23 Da (data not shown) The main focus of this work was to charac

23 Da (data not shown). The main focus of this work was to characterize the antimicrobial peptide microcin N. Microcins are

produced mainly in the stationary growth phase (Riley & Wertz, 2002), with the exception of microcin E492, which is produced during the exponential RAD001 supplier growth phase (de Lorenzo, 1984). Our results indicate that microcin N displays a behavior similar to that of microcin E492 in the exponential growth phase. However, its total activity does not diminish in the stationary phase (data not shown), showing a profile different from that of other bacteriocins, whose activities are expressed in the exponential or the stationary growth phase. Instead, the corrected microcin N activity has peaks in the exponential phase. The decrease in microcin N corrected activity observed in the late exponential phase could be http://www.selleckchem.com/products/PF-2341066.html related to a lower rate of translation or secretion, given that there was no difference between transcript levels in the two phases, using reverse transcription-PCR (data not shown). This could also explain the increase in corrected activity in the late stationary phase, owing to the continual accumulation of microcin N in the culture batch. Taking into consideration that all known microcins are soluble in organic solvents (Kolter & Moreno, 1992), hydrophobic reverse-phase columns (Sep-Pak C18) were utilized to concentrate and purify the microcin N from the supernatant

of the microcin N producer strain cultures. Microcin N was eluted using increasing concentrations

of methanol. As could be expected with a highly hydrophobic peptide, the microcin began to elute only from the fraction corresponding to 70% methanol. The high hydrophobicity of microcin N suggests a high propensity to form aggregates in aqueous solutions. Indeed, when microcin N was eluted with a more volatile solvent, such as acetonitrile, a tendency toward core aggregation was observed when the solvent evaporated (unpublished data). This property was also reported with extracts of microcin Edoxaban E492, which forms amyloid-like aggregates with cytotoxic properties on HeLa human tumor cells (Hetz et al., 2002). It remains unknown whether microcin N is capable of forming amyloid aggregates with cytotoxic properties, and so it would be interesting to study this effect on human cell lines. Tricine–SDS-PAGE performed on the fraction eluted from the Sep-Pak C18 column shows that a peptide of about 7 kDa was present in the fraction with microcin N activity (Fig. 3). The same results were observed when a second repurification step by HPLC was performed: a peptide of about 7 kDa was present in the fraction with microcin N activity (fraction between 15 and 21 min) (Fig. 4). The MS analysis of these fractions shows only one compound with a mass of 7274 Da, similar to the mass of other class II microcins, in agreement with mass determination by Tricine–SDS-PAGE.

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