Characterization of Two Aerobic Ultramicrobacteria Isolated from Urban Soil and a Description of Oxalicibacterium Solurbis Sp

RESEARCH LETTER Characterization of two aerobic ultramicrobacteria isolated from urban soil and a description of Oxalicibacterium solurbis sp. nov. Nurettin Sahin1, Juan M. Gonzalez2, Takashi Iizuka3 & Janet E. Hill4

1Egitim Fakultesi, Mugla Universitesi, Mugla, Turkey; 2Instituto de Recursos Naturales y Agrobiologia, IRNAS-CSIC, Sevilla, Spain; 3Central Research Laboratories, Ajinomoto Co. Inc., Kawasaki, Japan; and 4Department of Veterinary Microbiology, University of Saskatchewan, SK, Canada Downloaded from https://academic.oup.com/femsle/article-abstract/307/1/25/471128 by guest on 13 February 2020

Correspondence: Nurettin Sahin, Egitim Abstract Fakultesi, Mugla University, TR-48170 Kotekli, Mugla, Turkey. Tel.: 190 252 211 1826; fax: Two strains of aerobic, non-spore-forming, Gram-negative, rod-shaped bacteria T 190 252 223 8491; e-mail: (ND5 and MY14 ), previously isolated from urban soil using the membrane-filter [email protected] enrichment technique, were characterized. Analysis of their 16S rRNA gene sequence grouped strains ND5 and MY14T within the family Oxalobacteraceae Received 10 February 2010; revised 2 March (Betaproteobacteria). The highest pairwise sequence similarities for strain ND5 2010; accepted 2 March 2010. were found with members of the genus Herminiimonas, namely with Herminiimo- Final version published online 30 March 2010. nas saxobsidens NS11T (99.8%) and Herminiimonas glaciei UMB49T (99.6%). Although some fatty acid profiles, physiological and biochemical differences exist DOI:10.1111/j.1574-6968.2010.01954.x between strain ND5 and the respective Herminiimonas-type strains, DNA–DNA hybridization experiments confirm that strain ND5 is a member of the H. glaciei Editor: Aharon Oren genospecies. Taxonomical analyses revealed a wider range of variability within this

Keywords genus than considered previously. The highest pairwise nucleotide similarity for T Oxalicibacterium solurbis ; Herminiimonas ; strain MY14 was found with Oxalicibacterium ﬂavum (96.8%). Phylogenetic ultramicrobacteria; Oxalobacteraceae. analyses based on 16S rRNA and cpn60 gene sequences, DNA–DNA hybridization, fatty acid proﬁles, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain MY14T from other Oxalicibacterium species representing a new species, for which the name Oxalicibacterium solurbis sp. nov. (type strain MY14T = NBRC 102665T, = CCM 7664T) is proposed.

Herminiimonas arsenicoxydans (Muller et al., 2006), Hermi- Introduction niimonas saxobsidens (Lang et al., 2007) and Herminiimonas Extremely small free-living bacteria, showing biovolumes glaciei (Loveland-Curtze et al., 2009). The genus Oxalicibac- generally lower than 0.3 mm3 in situ (Koch, 1996), are known terium, with the type species Oxalicibacterium flavum, was to be present in a wide variety of natural environments and established by Tamer et al. (2002) and currently comprises have been classified with terms such as ultramicrobacteria three species. The species Oxalicibacterium horti and Oxali- (UMB), nanobacteria or picobacteria (Koch, 1996). With cibacterium faecigallinarum have been described recently the purpose of isolating copiotrophic and rapidly growing (Sahin et al., 2009). UMB, strains ND5 and MY14T were isolated from an urban The present paper deals with a polyphasic approach to soil sample using the 0.45-mm membrane filter enrichment describe strains ND5 and MY14T, which have been classified technique on 0.1 TSA (Iizuka et al., 1998). The site was in the genera Herminiimonas and Oxalicibacterium, respec- covered by a heap of fallen leaves and located in a grove in tively, and to propose a novel taxon for strain MY14T, the Tokyo metropolitan area. Analysis of the almost com- named Oxalicibacterium solurbis sp. nov. plete 16S rRNA gene sequence grouped strains ND5 and MY14T within the family Oxalobacteraceae (Betaproteobac- Materials and methods teria), most closely related to type strains of the genera

