Pathway substrates and some structural analogues directly activate
the regulatory protein DmpR to promote transcription of the
dmp operon genes encoding the (methyl)phenol degradative
pathway of Pseudomonas sp. strain CF600. While a wide range
of phenols can activate DmpR, the location and nature of substituents
on the basic phenolic ring can limit the level of activation and thus
utilization of some compounds as assessed by growth on plates. Here we
address the role of the aromatic effector response of DmpR in
determining degradative properties in two soil matrices that
provide different nutritional conditions. Using the wild-type system
and an isogenic counterpart containing a DmpR mutant with enhanced
ability to respond to para-substituted phenols, we
demonstrate (i) that the enhanced in vitro biodegradative capacity of
the regulator mutant strain is manifested in the two different soil types and (ii) that exposure of the wild-type strain to
4-methylphenol-contaminated soil led to rapid selection of a
subpopulation exhibiting enhanced capacities to degrade the
compound. Genetic and functional analyses of 10 of these derivatives
demonstrated that all harbored a single mutation in the
sensory domain of DmpR that mediated the phenotype in each case. These
findings establish a dominating role for the aromatic effector response
of DmpR in determining degradation properties. Moreover, the results
indicate that the ability to rapidly adapt regulator properties to
different profiles of polluting compounds may underlie
the evolutionary success of DmpR-like regulators in controlling
aromatic catabolic pathways.
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INTRODUCTION |
Naturally occurring soil and water
microorganism have an enormous metabolic versatility towards aromatic
compounds (2, 14, 45). However, many factors, including
the distribution of desired catabolic traits, incomplete degradation or
production of toxic dead-end products, biovailability, and inhibition
of microbial activity by toxicity of a polluting compound, can lead to
low efficiency of degradation by indigenous microflora. When degradation by indigenous microbial populations is unsatisfactory, bioaugmentation with more efficient exogenous organisms is often considered for bioremediation purposes (51). Generation of
new or improved microbial catabolic activities can be achieved by many different strategies, including modulation of (i) components of
the specific degradation pathway, (ii) properties that enhance bioavailability of the substrate, or (iii) survival and distribution traits of the bacteria in soils (9, 48, 49). The
regulatory circuits that control the expression of degradative pathways
lie at the top of the hierarchy of events that lead to efficient
biodegradation of aromatic compounds, since they determine under what
conditions and in response to what compound the catabolic enzymes are
expressed. The regulatory circuits therefore provide a prime target for
generating strains with enhanced degradative potential. Here we examine
the role of the DmpR-mediated regulatory circuit in bacterial
biodegradation in soil matrices using the
(methyl)phenol-degrading strain Pseudomonas sp. strain CF600.
Pseudomonas sp. strain CF600 is a natural isolate harboring
the self-transmissible IncP-2 catabolic megaplasmid pVI150
(40). The 15 genes encoding enzymes for the conversion of
(methyl)phenols to pyruvate and acetyl coenzyme A are organized in the
dmp operon of pVI150 (43). Expression
of the dmp operon is tightly controlled by the
divergently transcribed dmpR gene product (39,
41). The DmpR regulatory protein belongs to the
54-dependent family of transcriptional activators, which
have discrete domains involved in signal reception, transcriptional
activation, and DNA binding (Fig. 1)
(24). DmpR and XylR, a regulator of toluene and xylene
catabolism (1, 18), typify a subgroup of this family that
respond directly to the presence of aromatic effector molecules
(reviewed in reference 38). Proteins homologous to DmpR
and XylR are frequently found associated with catabolism of aromatic
compounds and include AphR (4), BphR (32),
HbpR (19), MopR (36), PheR (M. Takeo,
unpublished data [GenBank accession no. D63814]), PhhR
(27), PhlR Pseudomonas putida H
(5), PhlR from Ralstonia eutropha JMP134
(25), PhnR (23), PoxR (17), TbuT
(7), TmbR (12), and TouR (6).
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FIG. 1.
Schematic representation of the domain structure
of DmpR. The hatched box represents the extent of the NTP binding
motif found in this class of regulators (consensus, G--G-GKE---A---H--S
[24]). The position of the E135K mutation that confers
an enhanced response to para-substituted methylphenols is
indicated. The locations of restriction sites used in dmpR
manipulations are shown relative to the domain structure (Nd,
NdeI; P, PstI; Sn, SnaBI).
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Substrates of the dmp pathway and some structural analogues
serve as effector molecules that directly control the activity of DmpR
(41, 42). The aromatic effectors are bound through a
single common binding site on the signal reception A domain of DmpR
(28, 29). Binding of the aromatic compound releases interdomain repression, resulting in depression of the transcriptional activation property of the protein with consequent initiation of
transcription of dmp catabolic genes (26, 28, 29,
42). DmpR can be activated by a wide range of phenolics,
however, the location and nature of substituents on the basic phenol
molecule determine the level of activation, which can be limiting for
utilization of some compounds (29, 31).
