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Novel Isoprene-Degrading Proteobacteria From Soil and Leaves Identified by Cultivation and Metagenomics Analysis of Stable Isotope Probing Experiments - PubMed

  • ️Tue Jan 01 2019

Novel Isoprene-Degrading Proteobacteria From Soil and Leaves Identified by Cultivation and Metagenomics Analysis of Stable Isotope Probing Experiments

Nasmille L Larke-Mejía et al. Front Microbiol. 2019.

Abstract

Isoprene is a climate-active gas and one of the most abundant biogenic volatile organic compounds (BVOC) released into the atmosphere. In the terrestrial environment, plants are the primary producers of isoprene, releasing between 500 and 750 million tons per year to protect themselves from environmental stresses such as direct radiation, heat, and reactive oxygen species. While many studies have explored isoprene production, relatively little is known about consumption of isoprene by microbes and the most well-characterized isoprene degrader is a Rhodococcus strain isolated from freshwater sediment. In order to identify a wider range of bacterial isoprene-degraders in the environment, DNA stable isotope probing (DNA-SIP) with 13C-labeled isoprene was used to identify active isoprene degraders associated with soil in the vicinity of a willow tree. Retrieval by PCR of 16S rRNA genes from the 13C-labeled DNA revealed an active isoprene-degrading bacterial community dominated by Proteobacteria, together with a minor portion of Actinobacteria, mainly of the genus Rhodococcus. Metagenome sequencing of 13C-labeled DNA from SIP experiments enabled analysis of genes encoding key enzymes of isoprene metabolism from novel isoprene degraders. Informed by these DNA-SIP experiments and working with leaves and soil from the vicinity of tree species known to produce high amounts of isoprene, four novel isoprene-degrading strains of the genera Nocardioides, Ramlibacter, Variovorax and Sphingopyxis, along with strains of Rhodococcus and Gordonia, genera that are known to contain isoprene-degrading strains, were isolated. The use of lower concentrations of isoprene during enrichment experiments has revealed active Gram-negative isoprene-degrading bacteria associated with isoprene-emitting trees. Analysis of isoprene-degradation genes from these new isolates provided a more robust phylogenetic framework for analysis of isoA, encoding the α-subunit of the isoprene monooxygenase, a key molecular marker gene for cultivation-independent studies on isoprene degradation in the terrestrial environment.

Keywords: DNA-SIP; isoA; isoprene degradation; isoprene monooxygenase; metagenomics.

Copyright © 2019 Larke-Mejía, Crombie, Pratscher, McGenity and Murrell.

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Figures

FIGURE 1
FIGURE 1

Relative abundance of 16S rRNA genes at the family level in DNA isolated after DNA-SIP enrichments. The relative abundance of bacterial 16S rRNA genes retrieved by PCR from un-enriched (unfractionated) DNA, extracted after sampling, is shown on the left. Subsequent bars show the relative abundance of 16S rRNA genes retrieved from DNA-SIP experiments with isoprene-enriched willow soil samples after 6 and 7 days of enrichment with 12C- and 13C-labeled isoprene. DNA arising from willow soil samples enriched with 12C-isoprene and 13C-isoprene are designated as light (L) and heavy (H) fractions respectively (refer to Supplementary Figures S5, S6). Taxonomic affiliation of 16S rRNA genes is reported at the family level. Only 16S rRNA gene sequences with a relative abundance of greater than 1% are shown. 16S rRNA gene sequences with a relative abundance of less than 1% are grouped together as “others.” Families (Comamonadaceae, Burkholderiales unclassified, and Nocardiaceae) identified in these DNA-SIP experiments as putative isoprene-degrading bacteria according to 13C-labeling, are highlighted with a black border.

FIGURE 2
FIGURE 2

Phylogenetic analysis of isoA sequences obtained during this study. Trees were constructed with the neighbor-joining method. The analysis was carried out with 28 complete isoA sequences from extant isoprene-degraders, new isoprene-degrading isolates obtained in this study (filled circles, red for isolates from the willow soil DNA-SIP) and 11 complete isoA metagenome sequences [green triangles, wsMG (from willow soil SIP-metagenome); blue open triangles, plMG (from poplar leaf metagenome in blue from Crombie et al., 2018)]. Following removal of gaps and missing data, there were 1,428 bp in the alignment. Bin numbers are included following the isoA-containing contig identifications. Names of neighboring isoprene metabolic genes in the same contig are included in parentheses. Bootstrap values [1000 replications (Satola et al., 2013)] over 75% are shown as circles in the nodes. The scale bar indicates nucleotide substitutions per site.

FIGURE 3
FIGURE 3

Isoprene metabolic gene clusters. Isoprene metabolic cluster genes identified in isoprene-degrading isolates (bold) and representative iso gene-containing contigs obtained from metagenome co-assembly of 13C-DNA from isoprene-enriched willow soil (wsMG). The isoA gene that encodes for the α-subunit of the monooxygenase is shown in red along with the isoA taxonomic affiliation as determined by phylogenetic analysis shown in Figure 2. The % identity of iso gene-encoded polypeptides to the corresponding Iso polypeptides of the well-characterized Rhodococcus sp. AD45 are shown in Supplementary Table S2.

FIGURE 4
FIGURE 4

16S rRNA gene phylogenetic tree of representative members of the Comamonadaceae family and isolated strains. Eighteen 16S rRNA gene sequences were included in the Neighbor-joining analysis. Following removal of gaps and missing data, there were 1392 bp in the alignment. Bootstrap values [1000 replications (Satola et al., 2013)] over 75% are shown as circles in the nodes. Strains isolated in this study are shown with a red diamond. The scale bar shows nucleotide substitutions per site.

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