Polynucleobacter sphagniphilus sp. nov. a planktonic freshwater bacterium isolated from an acidic and humic freshwater habitat

Abstract

Strain MWH-Weng1-1^T, isolated from an acidic freshwater habitat located in the Wenger bog, Austria, was characterized by investigation of phenotypic, chemotaxonomic and genomic traits. Phylogenetic analyses based on 16S rRNA gene sequences placed the strain in the cryptic species complex PnecC within the genus Polynucleobacter. The strain has a genome of 2.04 Mbp with a G+C content of 45.6 mol%. The major fatty acids of the strain were C_16:1 ω7c, C_16:0 , and C_18:1 ω7c. In order to resolve the systematic position of the strain within the species complex PnecC, concatenated partial sequences of eight housekeeping genes were used for phylogenetic analyses. The obtained trees did not place strain MWH-Weng1-1^T close to any of the six previously described species within this cryptic species complex. Pairwise whole genome average nucleotide identity (gANI) comparisons with genome sequences of strains representing the six previously described species of the subcluster resulted throughout in values < 78%, which clearly suggested that strain MWH-Weng1-1^T (DSM 24018^T =CIP 111099^T) represents a novel species. We propose the name Polynucleobacter sphagniphilus sp. nov. and strain MWH-Weng1-1^T as the type strain for this new species.

The genus Polynucleobacter and the species Polynucleobacter necessarius were described by Klaus Heckmann and Helmut J. Schmidt [1] for bacterial endosymbionts of benthic freshwater ciliates affiliated with the genus Euplotes. Later, it was discovered that free-living strains, closely related to endosymbiotic strains, represent important planktonic freshwater bacteria [2–5]. Analyses of 16S rRNA gene sequences suggested that the genus Polynucleobacter can be subdivided in at least four subclusters designated PnecA, PnecB, PnecC, and PnecD [4]. Subcluster PnecC, which currently includes the endosymbiotic P. necessarius and five free-living species, was recognized as being a cryptic species complex, which means that this seemingly very species-rich subcluster cannot be resolved by 16S rRNA gene phylogenies [6, 7]. Taxonomic research on strains affiliated with subcluster PnecC is hampered by the lack of a type strain of P. necessarius or another pure culture representing this endosymbiotic specie [6]. Reference material of the type species of the genus was contained in the Euplotes aediculatus ‘stock 15’ culture (=E24 =ATCC 30859) (Heckmann and Schmidt, 1987), which is unfortunately not any more available. However, recently, it was shown [6] that the lost endosymbionts used for the species description are well represented by another endosymbiotic P. necessarius strain, i.e. STIR1, for which a complete genome sequence is available [8].

Here we describe strain MWH-Weng1-1^T affiliated with subcluster PnecC, which was isolated from a small acidic freshwater pond, and propose to establish for this strain the species name P. sphagniphilus sp. nov.

Strain MWH-Weng1-1^T was isolated from an acidic water body located at the margins of Wenger bog (approximate geographic coordinates 47.93 N 13.18 E) near Salzburg in Austria. The sampled water had a pH of 4.0, a conductivity of 63.4 µS cm^-1, and a temperature of 5.8 °C. The water was strongly stained by humic substances (OD_250nm of 0.2µm-filtered water of 1.77). The strain was isolated by using the filtration-acclimatization method and NSY medium [9, 10].

Cells of strain MWH-Weng1-1^T are short rods of small size (Table 1). The strain forms small circular, convex, colourless colonies with shiny surface on NSY agar plates. Growth at different temperatures and growth under anoxic conditions in an anaerobic chamber were examined by using NSY agar plates as described previously [11]. Salinity (NaCl) tolerance was determined using NSY agar supplemented with various NaCl concentrations as described previously [11]. The strain showed no anaerobic growth, grew at temperatures up to 31°C and tolerated salt concentrations up to 0.4% (Table 1).

Table 1.

