HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Durante-Rodríguez, G. Articles by Carmona, M. Search for Related Content PubMed PubMed Citation Articles by Durante-Rodríguez, G. Articles by Carmona, M. Agricola Articles by Durante-Rodríguez, G. Articles by Carmona, M.

New insights into the BzdR-mediated transcriptional regulation of the anaerobic catabolism of benzoate in Azoarcus sp. CIB

Gonzalo Durante-Rodríguez

M. Teresa Zamarro

Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain

Correspondence
Manuel Carmona
mcarmona{at}cib.csic.es

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
The expression of the bzd genes involved in the anaerobic degradation of benzoate in Azoarcus sp. CIB is controlled by the specific BzdR transcriptional repressor at the P_N promoter. This catabolic promoter is also subject to catabolite repression by some organic acids. In vivo and in vitro experiments have shown that BzdR behaves as a repressor of the P_R promoter by overlapping the transcription initiation site as well as the –35 and –10 boxes, benzoyl-CoA being the inducer molecule. In addition, by using a P_N : : lacZ fusion both in Azoarcus sp. CIB and in an isogenic strain lacking the bzdR gene, we have shown that the succinate-dependent catabolite repression requires participation of the BzdR repressor.

These authors contributed equally to this work.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Since aromatic compounds are widely distributed in the environment, they are a common carbon source for micro-organisms (Harwood & Parales, 1996). As many ecosystems are often anoxic, the anaerobic catabolism of aromatic compounds by micro-organisms is important in the global biogeochemical cycles (Gibson & Harwood, 2002; Lovley, 2003). Benzoate has been used as a good model compound for studying the anaerobic catabolism of aromatic compounds. Benzoate is a common carbon source in nature that is funnelled directly to the widely distributed benzoyl-coenzyme A (benzoyl-CoA) central pathway (Carmona & Díaz, 2005; Gibson & Harwood, 2002; Harwood et al., 1999). As a general rule, micro-organisms first activate benzoate to benzoyl-CoA, which is then converted to aliphatic intermediates through aromatic ring reduction, β-oxidation-like reactions and ring cleavage as the main steps. The biochemistry of the benzoyl-CoA central pathway has been well studied in some facultative anaerobic bacteria such as Rhodopseudomonas palustris, Azoarcus evansii and Thauera aromatica (Boll, 2005; Gibson & Harwood, 2002; Harwood et al., 1999) and to a lesser extent in strict anaerobes such as Desulfococcus multivorans (Peters et al., 2004), Geobacter metallireducens (Carmona & Díaz, 2005; Wischgoll et al., 2005) and Syntrophus aciditrophicus (Elshahed et al., 2001; Peters et al., 2007). Moreover, molecular microbial ecology studies suggest that the ability to degrade aromatic compounds under anaerobic conditions is more widespread among bacteria than previously thought (Jahn et al., 2005; Safinowski et al., 2006; Song & Ward, 2005). In contrast to the current knowledge on the biochemistry of the anaerobic benzoyl-CoA central pathway, few studies have been conducted to unravel the regulatory circuits that control the expression of the cognate genes, being mainly restricted to the regulation of the bad genes in Rhodopseudomonas palustris and the bzd genes in Azoarcus sp. CIB (Barragán et al., 2005; Durante-Rodríguez et al., 2006; Egland & Harwood, 1999, 2000; Peres & Harwood, 2006). In the latter organism, the bzd catabolic genes are organized as a single operon (bzdNOPQMSTUVWXYZA) controlled by the P_N promoter (López Barragán et al., 2004) and the bzdR gene, which is located immediately upstream of the catabolic operon and encodes the transcriptional repressor BzdR (Barragán et al., 2005). The BzdR-mediated repression of P_N is alleviated by the inducer benzoyl-CoA, the first intermediate of the catabolic pathway (Barragán et al., 2005) (Fig. 1). The BzdR protein is the prototype of a new subfamily of transcriptional regulators that exhibit a peculiar modular architecture. Thus, whereas the N-terminal region of BzdR (residues 1–130) shows significant similarity with members of the helix–turn–helix (HTH)-XRE family of transcriptional regulators (SMART release; available at http://smart.embl-heidelberg.de/), the C-terminal region (residues 131–298) shows similarity with shikimate kinases (PFAM accession no. PF01202) (Barragán et al., 2005). Although the BzdR subfamily of transcriptional regulators is actively growing, and currently BzdR-like proteins can be identified by sequence comparison analyses in benzoate degradation clusters of over 30 denitrifying proteobacteria from the and β-subgroups, to our knowledge there have been no reports on the expression of any of the bzdR genes.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Scheme of the bzd cluster and PR promoter from Azoarcus sp. CIB. (a) The bzdR regulatory gene and the bzdNOPQMSTUVWXYZA catabolic operon are shown as black and white boxes, respectively. The PR and PN promoters are indicated as thick white and grey arrows, respectively. The BzdR protein is represented as a black ellipse; benzoyl-CoA is represented as a white rectangle. The minus signs indicate repression of PR and PN by the BzdR protein. The ellipse within the rectangle represents a BzdR protein that is not able to repress the PR or the PN promoters. In the anaerobic catabolism of benzoate, this molecule is first activated to benzoyl-CoA by a benzoate-CoA ligase (BzdA) and is channelled to the central metabolism of the cell through a central pathway that involves the products of the genes bzdNOPQMSTUVWXYZ. (b) Expanded view of the PR promoter. The nucleotide sequence from position –241 to +42 is shown. The transcription start site (+1) and the inferred –10 and –35 boxes of the PR promoter are indicated. The ribosome-binding site (RBS) and the ATG start codon of the bzdR gene are shown in italic and bold letters, respectively. The BzdR-binding region (operator) is boxed. The TGCA and GCAC sequences within the operator region are underlined with a discontinuous line. A short palindromic structure is indicated by convergent arrows.