Pseudomonas strain CF375 is a derivative of
Pseudomonas strain CF600 harboring a single point mutation
in the signal reception-encoding region of dmpR, resulting in a Glu-to-Lys replacement of residue 135 (DmpR-E135K). Due to modulation of the regulatory properties of DmpR, the resulting strain
has a greatly enhanced ability to sense and degrade
para-substituted methylphenols such as 4-methylphenol and
3,4-dimethylphenol under laboratory conditions while retaining
wild-type ability to sense and degrade phenol and 2-methylphenol
(31). These properties of CF375 suggest that regulatory
mutants may serve as efficient bioaugmentation reagents. However, soils
provide heterogeneous nutritional environments, and microbial
degradation performance is dependent on varying physicochemical
properties of a given soil (50, 51). Here we address
whether the enhanced in vitro biodegradative efficiency of the CF375
regulatory mutant translates to increased biodegradative efficiency in
two different soil matrices that provide different nutritional
conditions. These experiments led to the isolation and analysis of
spontaneous mutants that arose from 4-methylphenol-amended soils. The
results from these studies provide evidence that CF600 naturally
rapidly adapts its suboptimal ability to degrade 4-methylphenol in soil
via enhancement of the ability of DmpR to respond to 4-methylphenol.
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MATERIALS AND METHODS |
General procedures.
Escherichia coli strains were
grown at 37°C, and Pseudomonas strains were grown at
30°C. E. coli DH5 (16) was used for
RSF1010-based plasmids, while the replication-permissive host
S17-1
pir (11) was used for propagation of R6K-based
suicide donor plasmids. Unless otherwise stated, Luria-Bertani medium
was used as rich medium and M9 salts supplemented with the indicated
carbon source was used as minimal medium (34). Kanamycin
(50 µg ml
1), rifampin (100 µg ml1),
streptomycin (1 mg ml1), and carbenicillin (100 µg
ml1 for E. coli strains and 1 mg
ml1 for Pseudomonas strains) were added to the
media as required.
Strain construction.
Spontaneous antibiotic-resistant
derivatives of the (methyl)phenol-catabolizing prototrophic
Pseudomonas sp. strain CF600 (40) and its
isogenic counterpart CF375 (carrying the DmpR-E135K mutation)
(31) were selected on rich medium supplemented with rifampin or streptomycin. The resident pVI150 plasmids of the resulting
Rifr derivative CF600.1 and Smr derivative
CF375.3 were then tagged by insertion of a kanamycin resistance gene
and a green fluorescent protein (GFP) variant or blue fluorescent
protein (BFP) gene. Insertion was made in each case at the
NruI site located 2.2 kb downstream of the last gene of the
dmp operon. This was achieved in a sequential
process schematically illustrated in Fig.
2. First, a 2.125-kb
XhoI-to-BglII fragment from downstream of the
dmp operon was cloned between the XhoI
and BamHI sites of pBluescript SK (Stategene) to generate pVI650. Second, a blunt-ended
HindIII-EcoRV-NotI linker was
inserted into the unique NruI site of this fragment,
simultaneously destroying the NruI site, to give plasmid
pVI651. A 2-kb HindIII fragment carrying a kanamycin
resistance gene from mini-Tn5Km (10) was then
inserted into the HindIII site of the linker to generate pVI652. A XhoI-to-EcoRI fragment, spanning the
entire XhoI-to-BglII fragment with the
Kmr insert, was then cloned between the SalI and
EcoRI sites of the Cbr R6K-based suicide plasmid
pGP704L (31), resulting in plasmid pVI653. NotI
fragments encoding the GFP mutant 3 derivative from pJBA27
(3) or BFP (Novo Nordisk, Bagsvæed, Denmark) from an analogous plasmid (pJBA68) (J. B. Anderson, unpublished data) were
then cloned into the NotI site of the linker, resulting in plasmids pVI654 (carrying a Km-GFP insert) and pVI655 (carrying a
Km-BFP insert). These insertions were introduced into the resident pVI150 plasmid of Pseudomonas strains by recombination from
the suicide plasmids following conjugation from E. coli
S17-1pir. Kmr was used to select for
first-site recombination, and second-site recombinants were detected by
screening for Cbs. The resulting strains were designated
CF600.1::Km-gfp, CF600.1::Km-bfp, CF375.3::Km-gfp, and
CF375.3::Km-bfp.
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FIG. 2.
(A) Schematic representation of the genetic organization
of dmp genes (open boxes) relative to the kanamycin
resistance and fluorescent protein genes (closed boxes) in tagged
pVI150 plasmids. Arrowheads indicate the direction of transcription.
The different illustrated DNA manipulation steps leading to the final
conformation of the DNA in the tagged strains are described in
Materials and Methods. Restriction sites: B, BglII; E,
EcoRV; H, HindIII; K, KpnI; N,
NotI; Nr, NruI; S, SauI; X,
XhoI. T0 and T1 indicate the locations of strong
transcriptional terminators within the cloned NotI fragments
(3). (B and C) Rates of CO2 production of the
indicated strains upon growth on minimal medium supplemented with 2.5 mM 2-methylphenol (B) or 4-methylphenol (C) as the sole carbon and
energy source. Values are the averages from duplicate experiments. Data
for CF600.1::Km-bfp and CF375.3::Km-bfp were indistinguishable
from those for their Km-gfp counterparts on both aromatic test
compounds (data not shown).
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DNA sequencing and plasmid generation.