Traits characterizing the six Polynucleobacter type strains affiliated with subcluster PnecC. All strains have the following characteristics in common. Motility not detectable; assimilation of acetic acid, pyruvic acid, and succinic acid; no assimilation of glycolic acid, oxalic acid, citric acid, and L-serine. 1, P. sphagniphilus sp. nov. MWH-Weng1-1^T; 2, P. wuianus QLW-P1FAT50C-4^T; 3, P. asymbioticus QLW-P1DMWA-1^T; 4, P. duraquae MWH-MoK4^T; 5, P. sinensis MWH-HuW1^T; 6, P. yangtzensis MWH-JaK3^T. +, increase in optical density (OD); w, weak increase in OD; -, no significant increase in OD.

	1	2	3	4	5	6
Cell morphology	short rods	short rods	short rods	curved rods	short curved rods	short rods
Cell length (µm)	0.6-1.0	0.6-1.7	0.7-1.2	0.9-2.9	0.6-1.4	0.5-1.5
Cell width (µm)	0.3-0.5	0.3-0.6	0.4-0.5	0.4-0.5	0.4-0.5	0.3-0.5
Temperature range of growth (°C)	5 - 31	5 - 34	5 - 34(w)	5 - 30	5 - 35	5 - 35
NaCl tolerance (%NaCl, w/v)	0 - 0.4	0 - 0.5	0 - 0.5(w)	0 - 0.3	0 - 0.5	0 - 0.3(w)
Anaerobic growth	-	-	+	-	-	+
Assimilation of:
Glyoxylic acid	-	w	w	-	-	-
Propionic acid	w	+	+	-	+	+
Malonic acid	+	w	w	-	+	w
Oxaloacetic acid	-	+	-	+	+	+
Malic acid	+	+	+	w	+	+
Fumaric acid	+	+	+	w	+	+
Levulinic acid	w	w	w	-	-	-
D-Galacturonic acid	+	w	w	w	w	w
D-Mannose	w	-	w	-	-	-
D-Glucose	-	-	w	w	-	-
D-Galactose	-	-	w	-	-	-
D-Lyxose	-	-	w	w	-	w
D-Fructose	w	w	w	w	-	w
L-Fucose	w	-	w	-	-	w
D-Sorbitol	w	-	w	-	-	-
L-Glutamate	+	+	+	-	+	-
L-Histidine	-	+	n.d.	n.d.	n.d.	n.d.
L-Aspartate	-	+	+	-	-	-
L-Cysteine	+	w	+	+	w	+
L-Alanine	+	+	w	-	-	-
L-Asparagine	-	w	w	-	-	-
L-Leucine	-	-	n.d.	n.d.	n.d.	n.d.
Betaine	-	-	w	-	-	-

Utilization of various substrates was investigated in the same way as for previously described Polynucleobacter species [11–15]. Briefly, growth enabled by utilization of a specific substrate was determined by comparison of OD at 575 nm established in liquid one tenth-strength NSY medium (0.3 g l^-1) with and without 0.5 g l^-1 test substrate, respectively. Differences of < 10 %, 10–50% and >50% of the OD obtained in the test treatments compared to the OD obtained without test substrate (i.e. in 0.3 g l^-1 NSY medium) were scored after 10 days of growth as no utilization (-), weak utilization (w) and good utilization (+), respectively (Table 1).

The analysis of the whole-cell fatty acid composition (Table 2) was carried out as described previously [12]. Biomass was harvested from cultures on R2A agar plates, which were inoculated with 1 ml cell suspension, incubated while keeping the agar surface moist and inspected for growth daily, starting the third day after inoculation. Once a biomass film was well visible, the cell mass was harvested.

Table 2.

Major fatty acid compositions of Polynucleobacter strains representing subcluster PnecC. Strains were grown on R2A agar. Data for strains QLW-P1DMWA-1^T, MWH-HuW1^T, MWH-JaK3^T were taken from [11], data for MWH-MoK4^T from Hahn et al. (2016b), and data for QLW-P1FAT50C-4^T from Hahn et al. (2017).

