2-DE was performed using the Immobiline/polyacrylamide system and 18 cm IPG strips (pH ranges 4 to 7) (Amersham Pharmacia Biotech, Sweden). Seven hundred microgram samples
were loaded, and isoelectric focusing was conducted at 20°C for 58,000 Vhrs (maximum check details voltage of 8,000 V) on IPGphor (Amersham Pharmacia Biotech, Sweden). For the second dimension, vertical slab SDS-PAGE (12.5%) was used (Bio-Rad protean II Xi, Bio-Rad laboratories, USA). Gels were stained using Colloidal Coomassie Blue G-2500 (5 g G-250, 170 ml CBL0137 in vivo methanol, 212.5 ml 40% ammonium sulfate, 15 ml phosphoric acid, and 102.5 ml purified water). Three sample preparations were made for every strain, and each sample was repeated at least twice. Images were analyzed using the Image-Master 2D Elite (Amersham Pharmacia Biotech, Sweden). In-gel protein digestion, MALDI-TOF-MS and protein identification Protein spots of interest
were excised from the gels. After destaining, gel pieces were digested with trypsin (Roche, Germany) for 12 h at 37°C. The extracts were dried and resolubilized in 2 μl of 0.5% TFA. Peptide mass fingerprinting (PMF) measurements were performed on a Bruker Reflex™ III MALDI-TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) working in reflectron mode with 20 kV of accelerating voltage and 23 kV of reflecting voltage. A saturated solution of α-Cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile and 0.1% trifluoroacetic acid (TFA) was used for the matrix. Mass accuracy for PMF analysis was 0.1–0.2 Da with external calibration; internal calibration was carried out using enzyme autolysis
peaks, and the resolution was 12,000. Database searches were performed AR-13324 using the software Mascot v1.7.02 (Matrix Science Ltd.) licensed in-house http://mascot.proteomics.com.cn/search_form_PMF.html against the database of V. cholerae N16961 (Version Vib CLEAN 040921, 3814 sequences). Monoisotopic peptide masses were used to search the databases with a mass tolerance of 100 ppm and one partial cleavage. Oxidation of methionine and carbamidomethyl modification of cysteine was considered. Scores greater than 48 were significant (p < 0.05), with more than five peptides matched and sequence 3-oxoacyl-(acyl-carrier-protein) reductase coverage greater than 15%. Sequencing of the gene VCA0518 The gene VCA0518 (designated in the genome of N16961, GenBank Accession Number NC002506), which corresponds to the fructose-specific IIA/FPR component (PTS system, FIIA), was amplified from all tested strains using primers 5′ GCG CTG GAT TTA AGG TGA TGG 3′ and 5′ TCG CCT ATA GAG GCA GAC AGG 3′ and sequenced. The sequences were searched in the CDD database (V2.16-27036PSSMs, http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) for conserved domain analysis. Quantitative real-time PCR (qRT-PCR) Total RNA from N16961 and JS32 cultured in sorbitol fermentation media was extracted at the inoculation time points 2, 4, 6 and 8 h with the RNeasy Mini Kit (QIAGEN).