In this work we have studied the regulatory circuit that drives the expression of a gene encoding a transcriptional regulator (BzdR) of a catabolic operon involved in anaerobic degradation of aromatic compounds. Moreover, we provide genetic evidence that the BzdR repressor is also required for the catabolite repression exerted by some carbon sources on the anaerobic benzoate-degradation pathway in Azoarcus sp. CIB.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Strains, plasmids, and growth conditions.
The Escherichia coli and Azoarcus strains, as well as the plasmids used in this work, are listed in Table 1. E. coli cells were grown at 37 °C on Luria–Bertani (LB) medium (Miller, 1972). Azoarcus strains were grown at 30 °C on MC medium as described previously (López Barragán et al., 2004). Where appropiate, antibiotics were added at the following concentrations: ampicillin, 100 µg ml^–1; gentamicin, 7.5 µg ml^–1; kanamycin, 50 µg ml^–1.

β-Galactosidase assays.
β-Galactosidase activities were measured with permeabilized cells as described by Miller (1972). Experiments were repeated at least three times for each sample.

Sequence data analyses.
Sequence comparison analyses with the Azoarcus sp. strain EbN1 complete genome dataset were done using the TBLAST algorithm (Altschul et al., 1990) at the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/BLAST/BLAST.cgi).

Purification of His₆-BzdR.
The recombinant plasmid pQE32-His₆BzdR (Table 1) was used for the overproduction of the His-tagged BzdR protein in E. coli strain M15 harbouring plasmid pREP4 (Table 1) as described previously (Barragán et al., 2005).

Gel retardation assays.
The DNA fragment used for gel retardation assays (P_R probe) was PCR amplified from plasmid pECOR7 (Table 1) by using oligonucleotides 5'PR-ScaI (5'-AAAAGTACTCGCGGTTCATGACGTTCATCTG-3'; an engineered ScaI site is underlined) and 3'PR-EcoRI (5'-CGGAATTCCGCCCAACATCAGCAGGTAGTTG-3'; an engineered EcoRI site is underlined). The amplified DNA was then digested with ScaI and EcoRI restriction enzymes, and the resulting 298 bp fragment was singly 3'-end labelled by filling in the overhanging EcoRI-digested end with [-³²P]dATP and the Klenow fragment of E. coli DNA polymerase as reported previously (Barragán et al., 2005). The retardation reaction mixtures in FP buffer (20 mM Tris/HCl, pH 7.5, 10 % glycerol, 2 mM β-mercaptoethanol and 50 mM KCl) contained 0.05 nM DNA probe, 500 µg BSA ml^–1 and purified His₆-BzdR protein in a final volume of 9 µl. After incubation of the retardation mixtures for 20 min at 30 °C, the mixtures were analysed by electrophoresis in 5 % polyacrylamide gels buffered with 0.5x TBE (45 mM Tris/borate, 1 mM EDTA). The gels were dried on Whatman 3MM paper and exposed to Hyperfilm MP (Amersham Biosciences).

DNase I footprinting assays.
The DNA probe used for DNase I footprinting assays was the same as that reported for the gel retardation assays (see above). For the assays, the reaction mixture contained 2 nM DNA probe, 1 mg BSA ml^–1 and purified protein in 15 µl FP buffer (see above). This mixture was incubated for 20 min at 37 °C, after which 3 µl (0.05 units) of DNase I (Amersham Biosciences) (prepared in 10 mM CaCl₂, 10 mM MgCl₂, 125 mM KCl and 10 mM Tris/HCl, pH 7.5) was added, and the incubation was continued at 37 °C for 20 s. The reaction was stopped by the addition of 180 µl of a solution containing 0.4 M sodium acetate, 2.5 mM EDTA, 50 µg calf thymus DNA ml^–1 and 0.3 µg glycogen ml^–1. After phenol extraction, DNA fragments were analysed as previously described (Barragán et al., 2005). A+G Maxam and Gilbert reactions (Maxam & Gilbert, 1980) were carried out with the same fragments and loaded on the gels along with the footprinting samples. The gels were dried on Whatman 3MM paper and exposed to Hyperfilm MP (Amersham Biosciences).

In vitro transcription assays.
Transcription assays were performed by a published procedure (Carmona & Magasanik, 1996). A 298 bp DNA fragment containing the P_R promoter was PCR amplified from plasmid pECOR7 (Table 1) by using oligonucleotides 5'PR-ScaI and 3'PR-EcoRI (see above), and it was cloned into the SmaI/EcoRI-restricted pJCD01 cloning vector (Marschall et al., 1998), yielding plasmid pJCD-P_R (Table 1). Reactions (50 µl mixtures) were performed in a buffer consisting of 50 mM Tris/HCl (pH 7.5), 50 mM KCl, 10 mM MgCl₂, 0.1 mM BSA, 10 mM dithiothreitol and 1 mM EDTA. Each DNA template (0.5 nM of supercoiled plasmid pJCD-P_R) was premixed with 50 nM ⁷⁰-containing E. coli RNA polymerase (Amersham) and different amounts of purified His₆-BzdR and benzoyl-CoA. For multiple-round assays, transcription was then initiated by adding a mixture of 500 nM (each) ATP, CTP and GTP; 50 mM UTP; and 2.5 µCi [-³²P]UTP (3000 mCi mmol^–1; 111 GBq mmol^–1). After incubation for 15 min at 37 °C, the reactions were stopped with an equal volume of a solution containing 50 mM EDTA, 350 mM NaCl and 0.5 mg carrier tRNA ml^–1. The mRNA produced was then precipitated with ethanol, redissolved in loading buffer (7M urea, 1 mM EDTA, 0.6 M glycerol, 0.9 mM bromophenol blue and 1.1 mM xylene cyanol), electrophoresed on a denaturing 7M urea–4 % polyacrylamide gel, and visualized by autoradiography. Transcript levels were quantified with a Bio-Rad Molecular Imager FX system. The reactions were done using as a template pJCD01 plasmid derivatives, which enabled an internal control mRNA of 105 nt to be obtained.

