Supplementary Figures
30.0
25.0
20.0
15.0
10.0
5.0 Isoprene concentration in the headspace (ppmv) 0.0 0 20406080100120140160
-5.0 Time (h)
OPL washings OPL swabs
Figure S1. Isoprene consumption in microcosms from oil palm leaf (OPL) washings and swabs.
Leaf microcosms were enriched and replenished with 12C-isoprene several times. Data points show the mean of triplicates and error bars show the standard deviation.
A
35.0
T1 T2 T3 30.0
25.0
20.0
15.0
10.0
5.0 Isoprene concentration in the headspace (ppm) headspace the in concentration Isoprene 0.0
-5.0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 Time (h) 12C-isoprene 13C-labelled isoprene
B
70.0
T1 60.0
50.0
40.0
30.0
20.0
10.0 Isoprene concentration in the headspace (ppmv) 0.0
-10.0 0 20 40 60 80 100 120 140 160 180 200 220 Time (h) 12C-isoprene 13C-labelled isoprene
Figure S2. Isoprene consumption in microcosms used in DNA-SIP experiments with oil palm soil (A) and leaf washings (B).
Soil (A) and leaf washings (B) microcosms were enriched and replenished with 12C- or 13C-labelled isoprene. Data points show the mean of triplicates and error bars show the standard deviation. B) Leaf washings enrichment are shown up to 220 h (T1) because of variation observed in the isoprene consumption between replicates. A
12 12 C-isoprene T1 12 C-isoprene T3 L C-isoprene T2 40 35 45 L L
35 30 40
35 30 25
30 25 20 25 20
15 20 15
15 DNA concentration(ng uL-1) DNA concentrationDNA uL-1) (ng
10 uL-1) DNA (ng concentration 10 10 5 5 5
0 0 0 1.397 1.399 1.401 1.403 1.405 1.407 1.398 1.399 1.4 1.401 1.402 1.403 1.404 1.405 1.406 1.407 1.398 1.4 1.402 1.404 1.406 Refractive Index Refractive Index Refractive Index 1 2 3 1 2 3 1 2 3
13 13 13 C-isoprene T3 C-isoprene T1 C-isoprene T2 L 45 L 45 30 L 40 40
25 35 35
30 30 20
25 25
15 20 20
15 15
10 uL-1) (ng concentration DNA DNA concentration uL-1) DNA (ng DNAconcentrationuL-1) (ng H 10 H 10 5 H 5 5
0 0 0 1.398 1.4 1.402 1.404 1.406 1.397 1.399 1.401 1.403 1.405 1.407 1.395 1.397 1.399 1.401 1.403 1.405 1.407 Refractive Index Refractive Index Refractive Index 1 2 3 1 2 3 1 2 3 B
Figure S3. Separation 12C and 13C DNA of oil palm soil (A) and oil palm leaf washings (B) throughout the DNA-SIP experiments.
DNA concentration and density of CsCl fractions obtained after ultracentrifugation of extracted DNA from enriched soil (A) and leaf washings (B) microcosms after enrichment with 12C-isoprene (top panel) and 13C-labelled isoprene (bottom panel) for each replicate (1, 2 and 3) throughout the DNA-SIP
13 12 experiments (T1, T2 and T3, refer to Table S1). The CsCl fractions chosen to represent C-labelled (heavy fraction) and unlabelled C-DNA (light fraction) are indicated: H arrow: heavy DNA; L arrow: light DNA.