Fatty acid	MWH-Weng1-1T	QLW-P1FAT50C-4T	QLW-P1DMWA-1T	MWH-MoK4T	MWH-HuW1T	MWH-JaK3T
	DSM 24018T	DSM 24008T	DSM 18221T	DSM 21495T	DSM 21492T	DSM 21493T
C12:0	4.4	3.3	3.4	3.8	5.5	3.7
C14:0	1.4	0.3	0.9	0.3	0.3	1.2
C15:0	-	0.2	0.3	-	0.3	-
C16:0	26.2	18.6	22.2	15.9	29.6	15.5
C17:0	-	-	-	-	0.5	-
C18:0	0.9	0.5	1.2	0.5	2.4	0.5
C20:0	-	-	1.1	-	-	-
C14:1 ω5c	0.5	-	-	-	0.2	0.6
C15:1 ω6c	-	-	-	-	0.6	-
C16:1 ω5c	0.4	0.4	0.9	0.4	-	0.4
C16:1 ω7c	35.8	40.8	41.3	38.6	45.0	35.6
C18:1 ω9c	-	-	-	0.3	0.4	-
C18:1 ω7c	15.1	20.4	12.9	19.8	1.1	20.4
11-methyl C18:1 ω7c	4.3	4.6	3.1	4.2	1.1	8.1
C12:0 2-OH	1.9	0.3	2.5	1.3	1.3	2.2
C16:1 2-OH	0.5	1.5	-	1.8	-	v
Feature 1	1.3	-	0.4	-	1.0	0.5
Feature 2	7.35	7.1	9.6	11.9	9.9	9.2
Feature 7	-	1.2	0.4	-	0.3	2.0

Summed features represent groups of two fatty acids which could not be separated by GLC and the MIDI system, such as summed feature 2 containing C_16:1 isoI and C_14:0-3OH and summed feature 7 containing C_19:1 ω6c and an unknown compound with an ECL of 18.846, or represent compounds with uncertain identity such as summed feature 1 (equivalent chain length 10.921) probably containing modified C_12:0.

Genome sequencing and analyses were performed for further characterization of strain MWH-Weng1-1^T. DNA used for sequencing was extracted from biomass grown in liquid NSY medium as described previously [16]. A fragment library was mate-pair sequenced by a Roche GS FLX system by using Titanium chemistry (Beckman Coulter Genomics SA, Grenoble, France). Sequencing resulted in 237 963 reads and de novo assembly of 234 897 reads by using the software MIRA [17] resulted in 20 contigs with a sequencing coverage of 38x. Three gaps could be closed by PCR amplification of gap regions and subsequent Sanger sequencing of amplicons. These efforts resulted in a reduction of the contig number to seventeen. The obtained genome sequence has a total length of 2.04 Mbp and a G+C content of 45.6 mol% (Table 3). The genome sequence was annotated using the IMG/ER annotation pipeline [18], as well as by the NCBI Prokaryotic Genome Annotation Pipeline. The annotation obtained by the latter pipeline was deposited in DDBJ/EMBL/GenBank (Accession Number MPIY01000000), while the analyses presented here refer to the IMG annotation (IMG genome ID 2574179738). Gene finding resulted in 2103 protein coding and 49 RNA genes.

Table 3.

Genome characteristics of the investigated Polynucleobacter strains.

Species	Strain	Life style	Genome size (Mbp)	Scaffolds	G+C content (mol%)	DDBJ/EMBL/GenBank accession number	Reference
P. sphagniphilus sp. nov.	MWH-Weng1-1T (=DSM 24018T)	FL	2.04	17	45.6	MPIY01000000	This study
P. wuianus	QLW-P1FAT50C-4T (=DSM 24008T)	FL	2.23	1	44.9	CP015922	Hahn et al., in press
P. asymbioticus	QLW-P1DMWA-1T (=DSM 18221T)	FL	2.16	1	44.8	CP000655	Meincke et al., 2012
P. duraquae	MWH-MoK4T (=DSM 21495T)	FL	2.03	1	45.2	CP007501	Hahn et al., 2016b
P. sinensis	MWH-HuW1T (=DSM 21492T)	FL	2.32	19	45.5	LOJJ01000000	Hahn et al., 2016a
P. yangtzensis	MWH-JaK3T (=DSM 21493T)	FL	2.05	42	45.4	LOJI01000000	Hahn et al., 2016a
P. necessarius	STIR1 [host, Euplotes aediculatus]	E	1.56	1	45.6	CP001010	Boscaro et al., 2013

FL, free-living; E, endosymbiotic.

