Leiden Institute of Chemistry, Gorlaeus
Laboratories, Leiden University, 2300 R.A. Leiden, The
Netherlands1 and Flanders
Interuniversity Institute of Biotechnology, Department of
Immunology, Paracytology and Ultrastructure, Vrije Universiteit
Brussel, B-1640 Sint Genesius Rode, Belgium2
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INTRODUCTION |
The Mn cycle in nature is
strongly determined by the redox state of the metal. Generally,
reduced Mn [Mn(II)] forms soluble salts, and oxidized Mn [Mn(III and
IV)] precipitates as highly insoluble oxides and oxyhydroxides.
Interconversions between reduced and oxidized forms are
usually catalyzed by microorganisms, which strongly
influence the Mn cycle (20).
Work in our laboratory has focused on the process of bacterial
Mn2+ oxidation, which is a widespread phenomenon that
occurs in many different environments and is catalyzed by a variety of
microbial species. Many different oxidation mechanisms are
recognized, including indirect mechanisms (which act through
changes in the pH or Eh of the environment) and direct
mechanisms (which involve the mediation of macromolecules, such as
proteins or protein-polysaccharide complexes). The fact that
macromolecules are directly involved in Mn2+ oxidation
suggests that this process has a physiological function, but its
functional significance remains unclear, even though several possibilities have been suggested. For example, Mn2+
oxidation may supply energy for growth (19, 28) or may be involved in scavenging of harmful oxygen species (4, 29), or
accumulated Mn oxides may serve as terminal electron acceptors that
support anaerobic growth (15, 16, 18). Other possible functions of Mn2+ oxidation include improving competition
for dissolved Mn2+, prolonging the viability of
manganese-encrusted cells (1), and lysing of complex
humic substances in order to provide small organic substrates for
growth (35). A major difficulty in elucidating the
mechanisms and functional significance of bacterial
Mn2+ oxidation stems from the diversity of the species
capable of Mn2+ oxidation. Common properties and unifying
principles that underlie the oxidizing processes were not found until recently.
Recent application of molecular genetic techniques appears to have
revealed a common element of the Mn2+-oxidizing
systems of two bacterial species. Leptothrix discophora SS-1, a freshwater proteobacterium, secretes an
Mn2+-oxidizing factor into media (2, 7). A
putative L. discophora SS-1 operon was identified in
which one of the genes, which supposedly encodes a structural component
of the oxidizing factor, encodes a protein homologous to multicopper
oxidases (13). Bacillus sp. strain SG-1, a
marine gram-positive spore-forming bacterium, produces spores
that are capable of oxidizing Mn2+ (16, 33). A
sporulation-dependent operon of SG-1 was found to encode,
inter alia, a protein homologous to several multicopper oxidases,
and transposon mutagenesis of this operon resulted in a
non-Mn-oxidizing phenotype (38).
Pseudomonas putida is a freshwater proteobacterial species,
and two strains of this species (MnB1 and GB-1) have been
shown to oxidize Mn2+ (14, 22, 31). When
supplied with Mn2+, the cells deposit Mn oxide outside the
outer membrane in the early stationary growth phase (31).
The oxidation appears to be catalyzed by an enzyme (31).
Transposon mutagenesis of these two P. putida strains
has yielded several mutants that are defective in Mn2+
oxidation or in secretion of the oxidizing factor(s) across the outer
membrane (8, 10, 17). An analysis of a number of these mutants indicated that cytochrome c is involved
in the oxidation of Mn2+ and that the specific protein
secretion pathway is involved in transport of the oxidizing factor.
Analysis of another nonoxidizing transposon mutant of strain
GB-1 localized the mutation in a partially sequenced open
reading frame (ORF) encoding, inter alia, two consensus
Cu2+-binding regions (17). This
finding suggests that GB-1 and other Mn2+-oxidizing
pseudomonads also depend on a Cu2+-binding protein
for Mn2+ oxidation. In this paper we describe a detailed
analysis of the mutation site in the transposon mutant and the effect
of Cu2+ on the Mn2+-oxidizing activity of
P. putida GB-1.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The bacterial strains
and plasmids used in this study are listed in Table
1. P. putida GB-1 (which
was first described by Corstjens et al. [11]) was
kindly provided by K. H. Nealson (Jet Propulsion Laboratory,
Pasadena, Calif.). This organism is resistant to ampicillin
(17) and chloramphenicol. P. putida GB-1-002
is a spontaneous streptomycin-resistant (Smr) mutant of
GB-1 (17) that was used to generate transposon mutants with
Smr as an extra phenotypic marker. The
transposon-containing plasmid pBR322::Tn5 was
constructed by T. Goosen (Department of Genetics, Wageningen
Agricultural University, Wageningen, The Netherlands). This construct
does not replicate in P. putida GB-1.
