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Methylosoma difficile gen. nov., sp. nov., a novel methanotroph enriched by gradient cultivation from littoral sediment of Lake Constance

LS Mikrobielle Ökologie, Fachbereich Biologie, Universität Konstanz, Fach M 654, 78457 Konstanz, Germany

Correspondence
Bernhard Schink
bernhard.schink{at}uni-konstanz.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
A novel methanotroph, strain LC 2^T, was isolated from the littoral sediment of Lake Constance by enrichment in opposing gradients of methane and oxygen, followed by traditional isolation methods. Strain LC 2^T grows on methane or methanol as its sole carbon and energy source. It is a Gram-negative, non-motile, pale-pink-coloured methanotroph showing typical intracytoplasmic membranes arranged in stacks. Cells are coccoid, elliptical or rod-shaped and occur often in pairs. Strain LC 2^T grows at low oxygen concentrations and in counter-gradients of methane and oxygen. It can grow on medium free of bound nitrogen, possesses the nifH gene and fixes atmospheric nitrogen at low oxygen pressure. It grows at neutral pH and at temperatures between 10 and 30 °C. Phylogenetically, it is most closely related to the genus Methylobacter, with the type strains of Methylobacter tundripaludum and Methylobacter psychrophilus showing 94 and 93.4 % 16S rRNA gene sequence similarity, respectively. Furthermore, the pmoA gene sequence of strain LC 2^T is most closely related to pmoA gene sequences of Methylobacter strains (92 % similar to Methylobacter sp. LW 12 by deduced amino acid sequence identity). The DNA G+C content is 49.9 mol% and the major cellular fatty acid is 16 : 17c (60 %). Strain LC 2^T (=JCM 14076^T=DSM 18750^T) is described as the type strain of a novel species within a new genus, Methylosoma difficile gen. nov., sp. nov.

The GenBank/EMBL/DDBJ accession numbers for the complete 16S rRNA gene sequence and partial pmoA and nifH gene sequences of strain LC 2^T are respectively DQ119050, DQ119047 and DQ665842.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Aerobic methane-oxidizing bacteria (MOB) or methanotrophs are a unique and important group of bacteria which act as natural filters, controlling the release of methane, an important greenhouse gas, from anoxic sediments and soils. Aerobic MOB in freshwater environments such as lake sediments are active at the zone where methane and oxygen meet. In a previous study from our lab, a specific cultivation set-up was developed in which methanotrophic bacteria were grown in opposing gradients of methane and oxygen (Bussmann et al., 2006). A novel bacterial strain, LC 2^T, was isolated which was found to be moderately related to the Methylobacter–Methylosarcina group (92–94 % similarity) and less related to the other genera of MOB (similarity 90–91 %), e.g. Methylomonas and Methylomicrobium, as determined by 16S rRNA gene sequence comparison. The closest relatives of this new strain were Methylobacter species such as Methylobacter psychrophilus (Tourova et al., 1999) and the recently described Methylobacter tundripaludum SV96^T (Wartiainen et al., 2006), which are either psychrophilic or have been isolated from cold habitats. In culture-independent studies, we found that littoral sediment of Lake Constance is dominated by type I MOB (Bussmann et al., 2006; Pester et al., 2004). Among these, Methylobacter-like MOB (clone group B1) dominated clone libraries of the partial pmoA gene (Bussmann et al., 2006) amplified with primers A189f–A682r (Holmes et al., 1995) as well as with primers A189f–mb661r (Costello & Lidstrom, 1999) and contributed to the major peaks observed in T-RFLP studies from the littoral sediment of Lake Constance (Pester et al., 2004). Recently, many pmoA and 16S rRNA gene sequences related to the genus Methylobacter have been reported as dominant groups in other freshwater habitats such as freshwater wetland marshes (Bodelier et al., 2005) and lakes such as Mono Lake (Lin et al., 2005) and Lake Washington (Nercessian et al., 2005), estuarine habitats (McDonald et al., 2005), as well as in chironomid larvae in lake sediments (Eller et al., 2005). To date, a total of 13 genera of methanotrophs have been described, which belong to either the class Alphaproteobacteria (type II MOB) or Gammaproteobacteria (type I MOB). Recently, it was discovered that Crenothrix polyspora Cohn, a well-known, uncultured filamentous bacterium, is a methane oxidizer, and its 16S rRNA genes (total of four clusters) formed a new group within the family Methylococcaceae which also branched near Methylobacter psychrophilus and related methanotrophs (Stoecker et al., 2006).