Strain MWH-Weng1-1^T shares with the five other free-living type strains representing species affiliated with subcluster PnecC a genome size in the range of 2.0-2.3 Mbp (Table 3), as well as a G+C value of DNA of about 45%. On the other hand, the genomes of the six free-living type strains differ largely in gene content (Table 4). Interestingly, regarding the listed selected accessory genes, the gene content of strain MWH-Weng1-1^T is most similar to P. wuianus QLW-P1FAT50C-4^T and a little less similar to P. asymbioticus QLW-P1DMWA-1^T, but much more distinct from the genomes of the type strains of the other three free-living species affiliated with subcluster PnecC. The genomes of strains MWH-Weng1-1^T and QLW-P1FAT50C-4^T differ only in two of the gene content features considered in Table 4, but the former genome differed in absence/presence from the genome of strain MWH-MoK4^T in 12 of the 16 considered accessory genes and gene clusters. It is worth mentioning that the two genomes most similar in gene content to strain MWH-Weng1-1^T share with this strains the origin from acidic freshwater systems, while the other three type strains considered in Table 4 were isolated from alkaline freshwater systems.

Table 4.

Comparison of the presence and absence of selected genes.

Genes putatively encoding	P. sphagniphilus sp. nov.	P. wuianus	P. asymbioticus	P. duraquae	P. sinensis	P. yangtzensis
	MWH-Weng1-1T	QLW-P1FAT50C-4T	QLW-P1DMWA-1T	MWH-MoK4T	MWH-HuW1T	MWH-JaK3T
Inorganic nutrients
ABC-type Fe3+ transport system	-	-	-	+	+	+
feoAB genes (uptake of Fe2+)	+	+	+	-	+	+
ABC-type Nitrate/Nitrite/Cyanate transporter	+	+	+	-	-	+
Nitrate reductase (assimilatory)	+	+	+	-	-	+
Nitrite reductase (assimilatory)	+	+	+	-	-	+
Cyanate lyase (releases NH3 and CO2 from cyanate)	+	+	+	-	-	+
Urease and ABC-type urease transporter	+	+	+	-	-	-
Oxidative phosphorylation/Energy metabolism
Cytochrome bd-I terminal oxidase (CydAB)	+	+	+	-	+	-
Fumarate reductase	+	+	-	+	-	+
Carbon monoxide dehydrogenase	+	+	-	2 clusters	-	+
Acetate permease actP	+	-	+	-	-	-
Anoxygenic photosynthesis
Photosynthesis gene cluster	-	+	-	+	-	-
Motility
Flagella genes	-	-	-	+	-	-
Oxidative stress
Catalase	1 gene	-	2 genes	-	-	1 gene
Other
Cellulose synthase operon protein C	-	-	+	-	-	-
Cellulose synthase catalytic subunit [UDP-forming]	-	-	+	-	-	-

The partial glutamine synthetase gene (glnA) and the 16S-23S internal transcribed spacer (ITS) sequences of strain MWH-Weng1-1^T determined by previous PCR amplicon sequencing (accession numbers FN823191 and FN429716, respectively) and by genome sequencing were identical, respectively.

