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Journal of Bacteriology, January 2000, p. 100-106, Vol. 182, No. 1
Department of Microbiology, University of
Iowa, Iowa City, Iowa 52242
Received 2 August 1999/Accepted 11 October 1999
Under anaerobic conditions, structurally diverse aromatic compounds
are catabolized by bacteria to form benzoyl-coenzyme A (benzoyl-CoA),
the starting compound for a central reductive pathway for aromatic ring
degradation. The structural genes required for the conversion of
4-hydroxybenzoate (4-HBA) to benzoyl-CoA by Rhodopseudomonas
palustris have been identified. Here we describe a regulatory
gene, hbaR, that is part of the 4-HBA degradation gene
cluster. An hbaR mutant that was constructed was unable to grow anaerobically on 4-HBA. However, the mutant retained the ability
to grow aerobically on 4-HBA by an oxygen-requiring pathway distinct
from the anaerobic route of 4-HBA degradation. The effect of the HbaR
protein on expression of hbaA encoding 4-HBA-CoA ligase, the first enzyme for 4-HBA degradation, was investigated by using hbaA::'lacZ transcriptional fusions.
HbaR was required for a 20-fold induction of A general strategy used by microbes
to degrade diverse aromatic compounds anaerobically is to first convert
them to benzoyl-coenzyme A (benzoyl-CoA), a compound that is the
starting point for a central pathway of aromatic ring reduction and
cleavage (18). Peripheral reactions leading to benzoyl-CoA
formation include modification and removal of benzene ring
substituents, often via 4-hydroxybenzoate (4-HBA) or
4-hydroxybenzoyl-CoA as an intermediate. The conversion of 4-HBA to
benzoyl-CoA is a relatively well-studied reaction sequence (Fig.
1). First, CoA is added to 4-HBA by
4-hydroxybenzoate-CoA ligase. Enzymes catalyzing this reaction have
been purified from the purple nonsulfur bacterium
Rhodopseudomonas palustris (16) and from the
denitrifying species Thauera aromatica (4). The R. palustris gene encoding this enzyme,
hbaA, has been cloned and sequenced (16).
4-Hydroxybenzoyl-CoA (4-HBA-CoA) is then dehydroxylated by 4-HBA-CoA
reductase to yield benzoyl-CoA (17). The genes encoding the
dehydroxylating reductase have been cloned from both R. palustris and T. aromatica, and sequenced, and this enzyme has been purified from T. aromatica (5).
0021-9193/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
HbaR, a 4-Hydroxybenzoate Sensor and FNR-CRP Superfamily Member,
Regulates Anaerobic 4-Hydroxybenzoate Degradation by
Rhodopseudomonas palustris
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-galactosidase activity
that was observed with a chromosomal
hbaA::'lacZ fusion when cells grown
anaerobically on succinate were switched to anaerobic growth on
succinate and 4-HBA. HbaR also activated expression from a
plasmid-borne hbaA-'lacZ fusion when it was
expressed in aerobically grown Pseudomonas aeruginosa
cells, indicating that the activity of this regulator is not sensitive
to oxygen. The deduced amino acid sequence of HbaR indicates that it is
a member of the FNR-CRP superfamily of regulatory proteins. It is most
closely related to transcriptional activators that are involved in
regulating nitrate reduction. Previously, it has been shown that
R. palustris has an FNR homologue, called AadR, that is
also required for 4-HBA degradation. Our evidence indicates that AadR
activates expression of hbaR in response to anaerobiosis
and that HbaR, in turn, activates expression of 4-HBA degradation in
response to 4-HBA as an effector molecule.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (16K):
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FIG. 1.
The hba gene cluster for 4-HBA degradation.
These genes are part of a 26-kb cluster of genes whose products are
involved in anaerobic degradation of aromatic compounds by R. palustris (12). The roles of the hba gene
products in the conversion of 4-HBA to benzoyl-CoA are also shown
(16, 17).
In previous work on anaerobic 4-HBA degradation, we identified a transcriptional activator, AadR, that is required for anaerobic growth of R. palustris on 4-HBA and for expression of hbaA (9). AadR is a member of the FNR family of transcriptional regulators, and based on the presence of the conserved cysteine residues shown to be essential for sensing anaerobiosis by FNR (23), we have proposed that AadR functions in oxygen sensing (9). In addition to being involved in expression of hbaA, AadR is required for expression of the benzoyl-CoA reductase genes, badDEFG (11). Here we describe HbaR, a second transcriptional activator and member of the FNR-CRP superfamily that is involved in regulating the anaerobic degradation of 4-HBA in response to 4-HBA as an effector molecule.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains and culture conditions.
