45-μm cellulose filter. One milligram of precipitated protein was dissolved in 100 μl of bacteriocin buffer (0.1 M Tris [pH 7.5], 0.01 M DTT, and 0.5 M MgCl2). To determine bacteriocin antibiotic activity, 100 μg/10 μl of the CaroS1K protein solution was added to an indicator plate containing Selonsertib order the Ea1068 strain growing on soft IFO-802 medium containing 0.65% agar. Growth inhibition zones at the point of addition were considered an indication

of Carocin S1 activity (Fig. 3). Figure 3 Analysis of the killing activity of purified Carocin S1. Intracellular solution was isolated from Hi-rif-8-6 (1) and TEW-7197 TH12-2 (3) strains. Extracellular solutions from Hi-rif-8-6 (2) and TH12-2 (4) strains were assayed for killing activity by addition to indicator plates containing strain Ea1068. Isolation of null alleles of the flhD, fliC, and flhA genes Since flagella assembly requires the expression of both the flhD and flhC genes,

we constructed the strain FlhD-KO (flhD::Kan). The linearized construct (containing the flhD::Kan DNA fragment) was transferred into H-rif-8-6, resulting in the homologous replacement of the native flhD gene PHA-848125 research buy and generating a null allele. The resultant kan and rif resistant transformants were screened by PCR with one set of primers (DY-SR1 and DY-SF1) representing the 5′ and the 3′ termini of the flhD/C operon. This set of primers generated a 1.3-kb product, if the transforming DNA was not integrated. Rapamycin in vitro However, a homologous replacement of the native flhD gene by the null allele yielded a 2.7-kb product. The observed PCR product was 2.7 kb, indicating that the flhD gene had been replaced by the null allele. The gene was therefore designated as ΔflhD (strain KH17). To confirm that Carocin S1 was actually secreted via T3bSS, we selected two components of T3bSS for deletion analysis, the fliC and flhA genes. The fliC gene encodes a FliC protein, which is an outer membrane component of T3bSS. The linearized construct (containing the fliC::Kan DNA fragment) was transferred into H-rif-8-6,

resulting in the homologous replacement of the native fliC gene and generating a null allele. The kan and rif resistant transformants were screened by PCR with one set of primers (fliC-sen and fliC-anti) representing the 5′ and the 3′ termini of the fliC operon. The gene was therefore designated as ΔfliC (strain FliC-KO). The flagellin-associated gene flhA encodes the inner membrane FlhA component of T3bSS. The same procedure was used to obtain the flhA knockout (KO) mutant, and the gene was designated ΔflhA (strain FlhA-KO). Complementation and analysis of flhD, flhC, fliC, and flhA genes Wild-type H-rif-8-6 was used as a control and transformed with plasmids containing the flhD (pBYL2D) and flhC (pBYL2C) genes as well as the flhD/C (pBYL2DC) operon. The effect of these transformations on the bacteriocin production and cell size of the wild-type strain was assessed.