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.     

Pyrophosphate increases the efficiency of enterobactin-dependent iron uptake in Escherichia coli.

Exogenous inorganic pyrophosphate increases the biomass yield of Escherichia coli. In this report, we show that the effect of pyrophosphate is related to iron uptake. We have found that addition of pyrophosphate, ammonium iron (III) citrate or iron (III) chloride, in M63 minimal medium containing 1.7 microM of iron, causes an increase in growth yield. In contrast to iron chloride or ammonium iron (III) citrate, exogenous pyrophosphate is deleterious to strains unable to synthesize enterobactin. Thus the positive effect of pyrophosphate is related to the enterobactin uptake system expressed in a low iron content medium. Pyrophosphate in minimal medium has a repressing effect on the expression of Fur-regulated genes. In iron rich medium where enterobactin synthesis is strongly decreased, addition of pyrophosphate increases expression of Fur-regulated genes. Furthermore, this latter regulatory effect of pyrophosphate in iron-rich medium is enhanced in the absence of enterobactin synthesis. It has also been shown that addition of pyrophosphate protects the cell against the oxidative stress caused by the presence of hydrogen peroxide in an iron-rich containing medium. These results indicate that pyrophosphate acts as an iron-chelating agent, could trigger the enterobactin-dependent iron uptake system and could promote an increased binding of iron to enterobactin.     

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.     

The activity of microcin E492 from Klebsiella pneumoniae is regulated by a microcin antagonist

Abstract Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae . Different growth conditions of the producer strain affect microcin activity. The production of a microcin antagonist is responsible for the changes in microcin activity. The microcin antagonist is induced when cells are iron-deprived, resulting in a low microcin activity. The microcin antagonist was purified using a procedure developed for the isolation of a catechol-type siderophore, and its activity was titrated using purified microcin. The inhibitory effect of the microcin antagonist is not observed when this compound is forming a complex with iron. The same inhibitory effect on microcin activity was obtained using purified enterochelin from Escherichia coli . The microcin antagonist was identified as enterochelin through thin-layer chromatography.     

The microcin J25 beta-hairpin region is important for antibiotic uptake but not for RNA polymerase and respiration inhibition

The antibiotic microcin J25 (MccJ25) was cleaved by hydrolysis with thermolysin giving a two-chain peptide (MccJ25-Th19) of 10 and 9 amino acid residues. MccJ25-Th19 with deep modifications in beta-hairpin region had no effect on Escherichia coli growth, but still inhibited RNA polymerase in vitro and oxygen consumption in Salmonella strains. MccJ25-Th19 showed antibiotic activity on E. coli transformed with plasmids containing either fhuA or sbmA genes, which code for proteins involved in MccJ25 transport. These results suggest that an intact beta-hairpin region is crucial for MccJ25 import but not for inhibition of E. coli RNA polymerase or oxygen consumption in Salmonella strains.     

Cloning of the determinants for microcin D93 production and analysis of three different D-type microcin plasmids

Three different microcin plasmids coding for D-type microcins were analyzed. Two of the plasmids (pMccD93 and pCP101) were small, multicopy plasmids and were closely related. The third plasmid (pCP106) was a conjugative, antibiotic multiresistance plasmid. Although plasmids pCP101 and pCP106 were previously classified as A-type microcin plasmids, we have determined that they are, in fact, D type. Furthermore, the determinants for microcin D93 production were cloned from plasmid pMccD93, and a DNA probe for the region implicated in the synthesis of microcin was obtained. This probe hybridized to plasmid C from Escherichia coli strain V517, indicating that this plasmid might be involved in the synthesis of a D-type microcin. The characteristics of replication of plasmid pCP106 were analyzed and appeared to be similar to those of ColEl plasmids, although pCP106 is a conjugative single-copy plasmid.     

Microcin R51 and a plasmid determining its synthesis

A new microcin produced by an Citrobacter R51 strain has been detected. This antibiotic has been shown to inhibit the growth of a number of Gram-negative as well as Gram-positive strains of bacteria on the minimal medium plates. The properties of partially purified microcin were characterized. Constitutive synthesis of microcin is determined by a conjugative plasmid. The genes of microcin synthesis and immunity were cloned on a plasmid and plasmid vehicles. A physical map of the 12 kb fragment coding for the production of microcin R51 and immunity to this antibiotic is presented.     

Inhibition of Salmonella enterica serovars by microcin J25

Escherichia coli microcin J25 (MccJ25) is a 2107-Da peptide antibiotic whose uptake into E. coli is mediated by the outer-membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. A survey of the sensitivity of several Salmonella enterica serovars showed that the antibiotic was highly active against some serovars, while S. Typhimurium, S. Derby, and some S. Enteritidis strains were completely resistant. Resistant strains became hypersensitive to MccJ25 when given the fhuA gene of E. coli, indicating that insensitivity is due to the inability of the FhuA protein to mediate penetration of MccJ25. Whereas in E. coli MccJ25 targets RNA polymerase, in S. Typhimurium it inhibits not only RNA synthesis but also cell respiration. Fluorescence viability staining showed that S. Typhimurium cells exposed to MccJ25 remain viable but are unable to form colonies.     

