Phenotypic characteristics
Strain CSW-27T was isolated from freshwater environment. Cells grew well on R2A agar, Luria-Bertani agar, trypticase soy agar and nutrient agar. Cells of strain CSW-27T were Gram-stain-negative, aerobic, oxidase-positive and catalase-negative. Transmission electron microscopy of strain CSW-27T showed a short rod-shaped bacterium that motile by flagella (Supplementary Fig. S2). Colonies were cream. The growth ranges of temperature, pH and NaCl concentration were at 20-40 oC, pH 5-9 and 0-4% NaCl, respectively. Detailed results from the phenotypic and biochemical analyses of strain CSW-27T are provided in the species description and Supplementary Table S1. Differential features between strain CSW-27T and two phylogenetically related strains, Rhizobium straminoryzae CC-LY845T and Rhizobium capsici CC-SKC2T were provided in Table 1.
Table 1
Differential characteristics of Rhizobium lacunae CSW-27T and the two phylogenetic related Rhizobium species
Characteristic
|
1
|
2
|
3
|
Isolation source
|
freshwater pond
|
surface of rice straw
|
root tumor of a green bell pepper
|
Motility
|
+
|
-
|
+
|
Temperature range for growth (oC)
|
20-40
|
25-40
|
25-37
|
pH range for growth
|
5-9
|
5-9
|
4-9
|
Glucose fermentation
|
+
|
-
|
-
|
Hydrolysis of :
|
|
|
|
Urea
|
+
|
-
|
-
|
Esculin
|
+
|
-
|
-
|
Tween 20
|
-
|
-
|
+
|
Enzymatic activities :
|
|
|
|
Catalase
|
-
|
+
|
+
|
Cystine arylamidase
|
+
|
+
|
-
|
N-Acetyl-β-glucosaminidase
|
-
|
+
|
+
|
Assimilation :
|
|
|
|
N-Acetyl-glucosamine
|
-
|
+
|
+
|
Adipate
|
+
|
-
|
-
|
Citrate
|
-
|
+
|
-
|
Carbon source utilization :
|
|
|
|
L-Leucine and L-aspartic acid
|
+
|
-
|
+
|
Tween 40
|
+
|
-
|
-
|
L-Arabinose, maltose, sucrose, D-mannose, L-rhamnose, D-raffinose, D-trehalose, dextrin, glycerol, D-adonitol, D-mannitol, acetate, gluconate, L-alanine, L-histidine, L-asparagine, L-ornithine, L-glutamic acid and L-proline
|
-
|
+
|
+
|
Citrate
|
-
|
+
|
-
|
L-Serine and L-threonine
|
-
|
-
|
+
|
DNA G+C content (% or mol%)
|
63.3
|
68.3
|
60.5
|
Strains: 1, CSW-27T; 2, Rhizobium straminoryzae CC-LY845T; 3, Rhizobium capsici CC-SKC2T. |
All data from this study except the G+C content of Rhizobium straminoryzae CC-LY845T (Lin et al. 2014) and Rhizobium capsici CC-SKC2T (Lin et al. 2015). +, Positive reaction; -, negative reaction. All strains are aerobic, rod-shaped and formed cream-colored colonies, and positive for oxidase, alkaline phosphatase, C4 esterase, C8 esterase lipase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase and β-glucosidase activities, hydrolysis of DNA, CM-cellulose and Tween 40, assimilation of glucose, arabinose, mannose, mannitol, maltose, gluconate and malate, utilization of D-glucose, D-fructose, D-galactose, D-cellobiose, N-acetyl-glucosamine, D-sorbitol as carbon sources. All strains are negative for: Gram staining; nitrate reduction; indole production; hydrolyses of gelatin, casein, starch, chitin, corn oil, lecithin and Tweens 60 and 80; arginine dihydrolase, C14 lipase, trypsin, α-chymotrypsin, β-glucuronidase, α-mannosidase and α-fucosidase activities; assimilation of caprate and phenyl-acetate; utilization of L-phenylalanine and Tween 80 as carbon sources. |
Chemotaxonomic characterizations
The fatty acid profiles of strain CSW-27T and two phylogenetically related strains were present in Table 2, and their profiles were similar. Their major fatty acid contained summed feature 8, which constitutes 55-70% of the total fatty acids, and they all had C16:0 3-OH as the predominant hydroxyl fatty acid. The predominant cellular fatty acid (> 50% of the total fatty acids) of strain CSW-27T was summed feature 8 (C18:1ω7c and/or C18:1ω6c; 57.3%).
