Limnohabitans planktonicus sp. nov., and Limnohabitans parvus sp. nov., two novel planktonic Betaproteobacteria isolated from a freshwater reservoir

Abstract

Two strains II-B4^T and II-D5^T, isolated from the meso-eutrophic freshwater Římov Reservoir (Czech Republic) were phenotypic ally, phylogenetically and chemotaxonomically characterized. Both strains are chemoorganotrophic, facultatively anaerobic, rod-shaped, non-motiles with identical G+C contents of their DNA of 59.9 mol%. Their major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, 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. Both strains contained Q-8 as the only respiratory quinone component. The 16S rRNA sequences of the two strains posses a similarity of 99.0%, however the DNA-DNA reassociation value was only 26.7%, and the strains can be discriminated from each other by several chemotaxonomic and biochemical traits. The 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 a narrow phylogenetic cluster (so-called R-BT065 cluster) formed by a large number of environmental sequences obtained from the pelagic zones of various freshwater habitats. We propose to place the two strains in separate new species within the genus Limnohabitans. The two proposed species are Limnohabitans planktonicus sp. nov. with the type strain II-D5^T (= DSM 21594^T = CIP 109844^T), and Limnohabitans parvus sp. nov. with the type strain II-B4^T (= DSM 21592^T = CIP 109845^T).

Betaproteobacteria frequently represent the numerically dominating group of bacterioplankton in a broad variety of freshwater habitats. Recent research demonstrated that only a few, phylogenetically narrow subgroups of the Betaproteobacteria are responsible for the ecological success of this group 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 FISH probe (Šimek et al., 2001). Application of this probe revealed that the targeted cells possess a planktonic lifestyle, and revealed that this cluster comprises typically 5-30 % (maximum ~ 50 %) of total bacterioplankton in non-acidic stagnant freshwater habitats (Šimek et al., 2001, 2005; Pérez and Sommaruga, 2006; Salcher et al., 2008; Šimek et al., unpub. data).

Ecological studies revealed that members of the R-BT065 cluster possess the ability to rapidly respond 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, this cluster usually represents the fastest growing segment of bacterioplankton in lakes (e.g. Šimek et al., 2006, Salcher et al., 2008). Thus, this cluster of bacteria obviously plays an important role in organic matter processing and transformation in freshwater systems. So far, the so-called R-BT065 cluster is lacking validly described species. Here, we describe two strains affiliated with this cluster and propose to establish for them two new species in the genus Limnohabitans (Hahn et al., in press).

The two strains, II-B4^T and II-D5^T, were isolated from the water column of Římov Reservoir (Supplemental Material Table S1 in IJSEM Online) using the filtration-acclimatization method (Hahn et al., 2004). Briefly, the whole water sample was filtered through a 0.8 μm polycarbonate membrane filter (Millipore) and was subsequently diluted with sterile medium in order to obtain cell concentrations suitable for inoculation of 24-well microplates with approximately 0.5 cell per well. The 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 media 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 optical density (OD) established in liquid one-tenth-strength NSY medium (0.3 g l⁻¹) with and without 0.5 g l⁻¹ test substance (pH of 7.2) as described previously (Hahn et al., 2009). 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. Growth at different temperatures (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 different NaCl concentrations (0, 0.05, 0.075, 0.1, 0.15, 0.2, 0.3, 0.5, 1.0 and 1.5% w/v).

Gram-staining was performed as described by Hucker and 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 l⁻¹ NSY medium at RT were inspected by 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 analyzed 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).

Cellular fatty acid (FA) contents of the strains 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 this analysis, biomass of replicated cultures of each strain grown in NSY medium (3 g l⁻¹) for 2 days at 21 °C was analyzed.

The determination of the G+C content of DNA, the analyses of major respiratory quinones, polar lipids, and the DNA–DNA hybridization of both strains 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-thermostated 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).

Both strains, II-B4^T and II-D5^T, are Gram-negative, rod-shaped and non-motile bacteria. Both grew well on NSY medium, R2A agar (Remel), Standard Methods agar (Remel), Peptone agar (BD-Difco), Casitone agar (BD-Difco), solidified Luria–Bertani broth (BD-Difco), solidified Brain Heart Infusion (BD-Difco), and solidified Tryptic Soy Broth (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 the phenotypic and chemotaxonomic investigations are presented in Tables 1 and 2. The strain II-D5^T was able to utilize 19 (plus 4 weakly) of 37 tested substrates, whereas the strain II-B4^T responded with negative growth in more than half of tests (22 substrates). The major polar lipids of both strains were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine (Supplemental Material Figure S2 in IJSEM Online), 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. Their major ubiquinone was Q-8, respectively, and they shared identical G+C contents of their DNA of 59.9 mol%.

