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Transcriptomics and lipidomics of the environmental strain Rhodococcus ruber point out consumption pathways and potential metabolic bottlenecks for polyethylene degradation Kevin GRAVOUIL, Romain FERRU-CLEMENT, Steven COLAS, Reynald HELYE, Linette Kadri, Ludivine BOURDEAU, Bouziane MOUMEN, Anne Mercier, and Thierry Ferreira Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00846 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017

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Environmental Science & Technology

1

Transcriptomics and Lipidomics of the Environmental Strain Rhodococcus ruber Point

2

out Consumption Pathways and Potential Metabolic Bottlenecks for Polyethylene

3

Degradation

4 5

Kévin Gravouil†, Romain Ferru-Clément§, Steven Colas†, Reynald Helye§, Linette Kadri†,

6

Ludivine Bourdeau†, Bouziane MoumenΓ, Anne Mercier†‡ and Thierry Ferreira†,§‡*

7 8 9 10 11 12 13 14 15 16 17 18 19

†

Cooperative laboratory ThanaplastSP-Carbios, Laboratory of Ecological and Biological Interactions, National Center for Scientific Research UMR 7267, University of Poitiers, Poitiers, France § Laboratory of Signalisation and Membrane Ionic Transports, National Center for Scientific Research STIM CNRS ERL 7368, University of Poitiers, Poitiers, France Γ Team Ecology, Evolution, Symbiosis, Laboratory of Ecological and Biological Interactions, National Center for Scientific Research UMR 7267, University of Poitiers, Poitiers, France

‡

These authors were equally involved in the management of this study.

20 21 22

ABSTRACT

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Polyethylene (PE), one of the most prominent synthetic polymer used worldwide, is very

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poorly biodegradable in the natural environment. Consequently, PE represents by itself more

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than half of all plastic wastes. PE biodegradation is achieved through the combination of

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abiotic and biotic processes. Several microorganisms have been shown to grow on the surface

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of PE materials, among which are the species of the Rhodococcus genus, suggesting a potent

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ability of these microorganisms to use, at least partly, PE as a potent carbon source. However,

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most of them, if not all, fail to induce a clear-cut degradation of PE samples, showing that

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bottlenecks to reach optimal biodegradation clearly exist. To identify the pathways involved

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in PE consumption, we used in the present study a combination of RNA-sequencing and

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lipidomic strategies. We show that short-term exposure to various forms of PE, displaying

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different molecular weight distributions and oxidation levels, lead to an increase in the 1 ACS Paragon Plus Environment


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expression of 158 genes in a Rhodococcus representative, R. ruber. Interestingly, one of the

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most up-regulated pathways is related to alkane degradation and β-oxidation of fatty acids.

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This approach also allowed us to identify metabolic limiting steps, which could be fruitfully

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targeted for optimized PE consumption by R. ruber.

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ABSTRACT ART

40 41 42 43 44 45 46

Keywords: plastic degradation, polyethylene (PE), Rhodococcus ruber, β-oxidation pathway,

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transcriptomics, lipidomics.

48 49

INTRODUCTION

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Polyethylene (PE) has become one of the most prominent synthetic polymers due to its

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excellent chemical and mechanical properties, its stability and durability as well as its low

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cost of production. Its worldwide annual production is around 80 million tons a year and the

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global demand is expected to rise 4.8% annually through 2024.1,2 The primary usage of this

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polyolefin is in the packaging industry, since it can be found as the main component of plastic

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bags, bottles and films. As a corollary to its intrinsic resistance, PE is generally considered as

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a chemically and biologically inert polymer, very poorly biodegradable if not submitted to

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previous chemical and/or physical treatments.3,4 PE thus tends to accumulate within the

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environment and it represents by itself 64.0% of all the plastic wastes. It has been estimated

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that hundreds of years would be required for a thin PE film to be degraded in the natural 2 ACS Paragon Plus Environment

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environment.5 PE degradation is a complex mechanism that requires two consecutive steps: i)

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an oxidation of the hydrocarbon chain to generate shorter aliphatic fragments and ii) a

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consecutive consumption of the generated fragments by specific microorganisms from the

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environment.6 Thermo- and photo-oxidization of PE (abiotic treatment) release fragments

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with a high chemical heterogeneity, displaying a wide-range of lengths and oxidation levels.

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Indeed, complex mixtures including alkanes, alkenes, ketones, aldehydes, alcohols,

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carboxylic acids, keto-acids, dicarboxylic acids, lactones and esters can be observed after PE

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oxidation steps.7 Certain additives, known as pro-oxidants, such as manganese (Mn)-, iron

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(Fe)- or cobalt (Co)-stearate facilitate the abiotic oxidation of the polymer and its subsequent

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fragmentation in the natural environment (oxo-biodegradable PE).8 However, the fate of the

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generated fragments, and specially their conversion to CO2 by microorganisms from the

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environment, remains a matter of debate. Indeed, if the average length of PE chains decreases

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drastically with the presence of pro-oxidants, to reach an average molecular weight (Mw)

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around 3,000 Da, most of the microorganisms do not seem to be able to consume aliphatic

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chains (i.e. alkanes) displaying a Mw over 600 Da.9-12 Therefore, only a minor fraction of the

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oxidized PE fragments is likely to be efficiently mineralized by environmental

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microorganisms.8

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Species of the Rhodococcus genus, which are known to thrive in a broad range of

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environments, including soil and water, have been associated to PE biodegradation in several

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studies.9,13,14 For example, Koutny and colleagues reported that, among twelve bacterial

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strains isolated from forest soils for their ability to adsorb and grow as a biofilm on the

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surface of oxidized PE films containing pro-oxidants, three appeared to be Rhodococcus

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strains.14 One species, assigned to R. ruber, also popped up in a screening using partially

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photolysed PE films as the sole carbon source to sustain growth on a minimal medium.13 This

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implies that R. ruber ought to be able to consume at least a fraction of the degradation

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products obtained from oxidized PE samples, but the molecular mechanisms of this

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consumption remain largely unknown.

