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

Abstract

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...

Protective Action of ppGpp in Microcin J25-Sensitive Strains

“…Thus, MccJ25 would inhibit transcription by clogging the conduit, thereby blocking the access of the substrates to the active center (1,25). Recently, we have found that in E. coli cells overproducing the microcin receptor, FhuA, the antibiotic also targets the respiratory chain and inhibits cell respiration (5). Although the mechanistic details of this action have yet to be defined, it seems to result from an increased superoxide production.…”

Sensitization of Microcin J25-Resistant Strains by a Membrane-Permeabilizing Peptide

“…Additionally, the inner membrane protein SbmA transports MccJ25 from the periplasmic to the cytoplasmic space (13). Once inside the sensitive cell, the peptide is able to inhibit E. coli RNA polymerase (RNAP) and membrane respiratory chain, which represent the MccJ25 targets (2,5,7,18). Several Salmonella enterica serovars showed high sensitivity against MccJ25, while others, like Salmonella enterica serovar Typhimurium, S. enterica serovar Derby, and some S. enterica serovar Enteritidis strains were completely resistant (16).…”

Potential Applicability of Chymotrypsin-Susceptible Microcin J25 Derivatives to Food Preservation

“…MccJ25 is active on gram-negative bacteria related to the producer strain, and among them are several human pathogens (11,12,16). It was previously shown that the E. coli RNA polymerase (5,18) and the bacterial respiratory chain (2,9) are the targets for MccJ25 action. MccJ25 is active on pathogenic strains of Salmonella spp., Shigella spp.…”