Table 1.

Phenotypic differentiation characteristics of type strains II-B4^T and II-D5^T from each other and from Limnohabitans curvus strain MWH-C5^T. The substrate utilization tests were performed for all three strains under the same conditions. All three strains are catalase-positive, oxidase-positive, non-motile and unpigmented and share the following traits: low salinity tolerance (0.5%) assimilate (all ‘+’) α-ketoglutarate, citrate, butyrate, D-glucose, D-glycerate, malate; no assimilation (‘−‘) of L-arginine, betaine, L-carnitine, DL-lactate, malonate, N-acetyl-glucosamine, oxalate, L-sorbose and spermidine. ‘+’, positive; ‘w’, weak; ‘−‘, negative assimilation. Data for L. curvus from Hahn et al. (in press).

	II-B4T	II-D5T	L. curvus
Cell morphology	short rods	rods	curved rods
Cell length (μm)	0.6 ± 0.14	0.9 ± 0.26	1-1.5
Cell width (μm)	0.3 ± 0.05	0.3 ± 0.07	0.4 - 0.5
Acetate	−	+	+
Ethanol	−	w	w
Fumarate	w	+	+
Galactose (D−)	−	−	w
Gluconate (D−)	−	−	+
Glutamate (L−)	+	+	−
Glutamine	w	+	−
Glycerol	w	+	−
Glycolate	−	w	−
Glyoxylate	−	w	−
Histidine (L−)	−	+	−
Mannose (D−)	w	+	+
Oxaloacetate	−	+	−
Phenylalanine (L−)	−	+	−
Proline (L−)	+	+	−
Propionate	−	−	w
Pyruvate	w	+	+
Ribose (D−)	−	−	w
Saccharose (D−)	−	−	w
Serine (L−)	−	+	−
Succinate	w	+	+
Tryptophan (L−)	+	w	−

Table 2.

Cellular fatty acid composition of both strains II-B4^T and II-D5^T and Limnohabitans curvus strain MWH-C5^T. Values are percentages of the summed fatty acids; nd – not detected. For unsaturated fatty acids, the position of the double bond is located by counting from the methyl (ω) end of the carbon chain. Cis and trans isomers are indicated by the suffixes c and t, respectively. Data for L. curvus from Hahn et al. (in press).

	II-B4T	II-D5T	L. curvus
C8:0-3OH	1.0	0.7	2.7
C10:0-3OH	nd	1.5	nd
C12:0	3.6	2.9	4.5
C12:0 -3OH	1.8	nd	nd
C14:0	0.4	0.5	1.0
C14:1ω5c	0.2	0.2	0.4
C15:1ω6c	1.3	nd	nd
C16:0	15.0	19.5	14.0
C16:1ω5c	0.5	0.7	0.2
C16:1ω7c/C16:1ω6c	66.4	62.4	76.7
C17:0	1.3	nd	nd
C17:0 cyclo	nd	0.7	nd
C17:1ω6c	2.6	nd	nd
C18:0	0.5	0.3	0.3
C18:1ω7c 11Me	nd	1.3	0.3
C18:1ω7c/C18:1ω6c	5.3	8.9	1.8
C18:1ω9c	0.3	0.5	0.2

The phylogenetic analyses of the 16S rRNA gene sequences by two independent algorithms resulted in trees with largely identical branching orders (Fig. 2). The two trees only differed in the phylogenetic positions of the taxa 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 within the family Comamonadaceae. Phylogenetic analyses with sequence sets including environmental sequences (data not shown) 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 important cluster of freshwater bacteria, thus the two investigated strains represent the first taxonomically described members of this phylogenetic cluster.

Fig. 2.

Neighbour-joining (NJ) tree based on almost complete 16S rRNA gene sequences, reconstructing the phylogenetic position of the two investigated strains. The NJ tree shared with a maximum likelihood (ML) tree (not shown) calculated with the same sequence set identical branching orders except of the branchings by P. vacuolata, C. koreensis, and V. paradoxus. Bootstrap values obtained by the NJ (first value) and the ML algorithm (second value) are shown. Nodes not reconstructed in the ML tree show instead of a bootstrap value a horizontal slash. Bar indicates 0.05 substitutions per nucleotide position

The affiliation of the two investigated strains to the genus Limnohabitans is 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 compositions, low salinity tolerance, lack of pigmentation, lack of motility, and their inability to assimilate lactate.