Mutations in the
A-domain-encoding DNA were initially identified by direct sequencing of
PCR-generated fragments from target strains (from 178 bp upstream of
the ATG start codon to codon 246 of dmpR) using
custom-designed oligonucleotides. Cloning of the mutant A domains was
performed using the plasmid pVI546, which is a Cbr
broad-host-range RSF1010-based plasmid that expresses a DmpR derivative
with an 8-amino-acid Flag epitope tag fused to the carboxy-terminal of
the protein. In this plasmid, dmpR is expressed from its
native promoter and has been manipulated to contain a silent
NdeI site overlapping the ATG initiation codon and a silent SnaBI site overlapping codons 221 and 222 of dmpR
to allow replacement of the entire A domain and regeneration of
full-length DmpR regulators (Fig. 1) (44). Introduction of
PCR fragments generated using primers that include these site and
template DNAs from the different mutants resulted in plasmids pVI656
(DmpR-F42Y), pVI657 (DmpR-R109C), pVI658 (DmpR-L113V), pVI659
(DmpR-D116N), pVI660 (DmpR-F122L), pVI661 (DmpR-E135K), pVI662
(DmpR-C137Y), and pVI663 (DmpR-179(CG)180). The nucleotide sequences of
all PCR-derived DNAs in these plasmids were confirmed. These plasmids
thus encode mutants of DmpR expressed from the same cis
elements as wild-type DmpR on plasmid pVI455 (42). To
introduce the mutations into pVI150 of Pseudomonas strain,
CF600, NotI fragments spanning the entire inserts of the above-described plasmids were cloned into the R6K-based Cbr
suicide vector pGP704L-NotI (31). The resulting
suicide plasmids, pVI664 (DmpR-F42Y), pVI665 (DmpR-R109C), pVI666
(DmpR-L113V), pVI667 (DmpR-D116N), pVI668 (DmpR-F122L), pVI669
(DmpR-C137Y), and pVI670 [DmpR-179(CG)180], were introduced from the
permissive host E. coli S17-1pir into a CF600
derivative (CF427 [31]) in which the internal
PstI fragment had been replaced by a Kmr
cassette (Fig. 1). The PstI fragment spans codons 40 to 422 of dmpR and thus all of the mutant codons present in the
donor suicide plasmids. Recombinants were identified on the basis of
the ability to restore growth on 2-methylphenol and sensitivity to
kanamycin and carbenicillin
CO2 production measurements.
For determination
of CO2 production upon growth with aromatic carbon sources
as the sole carbon and energy sources, cultures were grown overnight in
M9 medium supplemented with 2.5 mM test compound and 0.1% casein amino
acids. Cells were then washed in the same medium lacking casein amino
acids and diluted to an A650 of 0.2. Approximately 106 cells were plated on 50-mm-diameter M9
minimal plates containing the different aromatic compounds at 2.5 mM.
The plates were then incubated in a Respicon III respirometer (Norgren
Innovation AB, Umeå, Sweden), and CO2 production was
monitored every 30 min for up to 48 h.
Soil properties and sterilization.
Physical and chemical
properties of the soils were determined using standard procedures (Soil
Analysis Service Ltd., Helsinki, Finland). The sandy soil water content
was 9.1%, the organic carbon content was 1.6%, the total nitrogen
content was 0.13%, and the pH was 6.3. The pine forest humus had a
water content of 61.5, an organic carbon content of 22.4%, and a total
nitrogen content of 0.75%. Its original pH was 3.9; however, since
CF600 derivatives could not be established at this pH (data not shown),
the pH was increased to 6.4 by liming with CaCO3 (12 mg g
[fresh weight] of soil1). The forest humus (before
liming) and the sandy soil contained, respectively, 269 and 1,560 mg of
calcium (Ca) liter1, 4.1 and 31 mg of phosphorus (P)
liter1, 71.5 and 150 mg of potassium (K)
liter1, 38 and 107 mg of magnesium (Mg)
liter1, 11 and 50 mg of nitrate
(NO3) liter1 and 21 and <2 mg
of ammonia (NH4+) liter1. Quartz
sand (Sigma) had a pH of 7.0 when measured in sand-water suspension.
Soil samples were sterilized in 0.3- to 1-liter aliquots by autoclaving
(121°C, 20 min).
Soil inoculation and sampling.
Sterilized limed humus (1.5 g
[fresh weight]) and sandy soil (2 g [fresh weight]) were dispensed
in 14-ml polypropylene tubes and 20-ml glass scintillation vials.
Quartz sand was dispensed in 2-g aliquots, and its water content was
adjusted to 5.6%. The soils were amended with 595 µg of 2- or
4-methylphenol (Aldrich) made up as aqueous solutions prepared from 1 M
stock solutions in dimethyl sulfoxide (DMSO) (Merck). Thus, test
samples and controls received 5 µl of DMSO per 1.5 g (sandy
soil) or 2 g (limed humus) (fresh weight) of soil. This level of
DMSO did not affect the population numbers of controls (see Results).
Supplements were added to soils 1 to 1.5 h prior to initiation of the
experiment. Inverting and shaking before bacterial inoculation
vigorously mixed soils and the supplements. Bacteria were pregrown
overnight in semirich medium (1/4KSN-MYE [35]) in the
presence of 2 mM 2-methylphenol. Cell pellets were resuspended in
phosphate-buffered saline, and approximately 106 or
108 bacterial cells in 50 µl were inoculated into soils
as indicated. The amended soil samples were subsequently kept at room
temperature (20 to 22°C). After the additions, the final water
contents were 63% for humic soil, 15% for sandy soil, and 11% for
quartz sand.
The bacterial cell numbers were determined from three replicates for
each time point. Nine milliliters of phosphate-buffered saline solution
was added to each soil-containing tube and vortexed for 2 min.