Primer extension analysis.
Azoarcus sp. CIB cells harbouring plasmid pBBR5P_R (P_R : : lacZ) were grown anaerobically on benzoate-containing MC medium until the culture reached an OD₆₀₀ of 0.3. Total RNA was isolated by using an RNeasy Mini kit (Qiagen) according to the instructions of the supplier. Primer extension reactions were carried out with the avian myeloblastosis virus reverse transcriptase (Promega) and 10 µg total RNA as described previously (Martín et al., 1996), using oligonucleotide Lac57 (5'-CGATTAAGTTGGGTAACGCCAGGG-3') labelled at its 5'-end with phage T4 polynucleotide kinase and [-³²P]ATP (3000 Ci, 111 TBq mmol^–1; Amersham Biosciences). To determine the length of the primer extension products, sequencing reactions of pBBR5P_R were carried out with oligonucleotide Lac57 using the T7 sequencing kit and [³²P]dCTP (Amersham Biosciences) as indicated by the supplier. Products were analysed on 6 % polyacrylamide–urea gels. The gels were dried onto Whatman 3MM paper and exposed to Hyperfilm MP (Amersham Biosciences).

Real-time RT-PCR assay.
For this assay, RNA was extracted from Azoarcus sp. CIB cells grown on benzoate (3 mM), succinate (7.5 mM), or benzoate (3 mM) plus succinate (7.5 mM). Cells were harvested at the exponential phase of growth (OD₆₀₀ 0.2) and stored at –80 °C. Pellets were thawed and cells lysed in TE buffer (10 mM Tris/HCl pH 7.5, 1 mM EDTA) containing 5 mg lysozyme ml^–1 by a series of freeze/thaw cycles. RNA was extracted using the RNeasy Mini kit (Qiagen), including a DNase treatment according to the manufacturer's instructions, precipitated with ethanol, washed and resuspended in 40 µl RNase-free water. The concentration and purity of the RNA samples were measured by using an ND1000 spectrophotometer (Nanodrop Technologies) according to the manufacturer's protocols. Analyses of the transcript levels from the P_N or P_R promoters were carried out with 20 µl reverse transcription reactions containing 2 µg RNA, 0.5 mM of each dNTP, 200 U SuperScript II reverse transcriptase (Invitrogen) and 0.5 µM primer 3RTpN2 (5'-GTGTAGGTACACATCGTTGC-3') or 3RTpR2 (5'-GAGGATGTCCAGGAAGGATTG-3'), respectively, in the buffer recommended by the manufacturer. The reactions were incubated at 42 °C for 2 h, terminated by incubation at 70 °C for 15 min, and the template RNA was removed by incubation at 37 °C in the presence of 20 U RNase H (Amersham Biosciences) for 20 min. The cDNA obtained was stored at –20 °C. The IQ5 Multicolor Real-Time PCR Detection System (Bio-Rad) was used for real-time PCR in a 25 µl reaction containing 10 µl diluted cDNA, 0.2 µM 3RTpR2 primer, 0.2 µM 5RTpR1 primer (5'-GCTGTCATCGTGCTTCACG-3') for the P_R promoter, or 0.2 µM 3RTpN2 primer, 0.2 µM 5RTpN1 primer (5'-GCAACACATCAGAGGAGATAG-3') for the P_N promoter, and 12.5 µl SYBR Green mix (Applied Biosystems). PCR amplifications were carried out as follows: 1 initial cycle of denaturation (95 °C for 3 min), followed by 44 cycles of amplification (95 °C, 30 s; test annealing temperature, 57 °C, 30 s; elongation and signal acquisition, 72 °C, 30s). For relative quantification of the fluorescence values, a calibration curve of serial dilutions of the genomic DNA sample was made.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
BzdR auto-regulates its expression
Transcriptional regulators governing the expression of catabolic promoters from aromatic catabolic pathways usually also control their own expression (Díaz & Prieto, 2000; Tropel & van der Meer, 2004). However, there appear to be no studies on the auto-regulation of transcriptional regulators controlling anaerobic aromatic pathways. To gain some knowledge on this topic, we checked whether the BzdR regulator from the anaerobic bzd cluster of Azoarcus sp. CIB also controls the activity of its own P_R promoter. To accomplish this task, we fused the P_R promoter to the lacZ reporter gene to generate plasmid pBBR5P_R (Table 1). We then analysed the β-galactosidase activity when Azoarcus sp. CIBdbzdR, a mutant strain carrying a disrupted bzdR gene (Table 1), harbouring plasmid pBBR5P_R (P_R : : lacZ), was grown on benzoate, succinate, or benzoate plus succinate as carbon sources. In all conditions tested, the bzdR mutant cells showed similar levels of expression of the P_R : : lacZ fusion to those of the wild-type Azoarcus sp. CIB(pBBR5P_R) growing on benzoate (induction conditions) (Fig. 2a), which suggests that the bzdR gene product indeed behaves as a repressor of its own expression.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Expression of the bzdR gene under different growth conditions. (a) Activity of the PR promoter. β-Galactosidase activity of Azoarcus sp. CIB (white bars), Azoarcus sp. CIBdbzdR (black bars) and Azoarcus sp. CIBdbzdN (grey bars) cells harbouring plasmid pBBR5PR (PR : : lacZ) was assayed. Cells were grown anaerobically until mid-exponential phase on MC medium containing 3 mM benzoate (B), 7.5 mM succinate (S), or 3 mM benzoate plus 7.5 mM succinate (B+S). β-Galactosidase activity was measured as described in Methods. (b) β-Galactosidase activities of Azoarcus sp. CIB (white bars) or Azoarcus sp. CIBdacpR (hatched bars) cells grown aerobically (+O2) or anaerobically (–O2) until mid-exponential phase on MC medium containing 3 mM benzoate (B) or 7.5 mM succinate (S). (c) Activity of the PR promoter measured by real-time RT-PCR was determined as detailed in Methods. Relative changes in the PR transcript level are indicated as percentages of the values obtained from cells growing on benzoate plus succinate. Error bars in (a) and (b) indicate standard deviations (n=3).

