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Applied and Environmental Microbiology, January 2007, p. 232-242, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.01608-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Multiple-Level Regulation of Genes for Protocatechuate Degradation in Acinetobacter baylyi Includes Cross-Regulation{triangledown}

Simone Yasmin Siehler,{dagger} Süreyya Dal,{ddagger} Rita Fischer, Patricia Patz,§ and Ulrike Gerischer*

Microbiology and Biotechnology, University of Ulm, Ulm, Germany

Received 12 July 2006/ Accepted 23 October 2006


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The bacterium Acinetobacter baylyi uses the branched ß-ketoadipate pathway to metabolize aromatic compounds. Here, the multiple-level regulation of expression of the pca-qui operon encoding the enzymes for protocatechuate and quinate degradation was studied. It is shown that both activities of the IclR-type regulator protein PcaU at the structural gene promoter pcaIp, namely protocatechuate-dependent activation of pca-qui operon expression as well as repression in the absence of protocatechuate, can be observed in a different cellular background (Escherichia coli) and therefore are intrinsic to PcaU. The regulation of PcaU expression is demonstrated to be carbon source dependent according to the same pattern as the pca-qui operon. The increase of the pcaU gene copy number leads to a decrease of the basal expression at pcaIp, indicating that the occupancy of the PcaU binding site is well balanced and depends on the concentration of PcaU in the cell. Luciferase is used as a reporter to demonstrate strong repression of pcaIp when benzoate, a substrate of the catechol branch of the pathway, is present in addition to substrates of the protocatechuate branch (cross-regulation). The same repression pattern was observed for promoter pcaUp. Thus, three promoters involved in gene expression of enzymes of the protocatechuate branch (pobAp upstream of pobA, pcaIp, and pcaUp) are strongly repressed in the presence of benzoate. The negative effect of protocatechuate on pobA expression is not based on a direct sensing of the metabolite by PobR, the specific regulator of pobA expression.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The degradation of aromatic monomers by soil bacteria is a catabolic process of broad ecological relevance. One of the major pathways used by bacteria for this purpose is the ß-ketoadipate pathway (23). In all cases, these pathways consist of a set of central reactions which can be accessed by a large number of different aromatic substrates via short conversion pathways. This setup underlines the relevance of the central pathway for the organisms. It thus appears plausible to expect a sophisticated regulation of such a pathway to be able to react appropriately to a large number of different carbon source combinations (9, 17, 36, 40).

Here, we focus on Acinetobacter baylyi strain ADP1 (formerly referred to as Acinetobacter sp. strain ADP1), a versatile soil bacterium that has recently been recognized as a distinct species and which differs from close relatives by its unusually high competence for natural transformation (42). A. baylyi uses the ß-ketoadipate pathway, which allows it to grow on a large variety of aromatic compounds. The genes for the protocatechuate branch of the pathway (pca genes) are organized in an operon which includes the downstream qui genes encoding one of the funneling pathways, namely the conversion of the hydroaromatic compounds quinate and shikimate into protocatechuate (7). This large pca-qui operon underlies gene regulation at multiple layers. First, there is the specific induction by protocatechuate which is mediated by the IclR family member PcaU (4, 19, 30). The main effect of this regulator is activation of transcription at the promoter pcaIp upstream of the first pca gene, pcaI. In addition, it also represses the activity of promoter pcaIp by reducing the basal expression level three- to fourfold. Thus, PcaU is an activator/repressor protein with a dual function at the same promoter (39). Furthermore, it has a repressing effect on its own expression (39). The regulatory DNA is a 309-bp intergenic region between the pcaI gene and the pcaU gene facing in the opposite direction. Binding of the purified PcaU protein has been shown to occur to a 45-bp site between the two promoters pcaIp and pcaUp containing a characteristic arrangement of three perfect 10-bp repetitions (34). A related regulator binding site—but with less perfectly conserved repetitions—has been shown for close relatives of PcaU (12, 21). A higher level of gene regulation affecting pca-qui gene expression (besides other operons) is carbon catabolite repression (6). The presence of certain organic acids, which can serve as substrates for A. baylyi, overrides the effect of the inducer simultaneously present and causes a dramatic reduction of the expression level by 90% during the growth phase. The mechanism leading to this global regulatory effect, which has been documented at the level of enzyme activity and in transcript levels and promoter activity, is unknown. A third layer of regulation is the so-called cross-regulation between the two branches of the pathway. This type of regulation has been described in Pseudomonas putida, where benzoate is consumed prior to p-hydroxybenzoate when offered simultaneously (5, 31). Work by Neidle and collaborators has demonstrated that a similar situation can be found in A. baylyi. Benzoate is used first, and p-hydroxybenzoate is consumed thereafter (16). They were able to show that the expression of the pobA gene encoding the hydroxylase converting p-hydroxybenzoate into protocatechuate is repressed under these conditions. CatM and BenM, transcriptional regulators involved in the specific regulation of benzoate degradation, are necessary to bring about this ordered utilization (3, 14). Whether the central reactions of the pathway are also affected by cross-regulation was unknown.

