Thus all mutants were generated from V. parahaemolyticus VP53. Unless otherwise stated, bacteria were cultured in RAD001 mouse LB broth or LB agar at 37°C. Antibiotics

were added in the following concentration when needed: chloramphenicol at 10 μg/ml, and Kanamycin at 50 μg/ml for Escherichia coli and 100 μg/ml for V. parahaemolyticus. To induce rugose phenotype, a single colony was inoculated into 2 ml APW#3 broth [22], incubated at 37°C statically for 48 hours. Then 1 μl of culture was spotted on LB agar plate and incubated at 30°C for 48-72 hours. Pictures were taken when colony size reached Selleck 5-Fluoracil about half centimeter. Construction of Mutants Genetic regions to be targeted and primer sequences were determined based on the annotation of V. parahaemolyticus genome RIMD2210633 (GenBank Accession BA000031 and BA000032). Several mutants, including a mutation deleting the entire K-antigen structural gene operon on chromosome I (VP0219-0237), several partial deletion mutations in the region on chromosome I (VP0215-0218 and VP0220 gene), and a deletion mutation of exopolysaccharide region in chromosome

II (VPA1403-1406) as well as a deletion mutation in a separate region containing polysaccharide transport genes wza, wzb, and wzc were constructed (Table 1). Polymerase Chain Reaction (PCR) was performed using Taq DNA polymerase (Thermo Fisher, Waltham, MA). PCR products were purified on Qiagen PCR purification columns (Qiagen, Valencia, CA). Restriction enzymes were purchased the from New England Biolabs (Ipswich, MA). DNA was prepared for crossover recombination by overlapping PCR. First, three DNA fragments were amplified by PCR separately, including a fragment (500-1000 bp) upstream of targeted gene in V. parahaemolyticus, a fragment

(500-1000 bp) downstream of targeted gene in V. parahaemolyticus and a chloramphenicol resistant gene (Cm) in pKD3 [31]. The 3′ end of the reverse primer in the upstream DNA was complementary to the forward primer of Cm, and the 5′ end of the forward primer of downstream DNA was complementary to the reverse primer of Cm. Then the three fragments were mixed and assembled into one piece in a second PCR reaction where the product was amplified by primers at the two extremes. Genes deleted and primers used are listed in (Table 3). Two to four micrograms of PCR product were used to transform V. parahaemolyticus VP53.