At the opposite, the two investigated strains II-B4^T and II-D5^T can be discriminated from L. curvus by the presence or absence of minor fatty acids components, and some physiological and metabolical characteristics (Table 1). Furthermore, the two species can be differentiated from each other by their morphology and by several metabolic traits (see Table 1 for details), or by the absence or presence of seven minor fatty acids (Table 2). The strains differ, for instance, in assimilation of acetate, L-histidine, oxaloacetate, and in the presence/absence of C_17:0 and C_17:1ω6c fatty acids. Traits discriminating both strains from the type strain of L. curvus are, for example, assimilation of L-glutamate and L-proline, as well as, lack of assimilation of D-gluconate and propionate (Table 1).

Contrary to L. curvus, both newly described strains share a short diagnostic 16S rRNA gene sequence (E. coli positions 65-83) targeted by the FISH probe R-BT065 (5′-GTTGCCCCCTCTACCGTT-3′, Šimek et al., 2001). Comparative analysis of the almost full-length 16S rRNA gene sequences (1411 bp) of the strains II-B4^T and II-D5^T revealed a sequence similarity of 99.0 % whereas the similarities with L. curvus were 96.8 and 96.7%, respectively. DNA-DNA reassociation experiments with DNA extracted from strains II-B4^T and II-D5^T resulted in a value of 26.7%, which clearly justifies the proposal to place the two strains in separate species (Wayne et al., 1987).

Regarding to all explicit differences between the two strains and the type strain of L. curvus, as well as between the two strains, we propose to establish the two new 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.

Description of Limnohabitans parvus sp. nov.

Limnohabitans parvus [par’vus. L. masc. adj. parvus small, referring to the small cell size of the type strain].

Cells are Gram-negative, short rods, about 0.3 μm in diameter and about 0.6 μm length, non-motile, unpigmented, oxidase-positive and catalase-positive. Growth is evident at temperatures of 4–34 °C, well-growing under aerobic conditions, weak anaerobic growth. Good growth occurs on NSY, R2A, Standards Method Agar, TSB, LB Broth, Peptone, Brain Heart Infusion, and Casitone agar, not growing on Löwenstein Medium. 1–2 mm large colonies are formed within 2–5 days (RT). Growth occurs at a salinity range 0–0.5% NaCl (w/v). The only respiratory quinone is Q-8. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The fatty acids profile is largely composed of C_16:1ω7c/C_16:1ω6c (66.4 %), C_16:0 (15.0 %), C_12:0 (3.6 %), and C_18:1ω7c/C_18:1ω6c (5.3 %). The major detected hydroxy-fatty acids are C_8:0-3OH (1.0 %) and C_12:0-3OH (1.8 %). The type strain is II-B4^T (= DSM 21592^T = CIP 109845^T). The type strain was isolated from the freshwater meso-eutrophic Římov Reservoir, Czech Republic, and possesses a free-living planktonic lifestyle.

Description of Limnohabitans planktonicus sp. nov.

Limnohabitans planktonicus [plank.to’ni.cus. N.L. masc. adj. (from Gr. adj. planktos) planktonicus living in the plankton, planktonic].

Cells are Gram-negative, rod-shaped, about 0.3–0.4 μm in diameter and about 0.9 μm in length, non-motile, unpigmented, oxidase-positive and catalase-positive. Growth is evident at temperatures of 4 – 34 °C, well-growing under aerobic conditions, weak anaerobic growth. Good growth occurs on NSY, R2A, Standards Method Agar, TSB, LB Broth, Peptone, Brain Heart Infusion, and Casitone agar, not growing on Löwenstein Medium. 1–2 mm large colonies are formed in 2–5 days (RT). Growth occurs at a salinity range 0–0.5% NaCl (w/v). The only respiratory quinone is Q-8. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The fatty acids profile is largely composed of C_16:1ω7c/C_16:1ω6c (62.4 %), C_16:0 (19.5 %), C_18:1ω7c/C_18:1ω6c (8.9 %), and C_12:0 (2.9 %). The major detected hydroxyl-fatty acids were C_8:0-3OH (0.7 %) and C_10:0-3OH (1.5 %). The type strain is II-D5^T (= DSM 21594^T = CIP 109844^T). The type strain was isolated from the freshwater meso-eutrophic Římov Reservoir, Czech Republic, and possesses a free-living planktonic lifestyle.

Fig. 1.

Electron microscopy images illustrating the cell morphology and size of the type strains II-B4^T (A) and II-D5^T (B), respectively. 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.

Abbreviations

FISH

Fluorescent in situ hybridization

OD

Optical density

RT

room temperature