Limnohabitans curvus gen. nov., sp. nov., a planktonic bacterium isolated from a freshwater lake

Abstract

The chemoorganotrophic, aerobic, facultatively anaerobic, non-motile strain MWH-C5^T isolated from the water column of oligo-mesotrophic Lake Mondsee (Austria) was phenotypically, phylogenetically, and chemotaxonomically characterized. The predominant fatty acids of the strain were C_16:1ω7c/ω6c, 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%. The 16S rRNA gene similarity to the closest related type species were 96.6% (Curvibacter delicatus) and 95.7% (Rhodoferax fermentans). The phylogenetic analysis of the 16S rRNA gene sequences revealed the affiliation of the strain with the family Comamonadaceae (Betaproteobacteria), however, the revealed phylogenetic position of the strain did not indicate the affiliation to any previously described genus within this family. A family-wide comparison of traits revealed that the strain possesses a unique combination of G+C value, 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 the differences to the closest related genera, we propose to establish the genus Limnohabitans gen. nov. to accommodate this strain, and to place the strain in the new species Limnohabitans curvus sp. nov., with the type strain MWH-C5^T (DSM 21645^T, = CCUG 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 mainly composed of phylogenetic groups absent in marine bacterioplankton and in terrestrial habitats (Zwart et al., 2002; Hahn, 2006). Investigations with cultivation-independent methods 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; Zwart et al., 2002; Eiler & Bertilson, 2004). In this paper, we characterize a strain isolated from the pelagic zone of a freshwater lake, which is closely related to uncultured bacteria numerously detected in freshwater samples, and propose to establish for this strain the novel genus Limnohabitans sp. nov. and the novel species Limnohabitans curvus sp. nov. within the family Comamonadaceae (Betaproteobacteria).

Strain MWH-C5^T was isolated from deep, oligo-mesotrophic Lake Mondsee (47°50′2.92″N; 13°22′25.98″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 on growth of the strain on single carbon sources resulted only in weak growth. In order to avoid false negative results in such tests caused by too weak growth response on single substrate sources, tests on substrate utilization were performed in the presence of complex substrate mixtures as described previously (Hahn et al., in press). Briefly, growth enabled by utilization of specific substrates was determined by comparison of OD (575 nm) 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. The analysis of the phylogenetic positions of the strain was performed by means of 16S rRNA gene sequence analysis. Sequences were obtained, aligned and analyzed by the neighbour-joining approach as described previously (Hahn et al., in press). In addition, trees were constructed by using the maximum-likelihood and the Bayesian inference tree-building methods. For the maximum-likelihood tree calculation and bootstrap analysis we used the genetic algorithm GARLI (Zwickl, 2006) and a grid computing system (Cummings & Huskamp, 2005). A bayesian inference tree was calculated by using the program Mr Bayes (Ronquist & Huelsenbeck, 2003) performing one millions generations and subsequently explored by the AWTY system (Nylander et al., 2008). The determination of the G+C content of DNA and the analyses of major respiratory lipoquinone analyses were both carried out by the Identification Service and Dr.B.J.Tindall, DSMZ, Braunschweig, Germany. Fatty acid profiles of strain MWH-C5^T, as well as of the type strains of Curvibacter gracilis and Rhodoferax fermentans were characterized by using the MIS Sherlock automatic identification System (MIDI, Inc., Newark, DE) and the Sherlock Aerobic Bacterial Database (TSBA60) as described by Greenblatt et al. (1999). For each strain, biomass of replicated cultures obtained by growing the strains in NSY (3 g l⁻¹) for 2 days at 21 °C was analyzed.

The results of the phenotypic and chemotaxonomic investigations are presented in Tables 1 and 2. Strain MWH-C5^T is an aerobic, chemoorganotrophic, facultatively anaerobic, non-motile bacterium, and possess a cell morphology of small curved rods (Fig. 1). The predominant (> 5% of total) fatty acids of the strain were C_16:1ω7c/ω6c and C_16:0, the sole detected 3-hydroxy fatty acid was 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%.

Table 1.

Phenotypic traits of Limnohabitans curvus sp. nov. strain MWH-C5^T, and the type strains of the type species of the genera Curvibacter and Rhodoferax (C. gracilis strain CCUG 49445^T and R. fermentans strains CCUG 45364^T). The substrate utilization tests were performed for all three strains under the same conditions. All three strains assimilated acetate, pyruvate, fumarate, succinate, citrate, gluconate, and glucose, none of the four strains assimilated L-carnitine. All three strains are oxidase and catalase positive. −, negative; +, positive; w, weakly positive; n.d., not determined.

