A growth analysis with this strain was carried out in vz0825 supplemented LB-medium and in T-medium with different potassium and sodium ion concentrations (Figure 4). Overall, growth of the T283M Alvespimycin mutant was much less effected by vz0825 in comparison to the wild type strain. Sensitivity of the T283M mutant against compounds vz0500 and 1541–0004 did not differ from the wild type strain NM06-058 (data not shown). Figure 4 Growth determination. Growth of V. cholerae wild type strain NM06-058 (A) and the T283M exchange mutant (B) in the presence of vz0825 in media with different K+ and Na+ concentrations. Attempts to construct a kdpD knockout mutant For a further elucidation of the
effect of vz0825, the construction of a V. cholerae kdpD knockout HDAC inhibitor mutant was attempted. If KdpD is a major target of compound vz0825, the V. cholerae kdpD knockout mutant should be insensitive to the compound, unless the protein itself and its function are essential for the viability of the bacteria. The cloning procedure delivered the expected plasmid construct according to sequencing. The plasmid was successfully transformed into the
E. coli strain S17-1, according to the acquirement of ampicillin resistance, which is located on the plasmid pEX18Ap and also according to PCR amplification of the construct. The conjugation of the transformed E. coli with V. cholerae and the following selection on LB agar plates supplemented with carbenicillin (Carb) and Km did not lead to clones with learn more a deleted VC_A0531 gene, even after several modifications of the protocol. A possible explanation is that the gene product KdpD is indeed essential for V. cholerae, in agreement with KdpD being a prime target of vz0825. Discussion A HTS assay for small molecule inhibitors
of V. cholerae was developed and validated using a viability phenotype of V. cholerae that constitutively expresses green fluorescence. The assay is reliable, reproducible and simple to perform. Baricitinib During the development of the reporter strain, two reference strains of O1 serogroup belonging to biotypes O395 (classical) and N16961 (El Tor) were included along with the O139 strain MO10. The green fluorescence producing plasmid pG13 was electroporated into the three strains. During initial standardization experiments it was observed that the strain MO10 pG13 produced much greater level of green fluorescence as compared to other two strains (data not shown). For this reason strain MO10 pG13 was used in the screening experiments. A data bank search in SciFinder for the most active compounds vz0825 and vz0500 did not reveal pre-described antibacterial activities of the compounds with structural similarities above 70%. Compound 1541–0004, stemming from the commercial CDI collection, belongs to the group of styryl dyes, which have already in 1966 been shown to possess antimicrobial effects against the plant pathogen Xanthomonas oryza.