The current investigation focuses on different aspects of the expression control of the pca-qui operon and the respective regulator gene pcaU. We show that the complex (negative as well as positive) effects of PcaU on promoter pcaIp are intrinsic to this protein because they can be observed in an Escherichia coli background exactly as in Acinetobacter. Contradictory results concerning the expression control of pcaU itself are addressed here by applying different methods. It was observed that the pcaU expression follows the same pattern as the expression of the pca-qui operon. The gene encoding luciferase is introduced as a reporter for promoter activity and allows the very clear observation of expression differences due to the presence of additional carbon sources, which were less clear using ß-galactosidase (6). We are addressing the following questions: whether the amount of PcaU in the cell is important for its effect, and whether overexpression results in lower expression levels at pcaIp. We address cross-regulation of the pca-qui operon by demonstrating that cross-regulation by benzoate (or its metabolites) strongly affects the expression of this operon.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains and growth conditions.
Bacterial strains used in this study are listed in Table 1. Strains of A. baylyi were grown on mineral medium at 30°C as described previously (39). The carbon sources were used at the following final concentrations unless indicated otherwise: succinate, 10 mM; pyruvate, 10 mM; succinate plus acetate, 15 mM each; p-hydroxybenzoate, 5 mM; benzoate, 4 mM; and quinate, 5 mM. Protocatechuate was used for induction at 0.1 mM or 1 mM. Antibiotics for A. baylyi strains were used in the following concentrations: tetracycline, 6 µg/ml; gentamicin, 5 µg/ml; kanamycin, 12 µg/ml; and spectinomycin, 100 µg/ml. For growth curves, 0.6 ml of cells from a 5-ml overnight culture containing the same noninducing (but not the inducing) carbon source was inoculated into 60 ml of medium. E. coli strains were grown in LB medium at 37°C supplied with antibiotics when appropriate (tetracycline, 12.5 µg/ml; gentamicin, 10 µg/ml; kanamycin, 25 µg/ml; spectinomycin, 100 µg/ml; and ampicillin, 100 µg/ml).


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  		TABLE 1. Bacterial strains and plasmids used in this study

 
Transformation of Acinetobacter.
Acinetobacter strains were grown in 5 ml of mineral medium with succinate overnight. After addition of 10 µl of 1 M succinate and additional growth for 30 min, 50 µl of the culture was transferred onto a polycarbonate filter (Costar Nuclepore, Cambridge, MA) placed on a nonselective plate together with 1 µg of linear DNA. After incubation for 5 to 6 h at 37°C, the cells were washed off the filter and spread on a selective plate. Conjugation to move plasmids from E. coli S17/1 to strains of Acinetobacter was done as described previously (7).

Plasmid and strain construction.
Standard methods were used for plasmid isolation, DNA purification, restriction endonuclease cleavage, ligation, and transformation (35). To create plasmid pAC48, an EcoRI fragment of A. baylyi DNA containing pcaU was gained from plasmid pZR17 and cloned into plasmid pAC16 cut with EcoRI, resulting in pAC41. The lacZ cassette from pKOK6 was introduced using PstI. Plasmid pAC78 was made by using plasmid pAC16 representing a 798-bp NarI fragment from A. baylyi (containing the complete intergenic region between pcaI and pcaU and the 5' area of both genes) cloned into vector pRK415. The source of the Photinus pyralis luciferase gene was plasmid pFW11-luc containing a luc-aad9 cassette. It was cleaved using AccI and SmaI and cloned into pUC19, creating pUC19_luc. From this plasmid the luc-aad9 cassette could be gained as a PstI fragment and cloned into pAC16 linearized with PstI. The resulting plasmid pAC78 (identical to pAC17 except for the reporter gene cassette) contained a transcriptional pcaIp-luc fusion and an incomplete pcaU gene (approximately the 5' third of the gene). Plasmid pAC79 was made like plasmid pAC48, except that the PstI-prepared luc-aad9 cassette (from plasmid pUC19_luc) was used instead of a lacZ cassette. In all cases, the orientation of the cloned fragments was verified by restriction analysis.

A. baylyi strain ADPU12 was made by first inserting the above-mentioned 798-bp NarI fragment cut out from plasmid pZR9 into the BstBI restriction site of plasmid pZR102 (see Fig. 3). The orientation where the directions of transcription of recA and pcaU were opposite to each other was chosen to avoid measuring transcriptional activity from the recA promoter (plasmid pAC21). The lacZ-Kmr cassette from plasmid pKOK6 (SalI cut) was inserted into the XhoI site in the pcaU part of plasmid pAC21 (pAC33). Restriction analysis confirmed the correct orientation of the cassette (see Fig. 3). The plasmid was linearized using BamHI and used to transform A. baylyi strain ADP1. Initial selection was done on LB medium plates with kanamycin (15 µg/ml); subsequently, transfer to LB medium with ampicillin (200 µg/ml) was used to identify a strain that had not incorporated the vector part of plasmid pAC21. Southern blot analysis was employed to verify the correct integration of the construct into the recA gene locus on the chromosome of A. baylyi. Chromosomal DNA of the potential pcaU-lacZ fusion strain was digested with restriction endonuclease BamHI. Hybridization with a recA probe (insert of plasmid pZR102) showed an increase in size of the 3.5-kbp wild-type fragment to 8.8 kbp. Hybridization of HindIII-digested chromosomal DNA with the 798-bp NarI fragment containing the intergenic region between pcaI and pcaU resulted in signals at 3.5 kbp and 1.3 kbp. The 3.5-kbp signal is also observed in the wild type and represents the native pca gene cluster; the 1.3-kbp signal is not present in the wild type and is derived from the newly integrated pcaU-lacZ fusion in the recA gene (data not shown). The new strain was named A. baylyi strain ADPU12. A control strain containing only a lacZ cassette inserted into recA (opposite direction of transcription) verified that there was no detectable transcription under any of the conditions used (6).


Figure 3
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  		FIG. 3. Carbon source dependence of pcaU gene expression. (A) Transcriptional pcaU-lacZ fusion of A. baylyi strain ADPU12 located on the chromosome remote from the pca genes. The reporter gene cassette is not drawn to scale. (B) A. baylyi strain ADPU12 was grown on mineral medium containing the indicated carbon sources. The concentration of POB when given in addition to another carbon source was 1 mM; otherwise it was 5 mM. Succinate (succ), 10 mM; pyruvate (pyr), 10 mM; succinate plus acetate (S+A), 15 mM each; p-hydroxybenzoate (POB), 5 mM; benzoate, 4 mM; quinate (qui), 5 mM. Protocatechuate was used for induction at 0.1 mM or 1 mM.