Appropriate dilutions were plated on 1/4KSN-MYE containing kanamycin or
on minimal medium (KSN [53]) containing 2 mM 2- or
4-methylphenol as the sole carbon and energy source. In competition experiments, Luria-Bertani medium LB in the presence or absence of
antibiotic selection was used to distinguish the two strains (Kmr and Rifr for CF600.1::Km-gfp and
Kmr and Smr for CF375.3::Km-gfp). After
growth on the selection plates, the presence of the appropriate GFP or
BFP fluorescence was confirmed by visual inspection under UV light.
To determine the levels of methylphenols remaining in the soil samples
at the different time points, extractions of triplicate samples were
performed in glass scintillation vials. As internal standards, soil
samples were amended 1 h prior to the extraction with an
appropriate aromatic compound (2-methylphenol when determining 4-methylphenol levels and vice versa). Three milliliters of acetone amended with nitric acid was mixed with the samples for 1 h with rotation. Water was removed by adding dehydrated
Na2SO4, and the samples were then filtered
through nylon Acrodisc filters (0.45-µm pore size; Pall Gelman
Sciences). Analysis was performed with a gas chromatograph (5890A;
Hewlett-Packard) equipped with an HP-5MS column (30 m by 0.25 mm by
0.25 µm) using a flame ionization detector. The oven was programmed
so that after 4 min the initial temperature of 45°C was increased to
180°C at 10°C/min and then to 250°C at 35°C/min.
Luciferase assays.
Plate test screening was performed as
previously described (44). In brief, colonies of P. putida KT2440::Po-luxAB (31) harboring
various plasmids were replica plated and grown overnight on Luria agar
plates with antibiotic selection for the resident plasmid and
supplemented with the indicated quantities of the different aromatics.
Inverted plates were exposed to decanal vapor and the light emission
was recorded by placing a film over the plates. For quantitative
measurements, cells were grown and treated as previously described
(46), and values were determined using a PhL microtiter
plate luminometer (Mediators Diagnostika, Vienna, Austria).
Protein analysis.
Crude extracts of cytosolic proteins,
sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer to
nitrocellulose filters, and Western blot analysis with polyclonal
rabbit anti-DmpR sera were as previously described (42).
Antibody-decorated bands were revealed using chemiluminescence reagents
as directed by the supplier (Amersham Pharmacia Biotech). Differences
in expression levels were assessed by comparison of dilution series of
the test samples with those of the wild-type DmpR extract.
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RESULTS |
Growth of genetically tagged derivatives of Pseudomonas
strain CF600.
To enable identification of Pseudomonas
sp. strain CF600 and the dmpR-regulatory mutant strain CF375
from soil samples, differentially tagged derivatives were
generated as described in Materials and Methods. The resulting
antibiotic-resistant derivatives CF600.1 (Rifr) and CF375.3 (Smr)
harbor pVI150 plasmids carrying either Km-GFP or Km-BFP
insertions downstream of the dmp operon (Fig. 2A).
To test that these derivatives retained their respective parental
biodegradative properties, their ability to utilize 2-methylphenol and
4-methylphenol as sole carbon and energy sources was tested.
2-Methylphenol was chosen as the control aromatic compound since this
is the best effector of DmpR and strains harboring either wild-type
DmpR or DmpR-E135K respond and degrade this compound with equal
efficiency. As shown in Fig. 2B, the genetically tagged strains also
grow on 2-methylphenol equally well, with generation times like those found previously for the untagged derivatives (generation time of
approximately 1.5 h [31]). Importantly, the tagged
CF375.3 (DmpR-E135K mutant) strains retain the previously observed
phenotype of enhanced degradation efficiency with 4-methylphenol,
resulting in rates equivalent to those of the wild-type strain on
2-methylphenol. Hence, these results demonstrate that the genetic
markers do not confer any detectable detrimental genetic load under
these minimal growth conditions.
Propagation and biodegradation by CF600 derivatives in contaminated
soils.
Since the ability of microbes to degrade aromatic compounds
can be greatly influenced by abiotic factors determined by the soil
composition, we chose to analyze bacterial degradation of target
substrates in two different soil types. Limed forest humus (hereafter
referred to as humic soil) represents a comparatively nutrient-rich
soil, while the sandy soil is a nutrient-poor environment (see
Materials and Methods). Preliminary studies showed that monomethylated phenols were transformed and/or degraded by indigenous microflora in
these soils (data not shown). Therefore, in order to monitor removal of
the two test compounds, 2- and 4-methylphenol, by the added bacteria,
sterilized autoclaved soils were used.
The strains CF600.1::Km-gfp and CF375.3::Km-gfp were added to
both soil matrices with or without preamendment with the test aromatic
compounds. The same quantity of aromatic compound was added to each
soil type, which resulted in final concentrations of 1.0625 mg/g (dry
weight) for humic soil and 0.3324 mg/g (dry weight) for sandy soil. In
humic soil, the number of viable bacteria increased approximately
10-fold over the first 2 days of the experiment. This increase in
biomass was not dependent on the added phenols (Fig. 3A and
C) and, since no significant increase in
viable cell numbers was observed in unamended quartz sand (data not
shown), is attributable to the degradation of organic soil compounds. The same reason probably underlies the slight increase in biomass observed in the unamended sandy soil (Fig. 3B and D). In amended sandy soils, after an initial drop the viable counts of the introduced bacteria increased to approximately the initial inoculation density. In
addition, CF375.3::Km-gfp was observed to be more sensitive than
CF600.1::Km-gfp to the toxic effects of phenolics, notably 4-methylphenol (Fig. 3D), in the sandy soil. The difference in sensitivity to aromatic compounds in the two different soil types is
probably due to the quenching effect of components of the higher organic content of humic soil (21, 30, 37) and/or to the higher concentration of the added aromatic compounds in the water phase
of the sandy soil.