Figure S4. Relative abundance of bacterial orders during DNA-SIP enrichments Relative abundance of bacterial 16S rRNA genes (at the order phylogenetic level) obtained by PCR of enriched and un-enriched DNA extracted from oil-palm soil and leaf-washing samples. Results include an un-enriched (un-fractionated) sample and pooled heavy DNA fractions (Figure S3) for three time points T1,
T2 for one replicate (shown as T1.1, T2.1) and T3 for all three replicates (shown as T3.1, T3.2 and T3.3; refer to Table S1 for the time course). Soil samples, enriched towards the left, and leaf washings, enriched towards the right. Only 16S rRNA gene sequences with a relative abundance of greater than 2% are shown. 16S rRNA gene sequences with a relative abundance of less than 2% are grouped together as “others”. Representatives from the order Actinomycetales and
Sphingomonadales, highlighted with black border, were isolated from the DNA-SIP enrichments. 96 AJ344450.1 Gordonia polyisoprenivorans strain VH2
98 KY649295.1 Gordonia polyisoprenivorans strain i37
36 NR 026500.1 Gordonia polyisoprenivorans strain Kd2
NR 024639.1 Gordonia rhizosphera NBRC 16068 50 52 NR 074529.1 Gordonia bronchialis DSM 43247
36 NR 037030.1 Gordonia aichiensis strain E9028
100 NR 037031.1 Gordonia sputi strain 3884 55 NR 043330.1 Gordonia rubripertincta strain DSM 43248
RKME01 Gordonia strain OPL2
95 NR 118891.1 Gordonia terrae strain DSM 43249
NR 118597.1 Gordonia hydrophobica strain DSM 44015
NR 026297.1 Gordonia hirsuta DSM 44140 NBRC 16056
55 NR 118623.1 Gordonia amarae strain DSM 43392
0.0050
Figure S5. 16S rRNA gene phylogenetic tree of representative members of the Gordonia genus and the oil palm leaf isolate Gordonia strain OPL2.
Maximum Likelihood tree includes thirteen 16S rRNA gene sequences, twelve representatives from the
Gordonia genus and Gordonia strain OPL2. Following removal of gaps and missing data, there were
1,337 bp in the alignment. Bootstrap values (1000 replications) are shown. Confirmed isoprene- degrading isolates from the Gordonia genus are indicated with black diamonds, including Gordonia strain OPL2 isolated in this study. The scale bar shows nucleotide substitutions per site.
56 RKMD01 Sphingopyxis strain OPL5
42 CP019449.1 Sphingopyxis strain QXT-31
75 AB245353.1 Sphingopyxis panaciterrae Gsoil 124
NR 024631.1 Sphingopyxis chilensis strain S37 47 64 NR 137237.1 Sphingopyxis fribergensis strain Kp5.2
97 AY563034.1 Sphingopyxis granuli strain Kw07
71 NR 041366.1 Sphingopyxis ginsengisoli strain Gsoil250 17 NR 044973.1 Sphingopyxis alaskensis strain RB2256
HQ436492.1 Sphingopyxis rigui strain 01SU5-P 16 AY554010.1 Sphingopyxis flavimaris strain SW-151 52
65 100 DQ781321.2 Sphingopyxis litoris strain FR1093
NR 025248.1 Novosphingobium pentaromativorans US6-1 98 NR 043014.1 Sphingopyxis baekryungensis strain SW-150
NR 025838.1 Novosphingobium capsulatum strain GIFU 11526
99 NR 042934.1 Novosphingobium taihuense strain T3-B9
NR 026249.1 Sphingobium chlorophenolicum strain ATCC 33790
NR 044902.1 Sphingomonas haloaromaticamans strain A175
NR 152709.1 Sphingomonas naphthae strain DKC-5-1 95 100 EF467848.1 Sphingomonas polyaromaticivorans strain B2-7
80 NR 133865.1 Sphingomonas oligoaromativorans strain IMER-A1-28
68 AY453855.1 Sphingomonas phyllosphaerae NR 024700.1 Sphingomonas echinoides strain ATCC 14820 70
27 NR 024998.1 Sphingomonas koreensis strain JSS-26
0.020
Figure S6. 16S rRNA gene phylogenetic tree of representative members of the
Sphingomonadaceae family and the oil palm leaf isolate Sphingopyxis sp. OPL5
Maximum likelihood tree includes twenty-two sequences, twenty-one type strain representatives from
the Sphingomonadaceae family and Sphingopyxis strain OPL5. Following removal of gaps and
missing data, there were 1,370 bp in the alignment. Bootstrap percentages (1000 replications) are shown at the nodes. The strain isolated in this study is indicated with a black diamond. The scale bar shows nucleotide substitutions per site.