The purpose of the present study is to characterize strain LC 2^T formally and to determine its correct taxonomical position. Although this strain was isolated from a gradient culture, it could also grow on solid and in liquid media.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Isolation and growth conditions.
Strain LC 2^T was isolated from littoral sediment of Lake Constance after an initial enrichment in a gradient culture followed by transfer into a liquid dilution series and finally on solid agarose medium, as described in detail by Bussmann et al. (2006). Cells were grown in dilute mineral medium (Bussmann et al., 2006) either in liquid medium, on solid medium (with 1.2 % agarose NEEO, Ultraquality; Roth) with 0.01 % cycloheximide or in a gradient system (0.2 % agarose) with opposing gradients of methane and oxygen as described before (Bussmann et al., 2006). Growth on 1.5 % agar (BD Biosciences) was also checked. The diluted medium as described in Bussmann et al. (2004) contained the following salts (per litre): 0.1 g NaCl, 0.04 g MgCl₂.6H₂O, 0.05 g KCl, 0.015 g CaCl₂.2H₂O, 0.016 g Na₂SO₄ and trace element solution SL 10 (1 ml l^–1) (Widdel, 1988). Potassium/sodium phosphate buffer (pH 7.2) and KNO₃ were added to final concentrations of 150 µM and 50 µM, respectively, and the medium was buffered to pH 7.2 with 0.01 M HEPES. In addition, a seven-vitamin solution (Widdel & Pfennig, 1981) was added (1 ml l^–1). Strain LC 2^T was checked also for growth in: (i) classical nitrate mineral salt medium (Whittenbury et al., 1970) with the trace element solution replaced by SL 10 solution, (ii) low-nitrate mineral salt medium (1/10 concentration of potassium nitrate, pH 7.0 with HEPES buffer) and (iii) medium with 10-fold-concentrated version of the above-mentioned mineral medium (Bussmann et al., 2004). The effect of copper on growth was checked by inoculating the culture with additional 1–5 µM CuSO₄ in liquid medium. The basal concentration of copper in the mineral medium was very low (0.011 µM). Strain LC 2^T was grown in 100 ml flasks or 15 ml glass tubes with 25–30 or 5 ml medium, respectively, in closed desiccators. The strain was maintained by streaking single colonies on solid agarose plates or by inoculating a single colony in liquid medium and then streaking the grown liquid culture on agarose plates. Until about a year after isolation, strain LC 2^T was grown in closed desiccators under a gas atmosphere of 24 % methane, 2 % CO₂, 17 % O₂ and the balance N₂ (Bussmann et al., 2006). As the strain grew well in gradients and appeared to grow at low oxygen tension (Bussmann et al., 2006), the gas atmosphere for growing this culture was modified slightly to 20 % methane, 2 % CO₂, 30 % air (around 6 % O₂) and the balance N₂. All growth experiments performed in this study were performed with this modified gas atmosphere in the dark at 16 or 21 °C, unless otherwise mentioned. The strain was also tested for growth in closed 150 ml bottles with different volumes of liquid medium and a gas phase of methane and air (20 : 80). Possible presence of heterotrophic contaminants was checked every time by plating on 1 : 10-diluted nutrient agar plates with an additional 0.05 % yeast extract. For long-term storage, glycerol stocks were prepared and preserved at –70 °C. Survival of the strain after such preservation was checked by inoculation into fresh medium. Cultures were also preserved at 4 °C for long-term storage. Ten per cent inoculum was used in all growth experiments and the incubation period was 8 weeks. Since the bacteria tended to grow as a faint pink biofilm at the bottom, the tube or flask was shaken well to suspend the cells uniformly before the OD₅₇₀ was recorded. Growth was always confirmed by phase-contrast microscopy.