To confirm in vitro the regulation of the P_R promoter by the BzdR regulator, we first mapped the transcription start site of P_R by primer extension analysis performed with total RNA isolated from cells of Azoarcus sp. CIB(pBBR5P_R) grown on benzoate. Transcription initiation was located 39 nt upstream of the ATG translation initiation codon of the bzdR gene (Fig. 3), showing putative –10 (CACTAT) and –35 (TTGCAA) boxes separated by 17 bp (Fig. 1) that match significantly the –10 (TATAAT) and –35 (TTGACA) consensus sequences recognized by the ⁷⁰ subunit of RNA polymerase (⁷⁰-RNAP). We then performed in vitro transcription assays using purified His₆-BzdR protein, RNAP and plasmid pJCD-P_R, which contains a DNA fragment including the P_R promoter (Table 1), as supercoiled DNA template. As shown in Fig. 4, the formation of the expected 242 nt transcript due to the activity of the P_R promoter was inhibited by increasing concentrations of His₆-BzdR (Fig. 4, lanes 3–5). In the presence of 200 nM His₆-BzdR, the addition of benzoyl-CoA was able to increase the formation of the P_R transcript (Fig. 4, lane 6), suggesting that this CoA derivative acts as an inducer avoiding the repression effect of BzdR. These results are in agreement with the previous lacZ gene fusion experiments (see above), and they reveal a similar pattern of benzoyl-CoA-dependent induction of the BzdR-mediated repression at both the P_R and P_N (Barragán et al., 2005) promoters from Azoarcus sp. CIB.

View larger version (82K): [in this window] [in a new window]   Fig. 3. Identification of the transcription start site in the PR promoter. Total mRNA was isolated from Azoarcus sp. CIB cells harbouring plasmid pBBR5PR (PR : : lacZ), as described in Methods. The size of the extended product (lane PR) was determined by comparison with the DNA sequencing ladder (lanes A, C, G and T) of the PR promoter region. Primer extension and sequencing reactions were performed with primer Lac57 as described in Methods. An expanded view of the nucleotides surrounding the transcription initiation site (asterisk) in the noncoding strand is shown.

View larger version (26K): [in this window] [in a new window]   Fig. 4. In vitro activity of the PR promoter. Multiple-round in vitro transcription reactions were carried out by using pJCD-PR, a plasmid template that produces mRNA from PR of 242 nt. In vitro transcription reactions were performed with 50 nM E. coli RNAP in the absence of His6-BzdR (lane 2), or in the presence of 50 nM (lane 3), 100 nM (lane 4) or 200 nM (lane 5) purified His6-BzdR. In lane 6, 1 mM benzoyl-CoA was added to the transcription reaction containing 200 nM purified His6-BzdR. Heat-inactivated His6-BzdR (200 nM) was added as a control to the transcription reaction (lane 1).

View larger version (64K): [in this window] [in a new window]   Fig. 5. In vitro analysis of the interaction of BzdR with the PR promoter. (a) Gel retardation analysis of His6-BzdR binding to the PR promoter, performed as indicated in Methods. Lane 1, free PR probe; lanes 2–5, retardation assays containing 0.05, 0.1, 0.5 and 1 µM, respectively, of purified His6-BzdR. The PR probe and the PR–BzdR complex are indicated by arrows. (b) DNase I footprinting analysis of the interaction of purified His6-BzdR with the PR promoter region. The experiments were carried out using the PR probe as indicated in Methods. Lane 1, footprinting assay in the absence of His6-BzdR; lanes 2–5, footprinting assays containing 0.1, 0.2, 0.5 and 1 µM purified His6-BzdR, respectively; lane AG, A+G Maxam and Gilbert sequencing reaction. The protected region is marked by a bracket, and the phosphodiester bonds hypersensitive to DNase I cleavage are indicated by asterisks. The –10 and –35 boxes and the transcription initiation site (+1) of the PR promoter are also shown.

