Introduction

The family Rhizobiaceae (class Alphaproteobacteria) currently consists more than 100 species with validly published names (Parte 2014). Parallel to the accelerated description of new species in recent years, taxonomic revisions have also been performed: e.g. some Rhizobium species were reclassified in the genera Agrobacterium and Allorhizobium, and species formerly belonging to Rhizobium were proposed to be transferred into newly established genera, such as Pararhizobium and Neorhizobium (Mousavi et al. 2014, 2015). Considering the species with validly published names, the genus Rhizobium is clearly polyphyletic based on 16S rRNA gene sequences, and although genomic data are available for many species within the family Rhizobiaceae, the status of some Rhizobium species is still under debate (Carrareto Alves et al. 2014; Mousavi et al. 2015; Ormeño-Orrillo et al. 2015). The taxonomy of Rhizobium and related genera is complicated by the combination of several factors: in the case of many species, a large proportion of the genome is harbored in extrachromosomal replicons (chromids and plasmids), the loss or gain of which may significantly influence phenotypic results (Slater et al. 2009; Ormeño-Orrillo and Martínez-Romero 2013; Althabegoiti et al. 2014); 16S rRNA gene-based interspecies similarity values are high in many cases (Hunter et al. 2007), which hinders the species-level identification based on the most-widely used taxonomic marker gene; and the agricultural significance of many strains (Carrareto Alves et al. 2014) asserts the retention of classical systematics rather than modern taxonomy. As a result, strains of rhizobia (i.e. bacteria capable of nodulating leguminous plants and form nitrogen fixing symbioses) belonging or closely related to the genus Rhizobium are referred to as members of the ‘Rhizobium/Agrobacterium group’ (or cluster) in recent systematic works (Carrareto Alves et al. 2014; Ormeño-Orrillo et al. 2015).

Although almost all Rhizobium species were isolated from root nodules, new species belonging to family Rhizobiaceae have been described recently based on type strains originating from aquatic habitats, e.g. Rhizobium alvei was isolated from river water (Sheu et al. 2015), Shinella granuli, Rhizobium daejeonense and Rhizobium selenitireducens were isolated from bioreactors (Quan et al. 2005; An et al. 2006; Hunter et al. 2007), Rhizobium marinum was isolated from seawater (Liu et al. 2015) and Gellertiella hungarica from thermal bath (Tóth et al. 2017). This study presents the polyphasic characterization of a new aquatic strain, SA-276T, which was isolated from a freshwater lake and is closely related to members of the genus Rhizobium.

Materials and methods

Strain isolation and growth conditions

Strain SA-276T was isolated from the water of Lake St. Ana (a crater lake in the Ciomad Mountains, Harghita County, Romania; in Romanian: Lacul Sfânta Ana) in August 2012. A detailed site description including the physical and chemical characteristics of lake water is given by Felföldi et al. (2016). For isolation, plates containing lake water solidified with 20 g l−1 agar were used. The standard dilution plating technique was applied to obtain isolates by incubation at room temperature (20–22 °C). Subsequently, strain SA-276T was maintained on a modified R2A agar medium (pH 7.0), which contained only half amount of carbon sources as given in the original description (DSMZ medium 830, www.dsmz.de). Later, strain SA-276T showed effective growth on R2A agar, YMA agar (DSMZ medium 1031) and Rhizobium agar (DSMZ medium 98) media. For side-by-side analyses, the new strain and strains Rhizobium tubonense DSM 25379T (= CCBAU 85046T) and Rhizobium leguminosarum LMG 14904T (= USDA 2370T) were maintained on YMA agar at 28 °C.

Morphological and physiological analyses

Optimal temperature, pH and salt concentration values were determined based on the growth intensity observed at 4, 10, 20, 25, 30, 37, 45 and 55 °C, at pH from 4 to 11 (with intervals of 1) and with NaCl concentration from 0 to 5% (w/v, with intervals of 1%), respectively, as described previously (Felföldi et al. 2014). For testing the nitrate reduction of strains under anaerobic conditions, R2A liquid medium supplemented with 1 g l−1 KNO3 and Nitrate Broth (with Durham tubes; Barrow and Feltham 2003) was used. An anoxic atmosphere was created by using an Anaerocult A Mini (Merck) gas generator system.

Colony morphology of strain SA-276T was tested by direct observation of single colonies. Cell morphology was observed after Gram staining according to Claus (1992), while the presence of flagella was assessed as described by Heimbrook et al. (1989). Oxidase activity and catalase reaction were examined as given by Tarrand and Gröschel (1982) and Cowan and Steel (1974), respectively. Caseinase, urease and starch hydrolysis activities were determined as described by Smibert and Krieg (1994), while acid production from d-glucose was checked by the oxidative and fermentative tests according to Hugh and Leifson (1953). Additional metabolic tests were performed with API 50 CH, API 20 NE and API ZYM (bioMérieux) systems following the instructions given by the manufacturer. Susceptibility of the strains to antibiotics was studied on YMA plates using antibiotic-containing discs (Bio-Rad) after incubation for 3 days at 28 °C.