Morphological characterization and electron microscopy.
Cells were observed under a phase-contrast microscope (Axiophot; Zeiss) and photographed with a cooled charge-couple device camera (Magnafire; INTAS). For fixation for electron microscopy, 50 ml of an exponentially growing culture was spun down at 5000 g. The cell pellet was washed once with PBS (50 mM potassium phosphate buffer, pH 7.5, plus 0.9 % NaCl) and then suspended in 1 ml PBS. Glutaraldehyde (140 µl of a 25 % aqueous solution) was added to the cell suspension, which was then incubated overnight at 4 °C, before the cells were centrifuged again at 5000 g. The pellet was washed again in PBS and finally suspended in around 150 µl PBS. After chemical fixation, cells were embedded in 1.5 % (w/v) molten agar (final concentration). The agar block was cut into small pieces of 1 mm³ and the pieces were dehydrated in a graded methanol series (v/v: 15 and 30 % for 15 min; 50, 75 and 95 % for 30 min; 100 % for 1 h) under concomitant temperature reduction to –40 °C. The samples were infiltrated with Lowicryl K4M resin [in methanol (v/v): 50 % for 1 h; 66 % for 2 h; 100 % for 10 h] and then polymerized for 24 h at –40 °C and for 3 days at room temperature (Roth et al., 1981; Hoppert & Holzenburg, 1998). Resin sections of 80–100 nm thickness were cut with glass knives. Electron microscopy was performed in a Philips EM 301 transmission electron microscope at 80 kV with calibrated magnifications. To check for the presence of cellular appendages like flagella or pili, negative stainings were prepared with fresh cell material.

Utilizable carbon and nitrogen sources.
Utilization of various carbon sources was studied in liquid mineral medium supplemented with one of the following filter-sterilized substrates (0.1 %, w/v): formate, formamide, arabinose, raffinose, lactose, maltose, xylose, glucose, fructose and sucrose. The ability of the strain to grow on methanol and formaldehyde was tested at lower concentrations (10–50 mM methanol and 1–50 mM formaldehyde). Other substrates such as acetate (2 and 10 mM) were tested for growth in liquid medium to check for heterotrophic growth (Dedysh et al., 2005) of strain LC 2^T. Nitrogen sources were tested with liquid medium in which KNO₃ was replaced by one of the following compounds at 0.05 % (w/v): NH₄Cl, urea, glycine, serine, valine, asparagine, aspartate, L-glutamic acid, glutamate, peptone and yeast extract. To test the ability of the culture to fix atmospheric N₂, media free of bound nitrogen compounds were used. The acetylene reduction test was done with cultures grown with 10 mM methanol without any bound nitrogen source, as modified by Auman et al. (2001). Briefly, 25 ml of such a grown culture was transferred to a bottle with a rubber stopper and was gassed with nitrogen; air and acetylene were added (89 % N₂: 9 % air: 1 % acetylene) and incubated overnight. The gas phase was checked for ethylene production by gas chromatography.

Optimum pH, temperature and salt content.
The optimum pH and temperature ranges were determined in liquid medium. Growth at pH 2.6–9.0 was checked after buffering the medium with citrate/phosphate buffer (pH 2.6–6.6), HEPES buffer (pH 7 and 7.5) and glycine buffer (pH 8 and 9). Growth was also checked without using any buffer, but using only HCl or NaOH to adjust the pH. Strain LC 2^T was grown at a temperature range of 4–37 °C in liquid medium. To determine the optimum salt concentration, additional NaCl (0.5, 1, 1.5 and 2.0 %, w/v) was added to the mineral medium. The basal NaCl concentration in the mineral medium was 0.01 % (w/v).

Resistance to desiccation and heat.
Heat resistance was tested by heating cell suspensions at 50, 60, 70 or 80 °C for 10 min each followed by plating onto solid medium and incubating under optimal conditions for 2–3 weeks. Desiccation resistance was assessed according to Whittenbury et al. (1970) by air-drying suspensions of strain LC 2^T on glass slides and then inoculating into medium after an interval of 1–4 weeks. Formation of exospores was checked for by heating a 3–4 week old culture at 80 °C for 20 min and then looking for colony formation after incubation under standard conditions (Bowman et al., 1993). Cysts were stained according to Vela & Wyss (1964).

Cellular fatty acid analysis.
Phospholipid fatty acid analyses were performed at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). Strain LC 2^T was grown in 200 ml flasks, in closed desiccators. Cells were pelleted, freeze-dried and sent for phospholipid fatty acid analysis. There, the cells were saponified and methylated and the methyl esters were extracted and subjected to GC. The GC elution profile of the fatty acid methyl esters was compared with the fatty acid patterns stored in the fatty acid database of the Microbial Identification System (MIDI Inc.) and qualitative and quantitative compositions of the pattern were given.

Presence of soluble methane monooxygenase (sMMO) and nitrogenase.
The presence of sMMO was checked by PCR amplification of the mmoX gene with primers mmoXA–mmoXB (Auman et al., 2000) as well as by colorimetric assay (Graham et al., 1992). To check for the presence of nitrogenase, the nifH gene was amplified as described by Poly et al. (2001), partially sequenced and subjected to BLAST search at the NCBI (http://www.ncbi.nlm.nih.gov/) (Altschul et al., 1990).

DNA extraction, phylogenetic analysis and G+C content.
DNA was extracted and the complete 16S rRNA gene sequence was determined as described previously (Bussmann et al., 2006). 16S rRNA gene sequences of type I methanotrophs of the family Methylococcaceae along with newly described sequences of Crenothrix polyspora and sequences of some clones were obtained after BLAST search. Phylogenetic analysis was done using the ARB software package (version 2.5b) (http://www.arb-home.de; Ludwig et al., 2004). The new sequences were added to the ARB database and aligned using the FAST aligner tool as implemented in ARB. Alignments were checked and corrected manually where necessary. Sequences of 1419 nucleotides were used for alignment. Only those positions that were identical in at least 50 % of all sequences were used to create a filter. Phylogenetic analysis was done using the maximum-likelihood, neighbour-joining and maximum-parsimony algorithms as implemented in ARB (Ludwig et al., 2004). Phylogenetic distances were also determined by using the similarity matrix in ARB without using any filter and also with a sequence of Escherichia coli as the filter. Phylogenetic analysis of the pmoA gene was done as described earlier (Bussmann et al., 2006), and phylogenetic trees were constructed based on 164 amino acids. G+C content was measured at the DSMZ by HPLC by a method adapted from Tamaoka & Komagata (1984) and calculated according to the method of Mesbah et al. (1989).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Isolation and growth characters of strain LC 2^T
Strain LC 2^T was isolated after enrichment in a gradient culture system obtained after a final dilution of 8x10^–4 of the littoral sediment, after further transfers on liquid and solid media (Bussmann et al., 2006). The strain was isolated from a mixed culture in which it was associated with thin rods. When isolated, the colonies were much bigger and more mucoid and watery than after extended cultivation. This could be due to a tendency of the cells to form aggregates or to the presence of an initially contaminating bacterium. The strain grew in liquid culture at low oxygen tensions, up to 10 % air, i.e. 2 % oxygen. In gradients, it formed two bands, the deepest one being 32 mm from the air-exposed end (Bussmann et al., 2006). Strain LC 2^T also grew well on 1.5 % agar, although agarose was used routinely. The strain could not be maintained on solid medium by repeated transfers. Therefore, a single colony was always grown in liquid culture and then streaked on solid medium. No active motility could be detected 2 years after isolation. Cells occurred singly or in pairs or sometimes in chains of four or five cells (especially on solid medium) or in aggregates. In liquid medium, strain LC 2^T formed a mucoid pink biofilm at the bottom of the flask. If the flask was shaken, a whorl of mucoid biofilm appeared which was held together at a small point in the centre of the flask. After shaking, the cells could be suspended uniformly. Cells also became oval or elliptical in shape and changed from coccoid to rod-shaped forms in older cultures. Liquid cultures grew better in magnetically stirred flasks inside a desiccator. Strain LC 2^T resembled Methylobacter tundripaludum SV96^T in colony colour and cell size (Table 1). Comparison of strain LC 2^T with members of other methanotrophic genera is shown in Table 1.

Morphological characterization and electron microscopy
Strain LC 2^T formed pale-pink colonies, around 1–2 mm diameter on agarose plates and 2–3 mm diameter on agar, after incubation for 2–3 weeks (Table 1). Coccoid cells were observed (often in pairs) by phase-contrast microscopy which were 1.5–2 µm in length and approximately 1 µm in diameter (Fig. 1a). Intracytoplasmic membranes were arranged in stacks, mainly at the cell periphery (Fig. 1b), which is a feature typical of type I methanotrophs (Whittenbury et al., 1970). Cells contained large polyhydroxyalkanoate granules and, very probably, also glycogen granules, which stained dark.

View larger version (54K): [in this window] [in a new window]   Fig. 1. Morphology of strain LC 2T. (a) Phase-contrast micrograph of cells grown in liquid medium with methane. (b) Ultrathin sections showing membrane stacks (MS), polyhydroxyalkanoate (P) granules and glycogen (G) granules. (c) Ultrathin section of the cell periphery with the typical appearance of a Gram-negative cell envelope: two dark, thin layers of the outer membrane (OM), one dark, thick layer of peptidoglycan located in the periplasm (PP) and two dark layers of the cytoplasmic membrane (CM).

Utilizable carbon and nitrogen sources
Strain LC 2^T grew only on methane or methanol (10–50 mM). No other carbon substrates were utilized. Of the different nitrogen sources checked, it utilized nitrate, L-glutamine, L-glutamic acid, L-asparagine and L-aspartic acid. Growth on organic nitrogen sources such as peptone and yeast extract was better and faster than growth on nitrate. Strain LC 2^T grew without any bound nitrogen source under standard gas conditions. The acetylene reduction test was positive, although the ethylene peak was very small and the reaction required overnight incubation.

Effect of pH, temperature and NaCl concentration on growth
Strain LC 2^T grew at pH 5–9 when no additional buffers were used, with the best growth at pH 6–8. When the medium was buffered, growth was observed only around neutral pH, which might be due to the sensitivity of the strain to high concentrations of organic compounds. Strain LC 2^T grew in a temperature range of 16–30 °C, the optimum growth temperature being around 25 °C. The specific growth rates at 16, 25 and 30 °C were 0.0024, 0.0041 and 0.0065 h^–1. Although growth was fast at 30 °C, growth declined after reaching an OD₅₇₀ of around 0.15, which might be due to an imbalance of its metabolism. Little growth was observed at 10 °C, and no growth was observed at 37 or 4 °C. NaCl added to the medium to concentrations of 0.5–2 % inhibited growth.

Resistance to desiccation and heat and formation of exospores
Strain LC 2^T grew after a heat shock at 50 and 60 °C for 10 min, but did not grow after a heat shock at 70 °C. It formed neither microcolonies after exposure to 80 °C nor exospores. Even though cysts were frequently observed in older cultures, they were not resistant to desiccation for 1 week.

Cellular fatty acid analysis
Strain LC 2^T showed a pattern of fatty acids unique compared with representatives of the related type I methanotrophic genera Methylobacter, Methylosarcina, Methylomicrobium and Methylomonas, although the patterns of the genus Methylobacter were the closest (Table 2). Fatty acid patterns of Crenothrix polyspora were not available and thus could not be compared. The major fatty acid was 16 : 17c. Strain LC 2^T also contained 12 : 0 fatty acids, which have been observed only in the genus Methylosarcina. The proportions of 16 : 17c, 14 : 0 and 16 : 0 were comparable to those of members of the neighbouring genus Methylobacter. Strain LC 2^T contained 15 % 16 : 16c, which was similar to the values found in the genera Methylomicrobium and Methylomonas. There was no 16 : 15c or 5t fatty acid, as seen in almost all other genera except Methylosarcina, which has a very low percentage of 16 : 15c. In addition, strain LC 2^T also showed minor amounts of 15 : 0, 16 : 111c and 16 : 0 3-OH fatty acids, which have not been found so far in other type I methanotrophs.

Phylogenetic analysis and G+C content
The complete 16S rRNA gene sequence and partial pmoA gene sequence of strain LC 2^T were determined in previous studies; the 16S rRNA gene sequence was confirmed again after 1.5 years of cultivation, by cloning, screening of 20 clones by RFLP as described before (Bussmann et al., 2006) and sequencing of two clones. The sequences were 99.7 % identical to the deposited sequence, with only two or three bases different, confirming the sequence and the stability of the culture under the present growth conditions. In the phylogenetic tree, the position of strain LC 2^T was between the group of Methylobacter psychrophilus-like bacteria and C. polyspora (Stoecker et al., 2006) (Fig. 2). According to similarity values calculated using the ARB program (with or without E. coli as a filter), the closest cultured relatives are Methylobacter tundripaludum SV96^T (94 % similarity) and Methylobacter psychrophilus Z-0021^T (93.4 %). Different gene clusters of C. polyspora and other Methylobacter species showed similarity of 92–93 %. BLAST results revealed that the pmoA nucleotide sequence of strain LC 2^T differed by 14 % from the pmoA sequences of Methylobacter sp. strain LW12 and Methylobacter sp. strain BB5.1 and by more than 15 % from the other described species of Methylobacter. The amino acid similarities were 92 % to Methylobacter sp. LW 12 and Methylomicrobium buryatense 5B^T and 93 % to clone B67 and clone A55 from Lake Constance littoral sediment (Bussmann et al., 2006). The unique position of the strain LC 2^T sequence in the pmoA tree is shown in Fig. 3.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Maximum-likelihood tree of the 16S rRNA gene sequence of strain LC 2T in comparison with other methanotrophic cultured members and clones. Strain numbers are indicated for species and GenBank accession numbers are given in parentheses. Bar, 10 % sequence divergence.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Neighbour-joining tree based on the derived amino acid sequences of the pmoA gene of strain LC 2T and from other cultured and uncultured methanotrophs. Strain numbers are indicated for species and GenBank accession numbers are given in parentheses. Bar, 10 % sequence divergence.

Description of Methylosoma gen. nov.
Methylosoma [Me.thy.lo.so'ma. N.Gr. prefix methylo referring to methyl groups; Gr. neut. n. soma body; N.L. neut. n. Methylosoma a methyl group (-utilizing) body].

Obligately aerobic, C₁-compound-utilizing bacteria. Cells possess a typical membrane system with stacks of intracytoplasmic membranes, typical of type I methanotrophs. Gram-negative. Phylogenetic analysis and morphological characters place the genus close to the genus Methylobacter. The type and only known species is Methylosoma difficile.

Description of Methylosoma difficile sp. nov.
Methylosoma difficile (dif.fi.ci'le. L. neut. adj. difficile difficult, referring to difficulties in cultivating the type strain).

Large, coccoid cells, 1.5–2 µm in length and approximately 1 µm in diameter, often occurring in pairs, chains or aggregates; sometimes also slightly elongated rod-shaped forms. Non-motile; division by binary fission, forming cysts which are not resistant to heat or desiccation. Cells are pale-pink-pigmented and do not possess sMMO. Microaerobic, growing best at low (2 %) oxygen tensions. Uses methane or methanol (10–50 mM) as the sole carbon source and nitrate, L-glutamine, L-glutamic acid, L-asparagine or L-aspartic acid as the nitrogen source. Fixes atmospheric nitrogen; nifH gene present. Grows best at 16–30 °C and at neutral pH. Does not grow with enhanced concentrations of NaCl or copper. The major fatty acid is 16 : 17c, followed by 16 : 16c, 14 : 0 and 16 : 0. G+C content of the DNA of the type strain is 49.9 mol% (HPLC determination). Habitat: sediments of freshwater lakes, at the interface of oxic and methane-supplied sediment layers.

The type strain, LC 2^T (=DSM 18750^T=JCM 14076^T), was isolated from littoral sediment of Lake Constance in May 2004.

	ACKNOWLEDGEMENTS

 
This study was supported by Sonderforschungsbereich (SFB) 454 on the Deutsche Forschungsgemeinschaft, Bonn, and research funds of the Universität Konstanz. We wish to thank especially Dr Michael Hoppert, University of Göttingen, for performing the electron microscopic characterization.

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