Role of oxygen and additional carbon sources in the expression of the bzdR gene
Since the bzd genes are responsible for the anaerobic metabolism of benzoate in Azoarcus sp. CIB, the presence or absence of oxygen could play an essential role in the expression of such genes. In agreement with this, we have shown previously that the activity of the catabolic P_N promoter is inhibited by oxygen (Durante-Rodríguez et al., 2006). To determine whether oxygen controls the expression of P_R, we checked the activity of the P_R promoter driving the expression of the bzdR gene when Azoarcus sp. CIB was cultivated in the presence or absence of oxygen. To this end, we determined the β-galactosidase activity in Azoarcus sp. CIB(pBBR5P_R) cells that had reached, under aerobic or anaerobic conditions, mid-exponential growth phase on benzoate-containing minimal medium. As shown in Fig. 2(a, b), although the activity of the P_R promoter was almost twofold higher in cells growing aerobically than in cells growing anaerobically, oxygen does not appear to play a major role in the expression of the P_R promoter. To confirm this result, we checked the expression of the P_R promoter in Azoarcus sp. CIBdacpR, a strain that harbours a disruption of the acpR gene required for the oxygen-dependent activity of the catabolic P_N promoter and, therefore, unable to grow anaerobically on benzoate (Durante-Rodríguez et al., 2006). As shown in Fig. 2(b), the activity of the P_R promoter in Azoarcus sp. CIBdacpR(pBBR5P_R) cells growing anaerobically on succinate-containing minimal medium was similar to that observed in Azoarcus sp. CIB(pBBR5P_R) cells. Moreover, Western-blotting experiments using anti-BzdR antibodies revealed that the levels of BzdR protein in crude extracts of Azoarcus sp. CIB cells grown either aerobically or anaerobically were similar (data not shown). All these results taken together indicate that the P_R promoter is not subject to the AcpR/oxygen-dependent superimposed regulation that controls the activity of the catabolic P_N promoter in Azoarcus sp. CIB. The aerobic expression of the bzdR gene might contribute to the lack of expression of the bzd catabolic operon when the cells grow in the presence of oxygen, thus avoiding the production of some oxygen-sensitive enzymes such as benzoyl-CoA reductase (BzdNOPQ) (López Barragán et al., 2004).

The simultaneous presence of different carbon sources plays a major role in the superimposed regulation of aromatic catabolic clusters (Cases & de Lorenzo, 2005; Díaz & Prieto, 2000; Carmona et al., 2008). Whereas carbon catabolite repression of regulatory genes has been reported in aerobic degradation pathways (Canosa et al., 2000; Müller et al., 1996), such superimposed regulation of regulatory genes controlling aromatic anaerobic clusters has not been studied so far. As shown in Fig. 2(a), the activity of the P_R promoter in Azoarcus sp. CIB(pBBR5P_R) was almost twofold higher when the cells were grown on benzoate or benzoate plus succinate than when they were grown on succinate as sole carbon source. Therefore, in contrast to what had been observed for the bzd catabolic genes, whose expression becomes significantly reduced when the cells grow in the presence of benzoate and an additional carbon source such as succinate (López Barragán et al., 2004), the expression of the bzdR gene does not decrease when succinate is provided as an additional carbon source. To confirm that the activity levels of the P_R : : lacZ fusion are indeed the result of the transcriptional activity of the P_R promoter and are not due to a possible post-transcriptional regulation of the lacZ gene, we performed real-time RT-PCR experiments to monitor the P_R transcript levels directly. The results obtained using RNAs isolated from Azoarcus sp. CIB cells grown on succinate, benzoate or succinate plus benzoate were in good agreement with those reported by β-galactosidase assays with the P_R : : lacZ fusion (Fig. 2c). Therefore, all these results indicate that the expression of the bzdR gene is not subject to catabolite repression by organic acids, such as succinate, in Azoarcus sp. CIB.

BzdR is required for the catabolite repression of the bzd catabolic operon
As previously reported for Azoarcus sp. CIBlacZ, a strain that contains the P_N : : lacZ translational fusion integrated into its chromosome, the P_N promoter is subject to carbon catabolite repression by some organic acids such as succinate, malate and acetate (López Barragán et al., 2004). Whereas the molecular basis of the carbon catabolite repression has been studied in some aromatic aerobic clusters, there are no data on the factor(s) involved in catabolite repression of aromatic anaerobic clusters. To try to identify some of the factors involved in such catabolite repression, we analysed the expression of the P_N : : lacZ translational fusion in Azoarcus sp. CIB and Azoarcus sp. CIBdbzdR strains harbouring plasmid pBBR5P_N (P_N : : lacZ) (Table 1). As expected, the β-galactosidase activity levels in Azoarcus sp. CIB(pBBR5P_N) cells grown anaerobically in the presence of benzoate plus succinate were threefold lower than those obtained in cells grown on benzoate as the sole carbon source (Fig. 6a), which confirms the observed catabolite repression exerted by succinate at the P_N promoter when expressing the P_N : : lacZ fusion from the chromosome (López Barragán et al., 2004). However, it is interesting to note a stronger catabolite repression (about 500-fold repression) using the chromosome-inserted P_N : : lacZ fusion (López Barragán et al., 2004), suggesting that the element(s) responsible for the catabolite repression is present in Azoarcus sp. CIB at a concentration that cannot overcome the multicopy dosage effect of the P_N : : lacZ fusion in plasmid pBBR5P_N. To confirm that the activity levels of the P_N : : lacZ fusion are indeed the result of the transcriptional activity of the P_N promoter and they are not due to a possible post-transcriptional regulation of the lacZ gene, we performed real-time RT-PCR experiments to monitor the P_N transcript levels directly. The results obtained using RNAs isolated from Azoarcus sp. CIB cells grown on succinate, benzoate or succinate plus benzoate were in good agreement with those reported by β-galactosidase assays with the P_N : : lacZ fusion, and they confirmed that the catabolite repression caused by succinate on the P_N promoter is exerted at the transcriptional level (Fig. 6b).

View larger version (13K): [in this window] [in a new window]   Fig. 6. In vivo activity of the PN promoter. Azoarcus sp. CIB cells were grown anaerobically on MC medium containing 3 mM benzoate (B), 7.5 mM succinate (S), or a mixture of 3 mM benzoate and 7.5 mM succinate (B+S), until the cultures reached mid-exponential phase. (a) Expression of the PN : : lacZ reporter fusion. β-Galactosidase activity of Azoarcus sp. CIB (white bars) or Azoarcus sp. CIBdbzdR (black bars) cells harbouring plasmid pBBR5PN (PN : : lacZ) was assayed as described in Methods. Error bars indicate standard deviations (n=3). (b) Activity of the PN promoter measured by real-time RT-PCR. Total RNA was obtained from Azoarcus sp. CIB cultures, and real-time RT-PCR was performed as detailed in Methods. Relative changes in the PN transcript levels are indicated as percentages of the values obtained from cells growing on benzoate. The results of one experiment are shown, and the values were reproducible in three separate experiments with standard deviations of <10 % of the mean.

Concluding remarks
There are several reports showing that the downregulation of promoters from catabolic pathways when the cells grow in the presence of the particular substrate and a preferred carbon source is mediated by shifting the levels of the cognate transcriptional regulators. Thus, catabolite repression of the alkane degradation pathway (alk genes) encoded in the OCT plasmid from Pseudomonas putida strain GPo1 parallels a decrease of the expression of the AlkS activator (Canosa et al., 2000). Similarly, catabolite repression of phenol degradation (phl genes) in P. putida strain H occurs by interfering with the activating function of the PhlR transcriptional regulator (Müller et al., 1996). In this work, however, we show that a specific transcriptional regulator whose expression is not subject to catabolite repression (see above) mediates the catabolite repression of the cognate catabolic operon. Moreover, our results suggest the existence of additional factors that, together with BzdR, might account for the molecular basis of the catabolite repression of the anaerobic benzoate degradation pathway in Azoarcus sp. CIB. A possible scenario for catabolite repression that would explain our results is that BzdR might recruit an additional regulatory factor that binds to the P_N promoter in response to succinate, thus preventing the basal levels of bzd readthrough that allow the effector benzoyl-CoA to be generated. In this context, proteins such as Crc (catabolite repression control) (Morales et al., 2004), PtsN and PtsO (nitrogen phosphotransferase system) (Aranda-Olmedo et al., 2005, 2006; Cases & de Lorenzo, 2005; Marqués et al., 2006), and some o-type terminal oxidases (Cyo) that sense the redox state of the cell (Rojo & Dinamarca, 2004; Morales et al., 2006; Petruschka et al., 2001) have been reported to be involved in catabolite repression of several aromatic degradation pathways in Pseudomonas. Recently, a response regulator (BphQ) of a two-component regulatory system was shown to control catabolite repression of the bph operon for the degradation of polychlorobiphenyl/biphenyl in the β-proteobacterium Acidovorax sp. KKS102 (Ohtsubo et al., 2006). Orthologues of these carbon-repression mediators have been found in the genome of Azoarcus sp. EbN1, another Azoarcus strain that degrades aromatic compounds and whose complete genome is known (Rabus et al., 2005). Thus, Azoarcus sp. EbN1 has an orthologue of the Crc (EbA3323), CRP (EbA7043), PtsN/PtsO (EbA27937/EbA2794) and BphQ (EbA125) proteins, as well as three orthologues of the CyoB protein (EbA4228, EbA156 and EbA4554). The involvement of all or some of these proteins in carbon catabolite repression in Azoarcus sp. CIB, and the molecular mechanisms underlying the role of BzdR in the carbon catabolite repression, will be explored in future work and will allow us to increase our current view on the superimposed regulation of the bzd gene cluster, so far the best-studied regulatory system in anaerobic catabolism of aromatic compounds.

	ACKNOWLEDGEMENTS

 
This work was supported by Comunidad Autónoma de Madrid Grant P-AMB-259-0505, and Comisión Interministerial de Ciencia y Tecnología grants BIO2003-01482, BIO2006-05957, VEM2003-20075-CO2-02 and GEN2006-27750-C5-3-E/SYS. G. D.-R. is the recipient of a predoctoral fellowship from the Plan Nacional de Formación de Personal Investigador-MEC. M. C. is a holder of the Ramón y Cajal Program of the Spanish Ministerio de Educación y Ciencia. The technical work of M. Morales, M. Zazo and A. Valencia is greatly appreciated. The help of A. Díaz, S. Carbajo and M. Cayuela with sequencing is gratefully acknowledged.

Edited by: M. A. Kertesz

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Altschul, S. F., Gish, W., Miller, E., Myers, W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef][Medline]

Aranda-Olmedo, I., Ramos, J. L. & Marqués, S. (2005). Integration of signals through Crc and PtsN in catabolite repression of Pseudomonas putida TOL plasmid pWW0. Appl Environ Microbiol 71, 4191–4198.[Abstract/Free Full Text]

Aranda-Olmedo, I., Marín, P., Ramos, J. L. & Marqués, S. (2006). Role of the ptsN gene product in catabolite repression of the Pseudomonas putida TOL toluene degradation pathway in chemostat cultures. Appl Environ Microbiol 72, 7418–7421.[Abstract/Free Full Text]

Barragán, M. J. L., Blázquez, B., Zamarro, M. T., Mancheño, J. M., García, J. L., Díaz, E. & Carmona, M. (2005). BzdR, a repressor that controls the anaerobic catabolism of benzoate in Azoarcus sp. CIB, is the first member of a new subfamily of transcriptional regulators. J Biol Chem 280, 10683–10694.[Abstract/Free Full Text]

Boll, M. (2005). Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions. Biochim Biophys Acta 1707, 34–50.[Medline]

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]

Canosa, I., Sánchez-Romero, J. M., Yuste, L. & Rojo, F. (2000). A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway. Mol Microbiol 35, 791–799.[CrossRef][Medline]

Carmona, M. & Díaz, E. (2005). Iron-reducing bacteria unravel novel strategies for the anaerobic catabolism of aromatic compounds. Mol Microbiol 58, 1210–1215.[CrossRef][Medline]

Carmona, M. & Magasanik, B. (1996). Activation of transcription at sigma 54-dependent promoters on linear templates requires intrinsic or induced bending of the DNA. J Mol Biol 261, 348–356.[CrossRef][Medline]

Carmona, M., Prieto, M. A., Galán, B., García, J. L. & Díaz, E. (2008). Signaling networks and design of pollutant biosensors. In Microbial Biodegradation: Genomics and Molecular Biology, pp. 97–142. Edited by E. Díaz. London, UK: Horizon Scientific Press.

