–34 °C. They grew well under aerobic conditions, whereas anaerobic growth was only weak. The results of phenotypic and chemotaxonomic investigations are presented in Tables 1⇓ and 2⇓. Strain II-D5^T was able to utilize 62 % of 37 tested substrates (including four that were utilized weakly), whereas strain II-B4^T utilized only 41 % (including six that were utilized weakly) of the 37 tested substrates. The major polar lipids of both strains were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine (Supplementary Fig. S2), and their major fatty acids were C_16 : 1ω7c/C_16 : 1ω6c, C_16 : 0, C_18 : 1ω7c/C_18 : 1ω6c and C_12 : 0 (Table 2⇓). Their major ubiquinone was Q-8, and they shared identical DNA G+C contents of 59.9 mol%.

Phylogenetic analyses of 16S rRNA gene sequences by two independent algorithms resulted in trees with largely identical branching orders (Fig. 1⇓). The two trees differed only in the phylogenetic positions of the genera Polaromonas, Caenimonas and Variovorax, which are more distantly related to the two investigated strains than the other reference taxa. Both phylogenetic reconstructions indicated the affiliation of strains II-D5^T and II-B4^T with the genus Limnohabitans of the family Comamonadaceae. Phylogenetic analysis with a sequence set that included environmental sequences (Supplementary Fig. S3) confirmed the affiliation of the two strains with the R-BT065 cluster (Šimek et al., 2001). Note that L. curvus does not belong to this cluster of freshwater bacteria (Supplementary Fig. S3); thus, the two investigated strains represent the first taxonomically described members of this phylogenetic cluster of considerable ecological relevance (Šimek et al., 2010a).

The affiliation of the two investigated strains to the genus Limnohabitans was also supported by several chemotaxonomic and phenotypic traits shared by the two strains and the type strain of the type species of the genus Limnohabitans. These traits include the major fatty acids and quinone composition, low salinity tolerance, lack of pigmentation, lack of motility and their inability to assimilate lactate. However, strains II-B4^T and II-D5^T can be differentiated from L. curvus by the presence or absence of minor fatty acid components and some physiological and metabolic characteristics (Table 1⇑). Furthermore, the two strains can be differentiated from each other by their morphology and by several metabolic traits (Table 1⇑) and by the presence or absence of seven minor fatty acids (Table 2⇑). The two strains differ, for instance, in assimilation of acetate, l-histidine and oxaloacetate and in the presence/absence of C_17 : 0 and C_17 : 1ω6c. In addition, ecological differences between the two strains in their interaction with competitors, protistan predators and viruses have been revealed (Šimek et al., 2010b). Traits that differentiate the two strains from the type strain of L. curvus are, for example, assimilation of l-glutamate and l-proline as well as the lack of assimilation of d-gluconate and propionate (Table 1⇑).

In contrast to L. curvus, the two newly described strains share a short diagnostic 16S rRNA gene sequence (Escherichia coli positions 65–83) targeted by the FISH probe R-BT065 (5′-GTTGCCCCCTCTACCGTT-3′; Šimek et al., 2001). The 16S rRNA gene sequence similarity of strains II-B4^T and II-D5^T was 99.1 %, whereas the similarity with L. curvus MWH-C5^T was 97.0 and 97.2 %, respectively. To verify further the obvious differences between the strains, DNA–DNA reassociation experiments were performed with DNA extracted from strains II-B4^T and II-D5^T and L. curvus MWH-C5^T. These experiments resulted in a value of 26.7 % (25.2 % for a replicate) for strain II-B4^T against II-D5^T, 33.6 % (30.3 %) for strain II-B4^T against L. curvus MWH-C5^T and 33.0 % (24.3 %) for strain II-D5^T against L. curvus MWH-C5^T. These results clearly justify the proposal to place all three strains into separate species (Wayne et al., 1987).

Taking into account the differences observed between the two strains and the type strain of L. curvus, as well as between the two novel strains, we propose to establish the novel species Limnohabitans parvus sp. nov., with the type strain II-B4^T, and Limnohabitans planktonicus sp. nov., with the type strain II-D5^T, within the genus Limnohabitans. It is also necessary to emend the description of the genus Limnohabitans.