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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, November 2001, p. 3128-3131, Vol. 45, No. 11
Sección de Fisiología y
Genética Bacterianas, Facultad de Ciencias, Iguá 4225, Montevideo 11.400, and División Biología Molecular,
Unidad Asociada a Facultad de Ciencias, Instituto de
Investigaciones Biológicas Clemente Estable, Avenida Italia
3318, Montevideo 11.600, Uruguay
Received 22 March 2001/Returned for modification 27 June
2001/Accepted 14 August 2001
Microcin H47 is a gene-encoded peptide antibiotic produced by a
natural Escherichia coli strain isolated in Uruguay. In
order to identify cellular components necessary for its antibiotic
action, microcin H47-resistant mutants isolated in this work, as well as previously described mutants affected in membrane proteins, were
analyzed. These studies indicated that (i) receptor outer membrane
proteins for ferric-catechol siderophores would be involved in
microcin-specific binding to the cell surface, (ii) the TonB pathway is
needed for microcin H47 uptake, and (iii) the presence of the ATP
synthase complex is necessary for microcin action. The possibility that
this last structure contains the antibiotic target is discussed.
Microcins are a group of
antibiotic peptides produced by strains of gram-negative bacteria. They
are synthesized as gene-encoded precursors that thereafter undergo
posttranslation modifications, thus converting to mature molecules. In
general terms, they are considered to be narrow-spectrum antibiotics
that act against gram-negative bacteria, some microcins being
bactericidal while others are bacteriostatic (3, 17, 19).
Although at present only a few microcins have been thoroughly studied,
it is already clear that they exhibit a noteworthy diversity in their
mechanism of action. To exert their deleterious effect on sensitive
cells, they have to transit two steps: microcin uptake and interaction
with their targets. In some cases, uptake has been described as
starting by binding of microcin to a receptor protein in the cell
surface; the following outer membrane proteins were described as being
involved in such interactions: Cir for colicin V, the porin OmpF for
microcin B17, and FhuA for microcin J25 (8, 19, 20, 34).
To pass through the outer membrane barrier, the TonB pathway is used by
several microcins, e.g., E492, J25, and colicin V (28,
35). Finally, microcins B17 and J25 need the presence of a
cytoplasmic membrane protein, SbmA, to complete their uptake (20,
35).
It is the final interaction with their targets that points to microcins
as antibiotics with interesting modes of action: colicin V and microcin
E492 disrupt membrane potential (9, 42), microcin B17
inhibits DNA replication by irreversibly trapping DNA gyrase-DNA complexes (38), microcin C7 inhibits protein synthesis
(13), and microcin J25 was proposed to interfere with cell
division (33).
Here we present a study on the mode of action of microcin H47 (MccH47).
MccH47, which was found to be produced by a natural Escherichia
coli isolate, inhibits the growth of gram-negative strains. The
MccH47 genetic system was located in the chromosome, with an extension
of ca. 10 kb (21). In brief, it comprises four genes
involved in microcin synthesis: the microcin structural gene, two
further genes devoted to microcin secretion, and an immunity gene whose
product protects the cell against its own antibiotic production
(2, 12, 30, 31). MccH47 is ribosomally synthesized as a
peptide precursor which already possesses antibiotic activity, detected
as a toxic effect in cells carrying the microcin structural gene
mchB and lacking the remaining mch determinants. Although cells with such a genetic construction were nonviable, some
mutant clones grew which exhibited Atp Bacterial strains, plasmids, and bacteriophages.
The
strains, plasmids, and bacteriophages used in this study are listed in
Table 1.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.11.3128-3131.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
ATP Synthase Is Necessary for Microcin H47
Antibiotic Action
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
and
MccH47-resistant (MccH47r) phenotypes
(31). These results suggested that ATP synthase could be
involved in microcin action on susceptible cells. The present work
deals with the MccH47 mode of action and presents results that point to
ATP synthase as the possible target of this antibiotic.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
TABLE 1.
Bacterial strains, bacteriophages, and plasmids
Media and culture conditions. Luria-Bertani (LB)-rich medium, R medium for phage lysates, and M63 minimal medium supplemented either with glucose or succinate were used (24). Antibiotics were added to media at the following final concentrations: ampicillin (Ap), 50 µg/ml; chloramphenicol (Cm), 30 µg/ml; kanamycin (Km), 30 µg/ml; and tetracycline (Tc), 12 µg/ml. To prepare iron-chelated plates, ethylenediamine DI(o-hydroxyphenylacetic acid) (EDDHA) was added at a final concentration of 50 µM. All strains were grown at 37°C, except for those carrying Mucts, which were grown at 30°C.
