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Transcriptional Profiling of the Marine Oildegrading Bacterium Alcanivorax Borkumensis During Growth on Nalkanes

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Transcriptional Profiling of the Marine Oildegrading Bacterium Alcanivorax Borkumensis During Growth on Nalkanes RESEARCH LETTER Transcriptional pro¢ling ofthe marine oil-degrading bacterium Alcanivorax borkumensis during growth on n-alkanes Julia S. Sabirova1, Anke Becker2, Heinrich Lunsdorf¨ 3, Jean-Marc Nicaud4, Kenneth N. Timmis5 & Peter N. Golyshin6 1Department of Bioscience and Bioengineering, Ghent University, Ghent, Belgium; 2Institute of Biology III, Faculty of Biology, University of Freiburg, Freiburg, Germany; 3Department of Vaccinology and Applied Microbiology, HZI-Helmholtz Centre for Infection Research, Braunschweig, Germany; 4Laboratoire de Microbiologie et de Gen´ etique´ Moleculaire,´ INRA Centre de Grignon, Thiverval-Grignon, France; 5Department of Environmental Microbiology, HZI-Helmholtz Centre for Infection Research, Braunschweig, Germany; and 6School of Biological Sciences, Bangor University, Bangor Gwynedd, UK Correspondence: Julia S. Sabirova, Abstract Laboratory of Industrial Microbiology and Biocatalysis, Faculty of Bioscience The marine oil-degrading bacterium Alcanivorax borkumensis SK2 has attracted Engineering, Ghent University, Coupure Links significant interest due to its hydrocarbonoclastic lifestyle, its alkane-centered 653, B-9000 Ghent, Belgium. Tel.: 132 9 264 metabolism, and for playing an important ecological role in cleaning up marine oil 93 85; fax: 132 9 264 62 31; spills. In this study, we used microarray technology to characterize the transcrip- e-mail: [email protected] tional responses of A. borkumensis to n-hexadecane exposure as opposed to pyruvate, which led to the identification of a total of 220 differentially expressed Received 21 December 2010; revised 15 March genes, with 109 genes being upregulated and 111 genes being downregulated. 2011; accepted 31 March 2011. Among the genes upregulated on alkanes are systems predicted to be involved in Final version published online 20 April 2011. the terminal oxidation of alkanes, biofilm formation, signal transduction, and DOI:10.1111/j.1574-6968.2011.02279.x regulation. Editor: Michael Galperin Keywords Alcanivorax borkumensis; alkane; transcriptomics. alkanes by Pseudomonas putida GPO1, a bacterium that uses Introduction linear alkanes as a single carbon and energy source, where a Marine oil-degrading bacteria play an essential role in monooxygenase encoded by the alkB1 gene plays a central degrading crude oil and thus in cleaning up marine oil spills role in the terminal oxidation of alkanes (van Beilen et al., (Yakimov et al., 2007). Alcanivorax borkumensis has become 1994). Other alkane-degrading bacteria use for this initial a paradigm of marine ‘hydrocarbonoclastic’ bacteria, as it oxidation step enzymatic systems other than AlkB (for exclusively grows on alkanes and plays a predominant reviews, see van Beilen & Funhoff, 2007; Wentzel et al., ecological role in oil-degrading consortia that form follow- 2007). Our genome-wide study of alkane utilization by A. ing marine oil spills (McKew et al., 2007; Gertler et al., borkumensis using a proteomics approach has revealed 2009). Alcanivorax borkumensis SK2 metabolizes a wide several alternative systems for terminal oxidation of alkanes range of alkanes, such as linear alkanes, cyclo-alkanes, and by this bacterium, as well as major rearrangements of its isoprenoids (Dutta & Harayama, 2001; McKew et al., 2007). central carbon metabolism (Sabirova et al., 2006). However, Given its important ecological role in the removal of oil a number of specific questions intrinsically linked to alkane spills and with the availability of its full genome sequence utilization by this organism, for example how alkanes enter MICROBIOLOGY LETTERS MICROBIOLOGY (Schneiker et al., 2006), Alcanivorax may now serve as a the cell and which transport systems may be involved, how model organism to understand bacterial alkane metabolism. the cells physically interact with the hydrophobic substrate, Previous studies of bacterial alkane metabolism have been whether and how they attach to it, and which molecular mostly focused on studying the terminal degradation of mechanisms allow the cells to protect themselves against the c 2011 Federation of European Microbiological Societies FEMS Microbiol Lett 319 (2011) 160–168 Published by Blackwell Publishing Ltd. All rights reserved Transcriptomic responses of Alcanivorax borkumensis 161 toxic effect of alkanes, are left unanswered. Finally, the Carlsbad, CA) using the Ribolyser (Hybaid, Heidelberg, regulatory implications of alkane degradation on the overall Germany) (30 s, level 6.5) before spin column purification cellular activity could not be comprehensively studied using according to the RNeasy Mini Kit RNA purification protocol. the proteomic approach. To obtain a still more comprehen- Fluorescent-labeled amplified RNA was prepared using sive picture of alkane utilization, and in particular to be able the MessageAmp II-Bacteria RNA Amplification Kit (Ap- to look more closely into some of the