Chemotaxonomic analyses

Analyses of cell wall diamino acids, isoprenoid quinones, cellular fatty acids, polar lipids and the determination of DNA base composition were performed as described by Felföldi et al. (2011).

DNA sequence analyses

The 16S rRNA gene sequence of strain SA-276T was amplified and sequenced as described by Máthé et al. (2014) using the primers given previously (Felföldi et al. 2017). Amplification of protein-coding genes was performed with primers atpD-255F (5′-GCT SGG CCG CAT CMT SAA CGT C-3′) and atpD-782R (5′-GCC GAC ACT TCM GAA CCN GCC TG-3′) in the case of the beta subunit of ATP synthase (atpD gene), glnII-12F (5′-YAA GCT CGA GTA CAT YTG GCT-3′) and glnII-689R (5′-TGC ATG CCS GAG CCG TTC CA-3′) in the case of glutamine synthetase II (glnII gene), recA-41F (5′-TTC GGC AAG GGM TCG RTS ATG-3′) and recA-640R (5′-ACA TSA CRC CGA TCT TCA TGC-3′) in the case of recombinase (recA gene), following the protocols given by Vinuesa et al. (2005). Amplicons were purified with the PCR-M™ Clean Up System (Viogene, Sijhih, Taiwan), and sequencing of the PCR products was carried out through a service provided by the Biomi Ltd. (Gödöllő, Hungary).

Sequence alignment of the 16S rRNA gene with the closest related type strains was performed with the SINA Alignment Service (Pruesse et al. 2012). Sequence alignment of protein-coding genes was performed with the MEGA 7.0 software (Kumar et al. 2016), multiple fasta files were created with MergeAlign (Collingridge and Kelly 2012), and concatenation was conducted with SequenceMatrix 1.8 (Vaidya et al. 2011). Phylogenetic analyses (which included the search for the best-fit model parameters) were performed with the MEGA 7.0 software.

The presence of the nifH gene was assessed with the PCR-based method of Bürgmann et al. (2004).

Results and discussion

Morphological and physiological characteristics

Cells of strain SA-276T are Gram-stain negative, motile, facultatively anaerobic and mesophilic with a characteristic heterotrophic metabolism (Table 1). Based on the enzyme activities and substrates tested for utilization, the new strain could be distinguished from the closely related type strain, R. tubonense DSM 25379T, based on its broader substrate specificity (capable of utilizing d/l-arabinose, d-fructose, d-galactose, d-glucose, glycerol, d-lyxose, malic acid, d-maltose, d-mannitol, d-mannose, l-rhamnose, d-ribose, sucrose, trehalose, turanose, d/l-xylose), positive trypsin enzyme activity (Table 1) and penicillin sensitivity (Table S1, available in the online Supplementary Material).

Table 1 Differential phenotypic and biochemical characteristics of SA-276T and related type strains
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Chemotaxonomic characteristics

The major respiratory quinones of SA-276T were Q-10 and Q-9 in a ratio of 47:29. The fatty acid pattern of strain SA-276T was dominated by C18:1ω7c (41.0%) and cyclo C19:0ω8c (29.2%), and in lower amounts C14:0 3-OH (7.8%), C16:0 (7.5%) and other minor components (< 5%) (Table 2). Comparing these data with the related strains analyzed, their fatty acid compositions were similar and the dominance of fatty acids C18:1ω7c, cyclo C19:0ω8c and C16:0 has been reported in many Rhizobium, Pararhizobium and Shinella species by other authors (Lee et al. 2011; Zhang et al. 2011, 2015; Behrendt et al. 2016; Puławska et al. 2016), which confirmed that the new strain belongs to family Rhizobiaceae.

Table 2 Major fatty acids of SA-276T and related type strains
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The polar lipid pattern of strain SA-276T was dominated by phosphatidylmonomethylethanolamine (PMME), phosphatidylglycerol (PG) and phosphatidylcholine (PC), while phosphatidylethanolamine, an unidentified aminophospholipid and most likely diphosphatidylglycerol were detected as minor components (Fig. S1). Although Rhizobium species polar lipid data have not been previously reported for all species (Young et al. 2001; Kuykendall et al. 2005; Carrareto Alves et al. 2014), including recent descriptions (Saïdi et al. 2014; Zhang et al. 2015; Behrendt et al. 2016); PG, PC and/or PMME have been detected as characteristic polar lipids in various other Rhizobium and Shinella species (Liu et al. 2015; Sheu et al. 2015, 2016; Román-Ponce et al. 2016; Subhash and Lee 2016).

The cell wall of strain SA-276T contained the diagnostic diamino acid, meso-2,6-diaminopimelic acid.

The genomic G + C content value of strain SA-276T is 60.8 mol%, which falls within the range (57–66%) reported for the Rhizobium/Agrobacterium cluster (Carrareto Alves et al. 2014).