M63 glucose plates containing MccH47 were prepared as follows: the M63 plates were covered with a cellophane membrane, and about 107 E. coli RYC1000(pEX100) cells, which overproduce MccH47, were poured above with soft agar. After 24 h of incubation, the cellophane with the producing lawn was removed and the plates were exposed to chloroform vapors to kill cells that could have remained. These plates contained MccH47 that had passed through the cellophane membrane. These MccH47-containing plates were then used for selecting MccH47-resistant clones.Microcin, colicin, and bacteriophage 
80 sensitivity
assays.
The cross-streaking method was employed on LB plates: a
suspension of the microcin- or colicin-producing strain was seeded as a
band and incubated during 24 h; the clones to be assayed were then
cross-streaked, and the plates were further incubated. Sensitive clones
were unable to grow in the vicinity of the producing band, while growth
of resistant clones was not inhibited. The sensitivity to phage 80
was also assayed by cross-streaking. Mutants' sensitivity to MccH47
was rapidly screened by the cross-streaking method, and thereafter,
selected mutants were analyzed with more precision by a patch test
(29). This test was performed by seeding a lawn of each
strain to be assayed on minimal glucose plates and then stabbing the
lawn with a MccH47-overproducing strain, RYC1000(pEX100). After
incubation, halos of growth inhibition appeared when the strain assayed
was sensitive to MccH47, the level of sensitivity being estimated by
the diameter of the zone of inhibition.
Genetic techniques.
P1 transduction was performed as
described previously (24). Six independent Tn5
mutagenesis experiments were carried out by infecting BZB1011 with
phage 
467 as described previously (4).
MccH47r Tn5 insertion mutants were
selected on M63 glucose MccH47 Km plates.
MudII4042 in vivo cloning. The Tn5 insertion in FGB64 was cloned in vivo from the chromosome by means of lytic propagation of phage MudII4042 in FGB64 as described previously (14). For this purpose, FGB64 was first lysogenized with MuctsApr and then transformed with the MudII4042-containing plasmid pEG109, and the resultant strain was induced for phage propagation. The transducing phage lysate was used to infect MC4100 MuctsApr, and transductant clones were selected on LB plates containing Cm and Km.
Manipulation and sequencing of DNA. Routine DNA manipulations and plasmid vectors used were described previously (36). DNA sequence was elucidated by applying the chain termination method to double-stranded DNA using the Sequenase 2.0 kit from United States Biochemicals (Cleveland, Ohio) (37). Universal and reverse sequencing primers were used.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Isolation and preliminary characterization of MccH47-resistant
mutants.
Strain BZB1011 was mutagenized with transposon
Tn5 as described in Materials and Methods.
Kmr MccH47r mutants
appeared at a frequency of 105 of the viable
clones. Most mutants exhibited normal growth, similar to that of
BZB1011, while others grew poorly in LB and M63 glucose media. Their
level of MccH47 resistance was evaluated by a patch test as described
in Materials and Methods, finding two phenotypes: partially resistant
mutants and highly resistant mutants. The diameter of the growth
inhibition halos of the partially resistant mutants presented a mean
value of 6 mm, whereas no inhibition was observed with the highly
resistant mutants. In the same assays, the diameter of inhibition of
BZB1011 was 19 mm, on average.
tonB mutants.
Clones from the first group were
resistant to colicins M and V, which have in common the use of the TonB
pathway for their uptake (28). Since bacteriophage 80
is known to require the presence of TonB to infect cells
(27), 16 mutants were analyzed and were found to be
insensitive to this phage. The tonB gene maps to minute 28 of the E. coli K12 chromosomal map, near the trp
locus. The P1 transducing phage was propagated on four
MccH47r independent mutants from this group, and
the lysates were used to infect the trpA
MccH47s strain KS463, selecting prototrophic
transductant clones on minimal M63 glucose plates. Transductants from
each experiment were assayed for their sensitivity to MccH47, and 38 to
67% were resistant to this antibiotic. When tested for their
sensitivity to kanamycin, a strict correlation was found between the
MccH47r and Kmr phenotypes.
These results indicated that mutations conferring microcin resistance
mapped near trpA and that they had occurred by
Tn5 insertion. Finally, the plasmid pIM91, carrying only the tonB gene, restored microcin H47 sensitivity when introduced
into the same four mutant strains. One of these strains was selected as
the prototype of this group and was called FGB11. In sum, mutants from
the first group were shown to be TonB deficient, indicating that MccH47
makes use of the TonB pathway to cross the outer membrane of sensitive cells.
exb mutants.
Mutants classified in the
second group were resistant to colicin M and partially resistant to
MccH47. They also were sensitive to bacteriophage 80. This phenotype
could correspond to mutants affected in fhuA, which encodes
the outer membrane receptor of colicin M and of the iron carrier
ferrichrome (8, 16), or in exb, a locus
comprising two genes, exbB and exbD, whose
products accomplish ancillary functions in the process of active
transport through the outer membrane mediated by TonB (1).
A well-known fhuA deletion mutant, H1857 (6),
proved to be sensitive to MccH47, thus ruling out FhuA involvement in
MccH47 uptake. On the contrary, the exb strain W3110-6
(11) presented a partial level of resistance to this
antibiotic, similar to that of MccH47r mutants
under study. The plasmid pCH9, carrying only the exbBD genes, was introduced into four MccH47r mutants
from the second group, and the transformant clones that resulted were
sensitive to MccH47. One of these mutants was chosen as the prototype
of this group and was called FGB46. Therefore, we conclude that these
mutants are affected in the exb locus, a finding that
further supports the need for the TonB pathway for MccH47 uptake.
atp mutants. Mutants from the third group were characterized by their high level of resistance to MccH47 and by their poor growth on rich and minimal glucose media. P1 transducing phage was grown on one of these mutants, and the lysate was used to infect BZB1011, selecting clones on LB plates supplemented with kanamycin. All of the resulting transductants were MccH47r, thus confirming that the Tn5 insertion, now called Tn5 64, was responsible for the MccH47r phenotype. One of these transductants was named FGB64 and was used in further studies.
To identify the locus affected in FGB64 by the Tn5 64 insertion, this mutation was cloned in vivo using the chimerical phage MudII4042, as described in Materials and Methods. Transductants of strain MC4100 MuctsApr should carry recombinant plasmids containing MudII4042 DNA, part or all of the Tn5 sequence, and presumably, chromosomal DNA adjacent to the transposon in FGB64. Plasmids from three transductant clones were extracted and physically analyzed with HindIII (data not shown). Their restriction pattern indicated that a HindIII fragment of ca. 1,350 bp would contain one of the ends of the Tn5 insertion. This fragment was cloned into the HindIII site of the pUC13 vector, and the recombinant plasmid obtained was used to sequence the ends of the insert. On one side, 170 nucleotides were read before reaching the Tn5 end. This sequence was compared with those in databases using the program FASTA (25) and was found to correspond to the atp operon. The Tn5 insertion was located after nucleotide 1038 of the reported atp sequences (41), interrupting the sixth codon of the atpB gene, which is the first gene of the atp operon coding for a polypeptide of the ATP synthase complex. Mutants affected in the atp locus are impaired for oxidative phosphorylation since they encode abnormal or no ATP synthase. They can grow in most media, albeit poorly, thanks to substrate-level phosphorylation. It is known that deficient atp mutants are unable to grow in minimal media supplemented with a nonfermentable carbon source, as is the case with succinate (5). FGB64 and 11 other MccH47r mutants from the third group were seeded on M63 succinate plates and did not grow, exhibiting an Atp phenotype. The plasmid pBJC917, carrying
the atp operon, was introduced by transformation into these
12 mutants. The strains constructed were MccH47s
and Atp+, i.e., the atp operon
complemented for both phenotypes. Thus, the atp locus
appeared to be involved in conferring MccH47 sensitivity. Other
atp strains previously described, such as CM1470
recA (40), which bears a deletion in the
atp operon, were assayed with MccH47 and were found to be resistant.