Media and culture conditions.
P. putida GB-1 was
grown at room temperature in LD (L. discophora) medium as
described previously for L. discophora SS-1 (7). To measure the effects of copper, nickel, and zinc on growth and/or Mn2+-oxidizing activity, CuCl2,
ZnCl2, and NiCl2 were added at concentrations ranging from 0 to 100 µM. Cell growth was monitored by determining the optical density at 600 nm. Escherichia coli DH5
and GJ23 were cultured in Luria-Bertani medium (27) at
37°C. Solid media contained 1.8% (wt/vol) agar (Gibco BRL).
Selection markers were used at the following concentrations:
ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; tetracycline, 25 µg/ml; streptomycin, 100 µg/ml; and chloramphenicol, 50 µg/ml.
Determination of Mn2+-oxidizing activity.
The
Mn2+-oxidizing activity of P. putida
GB-1-002 was determined quantitatively with the redox dye Leucoberbelin
blue (LBB) as described previously for L. discophora SS-1
(7). LBB is oxidized by Mn with valences of +3 or higher,
which results in a blue product. P. putida cells were
harvested from a liquid culture by centrifugation. They were rinsed
once with 1 volume of 10 mM HEPES buffer (pH 7.5). Eventually, the
cells were resuspended in 1 volume of 10 mM HEPES (pH 7.5). Oxidation
reactions were started by adding MnCl2 (final
concentration, 100 µM) to equal amounts of cells or lysate. At
regular intervals 100-µl samples were added to 500 µl of LBB. The
cell material was removed by centrifugation, and 200-µl aliquots of
the supernatants were transferred to a microtiter plate. The absorbance
at 620 nm was measured with a Titertek Multiskan apparatus.
KMnO4 was used as the standard. In the LBB assay, 240 µM
KMnO4 is equivalent to 600 µM MnO2. In all
cases, LBB was oxidized only after Mn2+ was added to the samples.
All Mn2+ oxidation assays were performed at room temperature.
Molecular genetic techniques.
Transposon mutagenesis of
P. putida GB-1-002 was performed as described
previously (17). One of the nonoxidizing mutants obtained
was designated GB-1-007. An 11.0-kb Tn5-containing
EcoRI fragment of the genomic DNA of mutant GB-1-007 was
cloned in pUC19, resulting in plasmid pPLH7 (Fig.
1). A 5.9-kb
BamHI-EcoRI fragment and a 5.1-kb
BamHI fragment (3.1 kb of Tn5 plus 2.8 kb of
Pseudomonas DNA and 2.7 kb of Tn5 plus 2.4 kb of
Pseudomonas DNA, respectively) were cloned in vector pUC19,
yielding plasmids pPLH114A and pPLH114B, respectively. The subcloning
procedure allowed the nucleotide sequences adjacent to Tn5
to be determined with a primer (5'-CCG-TTC-AGG-ACG-CTA-CTT-GT-3') specific for the inverted repeats of Tn5.
Subsequently, sequences were determined by using M13/pUC19 forward and
reverse primers and primer walking. A sequence analysis was performed
by using automated dideoxy chain termination technology. The
resulting nucleotide sequences were analyzed further with programs
of the Wisconsin Genetics Computer Group (version 8.1) and the
Pseudomonas Genome Project.
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FIG. 1.
Map of the Tn5-containing EcoRI
fragment from the P. putida mutant GB-1-007. The
triangle indicates the site of the Tn5 insertion (length of
Tn5, 5.8 kb). The open arrows indicate predicted gene
locations and orientations. The vertical bars in cumA
indicate consensus Cu2+-binding regions. The solid
arrowheads indicate the locations of the forward and reverse M15/pUC19
primers and the Tn5 sequence primer. The small vertical
arrows indicate restriction enzyme sites used in constructing pPLH7 and
pPLH114A+B. Abbreviations for restriction enzymes: E, EcoRI;
B, BamHI; Bs, BsaAI (site of kanamycin cassette
insertion). The BamHI restriction site in the MCS of pUC19
is indicated by B*. The GenBank accession number of the E. coli sequence is X61396.