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FIG. 3.
Degradation and survival in two soil types. Logarithms
of viable colony-forming units of CF600.1::Km-gfp (open symbols)
and CF375.3::Km-gfp (closed symbols) are shown with continuous
lines. Counts were determined from both amended soils (squares) and
non-amended controls (diamonds). The number of CFU of
CF600.1::Km-gfp mutants detected by virtue of comparatively large
colony formation on 4-methylphenol is shown with open triangles.
Concentrations of 2-methylphenol (A and B) and 4-methylphenol (C and D)
in the limed pine forest humic (A and C) and sandy (B and D) soils are
shown with dotted lines. The concentrations of methylphenols in control
soils without bacterial inoculation (dotted line, no symbols) are also
shown. Values are the averages of triplicate determinations. Error bars
indicate standard deviations.
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The observed decreases in concentrations of the methylated phenols in
soils are shown in Fig. 3 and are clearly attributed to the activity of
inoculated bacteria rather than to some abiotic factors related to
chemical or photochemical reactions (compare noninoculated and
inoculated soil samples). As expected, CF600.1::Km-gfp and CF375.3
degraded 2-methylphenol with comparable efficiencies in both soils
(Fig. 3A and B). Degradation of 2- and 4-methylphenol was more
efficient in the humic soil than in sandy soil, consistent with the
high bacterial population achieved in humic soil. Most importantly, the DmpR-E135K regulatory mutant CF375.3::Km-gfp showed a clearly enhanced ability to degrade 4-methylphenol compared with its wild-type counterpart CF600.1::Km-gfp in both soil types (Fig. 3C and D). These results show that the enhanced ability of the
regulatory mutant to degrade 4-methylphenol on plates (Fig. 2) is also
observed under the two different nutritional conditions provided by the
two soil matrices.
Propagation of CF600.1::Km-gfp into soils amended with
4-methylphenol resulted in accumulation of a subpopulation which formed large colonies on minimal 4-methylphenol plates (Fig. 3C and D) and
that also showed improved growth on 3,4-dimethylphenol. The rapid
accumulation of this population in humic soil accompanied 4-methylphenol degradation. In sandy soil inoculated with
CF600.1::Km-gfp, the maximal number of this subpopulation was 2 orders of magnitude less than that detected in humus (compare Fig. 3C
and D), and no change in 4-methylphenol concentration was observed over
the time course of the experiment. No CF600.1::Km-gfp derivatives exhibiting the phenotype of enhanced growth on 4-methylphenol or
3,4-dimethylphenol were observed in unamended soils or in soils amended
with 2-methylphenol (Fig. 3A and B and data not shown). These results
suggest that this subpopulation was directly selected in response to
the presence of 4-methylphenol.
Competition between CF600.1::Km-gfp and CF375.3::Km-gfp
strains in soils.
The specific DmpR-mediated regulatory circuit is
subservient to global regulation in response to the availability of
alternative carbon sources in the medium (46, 47), leading
to various degrees of silencing depending on the nutrients supplied.
Therefore, to test if the conditional growth advantage of the
regulatory mutant strain leads to enhanced competitive growth in the
two soil types, 106 cells of strains CF600.1::Km-gfp
and CF375.3::Km-gfp were coinoculated into humic and sandy soils
with or without methylphenol amendment. In the experiment shown in Fig.
4, the two strains were directly distinguished on the basis of their differential antibiotic resistance markers (Rifr and Smr). Growth of
CF375.3::Km-gfp and CF600.1::Km-gfp in sandy soil with or
without 2-methylphenol was indistinguishable (Fig. 4A and B). However,
the DmpR-E135K regulatory mutant strain CF375.3::Km-gfp had a clear
advantage over CF600.1::Km-gfp in the presence of 4-methylphenol.
After an initial decrease, the cell numbers of CF375.3::Km-gfp rose
to 10 times higher than those of CF600.1::Km-gfp (Fig. 4C). In
humic soil, the DmpR-E135K mutant strain showed a growth advantage over
the wild-type strain under all conditions studied, i.e., in the absence
and presence of either 2-methylphenol or 4-methylphenol, and there was
no significant difference in bacterial cell numbers between the
treatments (Fig. 4D to F). This result may be attributable to the
natural phenolic content of humic soil, which can contain up to 556 µg of water-soluble phenolic compounds per g, i.e., approximately
50% of the specific phenolic amendments used (15, 22). As
seen previously, accumulation of CF600.1::Km-gfp derivatives with
improved growth on para-substituted phenols was observed in
humic soil amended with 4-methylphenol (Fig. 4F), again suggesting
that the presence of 4-methylphenol selects for variants with an
enhanced capacity to grow and/or survive in the presence of
4-methylphenol. Similar results were obtained in competition
experiments between CF600.1::Km-bfp and CF375.3::Km-gfp in
which bacteria were plated in the absence of selection or selected on
the basis on their common antibiotic resistance marker
(Kmr) and subsequently distinguished on the basis of their
differential fluorescent protein tags (data not shown). The fact that
the results obtained with the various strategies were similar
demonstrates that the different antibiotic selections for the two
strains in this and the preceding experiment has no significant effect
on bacterial count determinations.
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FIG. 4.