MICROBIOLOGY LETTERS MICROBIOLOGY Herminiimonas and Oxalicibacterium, respectively. Phenotypic characterization The genus Herminiimonas presently comprises ﬁve validly described species: Herminiimonas fonticola (Fernandes et al., Physiological and biochemical tests were carried out at 2005), Herminiimonas aquatilis (Kampfer¨ et al., 2006), 30 1C. Conventional biochemical tests were performed

FEMS Microbiol Lett 307 (2010) 25–29 c 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 26 N. Sahin et al. according to standard methods (Smibert & Krieg, 1994). 60 1C and 60 s at 72 1C, and a ﬁnal extension period of Bacterial growth at different pH values (6–9.5), tempera- 10 min at 72 1C. PCR reactions were conducted using an tures ( 5to421C) and NaCl concentrations (0–5%) was Eppendorf Mastercycler EP thermocycler. determined in basal mineral medium supplemented with PCR products were sequenced using sequencing primers 1 glycolate and lactate that contained (L ): 1 g L-glycolate, 1 g M13(-40)F and M13(48)R described above. Sequence data DL-lactate, 0.1 g yeast extract (Difco), 100 mL RM1-mineral were assembled and edited using the STADEN PACKAGE (Staden, solution, 1 g (NH4)2SO4, 0.5 g KH2PO4 and 0.5 g K2HPO4. 1996). Finished sequences were deposited in GenBank and The pH of the medium was adjusted to 6.8 with NaOH. cpnDB (http://cpndb.cbr.nrc.ca) sequence databases (Hill 1 RM1-mineral solution contained (L ): 2.0 g MgCl2 6H2O, et al., 2004).

0.4 g CaCl2 2H2O, 2.0 g NaCl and 10 mL trace element Downloaded from https://academic.oup.com/femsle/article-abstract/307/1/25/471128 by guest on 13 February 2020 solution (Iizuka et al., 1998). API 20NE, API 20E strips (bioMerieux)´ and Biolog GN microplates were used accord- Chemotaxonomic characterization ing to the manufacturer’s instructions, and reactions were For the analysis of fatty acids, cells were grown on R2A agar observed for 7 days. Additional utilization and assimilations at 28 1C for 4 days. Cells were saponified, methylated to of sugars, alcohols and amino acids were determined in the create fatty acid methyl esters and extracted as described above-indicated basal mineral medium with addition of previously (Kampfer¨ & Kroppenstedt, 1996). Peaks were 1 filter-sterilized solutions of the following substrates (g L ). automatically integrated and fatty acid names and percen- Sugars and alcohols: ethanol, 0.5; methanol, 0.5; n-propa- tages were determined using the Microbial Identification nol, 0.5; D-ribose, 2.0; xylose, 2.0. Organic acids: acetate, 0.5 standard software package MIDI (Sasser, 1990). Polar lipid and 2.0; benzoate, 0.5; caprylate, 0.5; oxalate, 0.5 and 2.0; profiles were examined by two-dimensional thin-layer chro- fumarate, 2.0; glycolate, 2.0; L-malate, 2.0; L-tartarate, 2.0. matography as described by Rowe et al. (2000). Amino acids: aminobutyrate, 2.0; L-arginine HCl, 2.0; L-glycine, 2.0; L-lysine, 2.0; and L-tryptophan, 2.0. DNA--DNA hybridizations 16S rRNA gene sequencing and phylogenetic The degree of DNA–DNA relatedness was determined by analysis measuring the divergence between the thermal denaturation The 16S rRNA gene sequences were analysed as described by midpoint of homoduplex DNA and heteroduplex DNA

Iizuka et al. (1998). Evolutionary distances were calculated (DTm) as described by Gonzalez´ & Saiz-Jim´ enez´ (2005). using Kimura’s two-parameter model (Kimura, 1980) without The G1C content of the DNA was determined according to taking into account the alignment gaps and unidentiﬁed base the ﬂuorimetric method described by Gonzalez´ & Saiz-´ positions. Phylogenetic trees were constructed from the dis- Jimenez´ (2002) using thermal denaturation temperature. tance data using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Fitch, 1971) methods with bootstrap values based on 1000 replications (Felsenstein, 1985). Results and discussion cpn 60 universal target sequencing Phenotypic characteristics Approximately 50–100 ng of genomic DNA was used as a The strains studied showed a limited substrate spectrum as template in PCR reactions (50 mL total volume) containing observed from the analysis of API 20 NE, API 20E and Biolog 1 PCR buffer (Invitrogen, Burlington, Canada), 2.5 mM GN microplates. Strains ND5 and MY14T utilized oxalate,

MgCl2, 200 nM dNTPs, 2.5 U Platinum Taq DNA polymer- formate, glycolate, lactate, pyruvate, succinate and malate. ase (Invitrogen) and 400 nM each of primers H1594 (50- Other carboxylic acids, alcohols and amino acids (except CGC CAG GGT TTT CCC AGT CAC GAC GAC GTC GCC alanine) were not utilized. Strain ND5 differs from H. glaciei 0 0 T GGT GAC GGC ACC ACC AC-3 ) and H1595 (5 -AGC UMB49 in its inability to utilize citrate and L-arabinose and GGA TAA CAA TTT CAC ACA GGA CGA CGG TCG CCG its capability to use acetate (Loveland-Curtze et al., 2009). AAG CCC GGG GCC TT-30). Ampliﬁcation primers in- On the other hand, strain MY14T utilized an even cluded annealing sites for standard M13 sequencing primers narrower range of substrates: fumarate, glycolate, lactate, M13(-40)F and M13(48)R (underlined). The primers am- malate, malonate (weak), pyruvate, succinate, oxalate, plify the universal target region of the cpn60 gene (encoding L-alanine and L-glycine. Although both strains utilized the universally conserved 60-kDa chaperonin, also known as oxalate, the cell yield was lower than those with fumarate, groEL or hsp60), corresponding to nucleotides 274–828 of glycolate, lactate or malate. Differential phenotypic charac- the Escherichia coli cpn60 gene. Reactions were incubated at teristics between MY14T and the type strains of the genus 94 1C for 3 min, followed by 40 cycles of 30 s at 94 1C, 60 s at Oxalicibacterium are given in Table 1.

Table 1. Differential characters of type strains of Oxalicibacterium Table 2. Whole-cell fatty acid composition of strain MY14 and type species strains of Oxalicibacterium species Characters 1 2 3 4 Fatty acid 1 2 3 4Ã

Mono-methyl succinate 11 1C14:0 – 0.9 6.0 4.2 cis-Aconitic acid w C16:0 37.2 24.3 17.8 20.1

Formic acid 11 1C17:0 2.5 3.0 2.9 1.3 a-OH-butyric acid 1 1 w C18:0 3.6 1.7 3.9 0.7 b-OH-butyric acid 1 C10:0 3-OH 6.0 7.0 8.9 4.6 a-Ketobutyric acid 1 C15:1 o6c – 1.4 – – a-Ketoglutaric acid w 11C18:1 o9c – – – 0.4 Downloaded from https://academic.oup.com/femsle/article-abstract/307/1/25/471128 by guest on 13 February 2020 Succinic acid w C18:1 o6c – – – 11.1