The bacterial
strains and plasmids used are described in Table
1. R. palustris cultures were
grown anaerobically in defined mineral medium (21) prepared
as described previously (19). Carbon sources were added at
the time of inoculation to a final concentration of 3 mM except for
succinate, which was added to 10 mM. Cultures were incubated at 30°C
and illuminated with a 40-W incandescent light bulb for phototrophic
growth or in the dark with shaking for aerobic growth.
Escherichia coli cultures were routinely grown in Luria
broth (LB) (7) at 37°C. For -galactosidase activity
assays, E. coli cultures were grown in LB containing 0.2%
glucose, either aerobically with shaking or anaerobically in
butyl-rubber-stoppered tubes incubated statically. Pseudomonas aeruginosa cultures were grown at 37°C in LB for routine
cultivation and in defined minimal medium (25) with 10 mM
succinate for cultures to be assayed for -galactosidase activity.
Antibiotics were used at the following concentrations (in micrograms
per milliliter): for R. palustris, gentamicin (100) and
kanamycin (100); for P. aeruginosa, kanamycin (100) and
gentamicin (20); and for E. coli, ampicillin (100),
gentamicin (10), kanamycin (100), and spectinomycin (100).
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Cloning and DNA manipulation. Standard protocols were used for cloning and transformations (30). Plasmid DNA was purified by using the QIAprep Spin Miniprep kit (Qiagen Inc., Chatsworth, Calif.). DNA fragments were purified from agarose gels by using the GeneClean spin kit from Bio 101 (La Jolla, Calif.). Chromosomal DNA was purified by using a variation on the method of Saito and Miura (29) as described previously (10).
Reporter plasmids pPE906 and pMD505 containing promoter-lacZ fusions were constructed by a two-step cloning procedure described previously (26). Briefly, promoter-containing DNA fragments were cloned directionally into
Spr-containing cohort
vectors (pHRP315 or pHRP316). Fragments containing the
Spr cassette and promoter region were then cloned
upstream of the promoterless lacZ gene ('lacZ) of
pHRP309. The fusion of the promoter-containing fragments and
'lacZ were then confirmed by sequencing.
Strains with mutations in hbaR (CGA612 and CGA614) were
generated by gene replacement with a cloned copy of hbaR
that had been interrupted with a gentamicin resistance
(Gmr) cassette. The mutagenesis construct was generated by
cloning a Gmr cassette from pUCGM (31) into the
unique AgeI site of hbaR in pPE604. The
hbaR::Gmr construct was then cloned
into pCF116 (14), which contains sacB for
counterselection. The resulting plasmid, pPE900, was mated into
R. palustris, and exconjugants were screened as described previously (10). The
hbaA::'lacZ strain (CGA507) was
generated by using a similar strategy. The hbaA-containing
fragment from pMD300 (16) was cloned into pJQ200mp18
(28) to generate pMD425. The hbaA reading frame
was then interrupted at a SalI site with a
'lacZ-Kmr cassette derived from
pUTminiTn5-lacZ (8) to generate pMD426 and mated
into R. palustris.
Expression of HbaR in E. coli.
HbaR was expressed by
using the T7 promoter system (3). DNA containing
hbaR was PCR amplified and cloned into the NdeI and SmaI sites of pT7-7 (35) to generate pPE905.
E. coli BL21(DE3) cells containing pPE905 were grown in LB
medium to an A660 of approximately 0.25. Cells
(1 ml) were harvested, washed, and resuspended in 1.0 ml of basal
medium supplemented with 0.02% concentrations of each of 18 amino
acids (no methionine or cysteine). After a 30-min incubation at 30°C,
isopropyl--D-thiogalactopyranoside (IPTG) was added to 1 mM, and incubation was continued for 30 min. Rifampin was added to a
final concentration of 0.5 mg/ml, and cells were incubated at 42°C
for 10 min to allow rifampin to enter the cells. After an additional 30 min at 30°C, cells were incubated with 10 µCi of
[35S]methionine for 2 min. Proteins were separated on
sodium dodecyl sulfate-15% polyacrylamide gels.