Table 2
Cellular fatty acid composition of Rhizobium lacunae CSW-27T and the two phylogenetic related Rhizobium species
Fatty acid
|
1
|
2
|
3
|
Saturated
|
|
|
|
C14:0
|
0.8
|
tr
|
tr
|
C16:0
|
9.2
|
7.4
|
6.8
|
C18:0
|
1.6
|
1.1
|
1.4
|
Unsaturated
|
|
|
|
C17:0 cyclo
|
0.8
|
0.9
|
tr
|
C18:1ω7c 11-methyl
|
4.0
|
1.4
|
4.3
|
C19:0 cycloω8c
|
6.9
|
3.2
|
5.2
|
Hydroxy
|
|
|
|
C16:0 3-OH
|
1.4
|
2.1
|
2.2
|
Summed features*
|
|
|
|
2
|
8.4
|
9.3
|
8.9
|
3
|
6.6
|
5.9
|
1.4
|
8
|
57.3
|
65.8
|
66.9
|
Strains: 1, CSW-27T; 2, Rhizobium straminoryzae CC-LY845T; 3, Rhizobium capsici CC-SKC2T. |
All strains were grown on R2A agar at 30 oC 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 suffix 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 C16:1ω7c and/or C16:1ω6c. Summed feature 8 comprises C18:1ω7c and/or C18:1ω6c. |
The polar lipids of strain CSW-27T consisted of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylmonomethylethanolamine (PME), phosphatidyldimethylethanolamine (PDE), phosphatidylcholine (PC), one uncharacterized aminophospholipid (APL1), three uncharacterized aminolipids (AL1-AL3) and two uncharacterized lipids (L1 and L2) (Supplementary Fig. S3). Strain CSW-27T contained spermidine (SPD, 99.5%) as the major polyamine and small amounts of putrescine (PUT, 0.5%) (Supplementary Fig. S4). The sole respiratory quinone detected in strain CSW-27T was ubiquinone-10 (Q-10) (Supplementary Fig. S5), as in all known members of the genus Rhizobium.
16S rRNA gene similarities and phylogenetic analysis
The almost complete 16S rRNA gene sequence of strain CSW-27T was obtained (1532 bp, GenBank accession number MH729077). Based on the16S rRNA gene sequence similarities, strain CSW-27T was closely related to the species of the genus Rhizobium and showed high sequence similarity with Rhizobium straminoryzae CC-LY845T (98.5%) followed by Rhizobium capsici CC-SKC2T (96.9%), Rhizobium helianthi Xi19T (96.8%), Rhizobium rhizoryzae J3-AN59T (96.4%), Rhizobium endolithicum JC140T (95.7%) and Rhizobium petrolearium SL-1T (95.2%). Sequence similarities < 95.1% were observed with the type strains of all other Rhizobium species. The phylogenetic trees manifested that strain CSW-27T formed a separate phylogenetic branch clustered with Rhizobium straminoryzae CC-LY845T, Rhizobium capsici CC-SKC2T and Rhizobium oryzicola ZYY136T 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-27T 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 confirmed 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 classified into 21 functional categories (Supplementary Table S2), and most of coding sequences were classified 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-27T and the strains of other related Rhizobium species were 13.3-22.1% (Supplementary Table S3), which are below the cut-off (70%) for species delineation (Goris et al. 2007). ANI values obtained from the comparison with available genomes were 73.4-86.5%. These values are lower than the threshold (95-96%) for species definition as described by Richter and Rosselló-Móra (2009). AAI values obtained were 66.0-88.8%, which is below the threshold of 90% for species boundary and above the threshold of 60% for genus boundary proposed by Rodriguez-R and Konstantinidis (2014). Therefore, strain CSW-27T 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-27T formed a distinct phylogenetic lineage cluster with Rhizobium straminoryzae SM12, Rhizobium rhizoryzae J3-AN59T and Rhizobium ipomoeae shin9-1T in the genus Rhizobium (Fig. 2), which supported that strain CSW-27T should be assigned to a novel species of the genus Rhizobium.