Bromosuccinic acid 11 1C18:1 o5c – 2.3 – –

Succinamic acid w w C17:0 cyclo 41.6 26.3 22.9 2.2

Alaninamide 11 w C19:0 cyclo o8c 6.4 16.1 19.8 1.1 D-Alanine 11 1Summed feature 3 2.8 14.5 14.2 37.3 L-Alanine 1 w Summed feature 7 – 2.5 3.8 17.0 L-Glutamic acid w 1 w Strains: 1, MY14T;2,Oxalicibacterium flavum TA17T;3,Oxalicibacterium L-Threonine w T T DNA G1C content (mol%) 63.3 63.5 59.7 55.6 horti OD1 ;4,Oxalicibacterium faecigallinarum YOx . Cells were grown on R2A agar at 28 1C for 4 days. Data are percentages of the total fatty Strains: 1, MY14T;2,Oxalicibacterium flavum TA17T;3,Oxalicibacterium acids. horti OD1T;4,Oxalicibacterium faecigallinarum YOxT. All strains were ÃData from Sahin et al. (2009). Summed feature 3 comprises 16:1o7c, positive for malate (API 20NE), and DL-lactic acid (BIOLOG GN), but 15 iso 2OH or any combination of these fatty acids. Summed feature 7 negative for the other substrates or reactions of the panels not men- comprises 18:1 o7c, 12t/9t or any combination of these fatty acids. tioned. , negative; 1, positive; w, weakly positive. clustered with the members of the genus Oxalicibacterium Chemotaxonomic characteristics (Fig. 1). The tree constructed based on the maximum- The major fatty acids of strain MY14T were C (37.2%) parsimony method showed a similar topology (see Fig. S2). 16:0 T and C cyclo (41.6%). In addition, C 3-OH (6%) was The highest pairwise nucleotide similarity for strain MY14 17:0 10:0 T the only hydroxylated fatty acid detected (Table 2). Further- was found with O. flavum (96.8%). Strain MY14 showed below 97.0% 16S rRNA gene and below 92% cpn60 nucleo- more, C16:0 and C17:0 cyclo seem to be present in strain MY14T in a significantly higher proportion than all other tide sequence similarity with other members of the genus Oxalicibacterium type species examined. Strain MY14T Oxalicibacterium. Phylogenetic analysis of translated cpn60 could be differentiated from the type strains of the other peptide sequences was consistent with the 16S rRNA gene- based phylogeny and supported the identification of MY14T Oxalicibacterium type species by its lack of C14:0 and summed feature 7 that comprises 18:1 o7c, 12t/9t fatty acids as a distinct species within the Oxalicibacterium genus (see (Table 2). The polar lipid profile consists of the predomi- Fig. S3). nant compounds phosphatidylethanolamine and phospha- The highest pairwise sequence similarities for strain ND5 T tidylglycerol, and a small amount of diphosphatidylglycerol were found with H. saxobsidens NS11 (99.8%) and T and one unknown polar lipid (see Supporting Information, H. glaciei UMB49 (99.6%). Strain ND5 also showed 98%, Fig. S1). 97.6% and below 94% cpn60 nucleotide sequence similarity T T Strain ND5 has some differences from the type strains of with H. saxobsidens NS11 , H. glaciei UMB49 and the rest of the members of the genus Herminiimonas, respectively. the four described Herminiimonas species by its C10:0 3-OH Peptide sequence identities for the ND5 cpn60 sequences fatty acid content being lower than 0.5% and C18:1o7c fatty acid content being significantly higher than the Herminii- were 4 97% to Herminiimonas spp. The G1C content of monas-type species examined. The major quinone system is the 555 bp cpn60 universal target region of ND5 was 52%. ubiquinone Q-8. The G1C content of DNA is 55.4 mol%. DNA--DNA hybridizations The DNA–DNA relatedness studies among strains MY14T 16S rRNA and cpn60 gene sequencing and and O. flavum TA17T, sharing the highest (96.8%) 16S rRNA phylogenetic analysis and cpn60 (92%) gene sequence similarity, showed that the T Phylogenetic analyses using the 16S rRNA gene sequences DTm values were 5.2 1C, which confirms that strain MY14 indicated that strain MY14T belongs to the family Oxalo- does not belong to the genospecies O. flavum. bacteraceae of the Betaproteobacteria. In the 16S rRNA gene The DNA–DNA relatedness studies between strains ND5 sequences based on neighbour-joining tree, strain MY14T and H. saxobsidens NS11T, the strain with the highest 16S

Fig. 1. Phylogenetic tree, based on neighbour-joining (Saitou & Nei, 1987), derived from an alignment comprising 16S rRNA gene partial region sequences (1379 bp). Percentage values at the nodes represent the result of 1000

bootstrap samples. The scale bar indicates the Downloaded from https://academic.oup.com/femsle/article-abstract/307/1/25/471128 by guest on 13 February 2020 number of substitutions per nucleotide position.