Gel mobility shift assays. Gel mobility shift assays were attempted by using several different protocols (15, 27). Binding buffers containing various salts at a range of concentrations were used. Target DNA fragments of different lengths were also tried.
Primer extension and reverse transcriptase PCR analysis. Primer extension analysis was used to determine the transcriptional start sites of hbaR and hbaA. The avian myeloblastosis virus reverse transcriptase primer extension system was used according to the protocol supplied by the manufacturer (Promega Corp., Madison, Wis.). The primer used to map the hbaR start site was complementary to nucleotides 13 to 33 of hbaR. The primer used to map the hbaA start site was complementary to bases 147 to 164 of hbaA. Primer extension products were analyzed on a 6% polyacrylamide gel next to a sequence ladder generated by using the same primers. Sequencing reactions were performed with the fmol DNA sequencing system from Promega.
DNA sequencing and analysis. DNA sequences were determined at the University of Iowa DNA Facility by using dye terminator cycle sequencing. The reactions were run on and analyzed with an Applied Biosystems model 373A stretch fluorescent automated sequencer. DNA sequences were analyzed with GENE Inspector, version 1.0.1 (Textco Inc., West Lebanon, N.H.). Similar sequences were identified from the SWISS-PROT 26 and GENPEP 78.0 databases by using the BLAST network service at the National Center for Biotechnology Information (Bethesda, Md.). The GAP program from the University of Wisconsin Genetics Computer Group software package, version 9.0, was used to make sequence comparisons and alignments. The AllAll program from the Computational Biochemistry Research Group (cbrg.inf.ethz.ch./section3_1.html) was used for phylogenetic analysis.
Enzyme assays. 4-HBA-CoA ligase activity was measured by using a spectrophotometric assay described previously (16). Briefly, reaction mixtures containing 50 mM Tris (pH 9.2), 5 mM MgCl2, 0.5 mM ATP, 0.8 mM reduced CoA, 0.25 mM 4-HBA, and cell extract were monitored for increase in absorbance at 330 nm due to formation of 4-HBA-CoA. Activity was calculated by using a millimolar extinction coefficient of 24.
-Galactosidase activity in E. coli and P. aeruginosa cultures was measured by the method of Miller
(24). For R. palustris cultures,
-galactosidase activity was measured by a variation of the method of
Miller (24), as described previously (11). Briefly, logarithmically growing cells were harvested, washed in Z
buffer, and sonicated. Cell extract and Z buffer were combined to a
volume of 1 ml, and 0.2 ml of a 4-mg/ml solution of
o-nitrophenylgalactopyranoside was added to start the
reactions. The rate of increase in absorbance at 420 nm due to
o-nitrophenol formation was measured spectrophotometrically. Activity was calculated by using a millimolar extinction coefficient of
4.5 for o-nitrophenol at 420 nm. Protein concentrations in cell extracts were determined by using the Bio-Rad (Richmond, Calif.)
protein assay kit.
Nucleotide sequence accession number. The DNA sequence of hbaR has been assigned GenBank accession no. AF172325.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Characteristics of hbaR. A 726-bp open reading frame designated hbaR was found next to the R. palustris genes encoding enzymes responsible for the conversion of 4-HBA to benzoyl-CoA (Fig. 1) (16, 17). HbaR was similar in its deduced amino acid sequence to members of the FNR-CRP superfamily of transcriptional regulators. It was most similar (52% similar, 42% identical) to NNR, a positive regulator of nitrite and nitric oxide reductase gene expression in Paracoccus denitrificans (37). The other proteins with the highest levels of amino acid sequence identity to HbaR were members of the DNR group of the FNR-CRP superfamily of transcriptional regulators, as described by Vollack et al. (39) (Fig. 2). HbaR had a low level of amino acid sequence similarity (36%) to the R. palustris protein AadR, an FNR-like regulator that contains the conserved cysteine residues shown to be involved in redox sensing by FNR (9, 23). HbaR does not contain these cysteine residues.
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70 consensus sequence (5'-TATAAT-3')
at four of six positions. The promoter region contained a 5-bp
inverted repeat centered at 
42.5 bases from the start site of
transcription (Fig. 3B). The sequence of this repeat matched that of
the consensus FNR binding site (33) as well as the inverted
repeat previously proposed to be the binding site of AadR (9,
11).
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Characterization of an hbaR mutant. An hbaR mutant (strain CGA612) was unable to grow on 4-HBA under anaerobic conditions, but it grew at wild-type rates on benzoate. The hbaR mutant also grew at wild-type rates on succinate and, like the wild-type parent strain, could grow aerobically on 4-HBA. The defect in anaerobic growth on 4-HBA was complemented in trans by a plasmid-borne copy of hbaR supplied on pPE901. The growth phenotype of this mutant suggested that hbaR is involved in regulating the enzymes that convert 4-HBA to benzoyl-CoA. The first step in this process is the addition of CoA to 4-HBA by 4-HBA-CoA ligase. The activity of 4-HBA-CoA ligase in extracts from wild-type cells grown anaerobically with 4-HBA (0.3 mM) plus succinate (10 mM) was 10.2 nmol/min/mg of protein, while the hbaR mutant had levels of 4-HBA-CoA ligase activity below the level of detection for the assay used (<0.25 nmol/min/mg of protein).
HbaR activates expression of 4-HBA-CoA ligase in response to
4-HBA.
To examine a possible regulatory role of HbaR, the
hbaR mutation was introduced into an R. palustris
strain containing a chromosomal hbaA::'lacZ fusion (CGA507). Levels of
expression of hbaA, the gene encoding 4-HBA-CoA ligase, in
wild-type and hbaR (CGA614) backgrounds were then compared.
Cells with an intact hbaR gene had 20-fold-higher levels of
hbaA expression when 4-HBA was present in anaerobic growth
medium than when they were grown on succinate only. In contrast,
expression of the hbaA::'lacZ fusion
was not induced by 4-HBA in hbaR mutant cells (Table
2). This 20-fold activation by HbaR is
consistent with the difference in 4-HBA-CoA ligase activity between the
wild type and hbaR mutant.
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-galactosidase
activity in the aerobically grown cells.
HbaR is active under aerobic conditions.
Primer extension
analysis was used to identify the promoter region of hbaA
(Fig. 4). The possibility that HbaR is
active in the presence of oxygen was investigated by expressing
hbaR in aerobically growing P. aeruginosa cells
containing a reporter plasmid (pMD505) that had the
hbaA promoter fused to a promoterless lacZ gene.
Such cells exhibited a fivefold increase in -galactosidase expression over the levels seen in the absence of hbaR (Fig.
5). When 4-HBA was present in the growth
medium, levels of -galactosidase activity increased twofold more
over levels in cells grown on succinate only, for a net 10-fold
induction. This suggests that HbaR acts directly at the hbaA
promoter and activates expression in response to 4-HBA. In addition,
this shows that, although hbaA is not expressed in
aerobically growing R. palustris cells (Table 2), HbaR is
able to activate transcription from the hbaA promoter in the
presence of oxygen.
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Expression of HbaR in E. coli. HbaR was expressed in E. coli cells from the plasmid pPE905 as described in Materials and Methods. The apparent molecular mass of the HbaR peptide (25 kDa) was close to that predicted based on the deduced amino acid sequence of HbaR (28.5 kDa). E. coli cell extracts containing overexpressed HbaR were tested to see if they would cause a shift in gel mobility of DNA fragments containing the hbaA promoter region. No change in the mobility of a fragment containing the hbaA promoter region was seen with these extracts or with extracts from cultures of R. palustris or P. aeruginosa expressing HbaR (data not shown). The addition of 4-HBA to the gel shift assay mixtures had no apparent effect on binding. An HbaR-His fusion peptide purified to near homogeneity was also tested in gel mobility shift assays. The purified peptide was mostly insoluble, precipitated out of solution easily, and bound nonspecifically to all DNA fragments tested including vector DNA (data not shown).
E. coli FNR can activate hbaR
expression.
The hbaR promoter region has an inverted
repeat centered at 42.5 bp from the start site of transcription that
matches the FNR consensus binding site (Fig. 3B). To determine whether
hbaR expression might be activated by an FNR-type regulator,
a 226-bp fragment containing the hbaR promoter region was
cloned in front of the promoterless lacZ gene in pHRP309 by
using a two-step cloning system described previously (26).
-Galactosidase expression from the PhbaR-'lacZ
plasmid (pPE906) was measured in wild-type and fnr mutant
E. coli cells. In wild-type cells, the levels of PhbaR-'lacZ fusion expression were threefold
higher when cells were grown anaerobically than when cells were grown
in the presence of oxygen (Table 3).
PhbaR-'lacZ activity was not induced in response
to anaerobiosis in the fnr mutant strain of E. coli. This suggests that hbaR could also be regulated
by an FNR-like protein in R. palustris. Similar experiments
were done to examine the possible effect of FNR on hbaA
expression. We found that -galactosidase expression from a
PhbaA-'lacZ plasmid present in E. coli
was not influenced by the fnr mutation.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Data presented here indicate that HbaR is a transcriptional activator that senses 4-HBA as an effector molecule and induces expression of hbaA, the gene encoding 4-HBA-CoA ligase and first enzyme of 4-HBA degradation. Although we were unable to demonstrate binding of HbaR to the hbaA promoter in vitro with gel mobility shift assays, experiments with HbaR expressed in P. aeruginosa indicate that HbaR activates expression of the hbaA promoter. These experiments also show that when expressed in P. aeruginosa, HbaR can activate expression from the hbaA promoter in the presence of oxygen (Fig. 5). In contrast, hbaA expression was not activated in aerobically grown R. palustris cells. This suggests that a second regulator is required for activation of hbaA expression in response to anaerobiosis. Our results showing that E. coli fnr influences hbaR expression and the fact that hbaR has an FNR binding site in its promoter region suggest that hbaR expression is activated in response to anaerobiosis in R. palustris by an FNR homologue. HbaR, in turn, activates hbaA expression in response to 4-HBA. The R. palustris FNR homologue in question is, presumably, AadR, since AadR is required for HbaA expression in this organism (9). This restriction of HbaR expression in R. palustris to anaerobically growing cells indicates that this organism must have a separate system for detecting 4-HBA under aerobic conditions and for activating the genes encoding enzymes of the aerobic 4-HBA degradation pathway.
HbaR is a member of the FNR-CRP superfamily of transcriptional regulators. This protein family has been divided into three classes based on sequence similarity to the reference proteins FNR, which activates gene expression in response to anaerobiosis, CRP, a regulator of catabolic functions, and NtcA, a regulator of genes involved in nitrogen and sulfur metabolism (13) (Fig. 2). The FNR class (which includes AadR of R. palustris) has been further subdivided into four groups based on the presence and spacing of the conserved cysteine residues required for assembly of the redox-sensitive iron-sulfur center. The sequence of HbaR places it in a group (DNR) of the FNR class that is comprised of proteins that lack the cysteine residues required for iron-sulfur center coordination and oxygen sensing (39). With the exception of HbaR, all the proteins within the DNR group are global regulators that control expression of genes involved in denitrification (1, 36-38).
A recently recognized theme among members of the FNR-CRP superfamily is the involvement of two members of the protein family in regulation of the same system. Transcriptional regulation of genes involved in denitrification in P. aeruginosa and Paracoccus denitrificans also involves control by two members of the FNR-CRP superfamily (2, 37). The involvement of multiple family members has also been demonstrated in Pseudomonas stutzeri, which has at least four FNR family members involved in regulating metabolic processes (39). In some cases, including the system we have described here, this multiplicity of regulators involves a regulatory cascade, with one protein acting as an oxygen sensor and, in turn, activating expression of a second regulator (2, 39). Hierarchical expression of two regulators that are members of the greater FNR family would be an effective strategy to prevent cross talk and provide regulation of relatively specialized target genes, as in the case of 4-HBA degradation, under conditions of oxygen deprivation. This study expands the range of functions regulated by the greater FNR-CRP superfamily to include aromatic carbon source utilization and expands the range of effectors sensed by members of this superfamily to include aromatic acids.
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ACKNOWLEDGMENTS |
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This work was supported by the Division of Energy Biosciences, Department of Energy (grant DE-FG02-95ER20184), and by the U.S. Army Research Office (grant DAAG55-98-0188).
We thank Walter Zumft for helpful discussions and Marilyn Dispensa for help with some strain constructions.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7783. Fax: (319) 335-7679. E-mail: caroline-harwood{at}uiowa.edu.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, January 2000, p. 100-106, Vol. 182, No. 1
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