Identification and characterisation of isoprene-degrading bacteria in an estuarine environment

Abstract

Approximately one-third of volatile organic compounds (VOCs) emitted to the atmosphere consists of isoprene, originating from the terrestrial and marine biosphere, with a profound effect on atmospheric chemistry. However, isoprene provides an abundant and largely unexplored source of carbon and energy for microbes. The potential for isoprene degradation in marine and estuarine samples from the Colne Estuary, UK, was investigated using DNA-Stable Isotope Probing (DNA-SIP). Analysis at two timepoints showed the development of communities dominated by Actinobacteria including members of the genera Mycobacterium, Rhodococcus, Microbacterium and Gordonia. Representative isolates, capable of growth on isoprene as sole carbon and energy source, were obtained from marine and estuarine locations, and isoprene-degrading strains of Gordonia and Mycobacterium were characterised physiologically and their genomes were sequenced. Genes predicted to be required for isoprene metabolism, including four-component isoprene monooxygenases (IsoMO), were identified and compared with previously characterised examples. Transcriptional and activity assays of strains growing on isoprene or alternative carbon sources showed that growth on isoprene is an inducible trait requiring a specific IsoMO. This study is the first to identify active isoprene degraders in estuarine and marine environments using DNA-SIP and to characterise marine isoprene-degrading bacteria at the physiological and molecular level.

Figure 1

Bacterial community composition of DNA‐SIP isoprene enrichments. The unenriched timepoint zero community is shown together with the labelled communities at timepoints one and two (12 and 15 days), retrieved from the heavy fractions of ¹³C‐isoprene enrichments. The data show the mean of two replicates, except timepoint zero (one sample). All genera present with a relative abundance > 1% at any timepoint are shown.

Figure 2

A. Phylogeny, based on 16S rRNA gene sequences, of known isoprene‐degraders (shown in bold), together with closely related non‐isoprene‐degrading strains. The tree was constructed in MEGA6 (Tamura et al., 2013) using the Neighbour‐joining method. All positions containing gaps and missing data were eliminated, and there were a total of 535 positions in the final dataset. B. Phylogenetic relationship of known isoprene‐degrading strains based on isoA sequences, together with alkene monooxygenase (xamoA) from Xanthobacter autotrophicus Py2 (Zhou et al., 1999). The tree was drawn in MEGA6 using the Maximum Likelihood method based on an alignment of isoA sequences. All positions containing gaps and missing data were eliminated and there were a total of 1010 nt in the final dataset. Scale bars indicate nucleotide substitutions per site. Bootstrap values (1000 replications) are shown at the nodes.

Figure 3

A. Arrangement of the isoprene gene clusters in Rhodococcus sp. AD45 (Crombie et al., 2015), Gordonia sp. i37 and Mycobacterium sp. AT1. B. The propane monooxygenase and associated genes in Gordonia sp. TY5, Gordonia sp. i37 and Mycobacterium sp. AT1. Open reading frames are coloured according to their homologues in Rhodococcus sp. AD45 (A) or Gordonia sp. TY5 (B). Hypothetical proteins and those with no close homologues adjacent to the gene clusters in Rhodococcus sp. AD45 or Gordonia sp. TY5 are shown in white. Regulatory genes are in black. CoA‐DSR, CoA disulfide reductase; Ph‐CoA‐DO, phytanoyl‐CoA dioxygenase.

Figure 4

Transcription of isoA or prmA in cells grown on isoprene or propane. The data show gene transcript abundance in Gordonia sp. i37, relative to cells grown on glucose (= 1). Data points represent the mean ± SD, n = 3.