 
To create strain ADPU47, the luc-aad9 cassette (cut with NdeI and XhoI) from plasmid pFW11-luc was cloned into the SnaBI site of plasmid pZR5. The plasmid that contained the luc gene in the same orientation as the pcaI gene was chosen (pAC64), and the insertion was separated from the vector by EcoRI and SalI cleavage. After transformation of A. baylyi strain ADP1, transformants were selected for on mineral medium with succinate and spectinomycin. The loss of the ability to grow on p-hydroxybenzoate as the sole carbon source and the presence of the luc-aad9 cassette in pcaI (as shown by PCR using primers directed against DNA outside of the fragment used for the transformation: pZR17-seq2, CATGTATAATATAACGCGG; and pcaJ-4293, TCGACGATGTATGCATG) verified strain ADPU47.

Strain ADPU53 was made by transforming A. baylyi strain ADP1 with SacI- and EheI-cleaved plasmid pZR430B-luc and selection on mineral medium with succinate and spectinomycin. Colonies were tested for their inability to grow on p-hydroxybenzoate as the sole carbon source. The presence of the luc-aad9 cassette was verified by PCR using primers targeting sequences outside of the DNA that had been used for the transformation (RA-19421, GCCTGCAATGTAATCTTC; and SS-21470, AGGTACCACCAGATCC).

Determination of ß-galactosidase and luciferase activities.
The protocol described by Miller was used to determine ß-galactosidase activity (29). Cells were permeabilized using chloroform and sodium dodecyl sulfate. The standard deviation for replicate assays from the same sample was no more than 2%. To characterize expression levels, we used the reporter gene activity at the onset of the stationary phase (39). The standard deviation between different cultures (same strain, same conditions) was up to 15%. For measurement of luminescence produced by luciferase activity, aliquots of cell suspension were withdrawn from a growing culture and the optical density was measured at 600 nm (OD600). After placing the sample tube with 150 µl of the cell suspension into the luminometer, 150 µl of 2.5x assay buffer (62.5 mM glycylglycin, pH 7.8, 25 mM MgCl2) and then 150 µl of 33 µM D-luciferin (freshly dissolved in water) were added and the luminescence was immediately measured for 15 s using a Flash'n Glow luminometer (Berthold Detection Systems, Pforzheim, Germany). The standard deviation for replicate assays from the same sample was no more than 2%. In all cases, activities of the enzymes were followed throughout the growth phase. The representative value characterizing a particular strain under a certain condition was taken in mid-exponential growth phase. The standard deviation for this value taken for independently grown cultures under identical conditions was up to 30%.

Northern blot analysis.
Cells for preparation of RNA were harvested within no more than 1 min, frozen in liquid nitrogen, and stored at –70°C. The preparation of total RNA and the Northern blot hybridization were done as described previously (7). For signal detection, a Bio Imager Fujix BAS 1000 (Fuji Photo Film Co., Ltd., Tokyo, Japan) was used. The software MacBAS (Fuji Photo Film Co., Ltd., Tokyo, Japan) was applied for quantitative exploitation. Rehybridization of the membranes to ensure application of equal amounts of total RNA with a 16S rRNA gene probe was done as detailed previously (7).

Southern blot analysis.
Total DNA from cells was extracted as described elsewhere (18). DNA was digested with restriction endonucleases and analyzed by Southern blotting and hybridization as described previously (6).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation as well as repression by PcaU at promoter pcaIp are intrinsic features of this regulator.
Previous investigations of the expression control of pcaIp were conducted with derivatives of A. baylyi strain ADP1 (7, 39). From these data, it cannot be excluded that additional components are required for the activation as well as the repression of expression observed for promoter pcaIp. A straightforward way to look at the effect of regulator PcaU on the pcaIp activity was to use a different cellular background. Therefore, a plasmid was constructed containing a fragment of A. baylyi DNA with the complete pcaU gene, the pcaU-pcaI intergenic region, and a portion of the pcaI gene transcriptionally fused to a lacZ reporter gene cassette (pAC48) (Table 1; Fig. 1). The construction was made in such a way that no vector-derived promoter activity would contribute to the reporter gene expression (Fig. 1). Growth experiments on E. coli DH5{alpha} with plasmid pAC48 were conducted in LB medium with and without various amounts of protocatechuate (0 to 5 mM), the inducer molecule activating the expression at the pcaIp promoter probably by directly interacting with the PcaU protein. The basal level of expression (70 ± 10 Miller units [MU]) increased when at least 10 µM protocatechuate was present in the medium, as observed earlier for the pcaIp expression in Acinetobacter (39). The highest expression was observed with 1 mM protocatechuate (2,740 ± 410 MU). The induction factor (39-fold) was comparable to Acinetobacter where the pcaIp regulation was also studied with pRK415-based plasmids (18-fold to 94-fold, depending on the carbon source [39]). These results show that (i) PcaU is produced because a protocatechuate-directed induction is detected, (ii) no additional protein besides PcaU is necessary for a concentration-dependent expression at pcaIp (results with plasmid pAC17 containing a truncated pcaU gene and not responding to the presence of protocatechuate [see the next paragraph] served as a control), and (iii) PcaU from A. baylyi and RNA polymerase from E. coli cooperate to allow this induction.


Figure 1
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  		FIG. 1. Structure of plasmids for the determination of the activity of promoter pcaIp. Overview of the A. baylyi DNA according to the annotation at GenBank (NC_005966; top). The restriction endonuclease sites given are the relevant sites for the construction of the plasmids. The black lines indicate the areas of the DNA drawn in the top of the figure that are contained in the respective plasmids. The reporter cassettes are not drawn to scale.

