1 Kmr Apr; Cloning vector Invitrogen, USA pNQ705-1 Cmr; suicide vector with R6K origin [22] pNQ705-vah1 Cmr; for

insertional vah1mutation [8] pNQ705-plp Cmr; for insertional plp mutation This study pNQ705-rtxA Cmr; for insertional rtxA mutation [9] pDM4 Cmr SacBCr; suicide vector with R6K origin [11] pDM4-rtxA5′-rtxA3′ Cmr SacBCr; for allelic exchange rtxA mutation This study pSUP202 Cmr Apr Tcr; E. coli – V. anguillarum shuttle vector [21] pSUP202-vah1 Apr Tcr; for complementation of vah1 This study pSUP202-plp Apr Tcr; for complementation Sirolimus of plp This study pQE-30 UA Apr; expression vector with N-terminal His6-tag QIAGEN, USA pQE30UA-plp Apr; for expression of rPlp that FK506 molecular weight is used to make anti-Plp This study pQE60 Apr; expression vector with C-terminal His6-tag QIAGEN, USA pQE-60-plp Apr; for expression of rPlp for enzymatic activity analysis This study Table 2 Hemolytic activity of culture supernatant from V. anguillarum wild-type and various V. anguillarum mutant

strains against rainbow trout blood cells V. anguillarum strain or treatment Hemolytic activity (Relative to wild-type control ± SD)a M93Sm 1.00 (±0.12) JR1 (vah1) 0.98 (±0.16) XM21 (vah1+) 1.20 (±0.28) S262 (plp) 0.28 (±0.09)b XM31 (plp+) 0.99 (±0.04) S123 (rtxA) 0.94 (±0.22) JR03 (plp vah1) 0.14 (±0.09)b S183 (vah1 rtxA) 1.51 (±0.29) XM62 (vah1+ rtxA)

0.73 (±0.03) S187 (plp rtxA) 0.12 (±0.09)b XM90 (vah1 rtxA plp) −0.04 (±0.09)b XM93 (vah1 rtxA plp+) 1.33 (±0.01) Water (positive control) 1.15 (±0.16) aHemolytic activity assays carried out using the tube assay method as described in the Methods. Hemolysis by M93Sm was given the value of 1.00. The data are representative of two independent experiments, each with three replicates, ± one standard deviation (SD). bStatistically different from hemolytic activity for M93Sm (P < 0.05). In contrast to the strong hemolytic activity against 5% rainbow trout blood mixed with culture supernatant from the wild type strain M93Sm, hemolytic activity of culture supernatant from strain S262 (plp) declined by >70% (Table 2). Additionally, all mutants containing a knockout of plp exhibited significant Clomifene declines (P < 0.05) in hemolytic activity. The triple hemolysin JSH-23 mutant, XM90 (plp vah1 rtxA) had no ability to lyse fish erythrocytes (Table 2). However, mutations in either vah1 or rtxA, but not plp, resulted in little or no decline in hemolytic activity against fish erythrocytes compared to supernatants from wild type cells (Table 2). Further, complementation of plp restored the hemolytic activity of supernatants from both the plp-complemented strains (XM31, plp + and XM93, vah1 rtxA plp+) (Table 2).