The mtfA and mtfB encode for an ABC-type I transporter system, and mdbA codes for a putative regulator. Microcin N has a bactericidal

activity against pathogenic strains, such as E. coli O157:H7, Salmonella enteritidis, and Salmonella enterica serovar Typhimurium, but it does not show antibacterial activity against strains of Campylobacter jejuni and Listeria monocytogenes (Wooley et al., 1999). Many properties of the microcin N system have not been characterized Tipifarnib order as yet, such as the spectrum of action against other bacteria, the identity of its receptor in the sensitive cell, its production kinetics, and the mechanism of action against the target cell. A key step in elucidating these properties is to purify microcin N to further perform biochemical and microbiological characterizations. In this work, we describe the DNA sequence of the microcin N genetic system, the purification and characterization of microcin N, and its expression pattern during bacterial growth. Escherichia coli DH5α was used as the indicator strain for the antimicrobial activity assays. The microcin N-producing strain used in all the experiments was E. coli MC4100 containing the

plasmid pGOB18. This plasmid is a pBR322 derivative that contains a fragment of 5.25 kb with microcin N genetic elements (O’Brien & Mahanty, 1994); mcnN and mcnI genes were amplified by PCR with primers IN1 (5′-CAA CAG ATT TAT CTG CTG GCC AGT-3′) and S2 (5′-TAT LDK378 nmr TCT ACC TTA ATG AAT CTT ATC CT-3′) and the PCR product was ligated to pGEM-T Easy (Promega Co., Madison, WI) to obtain pKAR. Table 1 summarizes the E. coli strains and plasmids used in this work. Plasmids pGOB18, pKAR, and pIN were purified using the EZNA Plasmid Minikit II (Omega Bio-Tek, Norcross, GA). The sequencing of the segment that encodes for the genetic elements that produce microcin N was carried out using the primer walking strategy, starting with a primer that anneals to the HindIII site of pBR322. Plasmids pIN

and pKAR were sequenced using the T7 and SP6 universal primers. The sequencing reactions Bcl-w were performed at Macrogen Co. (Seoul, South Korea). Liquid cultures of microcin N producer strains were grown in nutrient broth (Nut) (Difco, Franklin Lakes, NJ), Luria broth (LB) (MoBio, Carlsbad, CA), Müller–Hinton (MH) broth (Difco), and M63 minimal media supplemented with glucose (0.2%). For the antimicrobial-activity plate assay, the sensitive strain lawn (E. coli DH5α) was grown on nutrient agar (Difco). The antimicrobial assay was performed according to protocols described by Mayr-Harting et al. (1972). To prepare the sensitive strain lawn, 100-μL aliquots of a culture (OD600 nm∼0.6) were mixed with 4 mL of melted soft agar (0.7% w/v) and plated on nutrient agar. A culture of the strain E.