Chemical modification of microcin J25 with diethylpyrocarbonate and carbodiimide: evidence for essential histidyl and carboxyl residues

Bellomio, Augusto; Rintoul, María R.; Morero, Roberto D.

doi:10.1016/s0006-291x(03)00373-5

Cited by 24 publications

(28 citation statements)

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“…The biological activity of carbethoxylated MccJ25 was completely recovered after treatment with hydroxylamine, which gives the native MccJ25. Thus, it appeared that the polar histidyl residue is required for MccJ25 transport into the cell, its extrusion outside the cell, or RNAP inhibition (3). In the present paper, we offer evidence that the His 5 residue of the lariat ring is important for transport of the antibiotic into the cell through interaction with the inner membrane protein SbmA.…”

mentioning

confidence: 52%

“…To produce and purify the microcin variants, the mutant plasmids were transformed into a strain carrying the compatible plasmid pTUC341, containing the mcjBCD genes required for synthesis and export of the antibiotic (16). Native MccJ25 and MccJ25 analogues were purified by high-performance liquid chromatography as described previously (3). Amino acid substitutions in mutant peptides were confirmed by electrospray ionization mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

“…MccJ25 carries only two charged residues: a positively charged histidine (His 5 ) localized in the lariat ring and a negatively charged glycine (Gly 21 ) at the carboxyl terminus of the molecule. The two charged groups are close in the three-dimensional structure (12) and were shown to be important for MccJ25 activity (3). Amidation of MccJ25 Gly 21 specifically blocked RNAP inhibition but not transport inside the cell (17).…”

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confidence: 98%

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Microcin J25 Uptake: His⁵of the MccJ25 Lariat Ring Is Involved in Interaction with the Inner Membrane MccJ25 Transporter Protein SbmA

et al. 2006

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confidence: 52%

Section: Methodsmentioning

confidence: 99%

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confidence: 98%

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Microcin J25 Uptake: His⁵of the MccJ25 Lariat Ring Is Involved in Interaction with the Inner Membrane MccJ25 Transporter Protein SbmA

et al. 2006

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“…MccJ25 was purified from 2-liter cultures of strain MC4100 harboring pTUC202 as previously described (41). MccJ25 appeared homogeneous in two different systems of analytical reverse-phase high-performance liquid chromatography and showed a single peak of 2,107 Da on matrix-assisted laser desorption ionization-time of flight mass spectrometry (6). The chemical derivation and isolation of MccJ25-GA peptide was performed as described by Bellomio et al (6).…”

Section: Methodsmentioning

confidence: 99%

“…MccJ25 appeared homogeneous in two different systems of analytical reverse-phase high-performance liquid chromatography and showed a single peak of 2,107 Da on matrix-assisted laser desorption ionization-time of flight mass spectrometry (6). The chemical derivation and isolation of MccJ25-GA peptide was performed as described by Bellomio et al (6). Native MccJ25 and its amidated derivative were dissolved in methanol, and their concentrations were determined by absorbance at 278 nm (ε 278 ϭ 3,340 M Ϫ1 cm Ϫ1 ) (5).…”

Section: Methodsmentioning

confidence: 99%

Microcin J25 Has Dual and Independent Mechanisms of Action inEscherichia coli: RNA Polymerase Inhibition and Increased Superoxide Production