Genome comparative analysis
For further comparative analyses, the genome sequences of strain CSW-27T and four genome sequences from the genus Rhizobium were used, including Rhizobium straminoryzae SM12, Rhizobium rhizoryzae J3-AN59T, Rhizobium ipomoeae shin9-1T and Rhizobium petrolearium SL-1T. Genome characteristics of these strains is provided in Supplementary Table S4. Results from RAST showed that all five strains had very important characteristics for the gene compositions, and some genes are shared and some genes are different (Table 3).
Table 3
Comparison of the presence and absence of selected genes among Rhizobium lacunae CSW-27T and the four strains of the genus Rhizobium
Genes putatively encoding
|
1
|
2
|
3
|
4
|
5
|
Cofactors and vitamins
|
|
|
|
|
|
Ubiquinone biosynthesis
|
+
|
-
|
-
|
+
|
+
|
Chlorophyll biosynthesis
|
-
|
-
|
-
|
+
|
-
|
Coenzyme B12 biosynthesis
|
-
|
-
|
-
|
+
|
+
|
Cell wall and capsule
|
|
|
|
|
|
dTDP-rhammose synthesis, rhammose containing glycans
|
+
|
+
|
+
|
-
|
+
|
Capsular polysaccharide biosynthesis and assembly
|
-
|
-
|
-
|
-
|
+
|
Teichoic and lipoteichoic acids biosynthesis
|
+
|
-
|
-
|
-
|
+
|
Virulence, disease and defense
|
|
|
|
|
|
Adaptation to d-cysteine
|
-
|
+
|
+
|
+
|
+
|
Mercuric resistance
|
-
|
-
|
+
|
+
|
+
|
Zinc resistance
|
-
|
-
|
-
|
+
|
-
|
Fosfomycin resistance
|
-
|
-
|
+
|
-
|
-
|
Bile hydrolysis
|
-
|
-
|
-
|
-
|
+
|
Mycobacterium virulence operon possibly involved in quinolinate biosynthesis
|
-
|
-
|
+
|
+
|
+
|
Photosynthesis
|
|
|
|
|
|
Bacterial light-harvesting proteins
|
-
|
-
|
-
|
+
|
-
|
Photosystem II-type photosynthetic reaction center
|
-
|
-
|
-
|
+
|
-
|
Miscellaneous
|
|
|
|
|
|
Niacin-choline transport and metabolism
|
-
|
-
|
-
|
-
|
+
|
Dioxygenases (EC 1.14.12.-)
|
+
|
+
|
+
|
-
|
-
|
Muconate lactonizing enzyme family
|
+
|
-
|
+
|
+
|
+
|
Aromatic dioxygenase mess
|
+
|
-
|
+
|
-
|
+
|
Bacillus subtilis scratch
|
-
|
-
|
+
|
+
|
+
|
Dioxygenases (EC 1.13.11.-)
|
+
|
-
|
+
|
-
|
+
|
Phage, prophage, transposable element and plasmid
|
|
|
|
|
|
Transposable elements : CBSS-203122.12.peg.188
|
-
|
-
|
+
|
-
|
+
|
Phage replication
|
+
|
+
|
+
|
-
|
+
|
Phage tail fiber proteins
|
+
|
-
|
-
|
-
|
+
|
Phage lysis modules
|
+
|
-
|
+
|
-
|
-
|
Membrane transport
|
|
|
|
|
|
Protein secretion system, type I (for aggregation)
|
-
|
-
|
-
|
+
|
-
|
Protein and nucleoprotein secretion system, type IV (conjugative transfer)
|
+
|
+
|
-
|
+
|
+
|
Protein secretion system, type VI
|
+
|
+
|
-
|
-
|
-
|
TRAP transporter unknown substrate 5 and substrate 9