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Construction, screening of the genomic library of P. putida GB-1-002 to select constructs that hybridized with
digoxigenin-labeled pPLH7, and complementation were performed as
described previously (8, 17). To obtain a P. putida GB-1 cumB mutant (Fig. 1), the 5.2-kb
hybridizing EcoRI fragment from one of the constructs selected was cloned in pUC19, resulting in pPLH70 (which was identical to pPLH7 lacking Tn5 [Fig. 1]). A unique BsaAI
site in cumB was used to insert a 1.2-kb kanamycin cassette
(Pharmacia Biotech) after the 3' overhang was removed by using T4 DNA
polymerase. The EcoRI fragment containing the kanamycin
cassette was subcloned in pBR322, resulting in pPLH75. This construct
was mobilized to P. putida GB-1-002 by using E. coli GJ23 as described by Van Haute et al. (37). The
P. putida GB-1-002 cumB mutants were
selected on the basis of kanamycin resistance (recombination) and
chloramphenicol resistance (loss of E. coli GJ23).
Tetracycline sensitivity was used to select for double recombination
events. cumB gene replacement was confirmed by PCR by using
cumB-specific primers.
Nucleotide sequence accession numbers.
The nucleotide
sequences determined in this study have been deposited in the GenBank
database under accession no. AF086638.
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RESULTS |
Analysis of the Tn5 insertion site in the
nonoxidizing mutant GB-1-007.
Transposon mutagenesis of
P. putida GB-1-002 resulted in several classes of
nonoxidizing phenotypes, some of which were defective in secretion of
the Mn2+-oxidizing factor and some of which were completely
devoid of Mn2+-oxidizing activity (17). One of
the latter mutants was designated GB-1-007. The location of
Tn5 in GB-1-007 and the nucleotide sequences of the
adjacent regions were determined (Fig. 1). The translation products of
some of the ORFs detected are shown in Fig.
2. The transposon was inserted in an ORF
identified as a gene encoding a protein homologous to multicopper
oxidases based on the presence of predicted
Cu2+-binding regions (Fig. 2 and
3). This gene was designated
cumA (Cu protein involved in
manganese oxidation). Multicopper oxidases are found in a
wide variety of organisms and are characterized by their conserved
Cu2+-binding sites (Fig. 3). The predicted cumA
translation product is a 459-amino-acid protein that has a molecular
weight of approximately 50,500 and contains an N-terminal
signal peptide (Fig. 2). The overall level of identity with
an ORF of Pseudomonas aeruginosa (referred to below as
orfA), as determined on the basis of the amino acid
sequence, was 67%. cumA is preceded by a possible
Shine-Dalgarno sequence (Fig. 2). Upstream of cumA an ORF
designated orfX was detected. orfX exhibited
homology to guaA, which codes for GMP synthase (level of
identity with 400 amino acids encoded by the 3' end of the gene
encoding the Bacillus subtilis GMP synthase [GenBank
accession no. P29727] [25], 55%). Immediately
downstream from orfX strong secondary structures
indicate that a transcription termination site is present (Fig. 2).
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FIG. 2.
Nucleotide sequence of part of the 11.0-kb pPLH7
EcoRI fragment. The amino acid sequences of the potential
gene products are also shown. Possible Shine-Dalgarno sequences are
underlined. Inverted repeats which might represent transcription
terminators are indicated by arrows. The predicted CumA and CumB
signal peptides are enclosed in boxes, and the predicted copper-binding
regions are indicated by boldface type. The site of transposon
insertion is indicated by an open arrowhead, and the kanamycin cassette
insertion site in cumB is indicated by a solid arrowhead.
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FIG. 3.
Alignment of the amino acid sequences of the
copper-binding sites in CumA, the P. aeruginosa PAO1
homologue, and several other multicopper oxidases. The copper-binding
residues are designated 1, 2, and 3 on the basis of the types of copper
which they potentially bind. Abbreviations: CumA, P. putida GB-1 CumA (this study); Pcum, P. aeruginosa
PAO1 CumA homologue (Pseudomonas Genome Project); MofA,
L. discophora SS-1 MofA (GenBank accession no. Z25774)
(13); MnxG, marine Bacillus sp. strain SG-1 MnxG
(GenBank accession no. U31081) (38); Fet3, S. cerevisiae ferroxidase (GenBank accession no. P38993)
(5); CopA, P. syringae copper resistance
protein (GenBank accession no. M19930) (26); PcoA,
E. coli plasmid pRJ1004 copper resistance protein
(GenBank accession no. X83541) (9); Lacc, Neurospora
crassa laccase (GenBank accession no. P10574) (21);
Hcp, human ceruloplasmin (GenBank accession no. M13699)
(23).