Cases, I. & de Lorenzo, V. (1998). Expression systems and physiological control of promoter activity in bacteria. Curr Opin Microbiol 1, 303–310.[CrossRef][Medline]

Cases, I. & de Lorenzo, V. (2005). Promoters in the environment: transcriptional regulation in its natural context. Nat Rev Microbiol 3, 105–118.[CrossRef][Medline]

Cervin, M. A., Lewis, R. J., Brannigan, J. A. & Spiegelman, G. B. (1998). The Bacillus subtilis regulator SinR inhibits spoIIG promoter transcription in vitro without displacing RNA polymerase. Nucleic Acids Res 26, 3806–3812.[Abstract/Free Full Text]

Collier, D. N., Hager, P. W. & Phibbs, P. V., Jr (1996). The Bacillus subtilis regulator SinR inhibits spoIIG promoter transcription in vitro without displacing RNA polymerase. Res Microbiol 147, 551–561.[Medline]

de Lorenzo, V. & Timmis, K. N. (1994). Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol 235, 386–405.[Medline]

Díaz, E. & Prieto, M. A. (2000). Bacterial promoters triggering biodegradation of aromatic pollutants. Curr Opin Biotechnol 11, 467–475.[CrossRef][Medline]

Durante-Rodríguez, G., Zamarro, M. T., García, J. L., Díaz, E. & Carmona, M. (2006). Oxygen-dependent regulation of the central pathway for the anaerobic catabolism of aromatic compounds in Azoarcus sp. strain CIB. J Bacteriol 188, 2343–2354.[Abstract/Free Full Text]

Egland, P. G. & Harwood, C. S. (1999). BadR, a new MarR family member, regulates anaerobic benzoate degradation by Rhodopseudomonas palustris in concert with AadR, an Fnr family member. J Bacteriol 181, 2102–2109.[Abstract/Free Full Text]

Egland, P. G. & Harwood, C. S. (2000). HbaR, a 4-hydroxybenzoate sensor and FNR-CRP superfamily member, regulates anaerobic 4-hydroxybenzoate degradation by Rhodopseudomonas palustris. J Bacteriol 182, 100–106.[Abstract/Free Full Text]

Elshahed, M. S., Bhupathiraju, V. K., Wofford, N. Q., Nanny, M. A. & McInerney, M. J. (2001). Metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by "Syntrophus aciditrophicus" strain SB in syntrophic association with H₂-using microorganisms. Appl Environ Microbiol 67, 1728–1738.[Abstract/Free Full Text]

Gibson, J. & Harwood, C. S. (2002). Metabolic diversity in aromatic compound utilization by anaerobic microbes. Annu Rev Microbiol 56, 345–369.[CrossRef][Medline]

Harwood, C. S. & Parales, R. E. (1996). The β-ketoadipate pathway and the biology of self-identity. Annu Rev Microbiol 50, 553–590.[CrossRef][Medline]

Harwood, C. S., Burchhardt, G., Herrmann, H. & Fuchs, G. (1999). Genetic clues on the evolution of anaerobic catabolism of aromatic compounds. FEMS Microbiol Rev 22, 439–458.[Medline]

Heider, J., Boll, M., Breese, K., Breinig, S., Ebenau-Jehle, C., Feil, U., Gad'on, N., Laempe, D., Leuthner, B. & other authors (1998). Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica. Arch Microbiol 170, 120–131.[CrossRef][Medline]

Jahn, M. K., Haderlein, S. B. & Meckenstock, R. U. (2005). Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures. Appl Environ Microbiol 71, 3355–3358.[Abstract/Free Full Text]

Koudelka, G. B. & Lam, C. Y. (1993). Differential recognition of OR1 and OR3 by bacteriophage 434 repressor and Cro. J Biol Chem 268, 23812–23817.[Abstract/Free Full Text]

Kovach, M. E., Elzer, P. H., Hills, D. S., Robertson, G. T., Farris, M. A., Roop, R. M., II & Peterson, K. M. (1995). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175–176.[CrossRef][Medline]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]

López Barragán, M. J., Carmona, M., Zamarro, M. T., Thiele, B., Boll, M., Fuchs, G., García, J. L. & Díaz, E. (2004). The bzd gene cluster, coding for anaerobic benzoate catabolism, in Azoarcus sp. strain CIB. J Bacteriol 186, 5762–5774.[Abstract/Free Full Text]

Lovley, D. R. (2003). Cleaning up with genomics: applying molecular biology to bioremediation. Nat Rev Microbiol 1, 35–44.[CrossRef][Medline]

Magasanik, B. (1970). Glucose effects: inducer exclusion and repression. In The Lactose Operon, pp. 189–220. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Mandic-Mulec, I., Doukhan, L. & Smith, I. (1995). The Bacillus subtilis SinR protein is a repressor of the key sporulation gene spo0A. J Bacteriol 177, 4619–4627.[Abstract/Free Full Text]