|
+
|
-
|
-
|
-
|
+
|
TRAP transporter unknown substrate 4
|
+
|
-
|
-
|
-
|
-
|
Regulation and cell signaling
|
|
|
|
|
|
DNA-binding regulatory proteins, strays
|
+
|
-
|
+
|
-
|
+
|
HPr catabolite repression system
|
-
|
-
|
+
|
-
|
-
|
MazEF toxin-antitoxing (programmed cell death) system
|
+
|
-
|
+
|
-
|
-
|
Murein hydrolase regulation and cell death
|
+
|
-
|
-
|
-
|
-
|
Dormancy and sporulation
|
|
|
|
|
|
Persister cells
|
+
|
-
|
-
|
-
|
-
|
Respiration
|
|
|
|
|
|
Terminal cytochrome O ubiquinol oxidase
|
+
|
+
|
+
|
-
|
+
|
Carbon monoxide oxidation
|
-
|
-
|
-
|
+
|
-
|
Formate dehydrogenase
|
-
|
-
|
-
|
-
|
+
|
Stress response
|
|
|
|
|
|
Osmoregulation
|
+
|
-
|
-
|
-
|
+
|
Cluster containing glutathione synthetase
|
-
|
-
|
-
|
+
|
-
|
Heat shock dnak gene cluster extended
|
+
|
-
|
+
|
+
|
+
|
Flavohaemoglobin
|
-
|
-
|
+
|
-
|
-
|
SigmaB stress response regulation
|
-
|
-
|
-
|
-
|
+
|
Dimethylarginine metabolism
|
+
|
+
|
-
|
-
|
-
|
Iron acquisition and metabolism
|
|
|
|
|
|
Iron siderophore sensor and receptor system
|
+
|
-
|
+
|
+
|
+
|
Siderophore enterobactin
|
+
|
-
|
-
|
-
|
-
|
Siderophore anthraachelin
|
-
|
+
|
-
|
+
|
-
|
Siderophore assembly kit
|
-
|
+
|
-
|
+
|
-
|
ABC transporter [iron.B12.siderophore.hemin]
|
-
|
+
|
+
|
+
|
+
|
Nitrogen metabolism
|
|
|
|
|
|
Allantoin utilization
|
-
|
-
|
-
|
-
|
+
|
Denitrification
|
+
|
-
|
+
|
+
|
+
|
Phosphorus metabolism
|
|
|
|
|
|
Phosphoenolpyruvate phosphomutase
|
-
|
-
|
+
|
-
|
-
|
Sulfur metabolism
|
|
|
|
|
|
L-cystine uptake and metabolism
|
-
|
-
|
-
|
+
|
+
|
Utilization of glutathione as a sulfur source
|
-
|
-
|
-
|
-
|
+
|
Sulfur oxidation
|
-
|
-
|
-
|
-
|
+
|
RNA metabolism
|
|
|
|
|
|
tRNA modification position 34
|
-
|
-
|
-
|
+
|
-
|
Group II intron-associated genes
|
+
|
+
|
-
|
-
|
+
|
Protein metabolism
|
|
|
|
|
|
Protein chaperones
|
+
|
-
|
+
|
+
|
+
|
Selenocysteine metabolism
|
-
|
-
|
-
|
-
|
+
|
N-linked glycosylation in Bacteria
|
+
|
+
|
+
|
+
|
-
|
DNA metabolism
|
|
|
|
|
|
Nonhomologous end-joining in Bacteria
|
-
|
-
|
+
|
+
|
+
|
CRISPRs
|
-
|
+
|
-
|
-
|
-
|
Restriction-modification system
|
+
|
+
|
+
|
-
|
-
|
Metabolism of amino acids and derivatives
|
|
|
|
|
|
Glutamate and aspartate uptake in Bacteria
|
+
|
+
|
-
|
-
|
-
|
Histidine degradation
|
+
|
+
|
+
|
-
|
+
|
Cyanophycin metabolism
|
+
|
+
|
+
|
+
|
-
|
S-methylmethionine
|
+
|
+
|
+
|
-
|
-
|
Isoleucine, leucine, valine degradation and HMG-CoA metabolism
|
-
|
-
|
+
|
+
|
+
|
HMG-CoA synthesis
|
-
|
-
|
+
|
-
|