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In this study, we used RNA sequencing (RNA-seq) combined with mass-spectrometry-based

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lipidomics to unravel the mechanisms of oxidized PE mineralization by the environmental

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strain R. ruber C208. In this aim, R. ruber C208 was grown on mineral salt media

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supplemented with various forms of PE, displaying different molecular weight distributions

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and oxidation levels. The data presented here point out the alkane degradation pathway as

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being a central node in the degradation of the PE fragments generated by abiotic oxidation.

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Moreover, the present study also highlights potential bottlenecks in the PE mineralization

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process, which could become preferential targets for optimizing PE degradation by

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environmental microorganisms.

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MATERIALS AND METHODS

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Polyethylene Samples and Characterization. Four polyethylene (PE) samples were firstly

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selected, here named PE4K, PE4K-OX, PEfi and PEfi-OX. PE4K is a commercial powder of

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PE (Sigma-Aldrich 427772) with an approximate theoretical average molecular weight (Mw)

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of 4,000 g/mol. PE4K-OX corresponds to samples of PE4K that have been thermo-oxidized

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during 14 days at 120°C. PEfi and PEfi-OX are PE films with a 23 µm thickness provided by

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the Barbier Group (Sainte Sigolène, France) and containing 80% of linear low-density PE

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(LLDPE) and 20% of LDPE. PEfi-OX is an oxo-degradable film which has been stored at

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room temperature for 10 years. This film contains a pro-oxidant additive (organometallic

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cobalt salt) and it was fragmented during storage. By contrast to PEfi-Ox, PEfi did not contain

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any pro-oxidant additives. Each PE sample was characterized in terms of its molecular weight

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distribution and oxidation level (see Supporting Information SI for details).

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Growth Conditions. Five conditions were performed using Erlenmeyer flasks containing 100

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ml of a mineral salt medium (MSM) inoculated with the actinomycete Rhodococcus ruber 4 ACS Paragon Plus Environment

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C208 (DSM 45332) and 0.1% of mannitol (Alfa Aesar), 0.6% of PE4K, 0.6% of PE4K-OX,

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0.6% of PEfi-OX or without any carbon source addition (for details, see SI1). Three flasks per

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condition were prepared for RNA and lipid extractions. Before inoculation, PE4K, PE4K-OX

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and PEfi-OX were incubated with 70% ethanol for 30 min, and rigorously washed using

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sterile Milli-Q water three times. Cultures were incubated at 28°C, 110 rpm.

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RNA Extraction and Sequencing (RNA-seq). After short-term (3-day) PE exposure, RNA

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was extracted using the RNeasy Mini Kit (Qiagen) following manufacturer’s instructions (for

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details, see SI1). Illumina MiSeq 2×100 bp paired-end libraries with multiplex adaptors were

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prepared with an internal PhiX control by Genoscreen platform (http://www.genoscreen.fr/,

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Institut Pasteur-Lille, France). A quality check on raw data was performed using FastQC

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(version 0.11.2) among all the replicates and for each replicate. To be considered as good, the

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median

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sequences (http://ow.ly/OqFVY).

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DNA Extraction, Genome Sequencing and Assembly. The genomic DNA from R. ruber

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was extracted using the Wizard® Genomic DNA Purification Kit (Promega) according to the

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manufacturer’s instructions (for details, see SI). The genome sequencing was performed using

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454-sequencing™ on a Genome Sequencer (GS) Junior™ system after preparation of a

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sequencing library using the GS Junior Titanium Rapid Library Preparation Kit (454 Life

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Science) according to the manufacturer’s instructions. Genomic and transcriptomic data are

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available on the European Nucleotide Archive under study accession number PRJEB19207

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(http://www.ebi.ac.uk/ena/data/view/PRJEB19207).

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A

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http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) on raw sequences extracted from

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SFF file using sff2fastq tool (https://github.com/indraniel/sff2fastq). To be considered as

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good, the median quality score (Phred score) must be over 20 for the first 400 bases. Raw

quality

primary

Phred

quality

score

check

must

was

be

done

over

30

using

at

any

FastQC

position

(version

in

the

0.11.2;



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genome sequences were assembled de novo using the Roche GS de novo Assembler (Newbler

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3.0).

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RNA-seq Data Analysis and de novo Assembly. De novo transcriptome assembly in

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Rockhopper (version 2.02) proceeds in two stages (see SI for details).15 After this de novo

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assembly, transcripts were mapped to the genome contigs obtained after in-house sequencing

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of the R. ruber C208 strain (see above) using rnaQUAST (v0.1.1).16 Unaligned transcripts

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were not considered in the Differential Gene Expression (DGE) analysis, described below.

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Rockhopper was used to count mRNA occurrences thus defining an expression profile for

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each replicate. Bray-Curtis distances were calculated between each pair of samples then

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visualized after a principal coordinate analysis (PCoA) using the R package vegan.17 This was

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used as an additional quality check to ensure reproducibility of the experiment and to

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visualize if the response to the medium is consensual.

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Differential Gene Expression (DGE) Analysis. Rockhopper was also used to quantify the

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number of transcripts, to normalize the data and for evaluating differential transcript

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expression of each pair of conditions. Normalization was performed using upper quartile

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normalization. Finally, DGE analysis was performed using the DESeq.18 P-values were

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corrected using the Benjamini-Hochberg method, then reported as q-value.