 
Analysis of the regulation of gene expression at pcaIp in Acinetobacter has shown that the uninduced level of expression is higher in the absence of PcaU (2- to 3.5-fold) (39). To test whether PcaU expressed in E. coli has the same feature, we compared plasmids pAC48 and pAC17. These plasmids are identical except for the A. baylyi DNA. Plasmid pAC17 contains a truncated pcaU gene (Fig. 1). ß-Galactosidase activity produced in E. coli strains containing these plasmids was measured in the absence and presence of protocatechuate (Fig. 2). The culture with pAC17 produced ß-galactosidase at about 600 MU with and without protocatechuate, thus confirming that the induction observed in a strain containing plasmids with full-length pcaU is due to PcaU protein. In comparison, the uninduced reporter gene activity of plasmid pAC48 (complete pcaU gene) is much lower (ninefold; 70 MU). The difference in expression could be visualized by growth of the two strains on lactose MacConkey plates with and without 100 µM protocatechuate (data not shown). This difference is even stronger than that observed earlier in Acinetobacter and clearly shows that both the activating and repressing activities of PcaU take place in an E. coli background. These observations strengthen the evidence that PcaU itself has a repressing as well as an activating function without the contribution of additional factors.


Figure 2
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  		FIG. 2. Negative and positive effects of PcaU in E. coli. E. coli DH5{alpha} with plasmids pAC17 (truncated pcaU; empty squares) or pAC48 (native pcaU; filled diamonds) was grown in LB medium with the indicated concentrations of protocatechuate (PCA). ß-Galactosidase activity was determined at the onset of stationary phase.

 
The expression of the pcaU gene parallels the expression of the pca-qui operon.
Like many other regulator proteins, PcaU shows negative autoregulation (39). Whether it underlies a carbon source-dependent expression control has been analyzed in former investigations using different plasmids introduced in a recA derivative of the wild-type strain of A. baylyi with contradictory results (6, 19, 39). To clarify this issue, we used two approaches: first reporter gene technology and second quantitative Northern blot analysis. For the reporter gene approach, we established a chromosomal pcaU-lacZ transcriptional fusion in a remote location of the A. baylyi genome, resulting in an undisturbed protocatechuate metabolism. A pcaU-lacZ fusion in the original location would lead to a dysfunctional PcaU protein. In such a strain, regulation by PcaU could not be investigated. At the same time, the copy number of the pca regulatory region was increased by only one. Using extrachromosomal plasmids, as done in the past, increases the copy numbers of the respective Acinetobacter DNA by more than one and thus increases the likelihood of measuring a nonnative situation (increase of the ratio of pca intergenic region/PcaU protein). Strain ADPU12 contained a transcriptional fusion between the 5' 280 bp of the pcaU gene and a lacZ-Kmr cassette, the complete pca intergenic region, and the 5' 205 bp of the pcaI gene inserted into the recA gene (Fig. 3). The recA gene was chosen because (i) this allows the use of the strain as a recipient for plasmids containing Acinetobacter DNA and because (ii) recA-negative strains of Acinetobacter baylyi have been used in other investigations successfully (6, 10, 39). Strain ADPU12 therefore contained one additional copy of the pca regulatory DNA. Growth of strain ADPU12 on mineral medium with a number of different carbon sources was monitored along with the ß-galactosidase activity produced (Fig. 3). The expression level on aromatic compounds, which are channeled through the protocatechuate branch (p-hydroxybenzoate and quinate), is about seven times higher than on noninducing carbon sources (succinate and pyruvate). Combinations of noninducing carbon sources and p-hydroxybenzoate result in intermediate levels. To strengthen these results we used a second experimental approach, namely quantitative Northern blot analysis. The wild-type strain ADP1 was grown on mineral medium with an inducing carbon source (quinate) or a noninducing carbon source (succinate). RNA was isolated from cell samples taken in the course of growth and analyzed in a Northern blot hybridization with a pcaU probe. To ensure the even application of total RNA in this experiment, a subsequent hybridization of the same membrane with a 16S rRNA gene probe was performed. The signals were quantified using a phosphorimager and used to normalize the specific pcaU signal from the first hybridization (Fig. 4A). The results clearly show that the level of the pcaU transcript upon growth on succinate is severalfold lower than the level upon growth on quinate. In addition, the induction of the pca genes and the pcaU gene after inoculation of uninduced cells of A. baylyi strain ADP1 into mineral medium with p-hydroxybenzoate or after adding an inducing carbon source (p-hydroxybenzoate) to a noninducing medium (mineral medium with pyruvate) shows the parallel increase in the relative amounts of the respective transcripts (Fig. 4B). Taken together, the results for the two different approaches consistently show that the expression of the pcaU gene depends not only on the presence of its own gene product but also on the growth substrate. The pcaU gene is regulated according to the same scheme as the pca-qui genes.


Figure 4
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  		FIG. 4. Carbon source dependence of pcaU gene expression. Northern blot hybridization of total RNA (10 µg per lane) grown with the indicated carbon source (succinate, 20 mM; pyruvate, 20 mM). The graphs at the bottom of the figures show the growth curves of the cultures. The time points where RNA samples were taken are indicated by filled diamonds. The bars represent the quantification of the indicated signals in the blot shown above after normalization against the background as well as the 16S rRNA signal. The highest signal was set as 100%. All samples analyzed with one particular probe were blotted on the same membrane and hybridized in the same solution; therefore, the signals can be related to each other quantitatively. (A) Analysis of the pcaU transcript; (B) simultaneous analysis of the pcaU and pca-qui transcript in identical RNA samples. See Fig. 3 legend for abbreviations.

