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

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.

Isoprene Oxidation by the Gram-Negative Model bacterium Variovorax sp. WS11

“…Microorganisms 2020, 8, 349 7 of 18 was one of two Gram-negative isoprene-degrading bacteria isolated from this environment, together with Ramlibacter sp. WS9 [30], and was shown by 16S rRNA gene sequencing to be a novel species of Variovorax. Phylogenetic analysis of the 16S rRNA gene of Variovorax sp.…”

“…tBLASTn analysis was conducted using the translated iso amino acid sequences of Ramlibacter sp. WS9, a second Gram-negative isoprene degrader isolated from willow soil [30]. Alignment of amino acid sequences of isoA of known isoprene-degrading bacteria with reference oxygenase alpha subunits of other SDIMO reveals an interesting evolutionary relationship (Figure 2).…”

“…The alpha-oxygenase component of isoprene monooxygenase, isoA (see below), has been identified as an excellent target for molecular probes when investigating the molecular ecology of isoprene degraders [26,27]. Actinobacteria are among the most commonly identified isoprene degraders, and members of the α, β, and γ-proteobacteria and the Bacteroidetes have also been identified [27][28][29][30]. Rhodococcus is often observed during studies of isoprene-degrading communities.…”

“…Freshwater sediment enrichments contained approximately 50% relative abundance of isoA sequences related to Rhodococcus, while soil from beneath poplar, oil palm, and willow trees contained greater relative abundance of isoA relating to Variovorax spp., a member of the family Comamonadaceae (Betaproteobacteria) [26][27][28]30]. A recent study of willow soil reported that Comamonadaceae were enriched at 25 ppmv isoprene, a lower concentration than has previously been used in enrichment of isoprene degraders [30]. Members of the Comamonadaceae have also been identified following isoprene enrichment in soils from willow trees (Salix spp.)…”

“…Members of the Comamonadaceae have also been identified following isoprene enrichment in soils from willow trees (Salix spp.) and a tyre dump, and also from the phyllosphere of poplar trees using DNA-stable isotope probing (DNA-SIP) methods, indicating that members of this family may be significant in microbial cycling of isoprene [26][27][28]30].…”