YojI of Escherichia coli functions as a microcin J25 efflux pump

In the present study, we showed that yojI, an Escherichia coli open reading frame with an unknown function, mediates resistance to the peptide antibiotic microcin J25 when it is expressed from a multicopy vector. Disruption of the single chromosomal copy of yojI increased sensitivity of cells to microcin J25. The YojI protein was previously assumed to be an ATP-binding-cassette-type exporter on the basis of sequence similarities. We demonstrate that YojI is capable of pumping out microcin molecules. Thus, one obvious explanation for the protective effect against microcin J25 is that YojI action keeps the intracellular concentration of the peptide below a toxic level. The outer membrane protein TolC in addition to YojI is required for export of microcin J25 out of the cell. Microcin J25 is thus the first known substrate for YojI.     

Protective action of ppGpp in microcin J25-sensitive strains

As Escherichia coli strains enter the stationary phase of growth they become more resistant to the peptide antibiotic microcin J25. It is known that starvation for nutrients such as amino acids or glucose leads to increases in guanosine 3',5'-bispyrophosphate (ppGpp) levels and that the intracellular concentration of this nucleotide increases as cells enter the stationary phase of growth. Therefore, we examined the effects of artificially manipulating the ppGpp levels on sensitivity to microcin J25. A direct correlation was found between ppGpp accumulation and microcin resistance. Our results indicate that the nucleotide is required to induce production of YojI, a chromosomally encoded efflux pump which, in turn, expels microcin from cells. This would maintain the intracellular level of the antibiotic below a toxic level.     

In vitro inhibition activity of different bacteriocin-producing Escherichia coli against Salmonella strains isolated from clinical cases

Aims:  To compare in vitro the inhibitory activity of four bacteriocin-producing Escherichia coli to a well-characterized panel of Salmonella strains, recently isolated from clinical cases in Switzerland.
Methods and Results:  A panel of 68 nontyphoidal Salmonella strains was characterized by pulsed-field gel electrophoresis analysis and susceptibility to antibiotics. The majority of tested strains were genetically different, with 40% resistant to at least one antibiotic. E. coli Mcc24 showed highest in vitro activity against Salmonella (100%, microcin 24), followed by E. coli L1000 (94%, microcin B17), E. coli 53 (49%, colicin H) and E. coli 52 (21%, colicin G) as revealed using a cross-streak activity assay.
Conclusions:  Escherichia coli Mcc24, a genetically modified organism producing microcin 24, and E. coli L1000, a natural strain isolated from human faeces carrying the mcb -operon for microcin B17-production, were the most effective strains in inhibiting in vitro both antibiotic resistant and sensitive Salmonella isolates.
Significance and Impact of the Study:  Due to an increasing prevalence of antibiotic resistant Salmonella strains, alternative strategies to fight these foodborne pathogens are needed. E. coli L1000 appears to be a promising candidate in view of developing biotechnological alternatives to antibiotics against Salmonella infections.     

Growth and siderophores production in <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis> is regulated by metal ions

During stationary phase of growth under low stress of iron in succinic acid medium, Alcaligenes feacalis BCCM ID 2374 produced microbial iron chelators. Increase in iron concentration supported bacterial growth but suppressed siderophores production, 1 μM and 2 μM of iron was optimum for maximum siderophore yield, i.e. 354 and 360 μg/ml in untreated and deferrated medium, respectively. Threshold level of iron, which suppressed siderophores production in A. feacalis BCCM ID 2374, was 20 μM. Ten micromoles and above concentration of CuCl₂ and CoCl₂, and 20 μM of MgCl₂, MgSO₄, ZnCl₂ and ZnSO₄ severely affected siderophores production.     

Absence of ZnuABC-mediated zinc uptake affects virulence-associated phenotypes of uropathogenic Escherichia coli CFT073 under Zn(II)-depleted conditions

In an effort to uncover the role of the high-affinity Zn(II) uptake system in uropathogenic Escherichia coli CFT073, we deleted the znuB gene, which encodes for the transmembrane component of the ZnuABC transporter system. The null mutant for znuB did not grow on minimal medium unless supplemented with excess Zn(II) (50 μM ZnCl₂). In contrast, the E. coli K-12 Δ znuB cell line grew well on minimal medium that was not supplemented with Zn(II). The Δ znuB mutant was significantly deficient in the formation of biofilm under static conditions and also showed a substantially reduced migration front of swarm cells. Because motility and biofilm formation are important for E. coli CFT073 pathogenicity, we propose that the high-affinity Zn(II) uptake system may contribute to the virulence of this pathogen in the urinary tract.     

Modulation of alcohol dehydrogenase isoenzyme levels in Zymomonas mobilis by iron and zinc.