 
Use of luciferase as reporter for the expression of the pca-qui genes allows the unambiguous detection of carbon catabolite repression at the level of promoter activity.
Using the lacZ-encoded ß-galactosidase as a reporter has been very useful in characterizing the specific regulation in response to protocatechuate mediated by PcaU (39). Analysis of expression at promoter pcaIp under conditions which created less pronounced differences was difficult. The repression of expression at pcaIp by the presence of additional carbon sources (acetate plus succinate) has been clearly detected both at the level of protocatechuate 3,4-dioxygenase activity and at the level of transcript abundance (repression by at least 10-fold) (6). The effect could also be shown at the level of promoter activity by means of a transcriptional fusion with the lacZ gene, but it was less pronounced (repression factor of 4.7). Luciferase is supposed to be a less stable protein than ß-galactosidase, but to our knowledge there are no data available about this issue in bacteria. To compare the in vivo stabilities of the proteins ß-galactosidase and luciferase from Photinus pyralis, we used two plasmids which contained a transcriptional fusion between pcaIp and the respective reporter protein (pAC70 and pAC79) (Fig. 1) established in E. coli. We compared the rates of disappearance of the enzyme activity by inoculating an induced culture (LB plus 1 mM protocatechuate) into medium without inducer (LB). The time necessary to reduce ß-galactosidase by 50% was 35 min, which was the same time the culture needed to double its OD. This means that ß-galactosidase is very stable and is not degraded at observable rates under these conditions. In contrast, luciferase disappeared much faster than the culture grew; the time to reduce the activity of this enzyme by 50% was 19 min.

To find out if luciferase is a more powerful tool to characterize subtle expression differences, we compared the results of pcaIp promoter activity measurements in equivalent constructs using ß-galactosidase and luciferase from Photinus pyralis (Table 2). We evaluated the PcaU-specific induction, the repression by PcaU, and carbon catabolite repression and found in all cases much more pronounced effects when luciferase was used. The respective induction or repression factors were increased close to 10-fold or more. In particular, the two regulatory effects, repression by PcaU and carbon catabolite repression, display significant repression factors when luciferase is used (30-fold). The use of luciferase also allowed us to demonstrate much more clearly than in the past that the expression at pcaIp still underlies regulation in the absence of the specific regulator PcaU (Fig. 5). A combination of succinate and acetate decreased the basal level of expression by a factor of 10 under the level observed upon growth on pyruvate, a pattern also seen under carbon catabolite repression conditions. LB medium led to the highest level of expression in the absence of PcaU. These results clearly show (i) that luciferase appears more appropriate to characterize smaller expression differences and (ii) that the negative regulatory layers affecting pcaIp expression, like catabolite repression or repression by PcaU in the absence of inducer, appear much bigger when luciferase is used than when ß-galactosidase is used.


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  		TABLE 2. Comparison of ß-galactosidase and luciferase as reporter enzymes for the expression at pcaIp

 

Figure 5
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  		FIG. 5. Regulated expression at pcaIp in the absence of PcaU. A. baylyi strain ADPU59 was grown on mineral medium with the indicated carbon sources. The luciferase activity was taken from cultures in the absence (white columns) or in the presence (gray columns) of 1 mM protocatechuate. See Fig. 3 legend for abbreviations.

 
Enhanced copy numbers of pcaU lead to a decrease of expression at pcaIp.
The question of whether a change in the PcaU concentration in the cell is relevant for the expression at pcaIp could be investigated by using plasmids with additional copies of the pcaU gene. The plasmids were based on the pRK415 vector with a copy number of 2 per cell and contained a fragment with a complete pcaU gene (pAC79) or a truncated pcaU gene (pAC78), the pcaU-pcaI intergenic region, and a portion (205 bp) of the pcaI gene transcriptionally fused to a luciferase reporter gene cassette (Fig. 1). By introducing these plasmids into recA derivatives of the wild-type strain of A. baylyi (strain ADP197) and a mutant strain with a deleted pcaU gene in the chromosome (strain ADPU331), we established strains with no pcaU present and with one (chromosome), two (plasmid), and three (chromosome and plasmid) copies of pcaU per cell. To monitor the activity at promoter pcaIp in these strains, they were grown on mineral medium with three different noninducing carbon sources (succinate, pyruvate, and succinate plus acetate). Luminescence caused by the activity of pcaIp-driven luciferase expression was measured in the course of growth. The results show that with an increasing pcaU gene copy number in the cell, the expression at pcaIp decreases 12- to 30-fold (depending on the carbon source) in comparison to that for the strain with no pcaU present (Fig. 6). Under inducing conditions, pcaIp activity showed the expected high induction (for example, up to 1.4 x 105 relative light units [RLU]/OD600 for the strain with one copy of the pcaU gene on mineral medium with pyruvate and p-hydroxybenzoate), but the activities oscillated strongly in the course of growth so that the data under inducing conditions cannot be compared. (This was attributed to the location of the luc gene on the plasmid.) The data presented allow the conclusion that increased copies of the pcaU gene lead to a repression of the basal level of expression at promoter pcaIp that is stronger than that observed in the wild type. This is most likely due to an increased concentration of PcaU in the cell as a result of the additional copies of the gene.


Figure 6
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  		FIG. 6. Repression of expression at pcaIp by PcaU increases with increasing pcaU gene copy number. The strains of A. baylyi contained a plasmid-based pcaIp luciferase fusion and between zero and three copies of the pcaU gene. The strains were grown on succinate plus acetate (empty columns), pyruvate (gray columns), or succinate (black columns).