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The main question of the whole transcriptomic analysis performed in this study was the

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following: which genes are specifically involved in PE metabolization? We postulated that

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such genes should not be upregulated under optimal growth conditions (i.e. in the presence of

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a carbon source such as mannitol), nor under survival conditions (i.e. in mineral salt medium

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MSM). Therefore, replicates from MSM and mannitol conditions were merged into a single

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condition « MSM+mannitol », which was used as a standard to identify the genes displaying a

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dysregulated expression in R. ruber grown in the presence of the various PE samples. The

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transcripts, which were significantly over-expressed (q-value < 0.01), in at least one of the


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conditions of interest, were therefore considered as potential candidates for PE

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metabolization.

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Annotation of Over-Expressed Gene. Gene annotation was performed only on transcripts

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displaying a dysregulated expression in the presence of PE. These sequences were annotated

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using Argot2 and then Blast2GO (version 2.8).19-20 Following these annotation pipelines, up-

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regulated sequences were annotated with Gene Ontology (GO) terms, Enzyme Commission

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(EC) number and InterPro data, in the best case. EC were then mapped to known KEGG

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pathways using the « KEGG Mapper Search&Color Pathway » tool.21 Possible enzymatic

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reactions were highlighted, showing the most upregulated pathways. These pathways may

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contain some gaps in the reaction chain if one only considers the EC numbers. After manual

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review, gaps were filled up with putative enzymatic activities using GO annotations from both

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Argot2 and Blast2GO.

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Mass-Spectrometry-Based Lipidomics. Lipids were extracted by using a modified Folch

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method (for details, see SI).22 The MS analysis was performed on a Waters Synapt G2 HDMS

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with a nanoESI source operated in negative ionization mode (see SI for details). The analysis

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was performed using the Waters Masslynx v4.1 software.

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RESULTS

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Characterization of the PE Samples. The results from the Size Exclusion Chromatography

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(SEC) analyses for the four PE samples used in this study are displayed in Figure 1A. As

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shown, the oxidation treatments either obtained by heating (PE4K-OX) or by the action of

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pro-oxidants at room temperature (PEfi-OX) resulted in a dramatic reduction of the average

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molecular weights of the corresponding samples. The action of pro-oxidants shifted the

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weight average molecular weight (Mw) of the PE films from 94.1 kDa to 3.8 kDa for PEfi

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and PEfi-OX, respectively, and heat treatment induced a drop of PE4K Mw from 7.2 kDa to

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2.7 kDa for PE4K-OX. Interestingly, the molecular weight distribution of the PE4K-OX 7 ACS Paragon Plus Environment


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sample was very similar to the one of PEfi-OX. This shows that the “artificial” PE4K-OX

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samples recapitulated quite nicely the size distribution of the PE fragments, which are

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obtained under natural photo-oxidation of a commercial PE film containing pro-oxidants.

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The similarities between PE4K-OX and PEfi-OX were confirmed by the FTIR analyses

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(Figure 1B). As shown, among the four samples tested, PE4K-OX and PEfi-OX displayed the

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highest oxidation levels, and PE4K and PEfi appeared to be poorly oxidized, as reflected by

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the peak intensities at 1,715 cm, characteristic of ketone groups and the carbonyl index with

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values of 0.27, 2.50, 0.10 and 1.15 for PE4K, PE4K-OX, PEfi and PEfi-OX, respectively.

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From these results, R. ruber C208 was grown on mineral salt media (MSM) supplemented

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with the two oxidized forms of PE (PE4K-OX and PEfi-OX) and with PE4K. PEfi was not

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tested due to its very low oxidation level.

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Identification of Specific Pathways Induced in R. ruber under PE Supplementation. In

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order to get rid of misassembled transcripts from RNA Seq (see below), we first performed

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the full-genome sequencing of the R. ruber C208 strain. The quality check on raw data

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confirmed a very good quality for the sequencing process, with an overall median score over

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30 for the first 450 bases.

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The genome assembly process produced 181 long contigs (> 500 nt) from 154,147 sequences

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(ranging from 400 to 600 nucleotides), with an average contig size and a N50 contig size of

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31,309 and 65,423 nucleotides, respectively. The largest contig was 225,466 nucleotides long.

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The estimated total genome size was 5.67 Mb, which is consistent with the average size of

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known

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(http://www.ncbi.nlm.nih.gov/genome/?term=txid1827[Organism: exp]). A result of 99.7% of

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the bases used for the assembly had a Phred quality score of 40+. Finally, the coverage

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obtained was 14.4x, which is sufficient for our use of the data.

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The quality check on raw data obtained from RNA-Seq showed an acceptable level of quality

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with a median over 30 and a vast majority of 100nt-long sequences for the 14.6 x 106 paired-

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end sequences. The assembly process produced a total of 5,734 transcripts with an average

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length and a N50 of 405 and 423 nucleotides, respectively. The quality evaluation showed

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that 20 de novo assembled transcripts were not found in the reference genome, most of them

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being short sequences corresponding probably to chimeric assemblies. These 20 transcripts

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were therefore discarded.

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A principal coordinate analysis (PCoA) from a Bray-Curtis distance matrix was performed to

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examine the overall variation among the expression profiles (see Figure S1 and Table S1,

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Supporting Information). The three replicates corresponding to each condition were clustered

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closely, underscoring the reproducibility of the independent culture of R. ruber and the

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robustness of the molecular characterization of each transcriptome. The transcriptome from

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the five conditions were discriminated across the two first principal coordinates that explained

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76.1% of the variability, at the exception of the culture of R. ruber in MSM and in MSM

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supplemented with 0.6% of PE4K-OX that are clustered together.

