Rhizobium lacunae sp. nov., Isolated From a Freshwater Pond

Wen-Ming Chen National Kaohsiung College of Marine Technology: National Kaohsiung University of Science and Technology Nanzih Campus Che-Chia Yang National Kaohsiung Institute of Marine Technology: National Kaohsiung University of Science and Technology Nanzih Campus Chiu-Chung Young National Chung Hsing University Shih-Yao Lin National Chung Hsing University Shih-Yi Sheu (  sys816@webmail.nkmu.edu.tw ) National Kaohsiung Marine University https://orcid.org/0000-0002-4097-2037


Introduction
The genus Rhizobium (type species, Rhizobium leguminosarum), rst described by Frank (1889) and emended by Young et al. (2001), is a member of the family Rhizobiaceae within the class Alphaproteobacteria (2005). The genus Rhizobium comprises 91 species with validly published names so far stated on the List of Prokaryotic Names with Standing in Nomenclature (https://lpsn.dsmz.de/genus/rhizobium). Within the genus are free-living members as well as species that are capable of inducing the formation of plant hypertrophies, either as symbiotic nitrogen-xing nodules or as pathogenic tumors (Young et al. 2001;Kuykendall 2005). Members of the genus Rhizobium are characterized as Gram-stain-negative, non-spore-forming, rod-shaped, aerobic and chemoorganotrophic. Chemotaxonomically, members of the genus are characterized by summed feature 8 (C18:1ω7c and/or C18:1ω6c) as predominant fatty acid, Q-10 as the major ubiquinone and the DNA G+C content between 57 to 66 mol% (Gao et Fig. S1). The water sample was plated on R2A agar medium (BD Difco) through serial dilution technique. After incubation at 25 o C for 3 days, strain CSW-27 T was isolated as a single cream colony and subjected to detailed taxonomy analyses. Strain CSW-27 T was sub-cultured on R2A agar and stored at -80 o C in R2A broth with 20% (v/v) glycerol or stored following lyophilization. The phylogenetically related strains, Rhizobium straminoryzae CC-LY845 T and Rhizobium capsici CC-SKC2 T were maintained in our laboratory, and both strains were used as reference strains and evaluated together under identical experimental conditions to those for strain CSW-27 T .

Morphological, physiological, and biochemical characterizations
The cell morphology was observed by a phase-contrast microscopy (DM 2000; Leica) and a transmission electron microscopy (H-7500; Hitachi) using cells grown on R2A agar at 30 o C for 2 days. The Gram reaction was tested by Gram Stain Set S kit (BD Difco) and Ryu non-staining KOH method. Motility was tested by the hanging drop method , and the Spot Test Flagella Stain (BD Difco) was used for agellum staining. Poly-β-hydroxybutyrate granule accumulation was examined under light microscopy after staining of the cells with Sudan black (Schlegel et al. 1970) and visualized by UV illumination after directly staining growing bacteria on plates containing Nile red (Spiekermann et al. 1999). Colony morphology was observed on R2A agar under a stereoscopic microscope (SMZ 800; Nikon).
The pH range for growth was determined by measuring the optical densities (absorbance at 600 nm) of R2A broth cultures. The pH of the medium was adjusted prior to sterilization to pH 4-9 (at intervals of 0.5 pH unit) using the following biological buffers (Breznak and Costilow 2007): citrate/Na 2 HPO 4 (pH 4-5.5); phosphate (pH 6-7.5); and Tris (pH 8-9). The temperature range for growth was determined on R2A agar at 4-50 o C (4,10,15,20,25,30,35,37,40,45 and 50 o C). To investigate the tolerance to NaCl, R2A broth was prepared according to the formula of the BD Difco medium with NaCl concentration adjusted to 0-6% (w/v, at intervals of 1%). Growth under anaerobic condition was determined after incubating strain CSW-27 T on R2A agar and on R2A agar supplemented with nitrate (KNO 3 0.1%, w/v) in anaerobic jars by using Catalase activity was determined by bubble production in 3% (v/v) hydrogen peroxide and oxidase activity was assessed colorimetrically using tetramethyl p-phenylenediamine. DNA hydrolysis was investigated on DNase test agar (BD Difco). Hydrolyses of casein (2% skimmed milk, w/v), starch (2.5% soluble starch, w/v), lecithin (10%, w/v), corn oil (3%, w/v) and Tweens 20, 40, 60 and 80 (1%, w/v) was determined according to the methods described by Tindall et al. (2007). Chitin hydrolysis was assessed on chitinase-detection agar as described by Wen et al. (2002) and hydrolysis of carboxymethyl cellulose (CM-cellulose) was tested as the method described by Bowman using R2A agar as the basal medium (2000). Utilization of carbon sources was investigated in a basal medium containing (l −1 ): 0.4 g KH 2 PO 4 , 0.53 g Na 2 HPO 4 , 0.3 g NH 4 Cl, 0.3 g NaCl, 0.1 g MgCl 2 •6H 2 O, 0.11 g CaCl 2 and 1 ml trace element solution, pH 7 as described by Chang et al. (2004). Substrates were added at a concentration of 0.1% (w/v) and the tubes incubated under aerobic conditions at 30 ºC for 15 days. Additional biochemical tests were performed using API ZYM and API 20NE kits (both from bioMérieux) according to the manufacturers' recommendations.
Determination of cellular fatty acids, polar lipids, polyamines and isoprenoid quinones The fatty acid pro les of strain CSW-27 T and the two phylogenetic related strains were determined using cells grown on R2A agar at 30 o C for 3 days. The fatty acid methyl esters were prepared and separated according to the instructions of the Microbial Identi cation System (MIDI), analyzed by GC (Hewlett-Packard 5890 Series II) and identi ed by using the Aerobe (RTSBA6) database of the MIDI System (Sherlock version 6.0) (Sasser 1990). The polar lipid pro le of strain CSW-27 T was determined using cells grown on R2A agar at 30 o C for 3 days, and polar lipids were extracted and analyzed by two-dimensional TLC according to Embley and Wait (1994). Molybdophosphoric acid was used for the detection of total polar lipids, ninhydrin for amino lipids, Zinzadze reagent for phospholipids, Dragendorff reagent for choline-containing lipids and α-naphthol reagent for glycolipids. Polyamines were extracted from strain CSW-27 T and analysis was carried out as described by Busse and Auling (1988) and Busse et al. (1997).
After bacterial cells were cultivated in R2-PYE medium as described by Kämpfer et al. (2007) at 30 o C for 3 days, the polyamines were extracted and analyzed by using an HPLC with UV-VIS detector. The isoprenoid quinones of strain CSW-27 T were extracted and puri ed according to the method of Collins and analyzed by HPLC with a Spherisorb ODS column (1994).