 
Expression at promoters pcaIp and pcaUp under inducing conditions is strongly repressed by benzoate.
An additional regulatory level affecting the degradation of aromatic carbon sources through the ß-ketoadipate pathway has been identified and is referred to as cross-regulation (3). It results in a preferential utilization of substrates funneled through the benzoate branch over substrates metabolized via the protocatechuate branch. The response of the pobA promoter to this cross-regulation has been demonstrated, and the involvement of the transcriptional regulators BenM and CatM has been shown. It was unknown whether the promoter of the pca-qui operon, pcaIp, which is responsible for the central steps of the protocatechuate branch of the pathway, is also affected by this type of regulation. Therefore, we used the A. baylyi strain ADPU47 to find out if there is an effect of benzoate on the induced expression at promoter pcaIp. Strain ADPU47 (containing a transcriptional pcaI-luc fusion) showed the normal induced expression when grown in the presence of protocatechuate (Table 2; Fig. 7). Pyruvate was used as the carbon source in this case, because it is known that it does not act as a repressing substrate (6). When the organism was grown on a mixture of protocatechuate and benzoate, the expression was low throughout the growth phase (3% of the induced value) (Fig. 7). These data clearly establish that the pcaIp promoter underlies repression by benzoate or its metabolites. Since the promoter of the pcaU gene has been shown to reflect the activity pattern of promoter pcaIp under all conditions tested so far, we also tested the effect of the presence of benzoate on the expression of the PcaU protein. To this end, we used strain ADPU12, which has been described above. The activity of this promoter after growth of strain ADPU12 on a mixture of benzoate and protocatechuate was as low as on a noninducing carbon source (pyruvate), whereas the induced expression was 10-fold above this level (Fig. 7). In summary it can be said that the divergent promoters pcaIp and pcaUp both show a strong decrease in activity in the presence of benzoate despite the fact that the substrate inducer protocatechuate is included in the medium.


Figure 7
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  		FIG. 7. Effect of benzoate (ben) on the expression at promoter pcaIp (white columns) and pcaUp (hatched columns) measured in A. baylyi strain ADPU47 containing a pcaI-luc fusion or in A. baylyi strain ADPU12 containing a pcaU-lacZ fusion. The cells were grown on mineral medium containing the indicated carbon sources (protocatechuate [PCA], 1 mM). The induced expression on pyruvate plus POB was set as 100%. See Fig. 3 legends for other abbreviations.

 
Regulation by downstream metabolites in the protocatechuate branch.
Cross-regulation between the two branches of the ß-ketoadipate pathway has been characterized with respect to promoters pobAp (3) and pcaIp (see above). In addition to cross-regulation between the branches, there is also regulation within the protocatechuate branch: protocatechuate (the product of the reaction catalyzed by p-hydroxybenzoate hydroxylase [PobA]) represses pobA expression (3). One possible mechanism for this repression, which appears like a feedback inhibition, could be that PobR, being the specific regulator at pobAp, can sense the level of protocatechuate in the cell by binding it either competitively at the inducer binding site or at another site. To address this hypothesis, we established the PobR-dependent regulation at pobAp on a plasmid in E. coli (pZR430B-luc). The plasmid contains a transcriptional pobA-luc fusion, the intergenic region between pobR and pobA, and the complete pobR gene. Measurement of the pobAp-directed luciferase activity upon growth in LB medium resulted in a basal expression, which was increased by a factor of 20 in the presence of 200 nM p-hydroxybenzoate, thus showing that the inducer entered the cells and brought about a functional induction (2 x 106 RLU/OD). When p-hydroxybenzoate was added together with protocatechuate, there was no significant reduction of the induced activity seen with p-hydroxybenzoate alone. The entrance of protocatechuate into cells of E. coli has been shown above. Thus, protocatechuate cannot influence pobAp activity by binding directly to PobR.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the current investigation, we are presenting results allowing a more in-depth understanding of the control of gene expression applied to the protocatechuate branch of the ß-ketoadipate pathway of A. baylyi. PcaU-dependent regulation of pcaIp could be established in E. coli. All aspects of this level of regulation known from studies in A. baylyi could be observed in the E. coli background. Protocatechuate added to the medium must enter the cells at rates high enough to lead to a functional induction, which is supported by the observation of growth of E. coli with protocatechuate when provided with pca genes (32). A significant increase of expression occurs at 10 µM protocatechuate. The same threshold inducer concentration has been observed for A. baylyi (39), where the uptake of the inducer into the cells is mediated by the transporters VanK and PcaK, both belonging to the large major facilitator superfamily of transport proteins (8). Whether uptake of protocatechuate into E. coli is supported by transport proteins is unknown. If this is not the case, diffusion of the undissociated acid must be the way that brings protocatechuate into E. coli cells. Both functions of the PcaU protein observed earlier in A. baylyi, namely the protocatechuate-dependent induction and the repression of the basal expression level, can be observed, the latter effect being even stronger in E. coli (ninefold) than in A. baylyi (threefold). The fact that a plasmid with a truncated pcaU gene (pAC17) but a construction otherwise identical to plasmid pAC48 does not show either effect proves that PcaU alone is causing them. Furthermore, these results present evidence that PcaU interacts with RNA polymerase from E. coli in the same way that it does with the Acinetobacter enzyme, an observation that has been made before with the PobR activator from A. baylyi (10).

Contradictory results have been described with respect to the carbon source dependence of the regulator gene. The negative autoregulation had been clearly established, but whether or not there was an influence by the carbon sources had been determined with different results using two different plasmid-based reporter systems in A. baylyi (19, 39). Here, we used an approach for measuring the pcaUp activity where the fusion with the reporter gene was situated on the chromosome in a location remote from the native location of the pcaU gene, and therefore it had the lowest possible copy number. (An introduction of the reporter gene into the native pcaU gene was not possible, because this would create a strain without a functional PcaU.) Using this strain, it could be observed that the presence of aromatic compounds to be funneled through the protocatechuate branch of the pathway increased pcaU gene expression considerably (up to sevenfold) over a basal level observed upon growth on nonaromatic substrates. Application of quantitative Northern blot hybridization clearly confirmed the inducible quality of pcaUp. Thus, it is now thoroughly documented that the pcaU gene expression is not only autorepressed but also regulated in a carbon source-dependent manner. Furthermore, a parallel treatment of membranes with samples from identical RNA preparations with probes for pcaU or the pca-qui operon demonstrated that the induction occurs in a time-synchronized way. It thus appears as though the regulatory pattern determined by the pcaU-pcaI intergenic region affects both the promoters located within it and those facing in the opposite directions in the same way. In sum, the pca regulatory region represents a system in which the regulator protein PcaU binds to its binding site located between two promoters, pcaIp and pcaUp, and from this binding site affects expression of both promoters exactly in the same way without contribution by other proteins. This effect can have both a repressing and an activating nature, even though the location of the PcaU binding site with respect to the promoters would infer an activating function (no overlap, but spacing by 11 bp and 4 bp, respectively). The system is reminiscent of the SoxR- and MerR-dependent regulation in E. coli, where two divergent promoters depend on these regulators bound at a single DNA site in the regulatory region and the effect can be both negative and positive (24, 28).

Carbon catabolite repression of the pca-qui operon has been described as a mechanism of gene regulation at a higher level (6). At the level of promoter activity using the lacZ-based reporter construct, the effect could be demonstrated but at a fairly low level (4.7-fold repression). When luciferase was used instead of ß-galactosidase to determine promoter activity upon carbon catabolite repression, a much higher repression factor could be determined (30-fold). The transcriptional nature of this regulatory mechanism is well documented by this approach. The molecular mechanism responsible for this new type of carbon catabolite repression is still unknown.

In the course of the identification of PcaU, complementation experiments had indicated that the expression of the pca gene products is lower when increased amounts of PcaU are present in the cell (19). Here, results are presented that strongly indicate such a mechanism. The basal level of transcription at pcaIp is lowest when three copies of pcaU are present in the cells and highest when no pcaU is present. The additional copies of the gene must cause a higher level of PcaU. The mechanism leading to a reduction of expression may have to do with a higher occupancy of the PcaU binding site at higher concentrations. Taken together with the data presented above proving the differential expression of pcaU, it is strongly indicated that the PcaU levels in the cells differ and furthermore that different PcaU concentrations cause repression at pcaIp to be more or less pronounced. Under inducing conditions, overexpression of PcaU also causes a decrease of expression (19), indicating that there must be an optimal concentration of PcaU for the highest possible induction.

Cross-regulation between the two branches of the pathway has been shown earlier with respect to the pobA promoter, which is repressed if—in addition to the inducer of the pobA gene (p-hydroxybenzoate)—metabolites of the catechol branch of the pathway are present. cis,cis-Muconate could be identified as the regulatory active compound (3). Here, it could be shown that this type of regulation also applies to the pca-qui operon and therefore to the regulation of expression of the central reactions. The presence of a substrate of the parallel branch of the pathway (benzoate) strongly repressed the expression at pcaIp despite the presence of the inducer. Furthermore, the same repression pattern was observed at the promoter pcaUp. Thus it is established that the repression of the enzymes for p-hydroxybenzoate degradation by metabolites of the parallel branch is a mechanism of transcriptional regulation affecting all promoters involved in the expression of the respective enzymes, namely pcaIp, pcaUp, and pobAp. Cross-regulation seems to be an important mechanism, as it creates an order of degradation for carbon sources accessed by the two branches of the ß-ketoadipate pathway and is therefore established in multiple promoters. It remains to be seen whether or not the promoters of other funneling pathways preparing alternative aromatic compounds for the degradation through the protocatechuate branch also underlie this cross-regulation.

Concerning the molecular mechanism responsible for the repression, it is obvious that one might speculate that regulatory proteins that do bind metabolites representative of the benzoate/catechol branch could also affect the promoters necessary for p-hydroxybenzoate degradation. In fact, it was shown that the regulators BenM and CatM do bind to a 291-bp DNA fragment from the pcaU-pcaI intergenic region, and the binding was stronger in the presence of cis,cis-muconate. A putative binding site for this interaction was identified directly downstream of the transcriptional start site of the pcaU gene (3). In contrast, there was no binding of CatM or BenM in the pob regulatory DNA. In addition, deletions causing disruption of both the catM gene and the benM gene allowed the simultaneous degradation of substrates of both branches of the pathway (benzoate and p-hydroxybenzoate). CatM and BenM are LysR-type proteins positively regulating genes involved in benzoate degradation (14). These results clearly established the role of BenM and CatM in the cross-regulation, but the mechanism remained unclear. Brzostowicz et al. speculated that the binding of CatM or BenM a few base pairs downstream of the pcaU transcriptional start site might disturb the binding of PcaU to its binding site and therefore prevent high-level expression of the pca genes (3). Since pcaK encoding a transport protein bringing external p-hydroxybenzoate into the cells (PcaK) is part of the operon, it was thought that this transport might not happen at levels high enough to allow an induced pobA expression. This hypothesis is based on the assumption that another protein known to transport p-hydroxybenzoate in addition to PcaK, namely VanK, is also repressed under the conditions of cross-regulation (8). It was unclear what the effect of CatM and BenM binding in the pca intergenic region was. In the current investigation, we could show that the expression at both promoters, pcaUp and pcaIp, was as low as in the absence of inducing compounds. Due to the divergent nature of the pca intergenic region with the PcaU binding site centrally located, factors influencing PcaU binding may affect expression in either direction, which is supported by results presented in this paper. Following the suggestion that the binding site for CatM and BenM is located within the DNA encoding the pcaU RNA and thus 44 bp away from the PcaU binding site, it is unlikely that there is a direct disturbance between the two regulatory proteins. More likely is a physical hindrance of RNA polymerase binding to promoter pcaUp or a blocking of elongation of transcription. The result would be a strong decrease in the level of PcaU not sufficient to occupy the PcaU binding site in a way necessary for an efficient activation of pcaIp. Thus, binding of BenM and CatM upstream of the pcaU gene is very likely the molecular mechanism leading to the cross-regulation phenotype at promoter pcaIp. In contrast, the mechanism leading to cross-regulation of pobA expression is less obvious.

The absence of PcaU leads to an increased basal level of expression of the pca-qui operon as shown here and previously (39). This level is high enough to allow growth of cells with a truncated pcaU gene. Based on this observation, it can be assumed that under conditions where pcaU expression is repressed by cross-regulation, the pca-qui operon will be expressed at increased levels and thus PcaK should be present at levels high enough to bring p-hydroxybenzoate into the cells. Even under conditions where PcaU is present in the cells at wild-type levels, PcaK levels should be sufficient to bring enough p-hydroxybenzoate into the cells to allow efficient induction of pobA expression as shown using an Acinetobacter strain with a lacZ cassette inserted into the pobA gene (3). Since this strain does not convert p-hydroxybenzoate into protocatechuate, the inducer of the pca-qui operon is not created, but despite this the strain was successfully used to monitor effects of cell-internal p-hydroxybenzoate. Furthermore, results have been reported describing strains without PcaK and VanK that can grow on p-hydroxybenzoate like the wild type, indicating that these two transporters are not a necessary requirement for p-hydroxybenzoate uptake (8). In Pseudomonas putida a benzoate-mediated repression of pcaK expression by the regulator BenR, and furthermore a relief from the repression in a benR mutant as detected by measuring p-hydroxybenzoate transport, could be demonstrated (36). In contrast to A. baylyi, in this organism the pcaK gene has its own promoter region. We assume that BenR (an XylS family member) does not have a direct role in this repression.

If a repressed transport is not the mechanism for repression of pobA expression by cross-regulation, then what is a possible mechanism? Here, we evaluated the possibility that PobR senses the presence of protocatechuate by looking at PobR-dependent pobAp regulation in E. coli. PobR did not cause a reduction in p-hydroxybenzoate-dependent activation of pobAp when tested in E. coli in the presence of protocatechuate. This indicates that PobR is not integrating multiple signals like the level of protocatechuate in addition to the level of p-hydroxybenzoate. In contrast, there must be a different mechanism of bringing this information to the pobAp promoter which needs to be identified. Another possibility would be that PcaU, the natural sensor of protocatechuate, also binds to the PobR binding site. This scenario is convincing, since there is high similarity at the level of the proteins PobR and PcaU as well as at the level of their binding sites. A first indication that this is not the case came from gel retardation assays testing the purified PcaU protein for binding to the pobR-pobA intergenic DNA. These experiments could not detect any binding of PcaU to the PobR binding site (unpublished observation). Of course, the affinity of this interaction may be very low. Other possibilities would be (i) formation of a hybrid complex of PcaU and PobR monomers at the PobR binding site when protocatechuate is bound by PcaU, which does not allow a functional transcriptional activation, (ii) direct binding of CatM or BenM to a DNA site outside of the one tested earlier from which they may influence pobA expression, or (iii) the involvement of factors that have not been identified yet. These are models that need to be addressed.

To summarize, the mechanisms of cross-regulation, including regulation by a downstream metabolite (protocatechuate) at promoter pobAp, remain unknown, but one possible mechanism can now be excluded, namely the direct sensing of the repressing metabolites by PobR. Cross-regulation is probably not an uncommon mechanism for achieving the most efficient fine-tuning between different pathways. It was described recently between the pathways for biphenyl and salicylate degradation in Pseudomonas pseudoalcaligenes (15).

The results presented here help to unravel the complexity of regulation of the central pca-qui operon as well as of promoters influenced by cross-regulation (Fig. 8). It is intriguing that there is just one mechanism of activation of gene expression, whereas there are multiple mechanisms of repression, which make sure that gene expression is low under a variety of conditions and therefore help the organism to be highly economical with the available resources.


Figure 8
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  		FIG. 8. Schematic presentation of the regulatory levels known to contribute to gene expression within the protocatechuate branch of the ß-ketoadipate pathway of A. baylyi (the pobS gene located between pobR and the qui genes were not included in the scheme). The thick horizontal arrows represent promoters; thus, the double arrows stand for two divergent promoters. The vertical lines demonstrate regulatory mechanisms affecting positively (bottom) or negatively (top) the respective promoter. POB, p-hydroxybenzoate; PCA, protocatechuate.

 


   ACKNOWLEDGMENTS
 
This work was supported by DFG grant Ge 672/3-3 and by a grant from the Rudolf und Clothilde Eberhardt-Stiftung Ulm.

The technical assistance by Iris Steiner is gratefully acknowledged.


   FOOTNOTES
 
* Corresponding author. Mailing address: Microbiology and Biotechnology, University of Ulm, 89069 Ulm, Germany. Phone: 49-731-5022715. Fax: 49-731-5022719. E-mail: ulrike.gerischer{at}uni-ulm.de. Back

{triangledown} Published ahead of print on 3 November 2006. Back

{dagger} Present address: Universitätsfrauenklinik und Poliklinik, University of Ulm, Ulm, Germany. Back

{ddagger} Present address: Ratiopharm GmbH, Ulm, Germany. Back

§ Present address: Lilly Deutschland GmbH, Bad Homburg, Germany. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Applied and Environmental Microbiology, January 2007, p. 232-242, Vol. 73, No. 1
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