R. erythropolis R138 (Cirou et al., 2007) and P. atrosepticum CFBP6276 (Kwasiborski et al., 2013), as well as its expI KmR-derivative, which is defective for producing the NAHL signals and virulence factors (Latour et al., 2007), were routinely cultivated at 28 °C in TY medium (0.5% tryptone, 0.3% yeast extract) supplemented with kanamycin (50 μg ml−1) when appropriate. The NAHL-biosensor Agrobacterium tumefaciens NT1(pZLR4) was cultivated in Agrobacterium Broth (AB) medium supplemented with mannitol (2 g l−1), NH4Cl (1 g l−1) and gentamycin (25 μg ml−1) as described previously (Cha et al., 1998).
Cultures of R. erythropolis R138 in liquid AB medium with gamma caprolactone (2 g l−1) as a carbon source were washed twice in 0.8% NaCl. Bacteria were inoculated at OD600 value of 0.6 in fresh AB medium supplemented with inorganic (MgSO4) and organic (methionine) sulfur sources at 24 and 0.24 m. After 6 h of incubation at 30 °C, one volume of RNA protect Cell Reagent (Qiagen, Hilden, Germany) was added to the culture and was frozen at −80 °C until use. Each of the four tested culture conditions (two sulfur sources at two concentrations) was replicated three times.
Total DNA of R. erythropolis R138 was extracted from the pellet of a bacterial suspension adjusted to an optical density of 0.6 at 600 nm. The cell lysis was carried out at 37 °C during 2 h in 0.5 ml of TES buffer pH 8 (10 m Tris, 1 m EDTA and 150 m NaCl) supplemented with 20 mg ml−1 lysozyme (Eurobio, Les Ulis, France) and 133 μg ml−1 RNase (Sigma-Aldrich, St-Louis, MO, USA). Then, 100 μl of 20% w/v SDS were added before gentle mixing. One volume of phenol/choroform/isoamylic acid (25:24:1, Sigma-Aldrich) was added, mixed and centrifuged for 15 min at 15 000 g. The aqueous phase was washed twice by addition of one volume of Aquaphenol (MP bio, Santa Ana, California, USA). The genomic DNA was precipitated by one volume of isopropanol. After a centrifugation for 10 min at 15 000 g, the DNA was washed by an addition of a volume of 70% ethanol followed by centrifugation for 10 min at 15 000 g. The pellet was dried before the DNA was solubilized in TE buffer pH 7.5 (10 m Tris-HCl, 1 m EDTA). The DNA concentration was estimated using the Nanodrop 1000 spectrophotometer (Thermo Scientific, Whaltham, MA, USA) and frozen until used.
De novo genome assembly of R. erythropolis R138 was performed by combining Illumina and 454-Roche technologies as described by Kwasiborski et al. (2014). Reads were collected from three genomic libraries: two short-fragment libraries (300 bp and 380 bp) used for paired-end 2 × 72 Illumina-sequencing (ImaGif, Paris, France) and single read 454-sequencing (Eurofins MWG, Ebersberg, Germany), and a long-paired end library with an insert size of 8 kbp used for 454-sequencing (Eurofins MWG). Assembly was carried out using the CLC Genomics Workbench v5.1 (CLC bio, Aarhus, Denmark) with read length at 0.5 and similarity at 0.8 as parameters. The coding sequences and their functions of the genome were predicted using the Rapid Annotation using Subsystem Technology v4.0 (RAST) automated pipeline (Aziz et al., 2008). The genome of R. erythropolis R138 was deposited at NCBI (http://www.ncbi.nlm.nih.gov/) under the GenBank reference ASKF00000000.
Inoculation of bacteria on potato tuber slices was adapted from a protocol previously described by Kwasiborski et al., (2012). Bacteria cultivated in TY medium were washed in saline water (0.8% w/v NaCl) and the cell number was adjusted to 109 c.f.u. ml−1. Tuber slices (1 cm) of Solanum tuberosum variety Allians were covered with a membrane (Supor 450, diameter 25 mm, pore size of 0.45 μm, PALL, NY, USA) and inoculated by 300 μl of three cell suspensions: R. erythropolis R138 alone, a mix (ratio 10:1) between R. erythropolis R138 and P. atrosepticum CFBP6276, and a mix (ratio 10:1) between R. erythropolis R138 and the P. atrosepticum expI mutant. The 10:1 ratio between R. erythropolis and P. atrosepticum allowed a reduction of symptoms on potato tuber, as described previously (Cirou et al., 2007). The population dynamics of R. erythropolis and P. atrosepticum on potato tuber slices were measured every 3 hours over a period of 27 h. Bacteria on membranes were recovered by vortexing in saline water (2 ml). Serial dilutions of the bacterial suspensions were plated on TY medium supplemented with X-gal (40 μg ml−1) and incubated at 25 °C for 4 days before counting. R. erythropolis R138 appeared as white colonies, whereas P. atrosepticum CFBP6276 and its expI derivative appeared as blue colonies. Experiments were performed in triplicates.
