AgmR controls transcription of a regulon with several operons essential for ethanol oxidation in Pseudomonas aeruginosa ATCC 17933

Nicole Gliese, Viola Khodaverdi, Max Schobert{dagger} and Helmut Görisch

Fachgebiet Technische Biochemie, Institut für Biotechnologie der Technischen Universität Berlin, Seestraße 13, D-13353 Berlin, