Novel bacterial chloromethane degraders of a living tree fern evidenced by 13 C- 1 chloromethane incubations 2

(181 words) Background : Chloromethane (CH 3 Cl) is the most abundant chlorinated volatile organic 29 compound in the atmosphere and contributes to stratospheric ozone depletion. CH 3 Cl has 30 mainly natural sources such as emissions from vegetation. In particular, ferns have been 31 recognized as strong emitters. Mitigation of CH 3 Cl to the atmosphere by methylotrophic 32 bacteria, a global sink for this compound, is likely underestimated and remains poorly 33 characterized. Results and Conclusions : We investigated chloromethane-degrading taxa associated with 35 intact and living tree fern plants of the species Cyathea australis by stable isotope probing 36 (SIP) with 13 C-labelled CH 3 Cl combined with metagenomic DNA sequencing. Metagenome 37 assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified 38 as being involved in the degradation of CH 3 Cl in the phyllosphere, i.e., the aerial parts of the 39 tree fern, while a MAG related to Sorangium was linked to CH 3 Cl degradation in the fern 40 rhizosphere. The only known metabolic pathway for CH 3 Cl degradation, via a 41 methyltransferase system including the gene cmuA , was not detected in metagenomes or 42 MAGs identified by SIP. Hence, a yet uncharacterised methylotrophic cmuA -independent 43 pathway likely drives CH 3 Cl degradation in the investigated tree ferns. 44 45 47 48 49 50 51 52 53

labelled CH3Cl (Campro Scientific GmbH, Berlin, Germany; named hereafter [ 13 C]-CH3Cl) or 126 with CH 3 Cl at natural abundance (99%, Linde GmbH, Pullach, Germany; named hereafter 127 [ 12 C]-CH3Cl). Each labelling period lasted for about 6 hours during daylight, with plant growth 128 chambers opened and ventilated overnight until the next round of labelling took place at the 129 next day. At the start of the labelling experiment (August 17 th ), 20 mL CH3Cl ([ 13 C]-CH3Cl or 130 [ 12 C]-CH3Cl) was added to the chambers, so that the initial mixing ration in the chamber was 131 ~200 ppm. Chambers were closed and ventilation was turned on. For the following 18 days, 132 10 mL of CH 3 Cl ([ 13 C]-CH 3 Cl or [ 12 C]-CH 3 Cl) was added every day, providing an initial mixing 133 ration in the chambers of ~100 ppm. Gas samples were taken at the start of the incubation 134 period, after 2 and 4 hours and at the end of the incubation period with gas-tight syringes and 135 stored in 3 mL, pre-evacuated Exetainers (Labco Limited, England) for further analysis by gas 136 chromatography combined with single quadrupole mass spectrometry (GC MS). 13 C-labelling 137 [ 13 C]-CH 3 Cl and [ 12 C]-CH 3 Cl was carried out for 19 days. Soil humidity and salinity in the fern 138 soil was monitored throughout the experiment (data not shown).

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Analysis of carbon mass fraction (C %) and differential stable carbon isotope ratio (δ 13 C value) 151 From each pot, an aliquot of the plant biomass was sampled at the start of the experiment, 152 after 4 days and at the end of the incubation period (after 19 days). Samples were dried for 153 24h at 60°C and finely ground using a vibrating disc mill (RS200, Retsch, Germany). Stable 154 isotope ratios ( 13 C/ 12 C) were determined using an Elemental Analyzer (EA) Flash 2000 HT

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Stable carbon isotope ratios ( 13 C) are expressed in ( ) relative to the international 158 standard, as defined by the equation: where RSample is the isotopic ratio ( 13 C/ 12 C) of the sample and RReference is the known isotopic

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DNA extraction was carried out using the FastDNA TM Spin Kit for Soil (MP Bio Science Ltd., 181 D , K) . 13 C-labelled heavy DNA was separated 182 from unlabelled light 12 C-DNA using cesium chloride density gradient ultracentrifugation, as 183 described previously [53]. Density gradient 12 (250 L ) 184 confirmed by measuring refractive indexes using a digital refractometer (Reichert AR2000).

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To identify

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corresponding fractions were used for 16S rRNA amplicon sequencing.

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Methylobacterium was identified as the most abundant 13 C-labelled genus in the phyllosphere

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In the fern phyllosphere (leaf and leaf wash samples), other 13 C-labelled microorganisms than

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Pseudomonadaceae were detected in a CH3Cl-degrading mixed culture

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Compared to the phyllosphere, different taxa were enriched in the 13 C-DNA 336 rhizosphere samples (Figure 1), e.g. from Xanthobacteraceae and Myxococcales, but a 337 potential association of these families with degradation of CH3Cl has not yet been reported.

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Xanthobacteraceae were enriched in a CH 3 Cl-incubated forest soil in a previous study [

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In many methylotrophs, methylene-H4F is the entry point for carbon assimilation through the

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Dehalogenases can hydrolyse a broad range of haloalkanes to e.g. the corresponding 431 alcohols, and as a rule this will accompanied by the release of protons and halide ions.

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Searches using the BRENDA database led to the detection of putative genes encoding 433 halogenases (3) and dehalogenases (40)           OTUs are classified as 'labelled' by the following criteria: 1) 13 C-heavy > 12 C-heavy; 2) 13 C-heavy > 13 C-light; 3) 13 C-[heavy-light] > 12 C-[heavy-light]; 4) 13 C[Heavy-Light] > threshold, with threshold =0.005%. The taxonomic affiliation of each labeled OTU is indicated at the family, order and genus levels (4-6, respectively), with a specific color and capital letter (see legend box). Multiple sequence alignments of concatenated marker genes were constructed using PhyloPhlAn 3.0. Phylogenetic analysis was performed based on a matrix of pairwise distances estimated using the LG+F+G+I model with 500 bootstrap replications. Numbers at branch nodes refer to bootstrap values. (A) Phylogenetic tree of Methylobacterium species. Two metagenomic bins isolated from the LEAF fraction were classified as Methylobacterium (marked in pink and purple) and were placed within 19 selected reference genomes of Alphaproteobacteria.     Figure S1. Plant growth chamber setup. A) Details of gas-tight plant chamber. Gas-tight plant chambers were constructed with acryl glass (thickness: 5 mm). Butyl rubber stoppers at the top and the lower part of the chamber were used as ports for injecting CH 3 Cl and extracting gas samples. A small ventilator in the lower part of the chamber ensured an even distribution of the gases in the chamber. B) Gas-tight plant chambers with fern plants inside at the end of the incubation period. Aluminium foil was wrapped around the chambers during incubation with CH 3 Cl to reduce photosynthesis and microbial CO 2 consumption during the labelling experiment. Sunlight entered the chambers through the clear top of the chamber. Supplementary Figure S2. Monitoring of the SIP-incubation of ferns. Incubations were carried out over 19 days with three biological replicated intact fern plants in gas-tight incubation chambers per tested condition. At the start of the SIP-incubation, 20 mL CH3Cl was added to the chambers (initial mixing ratio of ~ 200ppm) and degradation was monitored with GC-MS. At the end of each day, incubation chambers were opened and ventilated overnight. From day 2 of the incubation, 10 mL CH3Cl was added to the chambers (initial mixing ratio of ~ 100ppm). (A) 13 C-labelled CH 3 Cl, and (B) 12 C-labelled CH 3 Cl. Red arrows indicate point of CH 3 Cl addition.

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Supplementary Figure S3. Analysis of carbon mass fraction (C %) and differential stable carbon isotope ratio (δ 13 C value) of fern leaves and washed leaf samples (T end washed) via EA/IRMS. Stable isotope ratios (δ 13 C) are expressed in permil (‰) relative to the international standard.