NAHL extraction and quantification procedures were adapted from Cha et al. (1998) Briefly, bacterial cell suspensions recovered from potato slices, were centrifuged for 10 min at 15 000 g. NAHLs were extracted from the supernatant by addition of one volume of ethyl acetate and air-drying the organic fraction. The extracted NAHLs were dissolved in 20 μl of ethyl acetate, of which 5 μl was spotted on TLC (thin layer chromatography) silica plates (Macherey-Nagel, Düren, Germany). TLCs were overlaid with the NAHL-biosensor strain A. tumefaciens NT1(pZLR4) in AB medium supplemented with agar (15 g l−1) and X-gal (40 μg ml−1). For the quantification of NAHLs, a calibration curve was obtained with pure NAHL, 3-oxo-octanoylhomoserine lactone (Sigma-Aldrich).
After 22-hour incubation of potato slices at 25 °C, bacteria were collected from membranes by vortexing in 1 ml of 50% RNAprotect Cell Reagent. After centrifugation for 10 min at 20 800 g, bacterial pellets were frozen at −80 °C until use. Each of the three conditions, R. erythropolis R138 alone and R. erythropolis R138 in association with either P. atrosepticum CFBP6276 or its expI derivative were replicated three times. The total RNA extraction was carried out using the NucleoSpin RNA II (Macherey-Nagel) according to the manufacturer’s recommendations. The RNAseq analysis was performed using ProfileXpert (University of Lyon I, Lyon, France). RNA control was carried out using the 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA, USA), then Ribo-Zero rRNA Removal Meta-bacteria kits (tebu-bio, Le Perray en Yvelines, France) was used for rRNA depletion. Directional RNA paired-end libraries were sequenced (2 × 50 bp) on Illumina Hiseq-2500. The number of aligned reads per predicted gene were counted and normalized according to the gene length to calculate for each gene the number of reads per Kilo base per Million mapped reads (RPKM). No interspecies hybridization was observed between the genome of the actinobacterium R. erythropolis R138 and that of the proteobacterium P. atrosepticum CFBP6276 (Kwasiborski et al., 2013).
The differentially expressed genes of R. erythropolis R138 in the presence vs absence of P. atrosepticum CFBP6276 and its expI mutant were identified using DEseq v1.9.6, an R program language package available through the Bioconductor platform (http://www.bioconductor.org/packages/release/bioc/html/DESeq.html). DEseq estimates the variance-mean dependence in count data from high-throughput sequencing assays and tests for differential expression based on a model using the negative binomial distribution (Anders and Huber, 2010). We also listed the highly expressed genes of R. erythropolis when it colonized the potato tuber slices alone. We considered only genes exhibiting a RPKM median value, which was over by three times the RPKM median value of all genes. All predicted genes of which the standard deviation of RPKM was over the RPKM mean value were discarded of the analysis.
Expression of several genes was quantified by qRT-PCR using biological triplicates. Sequences and characteristics of used primers are presented in the Supplementary Table S1. Reverse transcriptions were carried out using the protocol for high GC content bacteria from the Revert Aid Reverse Transcriptase according to the manufacturer’s recommendations (Fermentas, Whaltham, MA, USA). A Light Cycler 480 (Roche Applied Science, Penzberg, Germany) and Light Cycler 480 SYBR Green I Master (Roche Applied Science) were used for quantitative PCR. The 15 μl final-volume mix contained SYBR Green I Master (1x), forward and reverse primers (1 μ) and 0.01 μg of cDNA samples. After denaturation at 95 °C for 10 min, the amplification and quantification program was repeated 45 times as follows: 95 °C for 15 s, 60 °C for 15 s, 72 °C for 20 s, with a single fluorescence measurement, followed by the melting curve program (65 °C–95 °C with a heating rate of 0.1 °C s−1 and a continuous fluorescence measurement) and a final cooling step at 45 °C. The recombinase A (recA) gene was used as a reference gene.
