To produce antibodies against the NspC protein, the peptide GYDVEKLGAALKAFAERH corresponding to the amino acids 221–238 was synthesized and conjugated to KLH by Biosynthesis Inc. (Lewisville, TX). A male New Zealand White rabbit was immunized with an emulsion of 0.5 mL of a 2 mg mL−1 solution of the peptide in PBS and 0.5 mL of Freund’s Complete Adjuvant (Sigma) and boosted on weeks 3, 5, 7, 9, and 11. The animal was sacrificed at week 11, and the serum find more was used directly for the Western blots. This procedure was approved by the Appalachian State University IACUC committee (protocol number 07-3). For Western blotting, the serum and the horseradish peroxidase-conjugated goat anti-rabbit secondary

antibody were used at a 1 : 1000 and 1 : 10000 dilutions, respectively. ECL Plus chemiluminescent UK-371804 (GE Healthcare, Piscataway, NJ) or Super Signal West Femto (Thermo Fisher Scientific, Rockford, IL) HRP substrates were used for detection, and the X-ray films were developed manually. Total RNA was extracted from 5 mL of cells (AK 083 and AK103) grown to mid-log phase using Ambion RiboPure™-Bacteria kit (Applied Biosystems, Foster City, CA) and treated with DNase I for 2 h at 37 °C. One microgram of this RNA was reverse-transcribed using Protoscript® First Strand cDNA Synthesis kit (NEB, Ipswich, MA) with random primers. The cDNA from two biological

replicates was then used in quantitative real-time PCR (qRT-PCR) using gene-specific primers and SYBR Green PCR Master Mix (Applied Biosystems). Reactions were performed in triplicate. All PCR efficiencies were tested using a log dilution curve and were 100% efficient ±10%. All qRT-PCR products

were checked for accuracy using melt curve analysis. Data were analyzed using the relative expression software program, rest, which incorporates randomization and bootstrapping algorithms to analyze real-time quantitative PCR data (Pfaffl et al., 2002, available as freeware from www.qiagen.com). The rpoB gene encoding the RNA polymerase beta subunit was used as internal control (Quinones et al., 2005). A minimum of three biological replicates were performed for all experiments (unless otherwise noted) to ensure reproducibility of the results. Data were analyzed using Student’s t-test many (two-tailed, unpaired, unless otherwise noted) and differences were deemed statistically significant for P-values of 0.05 and below. Deletion of the nspC gene has been shown to be deleterious to biofilm formation in V. cholerae O1 El Tor (Lee et al., 2009). The inhibition of biofilm formation was attributed to the lack of norspermidine in the cell; however, the mechanism of this effect was not elucidated. Our repeated attempts to delete the nspC gene in V. cholerae O139 proved unsuccessful; therefore, we overexpressed the nspC gene from a multicopy plasmid (pACYC184::nspC, hereafter referred to as pnspC) to gain more insight into regulation of biofilm formation by polyamine synthesis pathways.