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Downstream from cumA, two other ORFs, designated
cumB and orfY, were identified (Fig. 1 and 2).
cumB, which is preceded by a possible Shine-Dalgarno
sequence, is located next to cumA. Since cumB and
orfY have the same orientation and because the short intergenic region does not contain a transcription termination site,
these genes are assumed to be part of an operon (designated Cum). The
predicted cumB translation product is a 145-amino-acid protein that has an estimated molecular weight of 16,000 and
contains a potential signal peptide (Fig. 2). CumB exhibited
homology (level of identity, 67%) with the protein encoded by the ORF
(orfB) downstream from orfA in P. aeruginosa (Fig. 1). It also exhibited homology with Orf178 of
E. coli (level of identity, 52% [32])
(Fig. 1) and Orf74 of Bradyrhizobium japonicum (level of
identity, 45%; GenBank accession no. L34743 [39]).
The orientation of orfY is opposite that of cumA
and cumB, and its predicted translation product (length, 485 amino acids; estimated molecular weight, 53,500) exhibited homology
(level of identity, 65%) with the product of the P. aeruginosa ORF (orfY) downstream from orfB
(Fig. 1). This protein also exhibited homology with Orf360 of
E. coli (level of identity, 38%
[32]), but in contrast to both P. putida and P. aeruginosa, E. coli
does not contain a cumA homologue preceding orf178.
Screening of the P. putida genomic library with
digoxigenin-labeled pPLH7 resulted in isolation of 12 positive clones.
The DNA inserts of nine of these clones contained a 5.2-kb
EcoRI fragment that hybridized with pPLH7. Mobilization of
these nine constructs to mutant GB-1-007 did not result in restoration
of Mn2+-oxidizing activity.
As complementation did not succeed, we could not eliminate the
possibility that inhibition of Mn2+ oxidation in the
mutant GB-1-007 resulted from a polar effect of the
Tn5 insertion in cumA on cumB. A gene
replacement study was performed to eliminate the possibility that
cumB is involved in the oxidation of Mn2+. The
mutant obtained (designated GB-1-010) was tested for
Mn2+-oxidizing activity, and the growth rate was compared
to the growth rates of the wild type and mutant GB-1-007. GB-1-010
retained the ability to oxidize Mn2+ (Fig.
4), but growth defects similar to those
of mutant GB-1-007 were observed (data not shown).
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FIG. 4.
(A) Manganese-oxidizing activities of P. putida GB-1-002 and GB-1-007 and cumB mutant GB-1-010
in LD medium containing 100 µM MnCl2. (B) Same three
samples after the redox dye LBB was added.
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Effect of Cu2+ on the Mn2+-oxidizing
activity of P. putida GB-1-002 and on the growth rates
of P. putida GB-1-002 and GB-1-007.
The effect of
a mutation in a gene (cumA) encoding a multicopper oxidase
on Mn2+ oxidation in P. putida may
indicate that Mn2+ oxidation is Cu2+
dependent. Therefore, we cultured cells with different
concentrations of exogenously added Cu2+ and determined the
Mn2+-oxidizing activities of the cultures in the early
stationary growth phase. We observed that Cu2+ had a clear
stimulating effect on the oxidation of Mn2+ (Fig.
5). A maximum Mn2+ oxidation
rate of 0.52 nmol/ml · min was observed in the presence of 40 µM Cu2+, which was approximately fivefold greater than
the rate observed in medium without extra Cu2+. At
Cu2+ concentrations greater than 40 µM the stimulating
effect decreased.
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FIG. 5.
Effects of different Cu2+ ( ),
Zn2+ ( ), and Ni2+ ( ) concentrations on
the Mn2+ oxidation rate of P. putida GB-1,
as determined by the LBB assay. For experimental details see the
text.
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To determine whether stimulation of the oxidizing activity was specific
for Cu2+ ions and not for addition of divalent cations in
general, the effects of Zn2+ and Ni2+ were
studied (Fig. 5). Stimulation of the oxidation of
Mn2+ was not observed. In contrast, at
Zn2+ concentrations greater than 20 µM a decrease in
oxidation was observed. The effect of Ni2+ was even more
pronounced. A decrease in Mn2+ oxidation was observed
at an Ni2+ concentration of 10 µM. These results indicate
that Cu2+ is specifically involved in oxidation of
Mn2+ in P. putida.