	MWH-C5T	C. gracilis	R. fermentans
Cell morphology	curved rods	curved rods	curved rods
Cell length (μm)	1.0 -1.5	1.1 - 2.8$	1.5 - 3.0$$$
Cell width (μm)	0.4 - 0.5	0.4 - 0.5$	0.6 - 0.9$$$
Min temp. of growth (°C)	4	9§	n.d.$$
Max temp. of growth (°C)	34	40§	n.d.$$
Max NaCl concentration (% NaCl)	0.5	< 3% $	n.d.$$
Anaerobic growth	+	n.d.$	+ $$
Ethanol	w	−	w
Glycerol	−	+	+
Glyoxylate	−	w	−
Gycolate	−	+	−
D-Glycerate	+	+	w
Oxalate	−	−	w
DL-Lactate	−	+	−
Propionate	w	+	w
DL-Malate	+	+	w
Malonate	−	w	−
alpha-Ketoglutarate	+	+	w
Oxaloacetate	−	+	w
L-Arginine	−	−	+
L-Glutamate	−	+	+
L-Glutamine	−	+	w
L-Histidine	−	+	−
L-Phenylalanine	−	+	−
L-Proline	−	+	+
L-Serine	−	+	+
L-Tryptophan	−	+	w
D-Ribose	w	w	+
L-Sorbose	−	w	−
D-Galactose	w	w	+
D-Mannose	+	w	+
D-Saccharose	w	+	w
N-acetyl-glucosamine	−	w	+
Betaine	−	−	w
Spermidine	−	−	w
Quinone type	Q8	Q8$	Q8 + RQ8 $$
DNA G+C content (mol%)	55.5	66.0$	60 $$

^$data from Ding and Yokota (2004)

^$$data from Hiraishi et al. (1991)

^$$$Madigan et al. (2000)

^§data presented by Ding and Yokota (2004) in the description of the genus Curvibacter

Table 2.

Whole cell fatty acid composition of Limnohabitans curvus sp. nov. strain MWH-C5^T and the type strains of the type species of Curvibacter and Rhodoferax (C. gracilis strain CCUG 49445^T and R. fermentans strains CCUG 45364^T). All strains were cultivated under identical conditions (NSY medium (3 g l⁻¹) at 21 °C for 2 days). The presented data were obtained from replicated cultures. nd, not detected.

Fatty acid	MWH-C5T	R. fermentans	C. gracilis
C8:0-3OH	2.7	3.9	5.1
C9:0-3OH	nd	0.1	nd
C10:0-3OH	nd	nd	0.2
C12:0	4.5	nd	5.6
C12:0-3OH	nd	nd	nd
C14:0	1.0	0.2	1.2
C14:1ω5c	0.4	nd	0.3
C15:0	nd	2.0	nd
C15:1ω6c	nd	0.1	0.1
C15:1ω8c	nd	0.2	nd
C16:0	14.0	46.3	15.2
C16:1ω5c	0.2	0.3	0.8
C16:1ω7c/ω6c	76.7	44.1	49.2
C17:0	nd	0.4	nd
C17:0 cyclo	nd	nd	nd
C17:1ω6c	nd	0.6	0.6
C17:1ω8c	nd	0.2	nd
C18:0	0.3	nd	nd
C18:1ω7c 11Me	0.3	0.2	0.7
C18:1ω7c/ω6c	1.8	1.3	20.7
C18:1ω9c	0.2	nd	nd
C19:1ω6c/C19:0 cyclo	nd	0.1	nd

Fig. 1.

Electron microscopy image illustrating the cell morphology and size of the strain MWH-C5^T. Cells of a liquid culture were concentrated by centrifugation and fixed with 2.5% glutaraldehyde, post-fixed with OsO₄ and embedded with Spurr resin. Ultra-thin sections were counterstained with uranyl acetate and lead citrate. The image was obtained by transmission electron microscopy at a 20000-fold magnification.