+
|
Aromatic amino acid interconversions with aryl acids
|
-
|
+
|
-
|
-
|
+
|
Indole-pyruvate oxidoreductase complex
|
-
|
-
|
-
|
-
|
+
|
Metabolism of aromatic compounds
|
|
|
|
|
|
Central meta-cleavage pathway of aromatic compound degradation
|
+
|
-
|
-
|
-
|
-
|
Homogentisate pathway of aromatic compound degradation
|
-
|
-
|
-
|
-
|
+
|
Biphenyl degradation
|
-
|
+
|
-
|
-
|
-
|
Carbohydrate metabolism
|
|
|
|
|
|
One-carbon metabolism: methanogenesis
|
-
|
-
|
-
|
-
|
+
|
CO2 fixation: CO2 uptake, carboxysome, photorespiration (oxidative C2 cycle), Calvin-Benson cycle
|
-
|
-
|
-
|
-
|
+
|
Di- and oligosaccharide: lactose and galactose uptake and utilization
|
+
|
+
|
-
|
+
|
+
|
Di- and oligosaccharide: maltose and maltodextrin utilization
|
+
|
+
|
+
|
-
|
+
|
Sugar alcohol: erythritol utilization
|
-
|
-
|
-
|
+
|
-
|
Fermentation: acetoin and butanediol metabolism
|
+
|
+
|
+
|
+
|
-
|
Monosaccharide: D-galacturonate and D-glucuronate utilization
|
+
|
+
|
+
|
+
|
-
|
Monosaccharide: L-rhamnose utilization
|
+
|
+
|
-
|
+
|
-
|
Monosaccharide: L-fucose utilization temp
|
+
|
+
|
+
|
-
|
+
|
Monosaccharide: D-galactarate, D-glucarate and D-glycerate catabolism
|
+
|
+
|
+
|
+
|
-
|
Strains: 1, CSW-27T; 2, Rhizobium straminoryzae SM12; 3, Rhizobium rhizoryzae J3-AN59T; 4, Rhizobium ipomoeae shin9-1T; 5, Rhizobium petrolearium SL-1T. +, Present; -, absent. |
The primary differences are that only strain CSW-27T had genes related to membrane transport e.g. TRAP transporter unknown substrate 4, related to programmed cell death and toxin-antitoxin systems (regulation and cell signaling) e.g. murein hydrolase regulation and cell death, related to dormancy and sporulation e.g. persister cells, related to iron acquisition and metabolism e.g. siderophore enterobactin, related to metabolism of central aromatic intermediates e.g. central meta-cleavage pathway of aromatic compound degradation, but the other four strains had not these genes (Supplementary Table S5). Other features are that only Rhizobium straminoryzae SM12, Rhizobium rhizoryzae J3-AN59T, Rhizobium ipomoeae shin9-1T and Rhizobium petrolearium SL-1T existed genes putatively encoding for adaptation to d-cysteine e.g. cystine ABC transporter, permease protein related to virulence, disease and defense; genes putatively encoding for ABC transporter (iron.B12.siderophore.hemin) e.g. ATP-binding component, periplasmic substrate-binding component, permease component related to iron acquisition and metabolism, but the novel strain had not the related genes.
