Cytochromes bd-I and bo3 are essential for the bactericidal effect of microcin J25 on Escherichia coli cells.

Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo3 was analyzed. The mutant strains lacking cytochrome bo3 or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo3 and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo3 are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25.

The antibacterial action of microcin J25: evidence for disruption of cytoplasmic membrane energization in Salmonella newport.

Microcin J25 (MccJ25) is a cyclic peptide of 21 unmodified amino acid residues produced by a fecal strain of Escherichia coli. It has previously been shown that the antibiotic activity of this peptide is mainly directed to Enterobacteriaceae, including several pathogenic E. coli, Salmonella and Shigella strains. In this paper we show that MccJ25 acts on the cytoplasmic membrane of Salmonella newport cells producing alteration of membrane permeability, and the subsequent gradient dissipation, that initiate the inhibition of process, such as oxygen consumption. These results, taken together with our in vitro observations [Rintoul et al. (2000) Biochim. Biophys. Acta 1509, 65-72], strongly suggest that the disruption of the cytoplasmic membrane gradient is closely related to the bactericidal activity of MccJ25 in S. newport.

Escherichia coli RNA polymerase is the target of the cyclopeptide antibiotic microcin J25.

Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in the rpoC gene, encoding the beta' subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-type rpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoC gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.

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

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.

Sequence analysis of the four plasmid genes required to produce the circular peptide antibiotic microcin J25.

A 4.8-kb plasmid region carrying the four genes mcjABCD necessary for production of and immunity to the cyclic peptide antibiotic microcin J25 (MccJ25) has been sequenced. mcjA encodes the primary structure of MccJ25 as a precursor endowed with an N-terminal extension of 37 amino acids. The products of mcjB and mcjC are thought to be involved in microcin maturation, which implies cleavage of McjA and head-tail linkage of the 21-residue pro-MccJ25. The predicted McjD polypeptide, which is highly similar to several ABC exporters, was found to be required for MccJ25 secretion, thus explaining its ability to confer immunity to MccJ25.

The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli.

Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non-SOS-dependent pathway [R. A. Salomon and R. Farias (1992) J. Bacteriol. 174, 7428-7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin-overproducing Escherichia coli strain by reverse-phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two-dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21-residue peptide, cyclo(-Val1-Gly-Ile-Gly-Thr- Pro-Ile-Ser-Phe-Tyr-Gly-Gly-Gly-Ala-Gly-His-Val-Pro-Glu-Tyr-Phe21- ). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe21-Val1 bond and led to a single peptide, MccJ25-L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 microgram.mL-1), the thermolysin-linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N-terminal extremity, the present study shows that removal of this extension and head-tail cyclization of the resulting propeptide are the only post-translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.

Escherichia coli outer membrane protein TolC is involved in production of the peptide antibiotic microcin J25.

A Tn5 insertion in tolC eliminated microcin J25 production. The mutation had little effect on the expression of the microcin structural gene and presumably acted by blocking microcin secretion. The tolC mutants carrying multiple copies of the microcin genes were less immune to the microcin. TolC is thus likely a component of a microcin export complex containing the McjD immunity protein, an ABC exporter.

Genetic analysis of plasmid determinants for microcin J25 production and immunity.

Microcin J25 (MccJ25) is a small peptide antibiotic produced by an Escherichia coli strain isolated from human feces. The genetic determinants for MccJ25 synthesis and immunity have been cloned from the low-copy-number wild-type plasmid pTUC1OO into the compatible vectors pBR322 and pACYC184. Physical and phenotypical analysis of insertion mutations and complementation tests defined three contiguous genes involved in MccJ25 production which span a region of about 2.2 kb. Immunity to the antibiotic is provided by an additional gene adjacent to the production region.

Fibrogenic potentials of coal slags used as abrasive blasting substitutes.

This study was designed to examine the fibrogenic potentials of four coal slags that are being used as substitutes for silica sand in abrasive blasting. Six groups of 100 male Sprague-Dawley rats, including four coal slag groups, a vehicle control, and a positive control for fibrosis (Minusil quartz), were used. Each dust treatment group was given a single 40-mg dose of test agent via intratracheal instillation. Interim sacrifices of 15 animals per group were performed at 2 d, 3 mo, and 6 mo posttreatment, with the terminal sacrifice conducted at 12 mo. Hematoxylin and eosin stained histologic sections were prepared from designated formalin-fixed tissues collected at each necropsy and examined microscopically. Pulmonary silicon analyses were performed for each group at the 2-d and 12-mo sacrifices. Pulmonary function analyses were conducted for each group at the 3-, 6-, and 12-mo sacrifices. Lung hydroxyproline analyses were conducted for 15 animals in each group at the terminal sacrifice. The pulmonary fibrogenic potentials of the four coal slag groups were compared histologically with the Minusil and vehicle controls. A mild to moderate interstitial fibrosis, which was progressive with time, was noted in each of the coal slag groups. However, the coal slag-induced lung fibrosis was much less than that produced by Minusil. Differences in fibrosis among the individual coal slags were relatively minor and certainly not as striking as those between the slags and Minusil. Other data derived from this study, such as lung hydroxyproline content, pulmonary particulate burdens, pulmonary function, and animal body weights, provided further evidence of a reduced toxicity for the coal slags compared to Minusil.