Phylogenetic reconstructions based on 16S rRNA gene sequences place strain MWH-Weng1-1^T in subcluster PnecC [4] of the genus Polynucleobacter (data not shown). As reported previously, strains affiliated with this species complex share 16S rRNA genes with sequence similarities ≥ 99%. In order to better resolve the phylogenetic position of strain MWH-Weng1-1^T a tree based on concatenated multilocus sequences was calculated (Fig. 1). Partial sequences of eight loci (rpoB, trpE, icdA, glnA, mdh, fbp, msbA, and gyrA) defined previously [6] were extracted from genome sequences (Table 3), concatenated and aligned by using the software MEGA7 [19]. This resulted in a total alignment length of 6359 bp. The same software was used for calculation of neighbour-joining (NJ), maximum likelihood and maximum parsimony trees (Fig. 1). In previous pairwise comparisons, sequence similarity values of these concatenated sequences correlated well with average nucleotide identify (ANI) values of whole genome sequences [6]. This suggests that this locus selection is somehow representative for the entire genomes. The phylogenetic reconstruction based on this multilocus sequence set also placed strain MWH-Weng1-1^T in subcluster PnecC but did not suggest that the strain is affiliated with any previously described species.

Fig. 1.

Phylogenetic position of strain MWH-Weng1-1^T. Neighbour-joining (NJ) tree calculated with concatenated multilocus sequences of eight protein-encoding genes. The tree was rooted with sequences of two Cupriavidus strains. All included Polynucleobacter strains are affiliated with subcluster PnecC. Bootstrap values are shown from left to right for NJ, maximum likelihood, and maximum parsimony trees calculated with the same sequence set.

Polynucleobacter strains affiliated with subcluster PnecC are known to either be obligate endosymbionts of ciliates [1, 20] or free-living organisms possessing a planktonic lifestyle [6, 21]. Due to the isolation of strain MWH-Weng1-1^T from a small freshwater system, due to its ability to grow in artificial media in absence of a potential host [20], and due to the lack of a large number of pseudogenes in the genome of the strain [8], it is assumed that this strain represents a free-living bacterium with a planktonic lifestyle. Cultivation-independent investigations on 56 European freshwater systems obtained priB sequences identical with the reference sequence of strain MWH-Weng1-1^T with high copy numbers from several acidic systems but no sequences or only very small copy numbers from all investigated alkaline systems (Huemer, A., Schmidt, J. & M.W. Hahn, unpublished data). Habitats in which the taxon represented by strain MWH-Weng1-1^T was detected with high abundance usually represented small bog ponds or humic lakes receiving water from neighbouring bogs. Typically, these habitats were characterized by the presence of peat moss (Sphagnum spp.) forming floating mats along the shore lines (Schwingrasen) and/or occurring in the catchment with high abundance. The pattern of environmental detection of the taxon suggests that the strain and the taxon represented by the strain are adapted to acidic freshwater systems. These detections fit very well to the environmental conditions in the home habitat of the type strain. A preference of strain MWH-Weng1-1^T for acidic habitats is also suggested by the presence of genes putatively encoding a Fe(II) transporter (Table 4) and the absence of genes encoding Fe(III) transporters (Hahn et al., 2016b). Furthermore, the above mentioned much higher similarity in gene content between the three PnecC strains obtained from acidic systems suggests a common adaptation to acidic environments.

We tested if strain MWH-Weng1-1^T has to be considered to be affiliated with one of the five previously described free-living species affiliated with subcluster PnecC [22] by performing average nucleotide identity (gANI) analyses with whole genome sequences by using the IMG/ER system [18]. This resulted in all pairwise comparisons in gANI values of 76.7 - 77.1% (Table 5), which suggests that the strain is not affiliated with any of these four species [23–27]. A comparison of strain MWH-Weng1-1^T with the type material of the species P. necessarius cannot be performed due to the lack of a genome sequence or pure genomic DNA of the endosymbionts, however, previous investigations suggested that the genome sequence of the endosymbiont P. necessarius STIR1 has to be considered to share a highly similar genome sequence with the endosymbiont described as P. necessarius [6]. An ANI comparison of the genomes of MWH-Weng1-1^T and STIR1 resulted in an ANI value of 79.6%, which is very similar to those values obtained for the four free-living type strains (Fig. 1). Thus, strain MWH-Weng1-1^T has to be considered to represent a new species affiliated with subcluster PnecC of the genus Polynucleobacter.

Table 5.