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After DGE analysis performed by Rockhopper, 158 genes appeared to be significantly

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up-regulated in at least one of the PE conditions versus the reference «MSM+mannitol»

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condition (q-value < 0.01; Figure 2A, Tables S1 and S2). Fold-changes varied from 4.4 to

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55.4 among these up-regulated genes. Only 11 transcripts were commonly overexpressed in

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R. ruber grown in the presence of the three types of PE (Core transcriptome; Table S3),

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whereas 22 to 33 transcripts were specifically up-regulated in a given culture condition

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(Figure 2B, Table S2).

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The annotation process allowed the annotation of 132 transcripts (84% of the total up-

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regulated transcripts) by a combination of the Argot2 (140 transcripts annotated (89%)) and

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Blast2GO (110 transcripts annotated (70%)) software tools. In the end, only 26 transcripts

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(16%) remained without any additional information (Tables S2, S4 and S5).

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After annotation, 50 transcripts (37% of the total up-regulated transcripts) were assigned with

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an EC number. These enzyme codes were mapped to all KEGG pathways using the 9 ACS Paragon Plus Environment


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"KEGG Mapper – Search&Color Pathway" tool. Interestingly, one of the most up-regulated

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pathways was related to fatty acid degradation (KEGG reference pathway: map00071), which

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contains both the alkane degradation and β-oxidation pathways (Figure 3A and Table S6).

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However, several gaps could be observed within this catabolic cascade, probably because of

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the limited number of enzyme codes provided by the annotation process (see above).

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Therefore, these pathways were manually completed according to GO annotations. This

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operation allowed us to fill all the gaps within the pathways, supporting the idea that the

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alkane and fatty acid degradation are globally up-regulated in the presence of PE. Overall, a

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total of 39 transcripts could be directly assigned to alkane degradation and β-oxidation

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(Figure 3A). These data nicely fit previous observations showing that some alkane degrading

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strains, such as Rhodococcus species indeed display the property to grow on the presence of

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PE.8,23

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The β-oxidation pathway accomplishes the complete degradation of saturated fatty acids

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having an even number of carbon atoms. However, certain fatty acids require additional steps

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for degradation. For example, the degradation of odd-chain fatty acids yields propionyl-CoA,

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which needs to be further carboxylated by a propionyl-CoA carboxylase (EC: 6.4.1.3) to

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produce succinyl-CoA. Interestingly, a gene encoding a putative propionyl-CoA carboxylase

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(RRUB_S0078_04852) was up-regulated under all three PE-supplementation conditions

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(Table S2 and Figure 3B).

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A closer look to the RNA-Seq data revealed that oxidized forms of PE (PE4K-OX and PEfi-

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OX) were more efficient than non-oxidized PE (PE4K) in inducing the mineralization

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pathway via the β-oxidation pathway in R. ruber, at least in terms of the number of induced

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genes. As an example, incubation with PE4K-OX resulted in the up-regulation of 22

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candidates in the pathways described in Figure 3B, with at least one candidate for each

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reaction being successfully identified. By contrast, incubation with PE4K correlated with the

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induction of 12 candidates only, and 2 “gaps” could be noted within the pathway, 10 ACS Paragon Plus Environment

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corresponding to aldehyde dehydrogenase and L-hydroxyacyl-CoA dehydrogenase activities,

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respectively (Figure 3B). From these observations, it appears that short oxidized PE fragments

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are likely more potent inducers of this pathway than non-oxidized PE.

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Potential Bottlenecks for Optimal PE Consumption by R. ruber. Several additional steps

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ought to be considered in order to account for an efficient mineralization of high-molecular

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weight PE, via the β-oxidation pathway (Figure 3A, Table S2). First, one may consider that

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large PE fragments are not likely to be efficiently internalized by R. ruber. In this scenario,

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the secretion of extracellular oxidases, to reduce the average molecular mass of external PE,

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could be a good alternative (Step 1). Second, the existence of dedicated transport systems for

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short length-PE fragments could be hypothesized (Step 2). Finally, additional cytoplasmic

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oxidation steps to provide small PE fragments compatible with their entry in the alkane

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degradation process, could be involved (Step 3). According to these hypotheses, several

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bottlenecks to an optimal mineralization of oxidized PE fragments clearly emerge.

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Concerning step 1, secreted laccases (EC 1.10.3.2) have been reported to play a key role in PE

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biodegradation, probably by promoting biotic oxidation.24 We could identify three highly

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homologous sequences in the R. ruber transcriptome, related to laccases/multicopper oxidases

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(namely RRUB_S0078_04240, RRUB_S0078_03834 and RRUB_S0078_04261), but,

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interestingly, none of them appeared to be either up- or down-regulated in the presence of PE,

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whatever the form considered (Table S1).

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A second potential bottleneck in this model is PE internalization (Step 2). Indeed, only small

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PE fragments are likely to enter the cell, and due to their high dispersity in terms of size and

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oxidation levels, they probably require dedicated transport systems. Within the 19 putative

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transporters identified in this study to be up-regulated in at least one condition of PE

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supplementation, 10 of them belong to the major facilitator superfamily (MFS) and 4 belong

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to the ATP binding cassette (ABC) family (Table 1). Interestingly, one transcript was reported

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to potentially encode a protein with a transport function but also as a NADH dehydrogenase 11 ACS Paragon Plus Environment


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activity. This suggests that this protein could be involved in the coupling of both the oxidation

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and the transport of its substrate. Finally, 34 different transcripts encoding putative

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cytoplasmic oxidases were identified in this study. Some of them could well participate to

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further steps in intracellular fragmentation of oxidized PE.

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Redistributions within Phospholipid Species during Incubation of R. ruber with PE. In

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addition to the genes involved in alkane degradation, an interesting observation was also the

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up-regulation of genes involved in fatty acid elongation (KEGG reference pathway:

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map00062) and of a key enzyme of the glycerolipid metabolism (KEGG reference pathway:

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map00561), namely a diacylglycerol kinase (EC: 2.7.1.107; RRUB_S0078_05576), which

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catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA), a central

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intermediate of the phospholipid (PL) biosynthesis pathway (Table S2). Altogether, these

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observations suggested that PE fragments might also be, at least in part, incorporated within

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PLs. Relevant to this hypothesis, some bacteria have already been shown to efficiently

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incorporate and modify n-alkanes into the membrane fatty acid composition. As an example,

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it was demonstrated that the hydrocarbonoclastic bacterium Alcanivorax borkumensis SK2

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can efficiently incorporate exogenously-supplied odd-numbered fatty acids within its PLs.25

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To evaluate this hypothesis, PLs were extracted from R. ruber grown under the various

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conditions and further analyzed using mass spectrometry (Figure 4). Under our control

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growing conditions in MSM supplemented with mannitol, three main lipid species could be

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observed in R. ruber, namely Cardiolipids (CL), Phosphatidylethanolamine (PtdE) and

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Phosphatidyl inositol (PtdI) (Figure 4A). Such a phospholipid profile appears to be quite

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characteristic of the Rhodococcus genus.26 Tandem mass spectrometry analysis revealed that

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the most represented PtdI and PtdE subspecies, as defined by their fatty acyl content, were

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respectively PtdI 35:0 (a PtdI bearing both a 16:0 and a 19:0 acyl chains), PtdE 35:0 (16:0 /

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19:0) and PtdE 35:1 (16:1 / 19:0) (Figures 4B and S2). CL species, which bear 4 fatty acyl

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chains, displayed a less homogeneous profile, ranging from CL 67:4 to CL 70:0 (Figure S2), 12 ACS Paragon Plus Environment

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the most represented subspecies being CL 68:3 (16:1 / 18:1 / 18:1 / 16:0; Figure 4B). A first

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important observation was that no additional PL subspecies could be observed in R. ruber

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grown in the presence of PE, showing that short oxidized PE fragments failed to incorporate

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within PLs. Moreover, no significant redistribution between subspecies was observed within

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the various PL classes (Figure S2). Overall, the most prominent impact of PE samples on PLs

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was an accumulation of PtdI at the expenses of CL and PtdE, the effect being more

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pronounced with the oxidized samples PE, PE4K-OX and PEfi-OX (Figure 4A). Altogether,

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it appears from these experiments that oxidized PE fragments are not efficiently incorporated

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within PLs. However, the presence of PE chains, and most specifically of PE oxidized

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fragments, induce a redistribution of the PL pattern, with a significant increase of PtdI at the

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expense of the other species.

328 329

DISCUSSION

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In the present study, we have developed a transcriptomic approach to identify genes encoding

331

candidate enzymes involved in the consumption of PE by R. ruber (C208). In this aim, the

332

bacterial strain was grown on mineral salt media supplemented with various forms of PE,

333

displaying different molecular weight distributions and oxidation levels. Data from SEC and

334

FTIR analyses of the different PE samples studied confirmed that a clear correlation exists

335

between the size reduction of a PE sample and its oxidation level, an observation that has

336

already been made in previous studies9: the size reduction (Figure 1A) of PE4K after thermo-

337

oxidation (PE4K-OX) and of PEfi after pro-oxidant action (PEfi-OX) nicely correlated with

338

an increase in the oxidation rates of the corresponding samples (Figure 1B).

339

A first important conclusion of this work is that the PE samples used, including a non-

340

oxidized short chain PE (PE4K) and oxidized PE samples, resulting either from abiotic

341

degradation of a PE film by the action of pro-oxidants (PEfi-OX) or of a short chain PE

342

submitted to heat treatment (PE4K-OX), lead to a strong induction of the pathways related to 13 ACS Paragon Plus Environment


Page 14 of 27

343

alkane degradation and β-oxidation of fatty acids. This observation points out these pathways

344

as being central nodes for the consumption of PE by R. ruber. Alkanes have been shown to be

345

very efficient in inducing these pathways25,27 and, accordingly, several Rhodococcus species

346

were demonstrated to consume alkanes and specifically R. ruber.28-32 Therefore, a likely

347

explanation for the induction of these pathways is the presence in the samples of short

348

aliphatic fragments, which could be recognized as the natural substrates/inducers.

349

Accordingly, we could show that PE4K-OX and PEfi-OX samples, which contain shorter

350

fragments than PE4K due to a fragmentation of the PE chains by oxidation (Figure 1A), were

351

more efficient in inducing β-oxidation (Figure 3B).

352

If able to adsorb and grow on the surface of oxidized PE samples and likely able to consume

353

short PE fragments, Rhodococcus species appear to do little harm to oxidized PE films, since

354

the Mw and Mn of pro-oxidant-containing PE films remain unchanged after incubation with

355

R. rhodochrous even when cultured for several weeks with the samples after their prior

356

abiotic fragmentation.9 These observations therefore suggest that bottlenecks clearly subsist to

357

confer Rhodococcus species the ability to degrade high molecular weight PE. Among the

358

various limiting steps evoked in the present study, a further oxidation of the PE samples to

359

increase the amount of metabolizable short fragments (Figure 3A, Step 1) is probably a

360

prominent one. In this scenario, the use of laccases (EC 1.10.3.2), which have been reported

361

to catalyze the biotic oxidation of PE, could be a good alternative. Relevantly, Santo and

362

colleagues have reported the existence of a copper-binding laccase in R. ruber, which proved

363

to be able, when overproduced, to induce a reduction of the PE Mw and Mn of 20% and 15%,

364

respectively.24 We identified three close homologues to this candidate in our RNA library but,

365

interestingly, none of them was shown to be significantly up-regulated in the presence of the

366

PE samples, whatever their oxidation levels (Table S1). This is not a surprising observation,

367

since the expression of laccases is known to be preferentially induced by their substrates such

368

as lignin or, alternatively, by various aromatic compounds related to lignin or lignin14 ACS Paragon Plus Environment

Page 15 of 27


369

derivatives or by metals, such as Cu2+.33 In other words, if R. ruber appears to display most of

370

the machinery for PE degradation, an important bottleneck may well be the full-fledged

371

induction of the implicated pathways.

372

Finally, we could also show in the present study that incubation of R. ruber with PE induces a

373

redistribution of the phospholipid pattern, with an accumulation of PtdI at the expense of PtdE

374

and CL (Figure 4A). Interestingly and as already mentioned, incubation with the various PE

375

samples did not correlate with the apparition of new subspecies within PL, showing that PE

376

fragments are not efficiently channeled towards the phospholipid pathway. This suggests that,

377

at least under conditions where PE is the only carbon source provided in the medium, such

378

fragments preferentially serve as substrates for the β-oxidation pathway.

379

Some phospholipids such as PtdI or PtdC (PhosphatidylCholine) display an overall cylindrical

380

shape and tend to organize themselves into bilayers (referred to as lamellar lipids). By

381

contrast, other phospholipids such as PtdE and CL display conical shapes (Type II lipids; see

382

the schematic representations in Figure 4A), and tend to form nonlamellar phases with a

383

negative curvature, such as the hexagonal phase HII.34 In this context, the observed

384

accumulation of PtdI at the expense of hexagonal phase-promoting lipids PtdE and CL, is

385

likely to result in a stabilization of the R. ruber cytoplasmic membrane, by a better

386

compaction/higher packing of the phospholipid species within the bilayer leaflets (Murinova

387

and Dercova, 2014). One may assume that such a mechanism could constitute an efficient

388

adaptation process for R. ruber to counter the chaotropic effects of PE fragments on the

389

cytoplasmic membrane. Indeed, many organic pollutants, including alkanes, are able to

390

penetrate within the cytoplasmic membrane with, as a corollary, an increase of membrane

391

fluidity. Classical resistance mechanisms consist in a modulation of the fatty acyl content of

392

PL, i.e. incorporation of long chain saturates, cyclopropane and branched fatty acids and/or

393

cis to trans isomerization of unsaturated chains, all of them resulting in a an overall increased

394

packing of the PL species.35 Interestingly, none of these processes was observed in R. ruber 15 ACS Paragon Plus Environment


Page 16 of 27

395

when challenged with PE, therefore pointing out the modulation of the lamellar versus Type

396

II lipid ratio as a potent adaptation mechanism. Altogether, the present study highlights some

397

potential bottlenecks in the consumption of PE fragments by R. ruber. Overall, such

398

observations could account, at least in part, for the intrinsic resistance of PE to

399

biodegradation. Designing new strategies for circumventing these limiting steps will

400

undoutfully constitute a promising new challenge in order to reduce the detrimental impacts

401

of this widely used polymer on the environment.

402 403

ASSOCIATED CONTENT

404

Supporting Information

405

The Supporting Information is available free of charge on the ACS Publications website

406

Detailed description of SI files experimental procedure (PDF)

407

Figures S1−S2: Reproducibility of the transcriptomic profiles and percentage of the

408

various PtdI, PtdE and CL subspecies (PDF)

409 410

Tables S1−S6: Data analysis from RNA-seq (XLS)

411

AUTHOR INFORMATION

412

Corresponding Author

413

*Mailing address: Cooperative laboratory ThanaplastSP-Carbios, Laboratory of Ecological and

414

Biological Interactions, National Center for Scientific Research UMR 7267, University of

415

Poitiers, UFR SFA, Pôle Biologie Santé, 1 rue Georges Bonnet, bât B37, 86073 Poitiers,

416

cedex 9, France. E-mail address: [email protected]. Phone: +33 (5) 49 45 40

417

04. Fax: +33(5) 49 45 40 14 (T.F.).

418

ORCID

419

Anne Mercier: 0000-0002-6970-452X 665

420

Notes 16 ACS Paragon Plus Environment

Page 17 of 27

421


The authors declare no competing financial interest.

422 423

ACKNOWLEDGMENTS

424

The authors are sincerely thankful for the collaboration and support provided by Carbios

425

(Biopôle Clermont-Limagne) and the laboratory of Ecology and Biology of Interactions (EBI)

426

at the University of Poitiers. The authors are also sincerely grateful to Gérard Pichon (Barbier

427

Group, France) for providing the PEfi and PEfi-OX samples and for sharing his expertise

428

concerning the PE abiotic degradation processes.

429 430

REFERENCES

431

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pharmaceuticals. Second Edition Piringer O.G., and Baner A. L. (Eds). Wiley-VCH Verlag

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hydroxylase system in the metabolism. AMB Express 2014, 4, 73–81.

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the biodegradation of polyethylene by the actinomycete Rhodococcus ruber. Int. Biodeterior.

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(25) Naether, D. J.; Slawtschew, S.; Stasik, S.; Engel, M.; Olzog, M.; Wick, L. Y.; Timmis,

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K. N.; Heipieper, H. J. Adaptation of the hydrocarbonoclastic bacterium Alcanivorax

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borkumensis SK2 to alkanes and toxic organic compounds: a physiological and transcriptomic

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approach. App. Environ. Microbiol. 2013, 79 (14), 4282–4293. 19 ACS Paragon Plus Environment


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(26) De Carvalho, C. C.; Costa, S. S.; Fernandes, P.; Couto, I.; Viveiros, M. Membrane

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transport systems and the biodegradation potential and pathogenicity of genus Rhodococcus.

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(27) Sabirova, J. S.; Ferrer, M.; Regenhardt, D.; Timmis, K. N.; Golyshin, P. N. Proteomic

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insights into metabolic adaptations in Alcanivorax borkumensis induced by alkane utilization.

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(28) Cappelletti, M.; Fedi, S.; Frascari, D.; Ohtake, H.; Turner, R. J.; Zannoni, D. Analyses of

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both the alkB gene transcriptional start site and alkB promoter-inducing properties of

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Rhodococcus sp. strain BCP1 grown on n-Alkanes. Appl. Environ. Microbiol. 2011, 77 (5),

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1619–1627.

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(29) Sharma, S. L.; Pant, A. Biodegradation and conversion of alkanes and crude oil by a

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marine Rhodococcus. Biodegradation 2000, 11 (5), 289–294.

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(30) Whyte, L. G.; Hawari, J.; Zhou, E.; Bourbonnière, L.; Inniss, W. E.; Greer, C. W.

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Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic

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Rhodococcus sp. App. Environ. Microbiol. 1998, 64 (7), 2578–2584.

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(31) Amouric, A.; Verhé, F.; Auria, R.; Casalot, L. Study of a hexane-degrading consortium

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in a biofilter and in liquid culture: biodiversity, kinetics and characterization of degrading

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strains. FEMS Microbiol. Ecol. 2006, 55 (2), 239–247.

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(32) Amouric, A.; Quéméneur, M.; Grossi, V.; Liebgott, P. P.; Auria, R.; Casalot, L.

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Identification of different alkane hydroxylase systems in Rhodococcus ruber strain SP2B, an

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hexane-degrading actinomycete. J. Appl. Microbiol. 2010, 108 (6), 1903–1916.

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(33) Piscitelli, A.; Giardina, P.; Lettera, V.; Pezzella, C.; Sannia, G.; Faraco, V. Induction and

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transcriptional regulation of laccases in fungi. Curr. Genomics 2011, 12 (2), 104–112.

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(34) Jouhet, J. Importance of the hexagonal lipid phase in biological membrane organization.

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(35) Murínová, S.; Dercová, K. Response mechanisms of bacterial degraders to

524

environmental contaminants on the level of cell walls and cytoplasmic membrane. Int. J.

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Microbiol. 2014, 2014, 873081.

526 527

Table legend and figure caption

528

Table 1. The main classes of transport systems overexpressed in the presence of PE.

529 530

Figure 1. Characteristics of the PE samples used in this study. A) The molecular weight

531

distribution of the various PE samples was obtained by size exclusion chromatography, as

532

described in the supporting information SI. The Mn and Mw obtained for each sample are

533

summarized in the inserted Table. B) The oxidation rate of the various samples was

534

determined by Fourier Transform Infrared Spectroscopy (FITR), as described in the materials

535

and methods section. The peaks at 1,715 cm, characteristic of ketone groups, are highlighted.

536 537

Figure 2. Expression profiles in R. ruber under PE supplementation compared to the

538

"MSM+mannnitol" condition. A) Plots show the average expression logarithm (x axis) and

539

the fold change logarithm. Blue points are significantly dysregulated genes (q-value < 0.01)

540

and no significant differences were found for genes represented by red points. Blue points

541

under the X-axis are genes significantly up-regulated in a given PE condition (PE4K, PE4K-

542

OX or PEfi-OX respectively) against MSM and mannitol media. Afterward, we focused only

543

on these genes. B) The Venn diagram represents the complete set of up-regulated genes in R.

544

ruber. Few of them are common to all three conditions, whereas 22 to 33 transcripts are

545

specific to one of the condition.

546 547

Figure 3. Pathways induced in R. ruber under PE supplementation. A) A schematic diagram

548

representing the potential steps for an efficient PE mineralization by the R. ruber C208 strain. 21 ACS Paragon Plus Environment


Page 22 of 27

549

The alkane degradation and β-oxidation pathways are highlighted. The number of potential

550

candidates for each step, identified from RNA seq annotation, is indicated. B) The candidates

551

corresponding to the different steps in the alkane degradation and β-oxidation pathways are

552

listed, together with their relative induction in the presence of the various PE samples

553

(symbolized by a cross). The potential gaps in the pathway are also indicated (boxes in gray).

554 555

Figure 4. Rearrangements in the phospholipid pattern in R. ruber under PE supplementation.

556

A) Phospholipids were extracted from R. ruber after incubation under the indicated conditions

557

after three days of culture, as described in the materials and methods section. Representative

558

mass spectra obtained in the negative ion mode are shown. The peaks corresponding to CL,

559

PtdE and PtdI are indicated. B) Representative MS/MS spectra for the most abundant CL,

560

PtdI and PtdE species encountered in R. ruber under the various culture conditions. Peaks

561

representative of the fatty acyl chains (e.g. 16:0) and/or the polar headgroups (e.g. inositol)

562

are indicated for each species.


