Short communication: Bacteriocin KC24 produced by Lactococcus lactis KC24 from kimchi and its antilisterial effect in UHT milk.

The severity of Listeria monocytogenes infections emphasizes the need for prevention or elimination of the pathogen from dairy products. Lactococcus lactis KC24, isolated from kimchi, exhibited an antimicrobial effect against food pathogens, including L. monocytogenes ATCC 15313. Lactococcus lactis KC24 was cultured in a 5-L jar fermenter at 35°C, and bacteriocin activity was maximal at 4 h of incubation and persisted for 20 h. Bacteriocin KC24 was inactivated by protease XIV, indicating that it has a proteinaceous nature. Bacteriocin activity was maintained at pH 3.0 to 9.0 and at temperatures of 50 to 121°C. The mode of inhibition against L. monocytogenes ATCC 15313 was shown to involve a bactericidal effect by treatment with 100 and 200 arbitrary units (AU)/mL of bacteriocin KC24. To test the activity of bacteriocin KC24 in a food product, bacteriocin KC24 and nisin (100 and 200 AU/mL) with 4 log cfu/mL of a mixed culture of L. monocytogenes (ATCC 15313, ScottA, H7962, and H7762) were applied to UHT milk. Compared with the control, treatment with bacteriocin KC24 completely inhibited the growth of L. monocytogenes and resulted in no detectable L. monocytogenes after 14 d at 4°C, whereas nisin moderately inhibited L. monocytogenes, resulting in a final concentration after 14 d at 4°C higher than the initial inoculum. Bacteriocin KC24 may prove useful in improving the safety of dairy products.

Identification of multicopy suppressors of the pcnB plasmid copy number defect in Escherichia coli.

Plasmids containing a ColE1 origin of replication are widely used for cloning purposes in Escherichia coli. Among the host factors that affect the copy number of ColE1 plasmids is the E. coli protein poly(A) polymerase I (PAP I), which regulates the intracellular level of RNA I, a ColE1-encoded negative regulator of plasmid replication. In strains that lack PAP I, RNA I levels are elevated, resulting in reduced levels of ColE1 plasmids in the cell. PAP I is encoded by the gene pcnB. We devised a genetic approach, based on the identification of multicopy suppressor clones, to identify trans-acting factors that can help offset the ColE1 plasmid copy number defect in a pcnB (-) genetic background. Using this strategy, we identified suppressors that mapped to two regions of the E. coli chromosome. The suppressor activity of one of the chromosomal regions was localized to the rssB gene, a response regulator gene known to be involved in the turnover of the stationary-phase sigma factor, RpoS. The second suppressor maps to min 55.4 of the E. coli chromosome, and the factor responsible for the suppressor activity appears to be a novel RNA or protein.

Impact of targeted vector design on Co/E1 plasmid replication.

The demands for recombinant proteins, in addition to plasmid DNA, for therapeutic use are steadily increasing. Bacterial fermentation processes have long been and still are the major tool for production of these molecules. The key objective of process optimization is to attain a high yield of the required quality, which is determined, to a large extent, by plasmid replication rates, metabolic capacity and the properties of the specific gene construct. When high copy number plasmids are used, the metabolic capacity of the host cell is often overstrained and efficient protein production is impaired. The plasmid copy number is the key parameter in the exploitation of the host cell, and can be maximized by optimal control of the flux ratios between biosynthesis of host cell proteins and recombinant proteins.

Characterization of pEC156, a ColE1-type plasmid from Escherichia coli E1585-68 that carries genes of the EcoVIII restriction-modification system.

The complete 4312-bp sequence of the pEC156 plasmid from Escherichia coli E1585-68, which carries genes encoding the EcoVIII restriction-modification (R-M) system, an isoschizomer of HindIII from Haemophilus influenzae, has been determined. Two clustered and convergently oriented open reading frames, large enough to encode genes of the EcoVIII R-M system, were found. The transcriptional start points were mapped by the primer extension method. The relative molecular masses of the EcoVIII endonuclease and EcoVIII methyltransferase deduced from the nucleotide sequence are 35,554 and 33,910, respectively. Nucleotide sequence analysis of pEC156 suggests that this plasmid is a ColE1-type replicon. It consists of an origin of replication and two untranslated genes encoding RNA I and RNA II, both involved in the regulation of plasmid DNA replication. The replication region also contains the gene encoding a 64-aa Rom-like protein. Inactivation of the putative rom gene by insertion of a kanamycin-resistance cassette resulted in 4.5-fold increase in pEC156-derived plasmid copy number in E. coli cells. All of these elements (RNA I, RNA II, and rom) reveal a high level of similarity to ColE1 homologs. The replication of all ColE1-type plasmids is dependent on the activity of E. coli DNA polymerase I. It was shown that a pEC156 derivative (pIB8) carrying an antibiotic resistance gene indeed failed to replicate in an E. coli polA12(ts) mutant at 43 degrees C, and its copy number was reduced in the E. coli pcnB80 mutant. These results prove that pEC156 is a ColE1-type replicon.

Isolation and characterization of a priB mutant of Escherichia coli influencing plasmid copy number of delta rop ColE1-type plasmids.

The lethality induced by the overproduction in Escherichia coli of a heterologous protein was used to select bacterial mutants. In one of these, the mutation responsible was mapped to priB. We describe the isolation of this mutant, the sequencing of the mutated gene, and its in vivo effect on plasmid replication.

Structure of the ColE1 DNA molecule before segregation to daughter molecules.

The segregation of daughter DNA molecules at the end stage of replication of plasmid ColE1 was examined. When circular ColE1 DNA replicates in a cell extract at a high KCl concentration (140 mM), a unique class of molecules accumulates. When the molecule is cleaved by a restriction enzyme that cuts the ColE1 DNA at a single site, an X-shaped molecule in which two linear components are held together around the origin of DNA replication is made. For a large fraction of these molecules, the 5' end of the leading strand remains at the origin and the 3' end of the strand is about 30 nucleotides upstream of the origin. The 3' end of the lagging strand is located at the terH site (17 nucleotides upstream of the origin) and the 5' end of the strand is a few hundred nucleotides upstream of the terH site. Thus the parental strands of the molecule intertwine with each other only once. When the KCl concentration is lowered to 70 mM, practically all of these molecules are converted to daughter circular monomers or to catenanes consisting of two singly interlocked circular units.