Regarding the protein secretion systems of membrane transport, all five strains had Type II but none Types III, V and VII. And, the five strains showed different patterns for Types I, IV and VI. Only Rhizobium ipomoeae shin9-1T possessed genes encoding proteins associated with Type I, only strain CSW-27T and Rhizobium straminoryzae SM12 existed genes encoding proteins associated with Type VI, and except Rhizobium rhizoryzae J3-AN59T the other four strains had genes encoding proteins associated with Type IV (Supplementary Table S6). Concerning the nitrogen metabolism, all five strains had most related enzymes. Furthermore, only Rhizobium petrolearium SL-1T existed genes encoding methanol dehydrogenase, formate dehydrogenase and carbamate kinase, and only Rhizobium rhizoryzae J3-AN59T had formamidase related gene. And, only both strains, Rhizobium petrolearium SL-1T and Rhizobium rhizoryzae J3-AN59T, possessed gene encoding aspartate ammonia-lyase (Supplementary Table S7). When examining the prevalence of total phage in genomes of strain CWS-27T, Rhizobium straminoryzae SM12, Rhizobium rhizoryzae J3-AN59T, Rhizobium ipomoeae shin9-1T and Rhizobium petrolearium SL-1T, it is found that it is about 0.5 to 2.6 percent in the five Rhizobium strains (Supplementary Table S8). However, only strain CSW-27T existed the three phage types including intact, questionable and incomplete which sorted by the completeness. And, only strain CSW-27T, Rhizobium straminoryzae SM12 and Rhizobium rhizoryzae J3-AN59T had intact prophage e.g. Siphoviridae, Rhodobacter phage RcapNL in strain CSW-27T; e.g. Siphoviridae, Rhodobacter phage RC1 and Myoviridae, Aurantimonas phage AmM-1 in Rhizobium straminoryzae SM12; e.g. Myoviridae, Aurantimonas phage AmM-1 in Rhizobium rhizoryzae J3-AN59T.
Homology analysis of gene contents were performed between strain CSW-27T and Rhizobium straminoryzae SM12, Rhizobium rhizoryzae J3-AN59T, Rhizobium ipomoeae shin9-1T and Rhizobium petrolearium SL-1T. As a result, a total of 2265 genes common are shared among the five strains (Supplementary Fig. S7), and there are 724 genes present as specific genes in strain CSW-27T. 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-LY845T and Rhizobium capsici CC-SKC2T. However, strain CSW-27T could be clearly differentiated from these two phylogenetic related strains by its ability to grow at lower temperature (< 25 oC), 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-raffinose, 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-27T, Rhizobium straminoryzae SM12, Rhizobium rhizoryzae J3-AN59T, Rhizobium ipomoeae shin9-1T and Rhizobium petrolearium SL-1T, it is also found that although they have common characteristics, strain CSW-27T 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-27T occupies a distinct position within the genus Rhizobium that is supported by a unique combination of chemotaxonomic and biochemical characteristics. Strain CSW-27T 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 flagella, 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 oC 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 oC (optimum, 30-37 oC), at pH 5-9 (optimum, pH 6-7) and with 0-4% NaCl (optimum, 0%). Negative for poly-β-hydroxybutyrate accumulation. Positive for oxidase activity and negative for catalase activity. Capable of hydrolyzing DNA, CM-cellulose and Tween 40. Incapable of hydrolyzing casein, starch, chitin, lecithin, corn oil and Tweens 20, 60 and 80. Positive for glucose fermentation, urea and esculin hydrolysis, β-galactosidase (PNPG) activity, and assimilation of glucose, arabinose, mannose, mannitol, maltose, gluconate, adipate and malate. Cells present alkaline phosphatase, C4 esterase, C8 esterase lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase (ONPG), α-glucosidase and β-glucosidase activities. Positive growth under aerobic condition are observed for D-glucose, D-fructose, D-galactose, D-cellobiose, N-acetyl-glucosamine, D-sorbitol, Tween 40, Tween 60, adipate, L-aspartic acid and L-leucine. The major fatty acid is summed feature 8 (C18:1ω7c and/or C18:1ω6c). Polar lipids present are phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine, one uncharacterized aminophospholipid, three uncharacterized aminolipids and two uncharacterized lipids. The predominant polyamine is spermidine. The sole and major isoprenoid quinone is Q-10.
The genome size of strain CSW-27T is 5.66 Mb with 63.3% G+C content. The type strain CSW-27T (=BCRC 81244T =LMG 31684T) was isolated from a freshwater pond in the Xingang National Primary School, Xingang Township in Chiayi County, Taiwan. The GenBank accession numbers for the 16S rRNA gene sequence and the whole genome of Sphingomonas lacunae CSW-27T are MH729077 and NZ_JAHHZO000000000.