For expression studies
of the trh-like genes in A. veronii isolates, total RNA was isolated from cells grown at the mid-log phase (OD600 nm=0.6) and the early stationary phase (OD600 nm=1) using TRIzol® LS reagent as per the manufacturer’s instructions (Invitrogen). Reverse transcription (RT)-PCR was performed using trh5 and trh6 primers for the detection of trh mRNA. Vibrio parahaemolyticus strain AQ4037 (trh+, tdh−) was used as a positive control. To show that the RNA preparation contains mRNA suitable for RT-PCR, normal metabolic gene gyrB was targeted using gyrB3F and gyrB14R primers to amplify a fragment of approximately 1100 bp (Yanez et al., 2003). All the three A. veronii isolates were TSA HDAC concentration positive for gyrB PCR, suggesting that the RNA preparation contains mRNA suitable for RT-PCR.
Further, to confirm the native expression, Trh-like hemolysin Western blotting was performed using Trh polyclonal antibodies developed in our laboratory. This antibody was developed by immunizing rabbits with a purified recombinant Trh protein of V. parahaemolyticus (Raghunath, 2008) by an intramuscular injection at 10-day intervals for 4 weeks consecutively. Animals were bled a week after the last dose by a cardiac puncture and antibody titers were determined ABT 199 using plate ELISA as described by Engvall & Perlman (1971). Aeromonas veronii isolates grown in LB broth at 37 °C overnight with shaking were harvested by centrifugation at 10 000 g for 10 min. Fifteen percent sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed on the lysed pellet as well as the supernatant (Laemmli, 1970). Western blotting was performed as per the procedure of Towbin et al. (1979). Trh-producing V. parahaemolyticus (AQ4037) was used as a positive control. In this study, a total of
44 isolates of Aeromonas spp. were screened for the presence of the trh gene. Among the Aeromonas spp. tested, only three clinical isolates of A. veronii (NT3818, NT3871 and VTE599) tested positive for PAK5 the presence of this gene, and the results of duplex PCR (Fig. 2) confirm that the negative reaction in other strains was not due to the inhibition of PCR. All other Aeromonas spp. including the remaining seven clinical isolates of A. veronii did not harbor this gene. A positive reaction with colony hybridization using a digoxigenin-labelled probe further confirmed the presence of the trh homolog in the three A. veronii isolates. To rule out the possibility of misidentification of these isolates, PCR targeting the toxR gene of V. parahaemolyticus was performed (Kim et al., 1999). All the three isolates were negative for this PCR, thus confirming that they are not atypical strains of V. parahaemolyticus. A gyrB sequence analysis of the three A. veronii isolates showed that they were highly similar to each other and had about 98% identity to the A. veronii biovar veronii gyrB sequences available in GenBank. In a recent study, Gonzalez-Escalona et al.