10
3 2
1
1
0.1 log scale (Optical Density at 540 nm) at Density (Optical scale log
0.01 0 20 40 60 80 100 120 140 160 180 200 Time (h)
Figure S7. Growth of Sphingopyxis sp. OPL5 in a 4 litre fermenter with isoprene.
Sphingopyxis OPL5 cells were grown in minimal medium and supplied with isoprene vapour in air.
Arrows indicate the three harvesting times.
12
10 )
-1 8
6 (nmol min mg 0
V 4
2
0 0 5 10 15 20 25 30 35 μ [S0] ( M)
Figure S8. Rate of oxygen uptake (V0) as a function of isoprene concentration [S0] in solution for
Sphingopyxis strain OPL5 cells, determined using a Clark-type oxygen electrode [12].
Table S1 Incorporation of 13C-labelled carbon during DNA-SIP experiments with oil palm soil and leaf washings
13C isoprene spiked into the Sample Time course Time (h) headspace (μmol g-1) *
OPS SIP Native soil (T0) 0 0
OPS SIP Time point 1 (T1) 31.2 142
OPS SIP Time point 2 (T2) 53.1 166
OPS SIP Time point 3 (T3) 85.9 190
OPL SIP Native leaf washing (T0) 0 0
OPL SIP Time point 1 (T1) ~25 214-226^
OPL SIP Time point 2 (T2) ~50 274-285.5^
OPL SIP Time point 3 (T3) ~75 302-311.5^
*Consumption of isoprene in DNA-SIP microcosms was monitored by gas chromatography.
Incorporation of carbon was estimated assuming that 50% isoprene consumed was assimilated.
^There was variation in the consumption times and spiking of fresh isoprene for the leaf-washing replicates. Table S2. Identity of Iso polypeptides encoded by iso gene clusters when compared to the corresponding Iso polypeptides from Rhodococcus sp. strain AD45
Isoprene-degrading isolates are in bold. wsMG designation indicates an iso gene-containing contig retrieved from willow soil metagenomes [2]. Percentage identities (% amino acid identity) are with respect to the Iso polypeptides of the most well-characterised isoprene-degrader Rhodococcus sp. AD45 [3], except for AldH1 from Gordonia i37 which was chosen because of its position in the cluster.
100 99-90 89-80 79-70 69-60 59-50 49-35 34-0
Isolate/contig Rhodococcus sp. AD45 IsoG IsoH IsoI IsoJ - IsoA IsoB IsoC IsoD IsoE IsoF Gordonia sp. i37 79 79 83 71 AldH1 86 57 78 74 71 60 Gordonia sp. OPL2 78 81 79 68 84 87 66 81 73 68 61 Variovorax sp. WS11 60 48 48 55 50 73 38 40 58 53 41 Sphingopyxis sp. OPL5 61 61 45 58 50 72 44 54 50 53 44 wsMG4 (Sphingopyxis-like) - - 57 49 72 44 50 56 51 42
References
1. Klindworth, A.; Pruesse, E.; Schweer, T.; Peplies, J.; Quast, C.; Horn, M.; Glöckner, F.O. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 2013, 41, 1–11, doi:10.1093/nar/gks808. 2. Larke-Mejía, N.L.; Crombie, A.T.; Pratscher, J.; McGenity, T.J.; Murrell, J.C. Novel isoprene-degrading Proteobacteria from soil and leaves identified by cultivation and metagenomics analysis of stable isotope probing experiments. Front. Microbiol. 2019, 10, doi:10.3389/fmicb.2019.02700. 3. Crombie, A.T.; Khawand, M. El; Rhodius, V.A.; Fengler, K.A.; Miller, M.C.; Whited, G.M.; Mcgenity, T.J.; Murrell, J.C. Regulation of plasmid-encoded isoprene metabolism in Rhodococcus, a representative of an important link in the global isoprene cycle. Environ. Microbiol. 2015, 17, 3314–3329, doi:10.1111/1462- 2920.12793.