Because the Mn2+-oxidizing activity of P. putida is growth phase dependent (31), the effects of
Cu2+ on the growth rates of both the P. putida wild type and the mutant were determined (Fig.
6). No significant differences in the
growth rate of the wild type were detected with Cu2+
concentrations up to 100 µM. Cells entered the logarithmic growth phase at approximately the same time after inoculation and
reached the stationary growth phase simultaneously. Similar results
were obtained with the mutant. However, compared to the wild type, the
mutant had a longer lag phase and reached a lower maximum cell density
(independent of the Cu2+ added). Addition of
Zn2+ or Ni2+ at concentrations up to 100 µM
did not have any effect on the growth rate of either the wild type or
the mutant (data not shown).
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FIG. 6.
Effects of no Cu2+ () and 100 µM
Cu2+ ( ) on the growth rates of P. putida
GB-1-002 ( ) and GB-1-007 (---). For experimental
details see the text. OD600, optical density at 600 nm.
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DISCUSSION |
Previous studies have indicated that Mn2+
oxidation in P. putida GB-1 is catalyzed by an outer
membrane enzyme or, more likely, an enzyme complex (8, 17,
31). Electrophoretic analyses of cell extracts
(12, 31) revealed the presence of Mn2+-oxidizing
factors with molecular weights ranging from 250,000 to 130,000, which
were assumed to represent the oxidizing complex or parts of the
oxidizing complex. We propose that an important constituent of the
oxidizing complex is a Cu-dependent oxidase, the product of
cumA. The genomic organization of the regions adjacent to
cumA indicates that this gene constitutes an operon with the downstream ORF cumB. cumA is preceded by inverted repeat
sequences that are able to form a stem-loop structure, the
transcription termination site of the preceding gene, orfX,
which is a homologue of the GMP synthase gene. We propose that the DNA
region between orfX and cumA contains the
promoter sequence. As the short intergenic region between
cumA and cumB does not contain transcription
termination sequences, these genes are probably transcribed from the
same promoter. cumB is followed by an ORF (orfY)
that clearly belongs to another operon in view of its opposite
orientation. Transposon insertion in cumA abolished the
Mn2+-oxidizing activity of the organism, whereas
mutation of cumB had no such effect, clearly indicating
that cumA is involved in Mn2+ oxidation. The
gene product CumA contains a signal sequence, in accordance with the
outer membrane location of the oxidizing factor. We found that growth
of cells on media with exogenously added Cu2+ stimulated
the Mn2+-oxidizing activity compared to the activity of
cells grown with no Cu2+ addition, whereas neither
Zn2+ nor Ni2+ enhanced the activity. The
suggestion that a Cu2+-dependent oxidase is involved
in Mn2+ oxidation in P. putida is supported
by evidence that the multicopper oxidases MofA and MnxG are involved in
Mn2+ oxidation in two other oxidizing organisms, L. discophora SS-1 and Bacillus sp. strain SG-1,
respectively (13, 38). In the latter organism the oxidizing
activity could also be stimulated by adding Cu2+
(38). In L. discophora SS-1, the effect of
Cu2+ on Mn2+ oxidation has not been
studied yet.
We demonstrated that the opportunistic pathogen P. aeruginosa PAO1 contains an ORF (orfA) that is
very similar to cumA of P. putida
GB-1. Preliminary experiments in our laboratory showed that in
principle, logarithmic liquid cultures of P. aeruginosa are able to oxidize Mn2+ (data not shown), although
it was difficult to reproducibly demonstrate this activity. In
spite of the uncertainty, it is tempting to correlate this oxidizing
activity with the presence of the cumA homologue
orfA.
The data obtained in this study strongly support the notion that
involvement of multicopper oxidases in Mn2+ oxidation is
common in Mn2+-oxidizing bacteria (36). However,
several questions remain to be answered. The first question is related
to the fact that complementation of the mutant has not been successful
so far. In a previous study (17) we found a single
transposon insertion in the mutant GB-1-007, which showed that the lack
of complementation cannot be due to other possible insertions. The
transposon insertion is located near the 3' end of cumA
between two copper-binding regions. It is possible that this location
of the transposon still allows production of large amounts of almost
complete CumA which may compete with CumA expressed from the
complementing fragment (for instance, in the formation of the
oxidizing complex). Because the essential fourth copper-binding region
is missing in mutated CumA, a nonfunctional Mn2+-oxidizing
complex should be formed. We will use site-specific gene replacement in
cumA to resolve this question.
