O-Antigen Prevents Phagocytosis by Fish Skin Cells Table 1. Colonization frequency of the skin, intestines, and spleen in each infected fish group. Strain Uninfected NB10 KL18 KL19 KL20 KL18c KL19c KL20c Genotypea WT Dwzt Dwzm DwbhA Dwzt complemented Dwzm complemented DwbhA complemented aAll strains contain pNQFlaC4-lac:lux, which carries the luxCDABE operon and is integrated into the chromosome. doi:10.1371/journal.pone.0037678.t001 Colonized fish/total fish Skin 24 h 0/20 19/20 0/20 0/20 0/20 18/20 19/20 19/20 48 h 0/20 20/20 0/20 0/20 0/20 19/20 19/20 20/20 Intestines 24 h 0/20 17/20 4/20 7/20 2/20 18/20 19/20 20/20 48 h 0/20 19/20 19/20 16/20 19/20 19/20 19/20 20/20 Spleen 24 h 0/20 0/20 0/20 0/20 0/20 0/20 0/20 0/20 48 h 0/20 1/20 0/20 0/20 0/20 0/20 0/20 0/20 Materials and Methods Ethics statement The infection studies were performed in strict accordance with the Swedish Bioethical Guidelines for care and use of laboratory animals. The protocol was approved by the Umea˚ Committee on the Ethics of Animal Experiments (Permit Number: A-103-11). Bacterial strains, plasmids and culture conditions Bacterial strains and plasmids used in this study are given as supplementary information in Table S1. V. anguillarum strains were routinely grown in Trypticase soy broth containing 1% sodium chloride (TSB-1%) at 24uC with aeration, or on Trypticase soy agar (TSA) grown at room temperature. Escherichia coli was routinely grown at 37uC with aeration in Luria broth (per liter: Bacto Tryptone, 10 g; Bacto yeast extract, 5 g; sodium chloride, 5 g). Plasmid transfers from E. coli to V. anguillarum were performed as described previously [51]. The vibrio selective medium TCBS agar (Difco) containing 10 mg ml21 chloramphenicol was used after conjugation to select against E. coli. PCR conditions, sequencing, and DNA techniques PCR was performed as previously described [52]. When a PCR fragment required minimal errors, the high-fidelity KOD poly- merase (Novagen) was used. Unless otherwise stated, conditions for various DNA techniques are according to Sambrook et al. [53]. Reaction conditions for DNA-modifying enzymes and DNA restriction enzymes were performed according to the manufac- turer’s instructions. Genomic DNA sequencing was done by Eurofins MWG GmbH as part of a genome sequencing project (unpublished data; E. Hjerde, D.L. Milton, and N.P. Willassen). Accession number The DNA sequence data for the wzm-wzt-wbhA operon have been submitted to the DDB/EMBL/GeneBank databases under accession number JQ920376. In-frame deletion mutagenesis In-frame, single gene deletions of the wzm, wzt, and wbhA genes were made by allelic exchange using the R6K origin-based suicide vector pDM4 as described previously [51]. A mutant allele of each gene was created by overlap PCR fusing several codons from both the 59 and 39 ends of each gene (Table S1). Each allele was cloned into pDM4 creating plasmids pDM4-wzt-AD, pDM4- wzm-AD, and pDM4-wbhA-AD. The plasmids were mobilized into the wild type and after allelic exchange, the mutants KL18 (Dwzt), KL19 (Dwzm), and KL20 (DwbhA) were created. All mutations were confirmed by sequencing a DNA fragment that was amplified by PCR from the mutated chromosomal locus in each mutant strain. Primers used in the overlap PCR to make each allele are included as supplementary information in Table S2. For complementation of each mutation, reverse allelic exchange was done. The wild-type genes were amplified using the primer A and primer D that was used to make the mutant allele and cloned into pDM4 creating pDM4-wzm-wt, pDM4-wzt-wt, and pDM4- wbhA-wt. Each plasmid was mobilized into the respective mutant strain and reverse allelic exchange was performed as described for the allelic exchange. LPS and EPS analyses Lipopolysaccharides were isolated from each bacterial strain by a Proteinase K digestion of whole-cell lysates as described by Preston and Penner [54]. The LPS were fractionated by 15%- SDS-PAGE [55] and visualized using Pro-QH Emerald 300 Lipopolysaccharide Gel Stain Kit (Invitrogen) according to the manufacturer’s instructions and an ImageQuant LAS4000 biomolecular imager. Exopolysaccharides were quantified from spent culture super- natants of each strain using an Alcian blue technique described by Plante [56]. Bacterial cultures (10 ml) were grown for three days in TSB-1% at 24uC with aeration. After bringing the bacterial cultures to equal cell densities, cells from 1 ml of culture were pelleted using low speed centrifugation (12,000 g, 15 min, 4uC) and the supernatants were collected and used to quantify the EPS. Isolation of rainbow trout skin epithelial cells Using a fine tip forceps, single scales were picked from the skin of a 20 g rainbow trout (Oncorhynchus mykiss) and placed in a small petri dish containing Hank’s balanced salt solution (HBSS, Invitrogen) with PEST (Invitrogen) at a final concentration of 100 U of penicillin and 100 mg of streptomycin. To remove skin mucus, the scales were washed 5 times in HBSS containing PEST. For confocal 3D-imaging microscopy, a drop of HBSS containing PEST was spotted onto a collagen I coated coverslip (BD Biosciences) that was placed in a small petri dish and 5 fish scales were equally distributed within the buffer solution on the coverslip. A second coverslip of larger size was placed on top of the fish scales PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e37678