Competition between CF600.1::Km-gfp (open symbols)
and CF375.3::Km-gfp (closed symbols) in sandy soil (left panels)
and limed pine forest humus (humic soil) (right panels) with or without
2-methylphenol or 4-methylphenol as indicated. The number of
CF600.1::Km-bfp mutants with enhanced growth on 4-methylphenol is
shown with open triangles. Values are the averages of triplicate
determinations.
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CF600.1::Km-gfp derivatives with improved growth on
para-substituted phenols.
Ten mutant
CF600.1::Km-gfp derivatives isolated from 4-methylphenol-amended
soils that exhibited improved growth on 4-methylphenol and
3,4-dimethylphenol were selected for analysis. The colonies were
derived from both soil types and represent different sampling days.
Since the present study demonstrates that the regulatory mutant
DmpR-E135K enhanced degradation by CF600 in these soil matrices, we
reasoned that the derivatives might likewise harbor mutations in
dmpR. Indeed, DNA sequence analysis of the 10 derivatives of
CF600.1::Km-gfp revealed that all harbored mutations within the A
domain of dmpR (Table 1).
Seven different mutations, representing transitions and transversions
that result in single amino acid substitutions and a 6-bp insertion
(TGCGGC, a duplication of the preceding sequence) that results in a
2-amino-acid insertion between residues 179 and 180 of DmpR, were
found. Furthermore, in two cases the same mutation was found in
derivatives from both soil types (i.e., L113V and F122L [Table 1]).
To confirm that the identified changes in the dmpR gene were
responsible for the phenotype of improved catabolism of
para-substituted phenols, one representative of each of the
seven mutants was introduced by recombination into the pVI150 plasmid
of CF600 as described in Materials and Methods. The strategy used
ensures that the identified mutation is the sole mutation introduced
into the wild-type system. Plate tests showed that all the recombinants
grew better than wild-type CF600 on both minimal 4-methylphenol
and 3,4-dimethylphenol plates. To directly compare the enhanced
4-methylphenol-catabolizing abilities of the mutant
derivatives, the growth profiles of the original isolate and its
cognate regenerated strain were compared. Examples of two of these
comparisons are shown in Fig. 5. The growth profiles of the strain pairs on 4-methylphenol were
indistinguishable in each case, demonstrating that the identified
mutations conferred the phenotype. Six of the mutations [F42Y, R109C,
L113V, D116N, F122L, and the insertion 179(CG)180], exemplified by
F42Y in Fig. 5A, conferred growth profiles similar to that conferred by
the E135K mutation, while the C137Y mutant had a lower growth rate that
was nevertheless much higher than that of the wild type (Fig. 5B).
Experiments with 2-methylphenol as the sole carbon and energy source
showed that all mutants grew at the expense of this substrate with
rates similar to that of the wild type (data not shown).
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FIG. 5.
Effect of DmpR mutations on growth rates with
4-methylphenol. The rates of CO2 roduction by the indicated
strains were measured upon growth on minimal medium supplemented with
2.5 mM 4-methylphenol. The rate achieved for each original mutant
isolate of CF600.1::Km-gfp is compared with that of a regenerated
CF600 derivative harboring the detected DmpR mutation. The profiles for
CF375.3::Km-gfp (closed squares) and CF600.1::Km-gfp
(open squares) are also shown to aid comparison. Values are the
averages from duplicate experiments. Results similar to those shown in
panel A were obtained with strains harboring DmpR-R109C,-L113V,
-D116N, -F122L, and -179(CG) 180 (data not shown).
|
|
The above results indicate that the mutations identified in this study,
like DmpR-E135K, modulate the response to 4-methylphenol. To directly
test this idea, the previously constructed luciferase reporter strain
P. putida KT2440::Po-luxAB, which carries a
chromosomal copy of the luxAB genes under the control of the
dmp operon promoter Po, was used. Plasmids
expressing the different DmpR derivatives from its native promoter
complete the system. The results shown in Fig.
6 demonstrate that five of the mutations
(F42Y, R109C, L113V, D116N, and C137Y), like E135K, are expressed at
levels comparable to that of wild-type DmpR and show an expected
enhanced ability to respond to 4-methylphenol. DmpR-C137Y exhibits the lowest enhancement above the wild-type level in response to
4-methylphenol (Fig. 6A) and is the derivative which exhibits the
lowest enhancement of growth rates at the expense of 4-methylphenol
(Fig. 5B).
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FIG. 6.
In vivo transcriptional response mediated by DmpR
derivatives in the presence of 4-methylphenol. (A) The luciferase
transcriptional response of P. putida
KT2440::Po-luxAB harboring plasmids expressing the
indicated derivatives was measured in the presence or absence of
effector. Figures are the averages and standard deviations of
triplicate determinations in each of two independent experiments. LU,
luciferase units. (B) Western analysis of DmpR levels. Crude extracts
(30 µg of cellular protein) derived from the cells exposed to
4-methylphenol used for panel A were separated by Sodium dodecyl
sulfate-11% polyacrylamide gel electrophoresis and probed with
anti-DmpR serum as described in Materials and Methods.
|
|
Unexpectedly, DmpR-F122L, which represents the mutation in 3 of the 10 strains isolated, did not show an enhanced response to 4-methylphenol
in the luciferase reporter system, and the response with
DmpR-179(CG)180 was comparatively extremely low. All of the DmpR
derivatives are expressed from the same cis-acting
regulatory elements. However, we have previously observed that single
amino acid substitutions of the A domain can result in different
protein stabilities (26) and that disruption of the
integrity of the A domain can result in nonfunctional unfolded proteins
(44), which may be differentially targeted for proteolysis
under different growth regimes. From Western blot analysis (Fig. 6B),
it is clear that these two derivatives are present at lower levels, and
quantitative Western analysis showed that they are present at 30 to
50% of the levels of wild-type DmpR (data not shown). Therefore, it
appears that the protein level of the regulator can account for the
lower response observed with DmpR-F122L but not DmpR-197(CG)180 in the reporter system.