The peptide antibiotic microcin 25 is imported through the TonB pathway and the SbmA protein.

Selection of spontaneous mutants for insensitivity to the peptide antibiotic microcin 25 led to the isolation of five categories of mutants. Phenotypic and mapping studies showed the mutations to be located in the fhuA, exb, tonB, and sbmA genes. The latter encodes a cytoplasmic membrane protein which is also required for the penetration of microcin B17.

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Influence of iron on microcin 25 production.

The effect of the iron content of the medium on the yield of the peptide antibiotic microcin 25 was examined; synthesis was optimal in minimal media and was reduced by adding iron. Escherichia coli AY25, the wild-type producer of the antibiotic, showed a 95% decrease in microcin yield when grown in minimal medium containing 10 microM iron (high iron) as compared to 0.2 microM (low iron). Addition of chelators to Luria broth elicited microcin production, and there was a complete reversal of the effect of the chelators by adding iron. Studies with Escherichia coli mutants deficient in iron-regulated proteins (fur) suggested that factors other than Fur could mediate iron regulation of microcin synthesis.

The FhuA protein is involved in microcin 25 uptake.

A chromosomal Tn5 insertion resulting in complete resistance to the peptide antibiotic microcin 25 was mapped to the min 4 region of the Escherichia coli genetic map. Additional experiments showed that the insertion disrupted the fhuA gene, which encodes the multifunctional outer membrane receptor for ferrichrome, the antibiotic albomycin, colicin M, and bacteriophages T5, T1, and phi 80. Thus, microcin 25 and all of these agents share the same receptor.

Microcin 25, a novel antimicrobial peptide produced by Escherichia coli.

Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized. Composition analysis and data from gel filtration indicated that microcin 25 may contain 20 amino acid residues. It has a blocked amino-terminal end. Microcin synthesis and immunity are plasmid determined, and the antibiotic was produced in minimal medium when the cultures entered the stationary phase of growth. The peptide appears to interfere with cell division, since susceptible cells filamented when exposed to it. This response does not seem to be mediated by the SOS system.

Metabolism of nephrotoxic isopropylcyclohexane in male Fischer 344 rats.

The metabolism of isopropylcyclohexane and associated renal pathology were evaluated in male Fischer 344 rats exposed by oral gavage. The rats experienced moderate proximal tubular damage similar to that produced by acyclic, branched-chain hydrocarbons. The urinary metabolites of isopropylcyclohexane included cis-4-isopropylcyclohexanol, trans-4-isopropylcyclohexanol, 2-cyclohexylpropanoic acid, 2-cyclohexyl-1,3-propanediol, 2t-hydroxy-4t-isopropylcyclohexanol, 2c-hydroxy-4c-isopropyl-cyclohexanol, and 2c-hydroxy-4t-isopropylcyclohexanol. The extent and preferred sites of oxidative metabolism of nephrotoxic hydrocarbons could potentially prove useful in elucidating the pathogenic mechanisms.

The metabolism of t-butylcyclohexane in Fischer-344 male rats with hyaline droplet nephropathy.

The molecule t-butylcyclohexane is one of the first examples of a branched alkyl group attached to a hydrocarbon ring shown to be capable of producing renal damage at the corticomedullary junction of male rats. A metabolic study of t-butylcyclohexane yielded the following urinary metabolites: trans-4-t-butylcyclohexanol, 2c-hydroxy-4t-t-butylcyclohexanol, 2-methyl-2-cyclohexylpropanoic acid, 2c-hydroxy-4c-t-butylcyclohexanol, 2-methyl-2-cyclohexyl-1,3-propanediol, 2t-hydroxy-4t-t-butylcyclohexanol, and cis -4-t-butylcyclohexanol. As with other hydrocarbons of similar molecular weight that induce nephropathy in male rats, preferential sites of oxidative metabolism were observed that could potentially be related to the pathogenesis.

Phospholipid transfer activities in toad oocytes and developing embryos.

The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing 14C-labeled phospholipids and 3H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily after fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not be essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth.