The flhD/C DNA was detected as GW2580 mouse previously described. Construction of the null alleles of flhD, fliC, and flhA genes The flhD gene was isolated from pBYL2DC by digesting with BsmI, which cleaves at two sites in pBYL2DC and thereby conveniently deletes flhC from the operon. The resulting plasmid was designated pBYL2D. A kanamycin resistant gene from pACYC177 was isolated, made blunt-ended using a DNA-blunting kit (Takara Co., Tokyo, Japan), and inserted in the unique EcoRV site of the flhD gene. The resulting plasmid was designated pBYL2D-Kan. The pBYL2D-Kan

Selleck Nec-1s was re-isolated and linearized after HpaI and SspI restriction enzyme digestion, which deleted the ampicillin resistance gene and replication site of the plasmid. The linearized construct was transferred into H-rif-8-6, resulting

in the homologous replacement of the native flhD gene and generating a null allele. The DNA fragment of fliC was amplified by PCR from H-rif-8-6. After PCR amplification using two oligonucleotide primers (fliC-sen and fliC-anti), the partial fliC DNA fragment was purified, digested using AhdI and HindIII, and subcloned into plasmid pBR322 to generate the fliC plasmid. A kanamycin resistant gene from pACYC177 was isolated, made blunt-ended, and inserted into the unique SalI site of the fliC gene. The resulting plasmid was designated pfliC-Kan. The pfliC-Kan was linearized after AhdI and HindIII restriction enzyme digestion, which deleted the ampicillin resistance gene and replication site of the plasmid. The linearized construct HDAC inhibitor was transferred into H-rif-8-6 resulting in the homologous

replacement of the native fliC gene and generating a null allele. The DNA fragment of flhA was amplified by PCR from H-rif-8-6 using Molecular motor oligonucleotide primers flhA-sen and flhA-anti. The partial flhA DNA fragment was purified, digested using the restriction enzymes ClaI and EcoRI, and subcloned into plasmid pBR322 using T4 ligase to generate the flhA plasmid. A kanamycin resistant gene from pACYC177 was isolated, made blunt-ended, and inserted in the unique SalI site of the flhA gene. The resulting plasmid was designated pflhA-Kan. Computer analysis of sequence data The nucleotide sequence and the deduced amino-acid sequence of FlhD/C were compared using the BLAST and FASTA programs of the National Center for Biotechnology Information server. Sequence data were compiled by DNASIS-Mac software (Hitachi, Tokyo, Japan). RNA preparation and Northern hybridization Bacteriocin synthesis medium (BSM; 0.5% sucrose, 0.1% NH4Cl, 0.2% KH2PO4, and 0.02% MgSO4·7H2O [pH = 7.5]) was used to produce Carocin S1. Total RNA was extracted from cells (Pectobacterium carotovorum subsp. carotovorum harboring constructs) that were grown without drugs at 28°C. To determine the stability of H-rif-8-6, TH12-2, TH12-2/pBYL2C, KH17, and KH17/pBYL2D strains, culture samples (8 ml each; with rifampicin [0.