Zymomonas mobilis is an unusual microorganism which utilizes both iron-containing alcohol dehydrogenase (ADHII) and zinc-containing alcohol dehydrogenase (ADHI) isoenzymes during fermentative growth. This organism is obligately ethanologenic, and alcohol dehydrogenase activity is essential. The activities of ADHI and ADHII were altered by supplementing growth medium with iron or zinc salts and by iron starvation. Growth under iron-limiting conditions (chelators, minimal medium) reduced ADHII activity but did not prevent the synthesis of the ADHII protein. The inactive form of this enzyme appeared quite stable, was not renatured by iron addition, and persisted in the cell. The iron-induced increase in ADHII activity required de novo synthesis which was blocked by antibiotic additions. The ability of Z. mobilis to synthesize ADHII and ADHI may be advantageous in nature.     

Molecular characterization of a DNA fragment carrying the basic replicon of pTUC100, the natural plasmid encoding the peptide antibiotic microcin J25 system

The Escherichia coli plasmid pTUC100 encodes production of, and immunity to, the peptide antibiotic microcin J25. In the present study, an approximately 8-kb fragment immediately adjacent to the previously sequenced microcin region was isolated and its DNA sequence was determined. The main features of the newly characterized region are: (i) a basic replicon which is almost identical to that of the RepFIIA plasmid R100; (ii) two ORFs with 96% identity to two ORFs of unknown function on pO157, a large plasmid harbored by enterohemorragic E. coli, and a large ORF which does not show significant homology to any other reported nucleotide or protein sequence; and (iii) two intact insertion sequences, IS1294 and IS1. Sequence analysis, as well as that of the G+C content of both the 8-kb fragment and the previously sequenced microcin locus, lead us to propose that plasmid pTUC100 is a composite structure assembled from DNA elements from various sources.     

Peroxide sensitivity of cold-shocked Salmonella typhimurium and Escherichia coli and its relationship to minimal medium recovery

Cold-shocked Salmonella typhimurium displayed minimal medium recovery (MMR), viable counts on M9 minimal agar being much higher than those on tryptone soya yeast extract agar (TSYA). The addition of catalase to TSYA restored counts to the level found on M9 agar. Peroxide concentrations between 12 and 30 μmol/1 were measured in TSYB but none was detected in M9 medium. Cold-shocked cells were sensitive to reagent hydrogen peroxide at a concentration similar to that found in TSYB. The minimal medium recovery phenomenon of cold-shocked cells is thus a manifestation of peroxide sensitivity. Changing the composition of growth media affected both cellular catalase activity and the magnitude of the MMR effect but the two properties were not directly related. Factors additional to cellular catalase activity must therefore affect susceptibility to peroxide following cold shock. Muta tional loss of catalase, exonuclease III or recA -dependent DNA repair functions all increased the sensitivity of cold-shocked Escherichia coli to the inhibitory effects of peroxide present in rich medium. The peroxide resistant fraction of a cold-shocked population of Salm. typhimurium (i.e. those cells able to grow on TSYA) was more resistant to gamma radiation than the population as a whole. Cold shock thus sensitizes cells to more than one form of oxidative stress. Prior exposure of growing cells to 30 μ mol/1 hydrogen peroxide abolished their sensitivity to rich medium following cold shock implying that Salm. typhimurium contains an inducible system protecting against oxidative stress.     

Effect of auxin on alkaloids, K+ and free amino acid content in cultured tobacco callus

Callus cultures derived from the petiole of Nicotiana tabacum L. cv. Burley 21 were grown at 25°C in the dark on two different basal media containing: (1) 11.5 μ M α-naphthaleneacetic acid and 1 μ M kinetin, and (2) 1 μ M α-naphthaleneacetic acid and 1 μ M kinetin. The contents of alkaloids, K⁺ and free amino acids of callus tissues were determined. The tissues were also examined microscopically for organization when organogenesis was not apparent. The first medium limited nicotine synthesis and stimulated its N-demethylation to nornicotine. The second medium stimulated nicotine synthesis and limited tissue growth. Although significantly higher concentrations of K⁺ were observed in calli grown on the high-auxin medium, both cultures were K⁺ deficient. The fact that the low-auxin medium limited K⁺ uptake to a higher degree would account for the lower growth observed in calli cultured on this medium, and it is possible that the effect of auxin concentration on nicotine production may be mediated through its effects on K⁺ uptake by cells of the culture. The free amino acid concentration increased in the calli grown on the low-auxin medium. Glutamic acid and proline, known as initial precursors of nicotine, increased significantly. Histological examination showed that the occurrence of meristematic areas in calli without organogenesis promoted nicotine synthesis. The relation between the accumulation of nicotine and formation of roots or shoot-buds is discussed.     

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

In this paper we compared the antibacterial activity of native microcin J25, a peptide antibiotic, with the activities of two analogues obtained by chemical modifications. In the first analogue, the negative charge of glutamic carboxyl group was specifically blocked with an L-glycine methyl ester and in the second the histidine imidazole ring was carbethoxylated. Both analogues decreased notably its antibiotic activity against Escherichia coli and Salmonella newport, strains sensible to the native microcin J25. The biological activity of the carbethoxylated analogue was completely recovered after treatment with hydroxylamine. The extreme importance of both polar residues could be interpreted as specific structural features indispensable for the peptide transportation into the cell, extrusion outside the cell or alternatively to inhibit the RNA-polymerase.