Results And Discussion
Phenotypic characteristics Strain CSW-27 T was isolated from freshwater environment. Cells grew well on R2A agar, Luria-Bertani agar, trypticase soy agar and nutrient agar. Cells of strain CSW-27 T were Gram-stain-negative, aerobic, oxidase-positive and catalase-negative. Transmission electron microscopy of strain CSW-27 T showed a short rod-shaped bacterium that motile by agella ( Supplementary Fig. S2). Colonies were cream. The growth ranges of temperature, pH and NaCl concentration were at 20-40 o C, pH 5-9 and 0-4% NaCl, respectively. Detailed results from the phenotypic and biochemical analyses of strain CSW-27 T are provided in the species description and Supplementary Table S1. Differential features between strain CSW-27 T and two phylogenetically related strains, Rhizobium straminoryzae CC-LY845 T and Rhizobium capsici CC-SKC2 T were provided in Table 1. Table 1 Differential characteristics of Rhizobium lacunae CSW-27 T and the two phylogenetic related Rhizobium species

Chemotaxonomic characterizations
The fatty acid pro les of strain CSW-27 T and two phylogenetically related strains were present in Table 2, and their pro les were similar. Their major fatty acid contained summed feature 8, which constitutes 55-70% of the total fatty acids, and they all had C 16:0 3-OH as the predominant hydroxyl fatty acid. The predominant cellular fatty acid (> 50% of the total fatty acids) of strain CSW-27 T was summed feature 8 (C18:1ω7c and/or C18:1ω6c; 57.3%). Strains: 1, CSW-27 T ; 2, Rhizobium straminoryzae CC-LY845 T ; 3, Rhizobium capsici CC-SKC2 T .
All strains were grown on R2A agar at 30 o C for 3 days. Data are expressed as percentages of the total fatty acids. Only fatty acids representing more than 0.5% of the total fatty acids of at least one of the strains are shown. tr, traces (less than 0.5% of total); -, 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 isomer is indicated by the su x c. *Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed feature 2 comprises C14:0 3-OH and/or iso-C16:1 I. Summed feature 3 comprises C 16:1 ω7c and/or C 16:1 ω6c. Summed feature 8 comprises C18:1ω7c and/or C18:1ω6c.
16S rRNA gene similarities and phylogenetic analysis Rhizobium capsici CC-SKC2 T and Rhizobium oryzicola ZYY136 T within the genus Rhizobium in the neighbour-joining tree in Fig. 1. Similar tree topologies were obtained in the maximum-likelihood and maximum-parsimony trees.

Genomic features
The genome of strain CSW-27 T comprised a total size of 5.66 Mb (GenBank accession number NZ_JAHHZO000000000) with 101 contigs, and G+C content was 63.3% ( Supplementary Fig. S6). It had an average coverage of 158x and a N50 size of 323865 bp, and was con rmed to be free of contamination. The sequence of the 16S rRNA gene from the genome and that of PCR determined sequence is very close but not identical with several nucleotides different, and the original sequence determined by PCR has been corrected. The genome harbored 5244 protein encoding genes, 6 rRNA genes and 49 tRNA genes. The protein encoding genes were classi ed into 21 functional categories (Supplementary Table S2), and most of coding sequences were classi ed as functional unknown (S, 22.2% of all assigned eggNOG), amino acid transport and metabolism (E, 12.1%), transcription (K, 9.7%) and inorganic ion transport and metabolism (P, 7.7%).
The dDDH values between strain CSW-27 T  Therefore, strain CSW-27 T was considered a new bacterial species.
UBCG was utilized for construction of a genome-based phylogenetic tree. The phylogenetic tree based on the coding sequences of 92 protein clusters showed that strain CSW-27 T formed a distinct phylogenetic lineage cluster with Rhizobium straminoryzae SM12, Rhizobium rhizoryzae J3-AN59 T and Rhizobium ipomoeae shin9-1 T in the genus Rhizobium (Fig. 2), which supported that strain CSW-27 T should be assigned to a novel species of the genus Rhizobium.

Genome comparative analysis
For further comparative analyses, the genome sequences of strain CSW-27 T and four genome sequences from the genus Rhizobium were used, including Rhizobium straminoryzae SM12, Rhizobium rhizoryzae J3-AN59 T , Rhizobium ipomoeae shin9-1 T and Rhizobium petrolearium SL-1 T . Genome characteristics of these strains is provided in Supplementary Table S4. Results from RAST showed that all ve strains had very important characteristics for the gene compositions, and some genes are shared and some genes are different (Table 3).   Homology analysis of gene contents were performed between strain CSW-27 T and Rhizobium straminoryzae SM12, Rhizobium rhizoryzae J3-AN59 T , Rhizobium ipomoeae shin9-1 T and Rhizobium petrolearium SL-1 T . As a result, a total of 2265 genes common are shared among the ve strains ( Supplementary Fig. S7), and there are 724 genes present as speci c genes in strain CSW-27 T . In summary, by comparing genomic information, we can gain insights into how these rhizobia strains metabolize various nutrients, their resistance to pathogens or harmful substances, and their ability to adapt to environmental changes, which provides a basic theory. These capabilities may give various rhizobia a competitive advantage to adapt to diverse environments in a complex microbial ecosystem.

Taxonomic conclusion
Phenotypic examination revealed many common traits between the novel strain and Rhizobium straminoryzae CC-LY845 T and Rhizobium capsici CC-SKC2 T . However, strain CSW-27 T could be clearly differentiated from these two phylogenetic related strains by its ability to grow at lower temperature (< 25 o C), by its ability to ferment glucose, by its ability to hydrolyze urea and esculin, by the absence of catalase and N-acetyl-β-glucosaminidase activities, by its inability to assimilate N-acetyl-glucosamine, by its ability to assimilate adipate, by the ability to utilize Tween 40 as carbon sources and by the inability to utilize L-arabinose, maltose, sucrose, D-mannose, L-rhamnose, D-ra nose, D-trehalose, dextrin, glycerol, D-adonitol, D-mannitol, acetate, gluconate, L-alanine, L-histidine, L-asparagine, L-ornithine, L-glutamic acid and L-proline as carbon sources (Table 1).
From the comparative genomic analyses for strain CSW-27 T , Rhizobium straminoryzae SM12, Rhizobium rhizoryzae J3-AN59 T , Rhizobium ipomoeae shin9-1 T and Rhizobium petrolearium SL-1 T , it is also found that although they have common characteristics, strain CSW-27 T could be obviously discriminated from these strains by its unique abilities. Based on the data obtained from 16S rRNA gene sequence and whole genome sequence comparison, strain CSW-27 T occupies a distinct position within the genus Rhizobium that is supported by a unique combination of chemotaxonomic and biochemical characteristics. Strain CSW-27 T represents a novel species of the genus Rhizobium, for which the name Rhizobium lacunae sp. nov. is proposed.
Description of Rhizobium lacunae sp. nov.
Rhizobium lacunae (la.cu'nae. L. gen. n. lacunae of a pond, from where the type strain was isolated).
Cells are Gram-stain-negative, aerobic, motile by agella, rod-shaped (0.8-1.1 µm wide and 1.7-2.2 µm long) and chemo-heterotrophic. After 48 h of incubation at 30 o C on R2A agar, colonies are cream colored, convex, round, smooth with entire edges and approximately 0.9-1.5 mm in diameter. Cells grow at 20-40 o C (optimum, 30-37 o C), at pH 5-9 (optimum, pH 6-7) and with 0-4% NaCl (optimum, 0%  The authors received no speci c grant from any funding agency. Compliance with ethical standards Figure 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of Rhizobium lacunae CSW-27 T and type strains of species of the genus Rhizobium. Numbers at nodes are bootstrap percentages > 70% based on the neighbour-joining (above nodes) and maximum-parsimony (below nodes) tree-making algorithms. Filled circles indicate branches of the tree that were also recovered using the maximum-likelihood and maximum-parsimony tree-making algorithms. Open circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony algorithm. Kaistia soli 5YN9-8 T was used as an out-group. Bar, 0.01 substitutions per nucleotide position. Figure 2