The MccH47 structural gene mchB encodes a peptide precursor
that possesses antibiotic activity of the same specificity as that of
mature microcin. Cells transformed with the recombinant plasmid pMVD10
had been found to be nonviable (31). This plasmid carries
an mchB-phoA gene fusion whose product retains antibiotic activity exerted as a toxic effect on the plasmid-bearing cells. pMVD10
was now used to transform four MccH47r
Atp mutants from the third group, including
FGB64, as well as BZB1011 (wild type), FGB11 (tonB), and
FGB46 (exb), selecting the transformant clones on LB
supplemented with chloramphenicol. Abundant colonies appeared in the
experiments with mutant Atp strains, while no
clones grew in the remaining plates. These results showed that the
atp mutants analyzed are resistant to the toxic
intracellular effect exerted by the MccH47 precursor, besides being
resistant to extracellular microcin. On the contrary, tonB
and exb mutants only resisted MccH47 coming from outside, which is the expected phenotype for uptake mutants. Therefore, the
presence of ATP synthase would be necessary for MccH47 action, although
this complex does not appear to be involved in microcin uptake. The
present results lead us to consider ATP synthase as the possible target
of MccH47.
cir, fiu, and fepA mutants. MccH47r mutants so far isolated were not affected in any outer membrane determinant, a fact that could indicate that this antibiotic does not need to interact with a surface receptor as a first step in its uptake. However, a triple mutant receptor strain, H1876 (cir fiu fepA), was found to be completely resistant to MccH47. These genes code for iron-regulated outer membrane proteins which interact with the TonB pathway: FepA is the ferrienterochelin receptor, while Cir and Fiu are both receptors for the ferric complexes of the enterochelin-related compounds dihydroxybenzoylserine and dihydroxybenzoate; FepA also participates in dihydroxybenzoylserine recognition (15). In our case, mutants affected in one or two of these loci remained sensitive to MccH47, although a double-mutant fiu cir strain, H1728, displayed a slightly decreased sensitivity. These results suggest that MccH47 indeed interacts with outer membrane receptors but that any of the three catechol receptors, FepA, Fiu, or Cir, would be able to accomplish this function. This could explain our failure in the isolation of MccH47 receptor mutants.
Since production of FepA, Fiu, and Cir is induced under limited-iron conditions (10), the sensitivity of strain BZB1011 to MccH47 was assayed with and without the addition of the iron chelator EDDHA. The critical dilution method was employed as described in Materials and Methods. A fourfold increase in the sensitivity of BZB1011 was detected under low-iron conditions compared to results of the assay in the absence of EDDHA. This result is in accordance with the finding that the iron-regulated catechol receptors are involved in uptake of MccH47. The presented analyses on the MccH47 mode of action indicate that this peptide antibiotic probably starts its action by binding to the outer membrane receptor proteins FepA, Fiu, or Cir and then crosses the outer membrane through the TonB pathway. In addition, ATP synthase was found to be required for MccH47 action. atp mutants resisted both mature MccH47 proceeding from the external milieu and the intracellular toxic activity of the MccH47 precursor. This phenotype characterizes target mutants, pointing to the possibility that ATP synthase could be the subcellular structure where MccH47 exerts its final antibiotic effect. Previous results on MccH47 immunity, a function clearly assigned to an integral membrane peptide, are in accordance with a membrane localization of the MccH47 target (30). Several antifungal natural products that inhibit the eukaryotic mitochondrial FoF1-ATP synthase have been described, including the well-known polyketide oligomycin (26, 32). However, there are no precedents of antibiotics targeted on the prokaryotic ATP synthase complex. The results presented here suggest that ATP synthase could be the target of microcin H47. If this were the case, it would be the first description of an antibacterial effect exerted through interaction with this subcellular complex. More studies will be needed to test this hypothesis.| |
ACKNOWLEDGMENTS |
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This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas "Fondo Clemente Estable" grant 4059, Comisión Honoraria de Lucha Contra el Cáncer, International Foundation for Science grant F/1495, and European Community grant CI1*-CT92-0011. M. Trujillo and E. Rodríguez were Fellows of the Programa de Desarrollo de las Ciencias Básicas (PEDECIBA).
We thank Klaus Hantke for generously supplying strains affected in ferric iron transport and plasmids pIM91 and pCH9. We are also indebted to María Parente for excellent technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Sección de Fisiología y Genética Bacterianas, Facultad de Ciencias, Iguá 4225, Montevideo 11.400, Uruguay. Phone: (5982) 525 86 18, ext. 143. Fax: (5982) 525 86 29. E-mail: magela{at}fcien.edu.uy.
Present address: Arthur and Rochelle Belfer Gene Therapy Core
Facility, Weill Medicine College, Cornell University, New York, NY 10021.
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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