Marqués, S., Aranda-Olmedo, I. & Ramos, J. L. (2006). Controlling bacterial physiology for optimal expression of gene reporter constructs. Curr Opin Biotechnol 17, 50–52.[CrossRef][Medline]

Marschall, C., Labrousse, V., Kreimer, M., Weichart, D., Kolb, A. & Hengge-Aronis, R. (1998). Molecular analysis of the regulation of csiD, a carbon starvation-inducible gene in Escherichia coli that is exclusively dependent on ^s and requires activation by cAMP-CRP. J Mol Biol 276, 339–353.[CrossRef][Medline]

Martín, A. C., López, R. & García, P. (1996). Analysis of the complete nucleotide sequence and functional organization of the genome of Streptococcus pneumoniae bacteriophage Cp-1. J Virol 70, 3678–3687.[Abstract]

Maxam, A. M. & Gilbert, W. (1980). Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65, 499–560.[Medline]

Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Morales, G., Linares, J. F., Beloso, A., Albar, J. P., Martínez, J. L. & Rojo, F. (2004). The Pseudomonas putida Crc global regulator controls the expression of genes from several chromosomal catabolic pathways for aromatic compounds. J Bacteriol 186, 1337–1344.[Abstract/Free Full Text]

Morales, G., Ugidos, A. & Rojo, F. (2006). Inactivation of the Pseudomonas putida cytochrome o ubiquinol oxidase leads to a significant change in the transcriptome and to increased expression of the CIO and Cbb3-1 terminal oxidases. Environ Microbiol 8, 1764–1774.[CrossRef][Medline]

Müller, C., Petruschka, L., Cuypers, H., Burchhardt, G. & Herrmann, H. (1996). Carbon catabolite repression of phenol degradation in Pseudomonas putida is mediated by the inhibition of the activator protein PhlR. J Bacteriol 178, 2030–2036.[Abstract/Free Full Text]

Ohtsubo, Y., Goto, H., Nagata, Y., Kudo, T. & Tsuda, M. (2006). Identification of a reponse regulator gene for catabolite control from a PCB-degrading beta-proteobacterium, Acidovorax sp. KKS102. Mol Microbiol 60, 1563–1575.[CrossRef][Medline]

Peres, C. M. & Harwood, C. S. (2006). BadM is a transcriptional repressor and one of three regulators that control benzoyl coenzyme A reductase gene expression in Rhodopseudomonas palustris. J Bacteriol 188, 8662–8665.[Abstract/Free Full Text]

Peters, F., Rother, M. & Boll, M. (2004). Selenocysteine-containing proteins in anaerobic benzoate metabolism of Desulfococcus multivorans. J Bacteriol 186, 2156–2163.[Abstract/Free Full Text]

Peters, F., Shinoda, Y., McInerney, M. J. & Boll, M. (2007). Cyclohexa-1,5-diene-1-carbonyl-coenzyme A (CoA) hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: evidence for a common benzoyl-CoA degradation pathway in facultative and strict anaerobes. J Bacteriol 189, 1055–1060.[Abstract/Free Full Text]

Petruschka, L., Burchhardt, G., Müller, C., Weihe, C. & Herrmann, H. (2001). The cyo operon of Pseudomonas putida is involved in catabolic repression of phenol degradation. Mol Genet Genomics 266, 199–206.[CrossRef][Medline]

Prieto, M. A., Galán, B., Torres, B., Ferrández, A., Fernández, C., Miñambres, B., García, J. L. & Díaz, E. (2004). Aromatic metabolism versus carbon availability: the regulatory network that controls catabolism of less-preferred carbon sources in Escherichia coli. FEMS Microbiol Rev 28, 503–518.[Medline]

Rabus, R., Kube, M., Heider, J., Beck, A., Heitmann, K., Widdel, F. & Reinhardt, R. (2005). The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1. Arch Microbiol 183, 27–36.[CrossRef][Medline]

Rojo, F. & Dinamarca, M. A. (2004). Catabolite repression and physiological control. In Pseudomonas, vol. 2, pp. 365–387. Edited by J. L. Ramos. New York: Kluwer.

Safinowski, M., Griebler, C. & Meckenstock, R. U. (2006). Anaerobic cometabolism transformation of polycyclic and heterocyclic aromatic hydrocarbons: evidence from laboratory and field studies. Environ Sci Technol 40, 4165–4173.[Medline]

Sambrook, J. W. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Song, B. & Ward, B. B. (2005). Genetic diversity of benzoyl coenzyme A reductase genes detected in denitrifying isolates and estuarine sediment communities. Appl Environ Microbiol 71, 2036–2045.[Abstract/Free Full Text]

Tropel, D. & van der Meer, J. R. (2004). Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol Mol Biol Rev 68, 474–500.[Abstract/Free Full Text]

Weickert, M. J. & Adhya, S. (1992). A family of bacterial regulators homologous to Gal and Lac repressors. J Biol Chem 267, 15869–15874.[Abstract/Free Full Text]

Wischgoll, S., Heintz, D., Peters, F., Erxleben, A., Sarnighausen, E., Reski, R., van Dorsselaer, A. & Boll, M. (2005). Gene clusters involved in anaerobic benzoate degradation of Geobacter metallireducens. Mol Microbiol 58, 1238–1252.[CrossRef][Medline]

Received 6 July 2007; revised 6 September 2007; accepted 11 September 2007.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Durante-Rodríguez, G. Articles by Carmona, M. Search for Related Content PubMed PubMed Citation Articles by Durante-Rodríguez, G. Articles by Carmona, M. Agricola Articles by Durante-Rodríguez, G. Articles by Carmona, M.