Effector specificity profiles of DmpR mutants.
The effector
specificity mutant DmpR-E135K was independently isolated during a
genetic selection for mutants that respond to either
2,4-dimethylphenol or 4-ethylphenol. Upon screening, the
E135K mutation was found to confer an enhanced response to other
mono- or disubstituted phenols with substituents in the para
position (31, 42). Therefore, the mutants identified in
this study were screened using a simple semiquantitative plate test
assay for the ability to respond to a range of
para-substituted phenols. The results summarized in Table
2 show that all but DmpR-C137Y and
DmpR-179(CG)180 had gained a detectable ability to respond to one or
more of the compounds that are noneffectors of wild-type DmpR. The
mutations DmpR-R109C, DmpR-D116N, and DmpR-E135K show the broadest
spectrum of response to para-substituted compounds, which
includes the ability to respond to the priority-list pollutants 2,4-dimethylphenol, 2,4-dichlorophenol, and 4-nitrophenol. Thus, this
class of mutants have potential utility in whole-cell biosensor applications for pollution monitoring (44, 52).
| |
DISCUSSION |
Here, using the (methyl)phenol degradative system of
Pseudomonas strain CF600, we examined the potential to
improve bacterial biodegradative efficiencies in the soil via
modulation of the ability of a regulator to respond to the target
compound. Utilizing strains that differ only by a defined point
mutation in the dmpR regulatory gene, we could rigorously
test the assumption that enhanced biodegradative capacity under
laboratory conditions translates to improved performance and
competitiveness in structurally and nutritionally heterogeneous soil
matrix conditions. The DmpR-E135K regulatory mutant, which has an
enhanced ability to respond to para-substituted
phenols, exhibited both enhanced biodegradative and enhanced
competitive properties compared to the wild type in two very different
soil types amended with 4-methylphenol (Fig. 3 and 4). Interestingly,
the regulatory mutant strain CF375.3::Km-gfp has a
competitive growth advantage over the wild-type strain in the pine
forest-derived humic soil matrix irrespective of specific amendments
(Fig. 4). In soil from under pine trees, the humus layer is enriched in
phenolics as a result of decomposition and humification, and monomeric
para-substituted phenols are predominant in the
water-soluble phenolic fraction (22). Thus, the universal competitive advantage of CF375.3::Km-gfp in the pine
forest humus may be attributed to the more efficient degradation of a
range of para-substituted phenolic compounds already present
in the soil. The above findings indicate that manipulation of the
regulatory circuit can indeed have significant beneficial effects
on both biodegradative efficiencies and competitiveness under soil
matrix conditions. Consistent with this interpretation,
incubation of CF600.1::Km-gfp in soils amended with
4-methylphenol was accompanied by accumulation of spontaneous mutants
with an enhanced ability to degrade 4-methylphenol (Fig. 3 to 5). DNA
sequence analysis of 10 derivatives revealed seven different individual
mutations in the signal receptor domain of DmpR, and the enhanced
growth phenotypes in each case could be attributed solely to the single amino acid substitutions or insertion within DmpR (Fig. 5). A number of
different molecular genetic strategies have previously been used to
isolate mutations within the A domain of DmpR that positively or
negatively modulate the response to aromatic effectors. These include
positive genetic selection systems and random PCR mutagenesis to
generate mutants with enhanced sensitivity and/or novel sensory
capacity (31, 42, 52), genetic selection of second-site suppressors of constitutively active mutations of DmpR
(26), and DNA shuffling between the A domain of DmpR and that of the toluene-xylene response regulator XylR (44).
The locations of the mutations identified in this study relative to previously identified mutations are shown in Fig.
7. The mutations identified in this study
are from spontaneous mutants that were isolated from soils
contaminated, albeit deliberately, with a toxic aromatic compound, and
thus more closely reflect selection pressures found in the soil
environment. Of the seven different mutations identified, five target
residues that had not previously been implicated in effector
recognition. However, two mutations (F42Y and D116N) target residues
previously implicated by molecular genetic manipulations.
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FIG. 7.
Locations of the A-domain dmpR
mutations identified in the present study (upper arrowheads) in
relation to previously identified A-domain mutations (lower
arrowheads). Open arrowheads indicate mutations that positively affect
the response to aromatic compounds as identified by enhanced responses
to an effector(s) of wild-type DmpR and/or expanded specificity
range (31, 42, 52). Grey and black arrowheads indicate
mutations that negatively affect the aromatic response of DmpR. Grey
arrowheads represent second-site suppressors that restore aromatic
control to semiconstitutive mutants of DmpR by tightening the A-C
domain interaction (26) or mutations that constrict the
aromatic response profile (F132L [44]). Black arrowheads
indicate mutations that result in the inability to respond to aromatic
compounds (26; L.C.Ng and V. Shingler, unpublished data). The hatched
box indicates the region (residues 110 to 186) that distinguishes the
effector specificity profiles of DmpR and XylR (44).
