The normalized collision-induced dissociation was set to 35.0. All spectra were converted to mgf using Proteome Discover version 1.2 (Thermo-Scientific) and submitted to a

local MASCOT (Matrix Science, London, UK) server and searched against bacteria in the SwissProt (release 57.15) and MSDB databases (release 9.0) with a precursor mass tolerance of 10 ppm, a fragment ion mass tolerance of 0.6 Da and strict trypsin specificity allowing up to one missed cleavage, carbamidomethyl as fixed modification and oxidation of methionine residues as variable modification. Proteins were AZD6738 nmr considered positive if the MASCOT score was over the 95% confidence limit corresponding to a score > 35 for proteobacteria. RNA preparation and quantitative real-time PCR (qRT-PCR) Total RNA from X. a. pv. citri mature biofilms and planktonic cells was extracted using TRIzol® reagent (Invitrogen), according to the manufacturer’s instructions. After DNAse (Promega) treatment, cDNA was synthesized from 1 μg of total RNA using M-MLV RT (Promega) and the oligonucleotide dN6 was added as follows: 200 U of M-MLV RT (Promega, USA), 0.25 μg of primer dN6 and 0.5 mM of deoxynucleoside triphosphates (dNTPs) (reaction final volume: 20 μl) and incubated for 1 h at 42°C, and www.selleckchem.com/products/ve-822.html then for 10 min at 94°C. The qRT-PCRs were performed by combining 1 μl of cDNA template, 0.5 U of Go Taq DNA polymerase (Promega), 1 × reaction buffer, 0.2

mM dNTPs and 20 pmol of each primer (final reaction volume, 20 μl) in a Mastercycler ep realplex thermal cycler (Eppendorf) using

SYBR Green I (Roche) to monitor 10058-F4 mw double-stranded DNA (dsDNA) synthesis. The qRT-PCR conditions were set to 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s and 72°C for 40 s. The primer pairs used for qRT-PCR are provided in Additional file 2: Table S2. As a reference gene, a fragment of 16S rRNA was amplified using the same qRT-PCR conditions. Values were normalized by the internal reference (Ctr) according to the equation ΔCt = Ct – Ctr, and quantified as 2–ΔCt. A second normalization using a control (time=0 days) (Ctc), ΔΔCt = Ct – Ctc, produces a relative quantification: 2–ΔΔCt[63]. Values Urease are the means of four independent experiments. Results were analyzed using one-way ANOVA (p < 0.05) and Student t-test (p < 0.05). GO enrichment analysis Proteins were considered as differentially expressed when variations between planktonic and biofilm grown cells were at least 1.5-fold and the quantitation p-value of 0.05. The GO enrichment analysis was performed using Blast2GO [64–66]. Acknowledgements We thank Rodrigo Vena for assistance with the confocal microscopy facility and Microquin for the culture media, and the Proteomics laboratory from the Biosciences core laboratories, King Abdullah University of Science and Technology, for providing the facility and equipment for gel electrophoresis and mass spectrometry analyses.