The site-directed gene replacement in cumB eliminated the
possibility that cumB is involved in Mn2+
oxidation and confirmed that the decreased growth rate of the mutant
GB-1-007 was the result of a polar effect of the transposon on
cumB transcription, which supported the suggestion that
cumA and cumB are cotranscribed from the same
promoter. The involvement of cumB in growth is consistent
with the observation that a cumB homologue in B. japonicum, orf74, is required for optimal
free-living growth (39). Like the mutant GB-1-007 and
the cumB mutant, mutants in which orf74 was
disrupted had a longer lag phase and reached a lower cell density
than the wild type. In E. coli, another
cumB homologue (orf178) seems to be involved in
the cell-killing function of members of the gef gene
family in a manner that so far is not known (32).
P. putida GB-1 is sensitive to the gef
gene family (32), which may be the result of the
product of cumB. This gene has not been found in
P. putida GB-1 previously.
A second question to be resolved concerns the stimulating effect of
Cu2+ on Mn2+ oxidation in P. putida. We found that the presence of low amounts of
Cu2+ in the culture medium specifically enhanced the
oxidizing activity of the cells. However, it is not clear yet
whether the stimulating effect should be ascribed to
Cu2+-enhanced transcription of the oxidizing factor
(presumed to be encoded by cumA), to production of a more
active factor as a result of optimal Cu2+ incorporation, or
to a combination of these effects. Why stimulation of Mn2+
oxidation decreased at Cu2+ concentrations higher
than the optimum concentration (40 µM) also must be explained.
It is possible that at supraoptimal concentrations, Cu2+
ions also occupy Mn2+-binding sites. Competition for
Mn2+-binding sites and/or Cu2+-binding sites
may explain the inhibiting effects of Zn2+ and
Ni2+ on Mn2+ oxidation. Studies of the effect
of Cu2+ on the expression of cumA and the
effects of specific Cu2+-chelating agents on
Mn2+ oxidation followed by reconstitution experiments
should provide more insight into these questions.
Finally, it is possible to speculate about the physiological functions
of multicopper oxidases in Mn2+-oxidizing bacteria.
Multicopper oxidases occur in a wide variety of organisms and can have
different cellular functions. The multicopper oxidase family includes
the CopA proteins of Pseudomonas syringae (26)
and Xanthomonas campestris (24) and the PcoA
protein of the E. coli plasmid pRJ1004 (9),
all of which are involved in Cu2+ resistance. The
P. putida GB-1 CumA protein probably is not involved in
Cu2+ resistance, as mutation of the corresponding
gene did not result in Cu2+-sensitive growth of
mutant cells. Other members of the multicopper oxidase family are
the Saccharomyces cerevisiae Fet3 protein
(5) and the human ceruloplasmin (23), both of
which act as ferroxidases involved in high-affinity iron uptake. White
rot fungi produce the multicopper oxidase laccase, which produces
strongly oxidizing Mn(III) chelates that are used in the oxidation of
lignin compounds (3). Production of strong oxidizing agents
that release nutrients from resistant organic compounds may be an
important function of Mn2+-oxidizing multicopper oxidases
in nutrient-poor environments (35). However,
metabolically inert structures, like the Mn2+-oxidizing
spores of Bacillus sp. strain SG-1, do not obviously benefit
from such a process. It is possible that the multicopper oxidases of
Mn2+-oxidizing bacteria have primary cellular functions
other than Mn2+ oxidation. These oxidases may have the
ability to oxidize Mn2+, which allows the bacteria to use
the products depending on the organism and circumstances and thus to
gain a selective advantage. Such an advantage may be exploited at the
cellular level (by generation of nutrients, production of alternative
electron acceptors, etc. [see above]), but an advantage at the
ecological level may also be envisaged. In some environments, oxidation
and reduction of Mn2+ (and Fe2+) are coupled to
oxidation and reduction of carbon, which permits efficient cycling
of nutrients and reduction equivalents in stable ecosystems
(30). Mn2+-oxidizing bacteria play an
important role in such ecosystems by guaranteeing the supply of an
electron sink to the reducing zones. This may contribute to the
widespread occurrence of Mn2+-oxidizing bacterial species
in nature.
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Abstract
Introduction