Phylogenetic analyses with all three tree-building methods revealed consistently the affiliation of strain MWH-C5^T with the family Comamonadaceae (Fig. 2). The sequence of the strain did not cluster within any previously described genus in any tree generated with the three methods. The phylogenetic relationships reconstructed by using the three algorithms indicated in all three cases a close relationship of the strain with the genera Rhodoferax, Pseudorhodoferax, Curvibacter Polaromonas, Variovorax, Ramlibacter, and Caenimonas. Regarding the phylogenetic relationships of the strain MWH-C5^T to these seven genera, the topologies and branching orders of the three trees was almost identical (data not shown). The trees only differed in the position of the type species of the genus Caenimonas, which clustered in the maximum-likelihood (ML) and bayesian inference (BI) trees with the genus Ramlibacter, while it clustered in the neighbour-joining (NJ) tree with the genus Curvibacter. Pair-wise sequence similarity values of 16S rRNA genes of strain MWH-C5^T and type strains of these seven most closely related genera were in the range of 94.4% - 96.2% (average 95.3), which is very similar to the range of 93.0% – 96.5% (average 95.4) observed for pair-wise comparisons among the type species of the seven genera (Supplementary Table S1).

Fig. 2.

Neighbour-joining (NJ) tree (Kimura-2 correction) based on almost complete 16S rRNA gene sequences. The tree presents sequences of type species of almost all genera currently affiliated with the family Comamonadaceae. Only Caenibacterium thermophilum, which is proposed by Lütke-Eversloh et al. (2004) to be a later heterotypic synonym of Schlegelella thermodepolymerans (the type species of the genus Schlegelella) is not shown. The genera Polynucleobacter, Duganella, and, Herbaspirillum represent reference taxa not affiliated with the family Comamonadaceae. All Bootstrap values ≥ 60% (1000 iterations) are depicted. The tree was rooted by using archeal sequences (not shown). Bar, 5 nucleotide substitution per 100 nucleotides.

A family-wide comparison of traits of strain MWH-C5^T and all type species of the 30 genera (including Pseudorhodoferax described by Bruland et al., in press) currently belonging to the family Comamonadaceae was performed. This comparative analysis was limited by the heterogeneity of data sets available for the different type species, because only very few traits were described for the overwhelming majority of strains and virtually no test on substrate utilization was performed for almost all strains. However, comparisons of chemotaxonomic traits, i.e. type of quinone, major fatty acids, major 3-hydroxy fatty acid, and G+C value, revealed a unique combination of these traits for strain MWH-C5^T among all Comamonadaceae genera (Table S2). Strain MWH-C5^T differs from all but two genera by its G+C content of DNA of 55.5 mol%, which is > 3 mol% lower in MWH-C5^T than in other type strains. Among Comamonadaceae type strains only those of Gisbergeria species (Grabovich et al., 2006) and the type strain of the type species of the genus Polaromonas, i.e. P. vacuolata (Irgens et al., 1996), posses G+C values < 59 mol%. Interestingly, Polaromonas vacuolata differs significantly in this trait from the type strains of three other Polaromonas species, which have G+C content of 61.5 to 63.7 mol% (Weon et al., 2008). The G+C value of the fifth described Polaromonas species has not been determined (Kämpfer et al., 2006). Furthermore, the lack of C_10:0-3OH is rare among Comamonadaceae bacteria, and all genera sharing this trait with strain MWH-C5^T possess G+C values > 59 mol%.

A more detailed comparison with the seven most closely related genera (Fig. 2) revealed several differences to type species of these genera suitable for discrimination of the new taxon from these previously described taxa (Table 3). The G+C value is again an important discriminative trait, however the strain also differes in at least one more trait from all other type species. For instance by the absence of C_10:0-3OH fatty acids from Caenimonas, Pseudorhodoferax, and Variovorax, by its ability to grow facultatively anaerobic from strictly aerobic Polaromonas, by a much lower salinity tolerance from Curvibacter type strains, by the ability to assimilate glucose and citrate from Ramlibacter, and by the ability to utilize alpha-ketoglutarate as a carbon source and the lack of rhodoquinone from Rhodoferax type strains.

Table 3.

Phenotypic and chemotaxonomic characteristics that differentiate Limnohabitans curvus gen. nov., sp. nov. strain MWH-C5^T from the closest related genera as determined by the phylogenetic reconstruction presented in Fig. 2. Data for Caenimonas are from Ryu et al., (2008); for Curvibacter spp. are from Ding & Yokota (2004) and Tables 1 and 2; for Polaromonas spp. from Kämpfer et al. (2006), Sizova and Panikov (2007), Jeon et al. (2004), and Irgens et al. (1996); for Pseudorhodoferax are from Bruland et al., (in press); for Rhodoferax spp. from Madigan et al. (2000), Hiraishi et al. (1991), and Finneran et al. (2003), and from Tables 1 and 2; for Variovorax spp. from Yoon et al. (2006), Kim et al. (2006), and Miwa et al. (2008); for Ramlibacter from Heulin et al., (2003) and Ryu et al., (2008). Q, Quinone; RQ, rhodoquinone; +, positive; −, negative; NA, not available; d, variable; w, weak.

Character	MWH-C5T	Caenimonas	Curvibacter	Polaromonas	Pseudorhodoferax	Ramlibacter	Rhodoferax	Variovorax
Cell morphology	Curved rods	Rods	Slightly curved rods	Rods	Short rods	Pleomorphic (rods to cocci(cysts))	Curved rods	Oval or rod shaped
Flagella	None	None	None or polar	None or single, polar	Single, polar	None	Single, polar	Peritrichous
Psychrophilic growth	+	−	−	d	NA	−	−	−
Oxygen requirement	Facultatively anaerobic	Strictly aerobic	Aerobic or microaerophilic	Obligately aerobic	Aerobic	Aerobic	Facultatively aerobic	Strictly aerobic
Maximum salinity (%)	0.5	NA	3	6	NA	NA	1	7
Carbon source used for growth:
alpha-Ketoglutarate	+	NA	+	+	NA	NA	−	NA
Citrate	+	−	+	+	d	−	+	NA
Gluconate (D−)	+	−	d	d	+	−	d	+
Glucose (D−)	+	−	d	d	−	−	d	+
Glutamate	−	NA	+	+	NA	NA	d	NA
Glutamine	−	NA	+	NA	NA	NA	−	NA
Glycerol	−	−	+	d	NA	NA	−	+
Glycolate	−	NA	+	NA	NA	NA	−	NA
Histidine (L−)	−	NA	+	−	NA	NA	−	NA
Lactate (DL−)	−	NA	+	d	NA	+	+	NA
Malate (DL−)	+	−	+	d	+	−	+	NA
Malonate	−	+	w	d	NA	NA	−	d
Mannose (D−)	+	−	d	−	−	−	d	+
N-acetyl-glucosamine	−	−	w	NA	NA	−	+	NA
Oxaloacetate	−	NA	+	d	NA	NA	w	NA
Phenylalanine (L−)	−	NA	+	NA	NA	NA	−	NA
Proline (L−)	−	NA	+	+	NA	NA	+	NA
Pyruvate	+	NA	+	+	NA	+	+	NA
Serine (L−)	−	NA	+	d	NA	NA	+	NA
Tryptophan (L−)	−	−	+	−	NA	NA	w	d
Quinones	Q-8	Q-8	Q-8	Q-8, Q-9	NA	NA	Q-8, RQ-8	Q-8
Major fatty acids	16:0, 16:1	16:0,18:1, feat. 3	16:0, 16:1, 18:1	16:0, 16:1, 18:1	16:0,18:1, feat. 3	NA	16:0, 16:1	16:0, 16:1, 18:1
Major 3-OH acid	8:0	10:0	8:0	8:0, 10:0$	10:0	NA	8:0	10:0
DNA G+C content (mol%)	55.5	63	62 - 66	52 - 63$$	69 - 70	67 - 70	59 - 62	66 – 69
Habitats	Freshwater	Activated sludge	Wellwater	Freshwater, marine	Activated sludge or soil	Soil	Freshwater, activated sludge, marine	Soil

^$Only P. hydrogenivorans DSM 17735^T and P. naphthalenivorans CJ2^T contain C_10:03-OH as the major hydroxylated fatty acid (Jeon et al., 2004; Sizova & Panikov, 2007)

^$$Only the typestrain of Polaromonas vacuolata has a G+C vulue of 52 mol%, while the other type strains of Polaromonas species have G+C vulues of 62-64 mol%

