International Journal of Systematic and Evolutionary Microbiology (2015), 65, 195–200 DOI 10.1099/ijs.0.070375-0 Proposal of Novosphingobium rhizosphaerae sp. nov., isolated from the rhizosphere Peter Ka¨mpfer,1 Karin Martin,2 John A. McInroy3 and Stefanie P. Glaeser1 Correspondence 1Institut fu¨r Angewandte Mikrobiologie, Justus-Liebig-Universita¨t Giessen, D-35392 Giessen, Peter Ka¨mpfer Germany [email protected] 2Leibniz-Institut fu¨r Naturstoff-Forschung und Infektionsbiologie e. V., Hans-Kno¨ll-Institut., giessen.de D-07745 Jena, Germany 3Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849, USA A yellow, Gram-stain-negative, rod-shaped, non-spore-forming bacterium (strain JM-1T) was isolated from the rhizosphere of a field-grown Zea mays plant in Auburn, AL, USA. 16S rRNA gene sequence analysis of strain JM-1T showed high sequence similarity to the type strains of Novosphingobium capsulatum (98.9 %), Novosphingobium aromaticivorans (97.4 %), Novosphingobium subterraneum (97.3 %) and Novosphingobium taihuense (97.1 %); sequence similarities to all other type strains of species of the genus Novosphingobium were below 97.0 %. DNA–DNA hybridizations of strain JM-1T and N. capsulatum DSM 30196T, N. aromaticivorans SMCC F199T and N. subterraneum SMCC B0478T showed low similarity values of 33 % (reciprocal: 21 %), 14 % (reciprocal 16 %) and 36 % (reciprocal 38 %), respectively. Ubiquinone Q-10 was detected as the major respiratory quinone. The predominant fatty acid was C18 : 1v7c (71.0 %) and the typical 2-hydroxy fatty acid C14 : 0 2-OH (11.7 %) was detected. The polar lipid profile contained the diagnostic lipids diphosphatidylglycerol, phosphatidylethanolamine, sphin- goglycolipid and phosphatidylcholine. Characterization by 16S rRNA gene sequence analysis, physiological parameters, pigment analysis, and ubiquinone, polar lipid and fatty acid composition revealed that strain JM-1T represents a novel species of the genus Novosphingobium. For this species we propose the name Novosphingobium rhizosphaerae sp. nov. with the type strain JM- 1T (5LMG 28479T5CCM 8547T). Members of the genus Novosphingobium are widely (Xie et al., 2014), N. lentum (Tiirola et al., 2005), N. distributed in the environment including soil, coastal or lindaniclasticum (Saxena et al., 2013), N. malaysiense (Lee freshwater sediments (Balkwill et al., 1997; Sohn et al., et al., 2014a), N. mathurense and N. panipatense (Gupta et al., 2004; Liu et al., 2005), surface water layers of lakes (Glaeser 2009), N. naphthalenivorans (Suzuki & Hiraishi, 2007), N. et al., 2009; 2013a, b), activated sludge/wastewater treat- nitrogenifigens (Addison et al., 2007), N. pentaromativorans ment plants (Neef et al., 1999; Fujii et al., 2003), con- (Sohn et al., 2004), N. rosa (Takeuchi et al., 1995), N. taminated groundwater bioremediation reactors (Tiirola resinovorum (Lim et al., 2007), N. sediminicola (Baek et al., et al., 2002; 2005), and associated with plants (Lin et al., 2011), N. soli (Ka¨mpfer et al., 2011), N. stygium (Balkwill 2014). At the time of writing, 29 species of the genus et al., 1997), N. subarcticum (which is a later subjective Novosphingobium with validly published names had been synonym of N. resinovorum; Lim et al., 2007); N. sub- described: Novosphingobium acidiphilum (Glaeser et al., terraneum (Balkwill et al., 1997), N. taihuense (Liu et al., 2009), N. aquaticum (Glaeser et al., 2013a), N. aquiterrae 2005) and N. tardaugens (Fujii et al., 2000). A further species (Lee et al., 2014b), N. arabidopsis (Lin et al., 2014), N. proposal was published recently, but the species name aromaticivorans (Balkwill et al., 1997), N. barchaimii ‘Novosphingobium ginsenosidimutans’ (Kim et al., 2013) has (Niharika et al., 2013), N. capsulatum (Yabuuchi et al., not yet been validly published. 1990), N. chloroacetimidivorans (Chen et al., 2014), N. Strain JM-1T was isolated from the rhizosphere of a field- fuchskuhlense (Glaeser et al., 2013b), N. hassiacum (Ka¨mpfer grown Zea mays plant in Auburn, AL, USA. The strain et al., 2002), N. indicum (Yuan et al., 2009), N. kunmingense produced single cells, which formed small yellow colonies (,0.5 mm) showing a smooth surface after 48 h at 25 uC Abbreviations: pNA p-nitroanilide; pNP, p-nitrophenyl. on nutrient agar (NA). Cell morphological features were The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene investigated by phase-contrast microscopy with cells grown T sequence of strain JM-1T is KM365125. on NA at 25 uC. The rod-shaped cells of strain JM-1 were Downloaded from www.sgmjournals.org by 070375 G 2015 IUMS Printed in Great Britain 195 IP: 131.204.246.172 On: Tue, 04 Aug 2015 15:19:01 P. Ka¨mpfer and others 1.8±0.4 mm long and 1.0±0.2 mm wide and motile in the Aligner (SINA; v1.2.11) (Pruesse et al., 2012) and incorpo- early growth phase, as observed by light-microscopy. Cells rated into the All Species Living Tree (LTP) database. In stained Gram-negative and were positive for cytochrome the same manner, type strain sequences representing the oxidase as determined by using an oxidase test (Merck). genus Novosphingobium that were not included in the LTP Endospores could not be detected. The strain produced database were added by using sequences published in catalase, after testing 24-hour-old colonies with H2O2. GenBank (http://www.ncbi.nlm.nih.gov/genbank/). The The 16S rRNA gene of strain JM-1T was sequenced using alignment including all type strain sequences representing the universal bacterial 16S rRNA gene targeting primers 8F the genus Novosphingobium was checked manually before (59-AGAGTTTGATCCTGGCTCAG-39) and 1492R (59- further analysis. Sequence similarities were calculated using ACGGCTACCTTGTTACGACTT-39; Lane, 1991) for the ARB neighbour-joining tool, without the application of PCR-amplification and Sanger sequencing. The sequence an evolutionary model. Different treeing methods were was processed and manually controlled based on the applied for the calculation of phylogenetic trees including electropherograms using MEGA5 (Tamura et al., 2011). all type strains representing the genus Novosphingobium, After unclear 59 and 39 ends were removed, the final considering 16S rRNA gene sequences between sequence sequence was 1443 nt long spanning 16S rRNA gene positions 68 and 1450 (according to E. coli numbering; positions 16 to 1527 (according to Escherichia coli Brosius et al., 1978). A maximum-likelihood tree was numbering; Brosius et al., 1978). Phylogenetic analyses calculated using RAxML version 7.04 (Stamatakis, 2006) were performed in the software package ARB release 5.2 with GTR-GAMMA and rapid bootstrap analysis, a (Ludwig et al., 2004). Therefore, the 16S rRNA gene neighbour-joining tree using ARB neighbour joining with sequence of strain JM-1T was aligned against an expert- the Jukes–Cantor correction (Jukes & Cantor, 1969), based reference alignment using the SILVA Incremental and a maximum-parsimony tree using DNAPARS v 3.6 91 N. acidiphilum FSW06-204dT (EU336977) 0.10 ** N. nitrogenifigens Y88T (DQ448852) N. hassiacum W-51T (AJ416411) N. lentum MT1T (AJ303009) 100 N. fuchskuhlense FNE08-7T (JN399172) ** 89 ‘N. sediminis’ YG-17 (FJ938155) ** N. stygium NBRC 16085T (AB025013) * N. taihuense T3-B9T (AY500142) N. tardaugens ARI-1T (AB070237) N. subterraneum NBRC 16086T (AB025014) 99 N. aquiterrae E-II-3T (FJ772064) ** N. kunmingense 18-11HKT (JQ246446) * N. ginsenosidimutans FW-6T (JQ349046) * N. arabidopsis CC-ALB-2T (KC479803) 75 * N. aromaticivorans DSM 12444T (CP000248) N. rhizosphaerae JM-1T (KM365125) Fig. 1. Maximum-likelihood tree showing the ** phylogenetic position of JM-1T among all type 88 97 N. capsulatum GIFU 11526T (D16147) N. aquaticum FNE08-86T (JN399173) strains and currently proposed species of the genus Novosphingobium. The phylogenetic 75 N. sediminicola HU1-AH51T (FJ177534) tree is based on nearly full-length 16S rRNA N. rosa IAM 14222T (D13945) gene sequences and was calculated in ARB N. soli CC-TPE-1T (FJ425737) using the RAxML treeing method. Type strains * N. chloroacetimidivorans BUT-14T (KF676669) of species of the genus Sphingorhabdus were N. naphthalenivorans TUT562T (AB177883) used as an outgroup. Nodes marked with an N. barchaimii LL02T (JN695619) asterisk were also conserved in the trees * NCIMB 8767T (EF029110) N. resinovorum generated with the neighbour-joining and * T 76 N. lindaniclasticum LE124 (JN687581) maximum-parsimony methods; one asterisk 100 N. indicum H25T (EF549586) represents nodes that occurred with both of ** T 82 N. malaysiense MUSC 273 (KC907395) these alternative methods, two asterisks mark 88 N. panipatense SM16T (EF424402) nodes that occurred in both or at least in the 100 N. mathurense SM117T (EF424403) maximum-parsimony tree with high bootstrap ** T N. pentaromativorans US6-1 (AF502400) values (.70 %). Numbers at nodes represent 100 Sphingorhabdus marina FR1087T (DQ781320) bootstrap values based on 100 resamplings; ** 100 Sphingorhabdus litoris FR1093T ( DQ781321) only values .70 % are depicted. Bar, 0.1 ** Sphingorhabdus flavimaris SW-151T (AY554010) substitutions per 100 nucleotides. Downloaded from www.sgmjournals.org by 196 International Journal of Systematic and Evolutionary Microbiology 65 IP: 131.204.246.172 On: Tue, 04 Aug 2015 15:19:01 Novosphingobium rhizosphaerae sp. nov. (Felsenstein, 2005). All trees considered 100 resamplings Table 1. Physiological test results of strain JM-1T in (bootstap analysis; Felsenstein, 1985). Comparison of the comparison with the most closely related species