International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1859–1865 DOI 10.1099/ijs.0.062232-0 Nitrolancea hollandica gen. nov., sp. nov., a chemolithoautotrophic nitrite-oxidizing bacterium isolated from a bioreactor belonging to the phylum Chloroflexi Dimitry Y. Sorokin,1,2 Dana Vejmelkova,3 Sebastian Lu¨cker,4 Galina M. Streshinskaya,5 W. Irene C. Rijpstra,6 Jaap S. Sinninghe Damste´,6 Robbert Kleerbezem,2 Mark van Loosdrecht,2 Gerard Muyzer7 and Holger Daims4 Correspondence 1Winogradsky Institute of Microbiology RAS, Moscow, Russia Dimitry Y. Sorokin 2Department of Biotechnology, TU Delft, The Netherlands [email protected] 3Department of Water Technology, ICT Prague, Czech Republic 4Department of Microbial Ecology, Ecology Centre, University of Vienna, Vienna, Austria 5Microbiology Department, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia 6Department of Marine Organic Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, Den Burg, The Netherlands 7Department of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands A novel nitrite-oxidizing bacterium (NOB), strain LbT, was isolated from a nitrifying bioreactor with a high loading of ammonium bicarbonate in a mineral medium with nitrite as the energy source. The cells were oval (lancet-shaped) rods with pointed edges, non-motile, Gram-positive (by staining and from the cell wall structure) and non-spore-forming. Strain LbT was an obligately aerobic, chemolitoautotrophic NOB, utilizing nitrite or formate as the energy source and CO2 as the carbon source. Ammonium served as the only source of assimilated nitrogen. Growth with nitrite was optimal at pH 6.8–7.5 and at 40 6C (maximum 46 6C). The membrane lipids consisted of C20 alkyl 1,2-diols with the dominant fatty acids being 10MeC18 and C18 : 1v9. The peptidoglycan lacked meso-DAP but contained ornithine and lysine. The dominant lipoquinone was MK-8. Phylogenetic analyses of the 16s rRNA gene sequence placed strain LbT into the class Thermomicrobia of the phylum Chloroflexi with Sphaerobacter thermophilus as the closest relative. On the basis of physiological and phylogenetic data, it is proposed that strain LbT represents a novel species of a new genus, with the suggested name Nitrolancea hollandica gen. nov., sp. nov. The type strain of the type species is LbT (5DSM 23161T5UNIQEM U798T). Lithoautotrophic nitrite-oxidizing bacteria (NOB) are which was dominant in a lab-scale nitrifying bioreactor, important players in the global nitrogen cycle, closing the with an unusual phylogenetic position within the phylum oxidative branch. So far, NOB have been found in three Chloroflexi (Sorokin et al., 2012). This was also the first bacterial phyla: Proteobacteria (alpha, beta, and gamma), nitrifying prokaryote with a Gram-positive cell wall and is Nitrospirae (Spieck & Bock, 2005) and Nitrospinae (Lu¨cker described here as a new genus and species. et al., 2013). Recently we have isolated an NOB strain, Strain LbT was isolated from a nitrifying bioreactor with a high loading of ammonium bicarbonate as the only Abbreviations: DAP, diaminopimelic acid; NOB, nitrite-oxidizing bacteria. substrate, which was oxidized to nitrate via nitrite by a The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene consortium of aggregated Nitrosomonas and an unknown sequence of strain LbT is JQ345500. NOB (Vejmelkova et al., 2012). The reactor was inoculated A supplementary figure is available with the online version of this paper. with the sludge of a full-scale nitritation bioreactor operating Downloaded from www.microbiologyresearch.org by 062232 G 2014 IUMS Printed in Great Britain 1859 IP: 157.193.10.221 On: Mon, 08 Aug 2016 12:07:44 D. Y. Sorokin and others in Rotterdam-Sluisjesdijk WWTP. The isolation mineral general, similar to the cells grown chemolithoautotrophi- 21 21 medium contained 0.2 g l KH2PO4 and 50 mg l CaCl2 cally with nitrite except that many mixotrophic cells sterilized in closed bottles with 10 % liquid and the gas phase contained small mesosome-like membrane vesicles in the consisting of air/CO2 (90 :10 , v/v). After sterilization the polar regions (Fig. S1, available in the online Supplementary medium was supplemented with 1 ml l21 trace metal Material). solution (Pfennig & Lippert 1966), 0.2 g l21 MgSO ,0.4g 4 Structural characterization included membrane lipid l21 filter-sterilized NH HCO and 5–50 mM filter-sterilized 4 3 and cell-wall peptidoglycan analyses. The polar lipids were KNO . For mixotrophic growth, 40 mM sodium formate T 2 extracted from cell material of strain Lb by Bligh-Dyer was added. The pH was maintained at 6.9–7.4 by adding extraction and analysed further by liquid chromatography- CO2 into the gas phase. The bottles were incubated on a mass spectrometry as described previously (Kulichevskaya rotary shaker at 100–120 r.p.m. at 37 uC. Solid medium was et al., 2012). The predominant membrane lipids were prepared by 1 : 1 mixing of double-strength liquid medium uncommon and comprised C20 alkyl 1,2-diols with a polar and 4 % (v/v) washed Noble agar at 50 uC. The plates were head group (sugar or phosphorus-sugar moiety) attached incubated in closed jars containing air/CO2/N2 (50 : 10 : 40, to the first OH group, whereas the second OH group was by vol.) in the gas phase. Growth was followed by measure- predominantly esterified with 12 methyl-octadecanoic acid ments of OD600, cell protein (Lowry et al., 1951), nitrite (12Me-C17 : 0). Similar glycolipids have only been identified consumption (Griess-Romijn van Eck, 1966) and nitrate in thermophilic members of the phylum Chloroflexi including formation (Bhandari & Simlat, 1986). Formate consump- Thermomicrobium roseum,arelativeofNitrolancea (see tion was analysed by HPLC anionic chromatography below) and Roseiflexus castenholzii, and in members of the (column Aminex HPx-87H, 30067.8 mm, t560 uC, flow 21 genus Thermus (Pond et al., 1986; Pond & Langworthy, 1987; rate 0.6 ml min , eluent 1.5 mM H3PO4; detector – RI). van ver Meer et al., 2002; Wait et al., 1997). Phase-contrast microphotographs were obtained using a Respiratory lipoquinones were extracted with cold acetone Zeiss Axioplan Imaging 2 microscope (Go¨ttingen). For from cells disrupted by grinding in liquid N2 and further total cell electron microscopy, cells were fixed with glu- separated by thin-layer chromatography (TLC). The excised taraldehyde (final concentration 3 %, v/v) for 2 h at 4 uC, bands were analysed by tandem mass spectrometry (LCQ washed with 10 mM potassium-phosphate buffer, pH 7, ADVANTAGE MAX) and the compounds were identified and positively contrasted with 1 % (w/v) neutralized phos- from their ionized masses. The dominant quinone species photungstic acid. For thin sectioning, the cells were fixed in was identified as MK-8. 1 % (w/v) OsO4 for 24 h at room temperature, washed, stained overnight with 1 % (w/v) uranyl acetate, dehy- For peptidoglycan analysis, the cell-wall fraction was drated in an increasing ethanol series, and embedded in obtained and analysed according to Streshinskaya et al. Epon resin. Thin sections were stained with 1 % (w/v) lead (1979). A cell suspension in 1 % (w/v) SDS was disrupted citrate. by sonication and heated briefly (100 uC, 5 min). Cell walls were separated by fractional centrifugation and purified T Strain Lb was isolated on mineral medium with nitrite as using trichloroacetic acid and trypsin. Quantitative deter- the only energy source after optimization of the growth mination of cell-wall amino acids was performed with a LC medium by supplementation with ammonium bicarbonate 600 E amino acid analyser (Biotronic) after acid hydrolysis (5 mM) as an additional N-source. Consistently, later (6 M HCl, 100 uC, 18 h). Isomers of diaminopimelic acid genome analysis (Sorokin et al., 2012) indicated that the (DAP) were determined by TLC on cellulose (modified organism, in contrast to all known NOBs, lacked the method of Hasegawa et al., 1983). The results demonstrated potential to utilize N-oxides as an N-source. The isolation the absence of meso-DAP in the murein, a diagnostic di- approach included several rounds of serial dilutions amino acid characteristic for all Gram-negative bacteria. in liquid medium followed by colony isolation from Instead, the di-amino acids ornithine and lysine were solid medium. The final purified culture was checked for present, along with D-glutamic acid and alanine, which heterotrophic contaminants using the same medium is characteristic of Gram-positive bacteria (Schleifer & without nitrite and supplemented with 200 mg l21 yeast Kandler, 1972). This result, taken together with the extract. ultrastructural organization, classical Gram-staining results, The isolate had a highly characteristic cell morphology that and the absence of genes coding for lipopolysaccharide was different from all known NOB species (Fig. 1a–c). synthesis (as indicated by genomic information; Sorokin et al., 2012) indicate that the novel NOB, in contrast to all Gram-staining and thin sectioning revealed a Gram-stain- nitrifiers known so far, is a Gram-positive bacterium. positive type cell wall (Fig. 1d, e), which makes strain LbT unique among the functional group of NOB. The Growth was observed under autotrophic conditions with intracytoplasmic membranes (ICM), typical for the genera nitrite as the electron donor. Strain LbT tolerated up to Nitrobacter and Nitrococcus, were clearly absent from strain 75 mM nitrite, which was stoichiometrically oxidized LbT. Carboxysome-like structures were not observed under to nitrate (Fig. 2). None of the organic compounds tested the growth conditions used. The ultrastructure of cells (yeast extract, pyruvate, organic acids, sugars, alcohols) grown mixotrophically with formate and nitrite was, in stimulated growth in the presence or absence of nitrite. The Downloaded from www.microbiologyresearch.org by 1860 International Journal of Systematic and Evolutionary Microbiology 64 IP: 157.193.10.221 On: Mon, 08 Aug 2016 12:07:44 Nitrolancea hollandica gen.