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Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25.

ABSTRACT: Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by Escherichia coli. We have isolated lacZ fusions to mcjA (encoding the 58-amino-acid microcin precursor) and mcjB and mcjC (which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The mcjA gene was found to be dramatically induced as cells entered the stationary phase. Expression of mcjA could be induced by resuspending uninduced exponential-phase cells in spent supernatant obtained from an early-stationary-phase culture. Induction of mcjA expression was not dependent on high cell density, pH changes, anaerobiosis, or the buildup of some inducer. A starvation for carbon and inorganic phosphate induced mcjA expression, while under nitrogen limitation there was no induction at all. These results taken together suggest that stationary-phase induction of mcjA is triggered by nutrient depletion. The mcjB and mcjC genes were also regulated by the growth phase of the culture, but in contrast to mcjA, they showed substantial expression already during exponential growth. Induction of the microcin genes was demonstrated to be independent of RpoS, the cyclic AMP-Crp complex, OmpR, and H-NS. Instead, we found that the growth-phase-dependent expression of mcjA, mcjB, and mcjC may be explained by the concerted action of the positively acting transition state regulators ppGpp, Lrp, and integration host factor. Measurements of microcin J25 production by strains defective in these global regulators showed a good correlation with the reduced expression of the fusions in such mutant backgrounds.

SUBMITTER: Chiuchiolo MJ

PROVIDER: S-EPMC95062 | biostudies-literature | 2001 Mar

REPOSITORIES: biostudies-literature

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Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25.

Chiuchiolo M J MJ Delgado M A MA Farías R N RN Salomón R A RA

Journal of bacteriology 20010301 5

Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by Escherichia coli. We have isolated lacZ fusions to mcjA (encoding the 58-amino-acid microcin precursor) and mcjB and mcjC (which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The mcjA gene was found to be dramatically induced as cells entered the stationary phase. Expression of mcjA could be induced by resuspending ...[more]

PMID: 11160108

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Project description:The lasso peptide microcin J25 (MccJ25) possesses strong antibacterial properties and is considered a potential effective component of bacterial disease treatment drugs and safe food preservatives. Although MccJ25 can be heterologously expressed in Bacillus subtilis as we have previously reported, its regulation and accumulation are yet to be understood. Here, we investigated the expression level and stability of MccJ25 in B. subtilis strains with disruption in peptidase genes pepA, pepF, and pepT. Oligoendopeptidase F (PepF) was found to be involved in reduction of the production of MccJ25 by degradation of its precursor peptide. In the pepF mutant, the MccJ25 reached a concentration of 1.68 µM after a cultivation time exceeding 60 hours, while the wild-type strain exhibited a concentration of only 0.14 µM. Moreover, the production of MccJ25 in B. subtilis downregulated the genes associated with sporulation, and this may contribute to its accumulation. Finally, this study provides a strategy to improve the stability and production of MccJ25 in B. subtilis.ImportanceMccJ25 displays significant antibacterial activity, a well-defined mode of action, exceptional safety, and remarkable stability. Hence, it presents itself as a compelling candidate for an optimal antibacterial or anti-endotoxin medication. The successful establishment of exogenous production of MccJ25 in Bacillus subtilis provides a strategy for reducing its production cost and diversifying its utilization. In this study, we have provided evidence indicating that both peptidase PepF and sporulation are significant factors that limit the expression of MccJ25 in B. subtilis. The ΔpepF and ΔsigF mutants of B. subtilis express MccJ25 with higher production yield and enhanced stability. To sum up, this study developed several better engineered strains of B. subtilis, which greatly reduced the consumption of MccJ25 during the nutrient depletion stage of the host strain, improved its production, and elucidated factors that may be involved in reducing MccJ25 accumulation in B. subtilis.

Dataset Information

Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25.

Publications

Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25.

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