rRNA (99.8%) and cpn60 (98%) gene sequence similarity (weak), pyruvate, succinate, oxalate, L-alanine and L-glycine. with ND5, showed that DTm values were 6.6 1C. However, Other differential characteristics are given in Table 1. The T the DTm value between H. glaciei UMB49 and strain ND5, main fatty acids are C16:0,C17:0 cyclo, C19:0 cyclo o8c and sharing 99.6% 16S rRNA and 97.6% cpn60 gene sequence C10:0 3-OH. The major quinone system is ubiquinone Q-8. similarity, was 1 1C, which is well below the 5 1C cut-off The major phospholipids are phosphatidylethanolamine point recommended for the delineation of species (Wayne and phosphatidylglycerol. The G1C content of DNA is et al., 1987; Rossello-Mora´ & Amann, 2001). Similar ob- 63.3 mol%. servations of contrasting high 16S rRNA gene sequence The type strain, MY14T (= NBRC 102665T, = CCM similarity (99.6–99.8%) and low (3–57%) DNA–DNA relat- 7664T), was isolated from a soil sample using the mem- edness have been reported among all described Herminii- brane-ﬁlter enrichment technique. monas-type strains (Kampfer¨ et al., 2006; Muller et al., 2006; Lang et al., 2007; Loveland-Curtze et al., 2009). On the basis of the results described above, it can be Acknowledgements concluded that the strain ND5 (= NBRC 102664, = CCM We are grateful to Dr Jean P. Euzeby,´ for his help with the 7665) is another strain of H. glaciei and strain MY14T Latin nomenclature of the species epithets. Thanks are also represents a novel species within the genus Oxalicibacter- due to Dr J. Loveland-Curtze for supplying the type strain of ium, for which the name O. solurbis sp. nov. is proposed. H. glaciei for hybridization experiments.

Description of O. solurbis sp. nov. Statement Oxalicibacterium solurbis (sol.ur’bis. L. n. solum, soil; L. n. urbs urbis, a city; N.L. gen. n. solurbis, of city soil, where the The GenBank/EMBL/DDBJ accession numbers for the nu- type strain was isolated). cleotide sequences reported in this study are AB008503 T T Gram-negative, small rods 0.4–0.5 0.8–1.2 mm (mean (strain MY14 16S rRNA gene), FJ860274 (strain MY14 T cell volume 0.07 mm3), motile by polar flagella. At low partial cpn60 gene), FJ860273 (O. flavum TA17 partial temperatures around 4 1C, elongated cells are sometimes cpn60 gene), FJ860269 (O. flavum NS13 partial cpn60 gene), T observed. No spores were observed. Oxidase and catalase FJ860271 (O. horti OD1 partial cpn60 gene), FJ860270 T reactions are positive. Colonies on nutrient agar (Oxoid (O. faecigallinarum YOx partial cpn60 gene), AB008506 CM3) with lactate are slightly yellow pigmented. Forms (strain ND5 16S rRNA gene), GQ375149 (strain ND5 partial T smooth, glistening, raised, opaque with entire edges; the cpn60 gene), GQ375148 (H. glaciei UMB49 partial cpn60 T diameter is up to 0.5–1 mm after 3 days of incubation at gene), GQ375147 (H. saxobsidens NS11 partial cpn60 T 28–30 1C. Growth occurs at 4 1C and up to 37 1C, but not gene), GQ375146 (H. aquatilis DSMZ 18803 partial cpn60 T 42 1C. Optimum growth occurs at 37 1C and pH 8.0. Grows gene) and FJ860272 (H. fonticola S94 partial cpn60 gene). in media containing 5% NaCl. The specific growth rate (m) under optimum conditions with lactate was 0.14 h1.No acid produced from glucose. Nitrate is not reduced to References nitrite. Negative for indole production, arginine dihydro- Felsenstein J (1985) Confidence limits on phylogenies – an lase, urease, esculin, casein and gelatine hydrolysis and approach using the bootstrap. Evolution 39: 783–791. b-galactosidase. Sugars and alcohols were not utilized; Fernandes C, Rainey FA, Nobre MF, Pinhal I, Folhas F & da Costa utilizes fumarate, glycolate, DL-lactate, L-malate, malonate MS (2005) Herminiimonas fonticola gen. nov., sp. nov., a

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