aforementioned issues, plied Biosystems, Darmstadt, Germany). Starting from we have now used microarray technology to compare the 500 ng total RNA, cDNA carrying a terminal T7 promoter transcriptional profile of SK2 grown on n-hexadecane, as a was synthesized. Subsequent in vitro transcription resulted model alkane, as compared with pyruvate, one of the few in aminoallyl-modified RNA that was labeled with Cy3- or non-alkane substrates A. borkumensis is able to use. Cy5-N-hydroxysuccinimidyl ester dyes (GE Healthcare, Lit- tle Chalfont, UK). Uncoupled dye was removed applying the Materials and methods RNeasy MinElute Kit (Qiagen). Bacterial strains and growth conditions Microarray hybridization and image acquisition Alcanivorax borkumensis SK2 was used for all experiments. Processing of microarrays before hybridization included the Alcanivorax borkumensis was grown until the late-exponen- following washes: once in 0.1% Triton-X100 (5 min, 20 1C); tial stage of growth as described earlier (Sabirova et al., twice in 0.032% (w/v) HCl (2 min, 20 1C); once in 0.1 M KCl 2006). Bacteria from alkane- and pyruvate-grown cultures (10 min, 20 1C); once in H2O (1 min, 20 1C); once in 0.064% were centrifuged for 10 min at 8000 g, and the cell pellets (w/v) HCl, 1 Â Nexterion blocking solution (Schott AG) were immediately frozen in liquid nitrogen and conserved (15 min, 50 1C); and once in H2O (1 min, 20 1C). Micro- at À 80 1C until RNA was isolated. arrays were dried by centrifugation (3 min, 185 g,201C). Hybridization was performed in an EasyHyb hybridiza- tion solution (Roche, Mannheim, Germany) supplemented Oligonucleotide design, printing, and layout with sonicated salmon sperm DNA at 50 mgmLÀ1 in a final of the A. borkumensis microarray volume of 100 mL for 90 min at 45 1C using the HS 4800 The Abo3kOLI microarray used in this study is based on the hybridization station (Tecan Trading AG, Switzerland). sequenced genome of A. borkumensis (Schneiker et al., Before application to the microarrays, labeled samples were 2006). The array contains 2924 50mer to 70mer oligonu- denatured for 5 min at 65 1C. cleotides representing predicted protein-encoding genes. In After hybridization microarrays were washed once in addition, the array contains 15 stringency controls of the 2 Â SSC, 0.2% sodium dodecyl sulfate (SDS) (w/v) (5 min, genes gap, rpsA, rpsO, rpsP, and rpmI (70%, 80%, and 90% 42 1C), twice in 0.2 Â SSC, 0.1% SDS (w/v) (1 min, 21 1C), identity to the native sequence), 12 alien DNA oligonucleo- twice in 0.2 Â SSC (1 min, 21 1C), and once in 0.05 Â SSC tides, and five spiking control oligonucleotides. Oligonu- (1 min, 21 1C). Following the washes, slides were dried by cleotides were designed using OLIGODESIGNER software centrifugation (3 min, 185 g,201C) and scanned with a pixel (Bioinformatics Resource Facility, CeBiTec, Bielefeld Uni- size of 10 mm using the LS Reloaded microarray scanner versity). All oligonucleotide probes were printed in four (Tecan Trading AG). Four independent biological replicates replicates. Microarrays were produced and processed as including a dye swap were processed for each comparison. described previously (Brune et al., 2006). Oligonucleotides (40 mM) in 1.5 M betaine, 3 Â SSC (1 Â SSC is 0.15 M Transcriptome data analysis sodium chloride, 0.015 M sodium citrate) were printed onto Nexterion Slide E (Schott AG, Mainz, Germany) using the The mean signal and the mean background intensities were MicroGrid II 610 spotter (BioRobotics, Cambridge, UK) obtained for each spot of the microarray images using the equipped with 48 SMP3 stealth pins (TeleChem Interna- IMAGENE SOFTWARE 6.0 software (Biodiscovery Inc., Los An- tional, Sunnyvale, CA). DNA was cross-linked to the surface geles) for spot detection, image segmentation, and signal by incubation of the slides for 2 h at 85 1C. Microarray data quantification. have been submitted to ArrayExpress under accession num- Spots were flagged as ‘empty’ if R 0.5 in both channels, ber A-MEXP-1990. where R = (signal mean À background mean)/background SD. The remaining spots were considered for further analy- sis. After subtractions of the local background intensities RNA isolation and synthesis of labeled cDNA from the signal intensities and the introduction of a floor Total RNA was purified using the RNeasy Mini Kit (Qiagen, value of 20, the log2 value of the ratio of intensities was Hilden, Germany). Cells were disrupted in RLT buffer calculated for each spot using the formula Mi = log2(Ri/Gi). provided with the Kit in Fast Protein tubes (Qbiogene, Ri = Ich1(i) À Bgch1(i) and Gi = Ich2(i) À Bgch2(i), where Ich1(i) FEMS Microbiol Lett 319 (2011) 160–168 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 162 J.S. Sabirova et al. or Ich2(i) is the intensity of a spot in channel 1 or channel 2 a non-alkane-degrading organism. Our earlier proteomic and Bgch1(i) or Bgch2(i) is the background intensity of a spot study has already revealed the presence of several alternative in channel 1 or channel 2, respectively. The mean intensity ways for the terminal oxidation of alkanes by A. borkumensis 0.5 was calculated for each spot, Ai = log2(RiGi) (Dudoit et al., (Sabirova et al., 2006).
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