Page 23 of 27


Fig. 1 PE sample PE4K PE4K-‐OX PEﬁ PEﬁ-‐OX

Mn (Da) 4900 1162 26650 1600

A

Mw (Da) 7233 2734 94133 3820

PE sample PE4K PE4K-OX PEfi PEfi-OX

Normalized weight fracQon

0.2

0.1

0.0

Log molecular weight (Da) Log Molecular weight (Da)

B

0.20

0.10

Absorbance

0.4

0.00 1800

1750

1700

0.2

0.0

Wavenumber (cm) ACS Paragon Plus Environment

1650


Page 24 of 27

Fig. 2 A PE4K-OX vs. MSM+mannitol

PEfi-OX vs. MSM+mannitol

log(average expression)



log(fold change)

Overexpressed in MSM+mannitol

PE4K vs. MSM+mannitol

Overexpressed in PE condition

B

PE4K

8

PE4K-OX 33

28 11 42

14 22

PEfi-OX

ACS Paragon Plus Environment

Page 25 of 27


A 1-alcohol

Aldehyde dehydrogenase (NAD+)

Fatty acid

Aldehyde

Aldehyde dehydrogenase (PQQ)

Alkan-1-ol dehydrogenase

0 candidate

0 candidate

Alkane-1monooxygenase

2 candidates

Alkane

intracellular oxidases 34 candidates

5 candidates

n'

Enzyme

Acyl-CoA dehydrogenase 12 candidates

FAD

HS-CoA

-oxidation

NADH+H+

(n>3)

FADH2

Trans- -enoyl-CoA H2O

NAD+

Enoyl-CoA hydratase 2 candidates

L-3-hydroxybutyryl-CoA

transporters 19 candidates

including 2 putative dehydrogenases

B

7 candidates

Amp+PPi

L-3-hydroxyacyl-CoA dehydrogenase n'' 2 candidates

extracellular oxidases

Long-chain-fatty-acid CoA—ligase

Fatty acid-CoA

Acetyl-CoA C-acyltransferase 5 candidates

3-ketoacyl-CoA

n

CoA

Fatty acid-CoA(n-2)

ATP

PE

Fig. 3

1 candidate

3 candidates

Alcohol dehydrogenase

(integral component of) membrane BUT NOT transporters 9 candidates

Candidates

RRUB_S0078_02980 Alkane-1-monooxygenase (OR monooxygenase) RRUB_S0078_03232 RRUB_S0078_02824 Alcohol dehydrogenase RRUB_S0078_04850 RRUB_S0078_05728 Aldehyde dehydrogenase (NAD+) RRUB_S0078_03706 RRUB_S0078_02095 RRUB_S0078_03294 RRUB_S0078_03496 Long-chain-fatty-acid CoA—ligase RRUB_S0078_04852 RRUB_S0078_05189 RRUB_S0078_05618 RRUB_S0078_05662 RRUB_S0078_00917 RRUB_S0078_01964 RRUB_S0078_03141 RRUB_S0078_04295 RRUB_S0078_04402 RRUB_S0078_04960 Acyl-CoA dehydrogenase RRUB_S0078_05068 RRUB_S0078_05322 RRUB_S0078_05357 RRUB_S0078_05414 RRUB_S0078_05560 RRUB_S0078_05724 Enoyl-CoA hydratase RRUB_S0078_02426 RRUB_S0078_04877 L-3-hydroxyacyl-CoA dehydrogenase RRUB_S0078_03706 RRUB_S0078_04088 RRUB_S0078_04762 RRUB_S0078_04960 Acetyl-CoA C-acyltransferase RRUB_S0078_05565 RRUB_S0078_05702


PE4K ×

Induction PE4K-OX

PEfi-OX

× × × × ×

× × × ×

× × ×

× × × × × × × ×

× ×

× × ×

× × × ×

× × × × × × ×

× ×

× ×

× × × ×

×

× × ×

×


Page 26 of 27

Fig. 4

A PtdE

CL

PtdI

Mannitol

MSM

PE4K

PE4K-OX

PEfi-OX

CL 68:3

255.24 16:0

15

281.25 18:1

10

5

30

Relative intensities (%)


253.22 16:1

0 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

20

10

297.28 19:0

0 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

m/z 15

PtdE 35:0

20

15

10

297.28 19:0

5


255.24 16:0

PtdE 35:1

253.22 16:1

m/z 25


B

20

297.28 19:0

PtdI 35:0

10 241.02 Inositol

255.24 16:0

5

0 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

0 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350

m/z

m/z ACS Paragon Plus Environment

Page 27 of 27


Table 1

Candidates

Characteristics

RRUB_S0078_04921

3-(2-hydroxyphenyl) propionic acid transporter

RRUB_S0078_04331 RRUB_S0078_04551 ATP-binding cassette transporter (ABC transporter) RRUB_S0078_04611 RRUB_S0078_05525 RRUB_S0078_05709

daunorubicin resistance protein DrrA family ABC transporter

RRUB_S0078_05093

L-lactate permease

RRUB_S0078_05393

membrane protein

RRUB_S0078_00234 RRUB_S0078_02100 RRUB_S0078_02645 major facilitator superfamily transporter (MFS transporter) RRUB_S0078_04582 RRUB_S0078_05259 RRUB_S0078_05396 RRUB_S0078_05438

MFS transporter ; cysteine synthase

RRUB_S0078_03475

MFS transporter (Dha2 family)

RRUB_S0078_03557

MFS transporter; DsbA oxidoreductase

RRUB_S0078_04770

NADH dehydrogenase (electron transport)

RRUB_S0078_04025

small multidrug resistance protein


Transcriptomics and Lipidomics of the ... - ACS Publications

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