Results of DNA sequence analyses

Sequencing the 16S rRNA gene of strain SA-276T resulted in a stretch of 1408 nucleotides. Based on this data, the currently most closely related species (represented by type strains) were identified with EzBioCloud’s online service (Yoon et al. 2017). R. tubonense CCBAU 85046T (= DSM 25379T), R. leguminosarum USDA 2370T (= LMG 14904T), Rhizobium anhuiense CCBAU 23252T and Rhizobium laguerreae FB206T showed the highest, 97.65%, pairwise similarity value based on the 16S rRNA gene, while 22 other Rhizobium, 2 Ensifer and 3 Pararhizobium type strains shared lower similarities (but higher than 97.0%) to strain SA-276T (see details in Table S2). These values are higher than the value (95%) suggested for general genus delineation by Tindall et al. (2010). However, according to the recommendation of Kim et al. (2014), based on pairwise comparison of bacterial genomes, the species level threshold should be increased to the level of 98.65% 16S rRNA gene sequence similarity. Furthermore, it has been previously noted by others (Hunter et al. 2007) that some strains showing < 1% sequence dissimilarity based on their 16S rRNA gene sequences may represent different Rhizobium species.

Phylogenetic analysis of the 16S rRNA gene (Fig. 1; Fig. S2) revealed that strain SA-276T clustered with the two sensu stricto Rhizobium sub-clusters (the ‘Rhizobium tropici’ and the ‘Rhizobium leguminosarum’ groups; Mousavi et al. 2015). Housekeeping genes were also applied to aid resolving the phylogeny of rhizobia (Mousavi et al. 2014). Based on the sequence analysis of atpD, recA and glnII genes (Fig. 2), strain SA-276T was positioned in a separate clade from nodule-forming Rhizobium strains.

Fig. 1
figure 1

Phylogenetic tree of SA-276T and related type strains based on the 16S rRNA gene. Phylogenetic tree has been reconstructed based on 1319 nucleotide positions using the maximum likelihood method. Only bootstrap values > 70% are shown. GenBank accession numbers are given in parentheses. Filled circles indicate that the corresponding nodes were also recovered by the neighbor-joining method

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Fig. 2
figure 2

Phylogenetic tree of SA-276T and related type strains based on concatenated sequences of genes atpD, glnII and recA. Phylogenetic tree has been reconstructed based on 1236 nucleotide positions using the maximum likelihood method. Only bootstrap values > 70% are shown. GenBank accession numbers are given in parentheses (order: atpD, glnII and recA). Filled circles indicate that the corresponding nodes were also recovered by the neighbor-joining method

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In the case of the new strain, PCR failed to detect the nifH gene. Genes required for nitrogen fixation in legumes are encoded in unstable plasmids (Ormeño-Orrillo et al. 2015; Remigi et al. 2016), offering a possible explanation for the absence of nifH. Consequently, planktonic species may lose these traits since they probably do not establish nitrogen-fixing symbiosis with plants.

Taxonomic conclusion

Strain SA-276T shared the main physiological characteristics of family Rhizobiaceae (Carrareto Alves et al. 2014): aerobic, Gram-stain negative and rod-shaped. However, unlike many other members of this group, the new strain was not associated with soil and plants, and lacked the ability of nitrogen fixation. Chemotaxonomic data (polar lipid pattern, fatty acid composition) support the conclusion that strain SA-276T belongs to family Rhizobiaceae, and phylogenetic analyses have confirmed that it is closely related to members of genus Rhizobium.

In conclusion, based on the data discussed above, strain SA-276T is considered to represent a new species, for which the name Rhizobium aquaticum sp. nov. is proposed.

Description of Rhizobium aquaticum sp. nov.

Rhizobium aquaticum (a.qua’ti.cum. L. neut. adj. aquaticum living in water, aquatic; referring to the isolation source of the type strain).

Cells are rod-shaped (0.5–0.7 × 1.6–1.9 μm) and motile. Colonies on YMA agar medium are beige-coloured, circular and raised. Growth occurs after 1–2 days of incubation at 10–45 °C (optimum, 20–30 °C), at pH 6–10 (optimum, pH 7–9) and 0–2% (w/v) NaCl concentration. Capable of growth under anaerobic conditions with nitrate. Positive for acid phosphatase, aesculin hydrolysis, alkaline phosphatase, catalase (weak), esterase (C4), esterase lipase (C8), β-galactosidase, α-glucosidase, β-glucosidase, leucine arylamidase, N-acetyl-β-glucosaminidase, naphthol-AS-BI-phosphohydrolase, nitrate reduction to nitrite, trypsin and urease. Negative for the following enzyme activities: arginine dihydrolase, caseinase, α-chymotrypsin, cystine arylamidase, α-fucosidase, α-galactosidase, gelatine hydrolysis, glucose fermentation, β-glucuronidase, indole production, lipase (C14), α-mannosidase, oxidase and valine arylamidase. The major respiratory quinones are Q-10 and Q-9. The major fatty acids are C18:1ω7c (41.0%) and cyclo C19:0ω8c (29.2%). The major polar lipids are PMME, PG and PC. The cell wall contains meso-2,6-diaminopimelic acid. The G + C content of the genomic DNA of the type strain is 60.8 mol%.

The type strain is SA-276T (= DSM 29780T = JCM 31760T) which was isolated from the water of a crater lake. The TaxonNumber of strain SA-276T is TA00355.