BLAST searches with the 16S rRNA gene sequences of strain MWH-C5^T and subsequent phylogenetic analyses revealed that strain MWH-C5^T is closely related to a large number of uncultured strains (Supplemental Material Fig. S1) detected in a large number of cultivation-independent investigations on the diversity of freshwater bacteria (e.g., Zwart et al., 2002; Crump & Hobbie, 2005; Shaw et al., 2008). Some of these uncultured bacteria share 16S rRNA sequence similarities with strain MWH-C5^T of 99.9%. The phylogenetic cluster formed by these uncultured organisms was previously designated the “Rhodoferax sp. BAL47” cluster (Zwart et al., 2002). The high number of sequences affiliated with this cluster, as well as their geographically widespread origin indicates a significant contribution of bacteria affiliated with this cluster to bacterioplankton in many freshwater lakes and ponds. This is also indicated by an investigation of 15 diverse lakes in northern Europe for the presence of bacteria affiliated with the “Rhodoferax sp. BAL47”cluster or 14 other clusters, which also contain taxa frequently detected in freshwater habitats, by a cultivation-independent method (Lindström et al., 2005; Zwart et al., 2003). The “Rhodoferax sp. BAL47”cluster and an actinobacterial cluster were the only clusters detected in all 15 investigated habitats. A prominent and well-investigated subgroup of the “Rhodoferax sp. BAL47” cluster is the so-called R-BT065 group (Šimek et al., 2001; Šimek et al., 2005), which can be detected by a specific FISH probe. Investigations of several freshwater ponds and lakes by using this FISH probe revealed that the targeted cells possess a planktonic lifestyle, and that this taxon comprises typically 5-30 % (maximum ~ 50 %) of total bacterioplankton cells in non-acidic stagnant freshwater habitats (Šimek et al., 2001; Šimek et al., 2005; Šimek et al., unpubl. data). The origin of strain MWH-C5^T from the pelagic zone of a freshwater lake, as well as the close phylogenetic relationship with strains inhabiting the water column of freshwater systems indicates that this strain shares a planktonic lifestyle with R-BT065 bacteria and other members of the “Rhodoferax sp. BAL47” cluster.

Based on the reconstructed phylogenetic position of strain MWH-C5^T (Fig. 2), the phylogenetic distances to type species of closest related genera (Table S1), and the phenotypic and chemotaxonomic differences between the strain and type species of related genera (Table 3 and Table S2) we propose to establish the new genus Limnohabitans gen. nov., and the new species Limnohabitans curvus sp. nov. with strain MWH-C5^T as type species and type strain of the new genus and the new species, respectively.

Description of Limnohabitans gen. nov.

Limnohabitans (Lim.no.ha.bi’tans. Gr. n. limne, lake; L. part. adj. habitans, inhabiting; N.L. part. adj. used as a masc. n. Limnohabitans, lake dweller, referring to the type of ecosystem inhabited by these bacteria).

Aerobic, facultatively anaerobic, chemo-organotrophic, oxidase and catalase positive bacteria. Cells are non-pigmented, non-motile curved rods. Not halotolerant, do not grow at NaCl concentrations > 0.5%. Mesophilic. Major fatty acids (constituting >5 % of total fatty acids) are C_16:0 and C_16:1ω7c/ω6c. The major quinone is ubiquinone Q-8. Isolated from the water column of freshwater habitats. The genus is affiliated to the class Betaproteobacteria and to the family Comamonadaceae. The type species is Limnohabitans curvus.

Description of Limnohabitans curvus sp. nov.

Limnohabitans curvus (cur.vus. L. masc. adj. curvus curved or crooked).

Cell morphology of curved rods, 1.0–1.5 μm in length and 0.4–0.5 μm in width. Chemoorganotroph, aerobic, facultatively anaerobic, oxidase and catalase positive. Colonies grown on NSY agar are unpigmented, circular and convex with smooth surface. Growth occurs at 4–34 °C, and with 0–0.5 % (w/v) NaCl. Assimilates acetate, glycerate, alpha-ketoglutarate, pyruvate, fumarate, citrate, malate, succinate, gluconate, glucose, and mannose. Week assimilation was observed for several substances (Table 1). No assimilation of glycerol, glyoxylate, glycolate, oxalate, lactate, malonate, oxaloacetate, arginine, glutamate, glutamine, histidine, phenylalanine, proline, serine, tryptophan, sorbose, N-acetyl-glucosamine, betaine, spermidine, and carnitine. Major cellular fatty acids (> 5% of total) are C_16:0, and C_16:1ω7c/ω6c. The major quinone is ubiquinone Q-8, and the G+C value of the DNA is 55.5%. The type strain is MWH-C5^T (DSM 21645^T, = CCUG 56720 ^T) isolated from Lake Mondsee, Austria.

Abbreviations

OD

optical density

FISH

fluorescence in situ hybridization

NJ

neighbour-joining

ML

maximum-likelihood

BI

bayesian inference