(1:500 dilution). Lastly, the coverslips were mounted in Vecta- shield with DAPI (Vector Laboratories) and samples were examined on a Leica TCS SP5 (Leica Mycrosystems CMS GmbH, Mannheim, Germany) inverted microscope (DMI6000) with a 636 water (1.2 numerical apertures) apochromatic objective [37]. Images of Alexa 488 were captured using 458 nm line from an Ar+ laser and DsRed excitation was performed with the 514 nm line from an Ar+ laser. DAPI excitation was performed using a multiphoton Ti:Sapphire laser (Spectra-Physics Mai Tai BB, 710–990 nm, 100 femtoseconds, 80 MHz). Galleria Mellonella Killing Assays Galleria mellonella killing assays were based on the method described by Seed & Dennis [38]. Cultures of B. cenocepacia K56-2 and BCAM0223::Tp mutant were grown in microaer- ophilic conditions previously described. Cells were then harvested and ressuspended in 10 mM MgSO4 plus 1.2 mg/ mL ampicilin. A micrometer was adapted to control the injection volume of a micro syringe. This apparatus was used to inject 3.5 mL of bacterial suspension containing 10 CFU/ larvae into the haemocoel of the caterpillars via the hindmost left proleg. Control larvae were injected with the solution 10 mM MgSO4 plus 1.2 mg/mL ampicilin. Following injection, larvae were placed in glass Petri dishes and stored in the dark at 37uC. The survival and appearance was recorded at 24 h intervals until 96 h. Larvae were considered dead when they displayed no movement in response to shaking of the petri dish or touch with a pipette tip. Statistical Analysis All experiments were performed a minimum of three times. Relative comparisons were done between corrected values with ANOVA test for significance. A P value ,0.05 was considered statistically significant. Results Bioinformatic Analysis of BCAM0223, a New Member of the Trimeric Autotransporter Family The 4653-bp BCAM0223 gene from B. cenocepacia K56-2 encodes a protein with 1550 residues that contains several domains with homology to members of the trimeric autotran- sporter adhesin family of surface-attached oligomeric proteins, in particular to the C-terminal translocator domain of YadA, a Yersinia enterocolitica trimeric autotransporter adhesin [26]. We have used the web-based daTAA program (http://toolkit. tuebingen.mpg.de/dataa) to confirm that BCAM0223 has, like the other TAA proteins, a head-stalk-anchor modular organiza- tion [39]. Using the daTAA program, we also predict the presence of a signal peptide with a cleavage site between amino acids 42 and 43. A BLASTP sequence similarity searches indicated that BCAM0223 shares significant overall amino acid sequence identity with at least three other members of the TAA family. The highest identities (46%) were observed with a protein from B. cenocepacia MC0-3 (YP_001777862) and the autotransporter adhesin from B. cenocepacia PC184 (ZP_04942611.1) followed by the hemaggluttinin like protein from B. xenovorans (Bxe_B2271) (34%). A closer examination of the amino acid identity of BCAM0223 with its counterparts revealed that the higher degree of identity is restricted to the C-terminal domain regions (YadA- head-like domains). Characterization of B.cenocepacia BCAM0223 Mutant Transcriptional Organization of the Genes Linked to BCAM0223 As previous described, the TAA-encoding gene BCAM0223 belongs to a cluster of nine genes that we called TAA cluster [28]. To determine the transcriptional organization of this gene cluster, reverse transcription PCR (RT-PCR) experiments were performed in B. cenocepacia K56-2. As shown in Figure 1_A, we designed primer pairs that include intergenic regions between the desig- nated open reading frames, allowing the detection of co- transcripts. PCR products of 800 and 700 bp, comprising respectively the intergenic regions of BCAM0224-BCAM0223, BCAM019–BCAM0220 and BCAM0217–BCAM0218 were de- tected (Fig. 1_B). In contrast, no PCR products were obtained for the primer pairs designed to amplify the intergenic regions between BCAM0223-BCAM0222, BCAM0227–BCAM0224 and BCAM0220–BCAM0221 (Fig. 1_B). Our results thus indicate that the three TAA-encoding genes are organized as two subclusters, consisting of two divergently oriented bicistronic operons, BCAM0224-BCAM0223 and BCAM019–BCAM0220, flanked by regions harboring predicted promoter and terminator sequences. The neighbor genes BCAM0221, BCAM0222 and BCAM0227 are organized in monocistronic units (Fig. 1). Effect of the BCAM0223 Mutation on Hemagglutination Ability of B. cenocepacia K56-2 In silico analysis of the BCAM0223 amino acid sequence using Pfam protein database [40] revealed significant hits for the presence of eight noncontiguous coiled-coil clusters of HIM motif repeats (Pfam domain PF05662) and five clusters of Hep-Hag motif repeats (Pfam domain PF05658), which are located, respectively, on the neck and head2 domains (Fig. 2_A). HIM and Hep-Hag are short repeat motifs found in bacterial hemagglutinins and invasins [41]. To determine whether the BCAM0223 gene is related with the hemagglutination ability of B. cenocepacia K56-2, we constructed a knockout BCAM0223 mutant and examined its hemagglutinat- ing activity. The insertional mutation does not affect the growth rate of the mutant relative to the wild-type strain (data not shown). We found that B. cenocepacia K56-2 caused hemagglutination of sheep red blood cells after 1 h incubation at room temperature, whereas the B. cenocepacia K56-2 (BCAM0223::Tp) showed a greater decrease of hemagglutination activity (Fig. 2_B). Effect of the BCAM0223 Mutation on Adherence to Extracellular Matrix Proteins and Biofilm Formation Ability of B. cenocepacia K56-2 A typical feature of TAAs is their ability to adhere to extracellular matrix (ECM) proteins and to promote biofilm formation [12]. Therefore, we examined the ability of the mutant B. cenocepacia K56-2 (BCAM0223::Tp) to bind immobilized human ECM components, such as laminin, fibronectin, collagen type I and IV and vitronectin. As shown in Fig. 3_A, compared with the wild-type B. cenocepacia K56-2, the BCAM0223-negative mutant adhered significantly less (P,0.001) to vitronectin. In contrast, the wild-type and the mutant did not display significant differences in adherence either to fibronectin, laminin or collagen I and IV (Fig. 3_A). We next determined the capacity of both wild-type B. cenocepacia K56-2 and B. cenocepacia K56-2 (BCAM0223::Tp) mutant to form biofilms in vitro on polystyrene surfaces after 24 and 48 h of incubation in LB medium at 37uC. As shown in Fig. 3_B, either after 24 h or 48 h incubation, the BCAM0223 mutant strain exhibited a statistically significant reduction in biofilm formation. PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e41747