|
|
During aromatic effector activation of DmpR, productive binding of
aromatic effectors alleviates inhibitory interactions between the A and
C domains, thus releasing the transcriptional activating property of
DmpR (26, 28, 42). Not all effectors are equally efficient
in activating DmpR, and modulation of the A-C domain inhibitory
interaction through mutations in either the A or C domain can alter the
magnitude of the response to a given effector (26, 31). In
addition, mutations within the signal reception A domain can expand the
range of compounds that can activate DmpR to promote transcription
(31, 42, 52). As illustrated in Fig. 7, residues involved
in modulating effector specificity and maintaining the A-C domain
interaction are interdispersed on the linear sequence, and replacement
of some residues results in modulation of both the effector specificity
and A-C domain interaction (e.g., E135R/A/D and D140K [26,
42]). Thus, the phenotype of individual A-domain mutations
identified here could have arisen by influencing (i) the aromatic
effector binding properties, (ii) the repressive regulatory function of
the A domain and thus the functional consequences of binding, or (iii)
a combination of both, as was found for DmpR-E135K (29).
Given the interdependence of the A-domain-mediated properties, it is
difficult to infer the precise mechanism by which each of the A-domain
mutations may be mediating its effect. Nevertheless, it is interesting
(i) that five of the mutations [L113V, D116N, F122L, C137Y, and
179(CG)180] lie within residues 110 to 186, which were identified by
DNA shuffling to distinguish the effector activation profiles of
(methyl)phenol-responsive DmpR and toluene-xylene-responsive XylR
(44), (ii) that the R109C mutation lies directly adjacent to this region in a residue conserved between DmpR and XylR, and (iii)
that the F42Y mutation confers the ability to promote a low level of
transcriptional activation in the absence of effectors (Fig. 6), which
is indicative of loosening of the A-C domain interaction (26).
When the individual DmpR sensory mutations were introduced into a
luciferase reporter system, five of the mutations (F42Y, R109C, L113V,
D116N, and C137Y) mediated enhanced responses to 4-methylphenol,
consistent with their ability to promote enhanced rate of degradation
by their CF600 counterpart (Fig. 5 and 6). Unexpectedly, however, the
DmpR-F122L and DmpR-179(CG)180 mutants, while promoting enhanced growth
on 4-methylphenol (Fig. 5), did not give an enhanced response to
4-methylphenol in the luciferase reporter system (Fig. 6). These
apparently conflicting results have been rigorously checked with
independent isolates in both systems (data not shown). While we can
only speculate on the reason for the disparity of the results in the
two systems, a possible explanation may lie in the different
physiological states of the cells under the two experimental
conditions. The DmpR-mediated regulatory circuit is subservient to
global regulation in response to the nutritional and physiological
status of the cell, resulting in optimal expression under conditions
likely to prevail in the environment (46). One major
mechanism that links the physiological status of the cell to
DmpR-mediated transcriptional control has been identified and involves
the metabolic alarmone ppGpp (47). Since ppGpp regulates
the levels of transcription of many genes, including those of some
global regulatory proteins (8), it is plausible that
stability, folding, and/or action of a given mutant may differ
depending on the medium composition and growth phase of the cell.
The physiological status of the cell also appears to have profound
effects on mutational rates (33). The appearance of
mutants with different degrees of fitness as a result of the increased mutational rate in starved or stationary-phase bacterial cultures has
been shown (13, 20). Furthermore, a higher mutation rate creating desired catabolic phenotype was found with nongrowing bacterial cultures in the presence of potentially useful substrates (13). Therefore, the rapid appearance of the fitter
mutants able to efficiently utilize a polluting substrate might be
expected in natural soil environments, since these provide suboptimal
nutrient levels and growth conditions. As shown in Fig. 3C, incubation of CF600.1::Km-gfp in humic soil contaminated with 4-methylphenol for as little as 4 days results in a fitter mutant subpopulation comprising about 0.1 to 1% of the total population. Although we analyzed only 10 mutants, all were found to harbor mutations in the
sensory A domain of DmpR and mediate the enhanced ability to degrade
the pollutant. Thus, it follows that the majority of the observed
fitter population have gained their phenotype by virtue of modulating
the specific regulatory circuit. Hence, it is likely that mutation to
optimize the effector response to a given pollutant(s) is a major
adaptation mechanism for aromatic catabolism of compounds that are
controlled by DmpR-like regulators. Our results show that selection of
DmpR-regulatory mutants with an optimized response to the contaminant
present might be a natural ongoing process in polluted environments
that can occur in a short time frame. As outlined in the introduction,
DmpR- and XylR-like regulators are frequently associated with aromatic
catabolic pathways. Our findings suggest that genetic variation in the
A domains may be feature of this class of regulator and that the
concomitant ability to rapidly adapt transcriptional levels via the
regulator response to aromatics may account for the predominance of the DmpR-XylR family of regulators in controlling aromatic catabolic pathways.
This work was supported by grants from the Swedish Foundation for
Strategic Research, the Swedish Research Councils for Natural and
Engineering Sciences, and the Academy of Finland.
We thank Mirja Salkinoja-Salonen for advice and access to equipment for
the chemical analysis, Rainer Peltola and Irina Tsitko for advice and
help in performing the chemical analysis, Chun-Mei Li for technical
assistance, Jens B. Andersen for plasmids, and Martin Gullberg for
critical reading of the manuscript.
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Abstract
Introduction