Whole genome Average Nucleotide Identity (gANI) values of strain MWH-Weng1-1^T with the genomes of the five free-living type strains of species affiliated with subcluster PnecC and the endosymbiont P. necessarius STIR1, which is also affiliated with this subcluster. Analyses were performed by using the IMG/ER system [18]. Exchanging of subject and query genome resulted in all pairwise calculations in identical gANI values, however the obtained Alignment Fractions (AF) differed when query and reference genomes were exchanged.

Strain	gANI (%)	AF (%)
P. asymbioticus QLW-P1DMWA-1T	76.7	65a/68b
P. yangtzensis MWH-JaK3T	76.9	64a/64b
P. sinensis MWH-HuW1T	76.9	55a/63b
P. duraquae MWH-MoK4T	76.7	63a/62b
P. wuianus QLW-P1FAT50C-4T	77.1	63a/68b
P. necessarius STIR1 (Endosymbiont)	76.9	71a/54b

^agenome of MWH-Weng1-1^T served as subject genome

^bgenome of MWH-Weng1-1^T served as query genome

Strain MWH-Weng1-1^T can be discriminated from the type strains of Polynucleobacter species not affiliated with subcluster PnecC by chemotaxonomic traits. As for other species affiliated with subcluster PnecC, strain MWH-Weng1-1^T can be discriminated from the type strains of P. rarus [14], P. acidiphobus [15] and P. difficilis [12] based on the G+C content of their DNA [6]. The discrimination of strain MWH-Weng1-1^T from P. cosmopolitanus is possible by the absence of the fatty acid C_12:03-OH [13], which was so far only found in Polynucleobacter strains affiliated with the species P. cosmopolitanus. Furthermore, strain MWH-Weng1-1^T, like all other PnecC strains, contains the signature sequence 5’-GAGCCGGTGTTTCTTCCC-3’ at Escherichia coli position 445–463 of the 16S rRNA, which is absent in Polynucleobacter strains not affiliated with this subcluster [6]. A feature distinguishing strain MWH-Weng1-1^T from all previously described type strains of species affiliated with subcluster PnecC is the combination of negative results in assimilation tests with both oxaloacetate and L-aspartate (Table 1). The other type strains showed at least for one of these two substrates positive growth. Furthermore, strain MWH-Weng1-1^T is the only type strain within subcluster PnecC with a clearly positive result on growth with D-galacturonic acid.

Description of Polynucleobacter sphagniphilus sp. nov

Polynucleobacter sphagniphilus (sphag.ni'phi.lus. N.L. m. Sphagnum, generic name of sphagnum moss; N.L. adj. philus -a -um (from Gr. adj. philos -ê -on), friend, loving; N.L. masc. adj. sphagniphilus, Sphagnum-loving).

Contains free-living Polynucleobacter strains dwelling in the water body of acidic freshwater systems frequently characterized by Sphagnum moss growing along the shore lines and the terrestrial neighbourhood.

Cells are short sometimes slightly curved rods, 0.65-1.0 µm in length and 0.3–0.5 µm in width, depending on cultivation conditions. Chemo-organotrophic, aerobic, anaerobic growth was not observed. Colonies grown on NSY agar are non-pigmented, circular and convex with smooth surface. Growth occurs up to 31 °C and in 0–0.4% (w/v) NaCl. Assimilates acetate, pyruvate, malonate, malate, fumarate, succinate, D-galacturonic acid, L-glutamate, and L-alanine. Does not assimilate glycolate, glyoxylate, oxalate, oxaloacetate, citrate, D-glucose, D-galactose, D-lyxose, L-leucine, L-histidine, L-aspartate, L-asparagine, L-serine, or betaine. Major fatty acids of the strain are C_16:1 ω7c, C_16:0, and C_18:1 ω7c. The type strain is MWH-Weng1-1^T (=DSM 24018^T = CIP 111099^T), which was isolated from a small acidic bog pond located in Austria. The genome of the type strain is characterized by a size of 2.04 Mbp and a G+C content of 45.6 mol%. Genome and 16S rRNA gene sequences characterizing the type strain are available under the accession MPIY01000000.

Abbreviations

gANI

whole genome average nucleotide identity

NJ

neighbour-joining

OD

optical density