et al. 2007

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Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O 2˙؊ ) in bacterial cells, which could be the main reason for the damage produced on the membrane respiratory chain.MccJ25 is active on gram-negative bacteria related to the producer strain, including some pathogenic strains (43,44,55). Four plasmid genes, mcjABCD, are involved in MccJ25 production: mcjA, mcjB, and mcjC code for an MccJ25 precursor and two processing enzymes required for the in vivo synthesis of the mature peptide, respectively, and mcjD encodes the McjD immunity protein (53). McjD, a homologous ABC exporter family protein, participates in MccJ25 secretion (52). Thus, immunity is mediated by active efflux of the peptide, keeping its intracellular concentration below a critical level (53). Recently, it has been demonstrated that YojI, a chromosomal protein with ABC-type exporter homology (36), is also able to export MccJ25 from the cells (14). TolC, an E. coli outer membrane protein, is necessary for MccJ25 secretion mediated by either McjD or YojI (11,14). On the other hand, the uptake of MccJ25 by E. coli is dependent on the outer membrane receptor FhuA (15, 45) and the four inner membrane proteins TonB, ExbD, ExbB, and SbmA, the first three of which constitute the Ton complex (38), while the last one acts as a transporter (46).Convincing evidence showing that RNA polymerase (RNAP) is the target for MccJ25 action in E. coli was previously provided by our laboratory. The peptide inhibits the enzyme activity by obstructing the secondary channel and consequently preventing access of the substrates to its active sites (1,12,34,57). Later, it was demonstrated that MccJ25 can bind and penetrate into the phospholipid monolayer and disrupt the electric potential of liposomes composed of phospholipids from gram-negative bacteria (5, 40). These results encouraged the study of the effect of MccJ25 on the bacterial membrane. MccJ25 was found to disrupt the membrane potential inhibiting oxygen consumption in Salmonella enterica serovar Newport (41) and S. enterica serovar Typhimurium transformed with a plasmid carrying fhuA from E. coli (55), suggesting the presence of a se...

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Sequence Determinants Governing the Topology and Biological Activity of a Lasso Peptide, Microcin J25

Ducasse¹,

Yan²,

Goulard³

et al. 2012

ChemBioChem

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Microcin J25 is a potent antibacterial peptide produced by Escherichia coli AY25. It displays a lasso structure, which consists of a knot involving an N-terminal macrolactam ring through which the C-terminal tail is threaded and sterically trapped. In this study, we rationally designed and performed site-specific mutations in order to pinpoint the sequence determinants of the lasso topology. Structures of the resulting variants were analysed by a combination of methods (mass spectrometry, NMR spectroscopy, enzymatic digestion), and correlated to the antibacterial activity. The selected mutations resulted in the production of branched-cyclic or lasso variants. The C-terminal residues below the ring (Tyr20, Gly21) and the size of the macrolactam ring were revealed to be critical for both the lasso scaffold and bioactivity, while shortening the loop region (Tyr9-Ser18) or extending the C-terminal tail below the ring did not alter the lasso structure, but differentially affected the antibacterial activity. These results provide new insights for the bioengineering of antibacterial agents using a lasso peptide as template.

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Chemical modification of microcin J25 with diethylpyrocarbonate and carbodiimide: evidence for essential histidyl and carboxyl residues

Cited by 24 publications

References 17 publications

Microcin J25 Uptake: His⁵of the MccJ25 Lariat Ring Is Involved in Interaction with the Inner Membrane MccJ25 Transporter Protein SbmA

Microcin J25 Uptake: His⁵of the MccJ25 Lariat Ring Is Involved in Interaction with the Inner Membrane MccJ25 Transporter Protein SbmA

Microcin J25 Has Dual and Independent Mechanisms of Action inEscherichia coli: RNA Polymerase Inhibition and Increased Superoxide Production

Sequence Determinants Governing the Topology and Biological Activity of a Lasso Peptide, Microcin J25

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Chemical modification of microcin J25 with diethylpyrocarbonate and carbodiimide: evidence for essential histidyl and carboxyl residues

Cited by 24 publications

References 17 publications

Microcin J25 Uptake: His5of the MccJ25 Lariat Ring Is Involved in Interaction with the Inner Membrane MccJ25 Transporter Protein SbmA

Microcin J25 Uptake: His5of the MccJ25 Lariat Ring Is Involved in Interaction with the Inner Membrane MccJ25 Transporter Protein SbmA

Microcin J25 Has Dual and Independent Mechanisms of Action inEscherichia coli: RNA Polymerase Inhibition and Increased Superoxide Production

Sequence Determinants Governing the Topology and Biological Activity of a Lasso Peptide, Microcin J25

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Microcin J25 Uptake: His⁵of the MccJ25 Lariat Ring Is Involved in Interaction with the Inner Membrane MccJ25 Transporter Protein SbmA

Microcin J25 Uptake: His⁵of the MccJ25 Lariat Ring Is Involved in Interaction with the Inner Membrane MccJ25 Transporter Protein SbmA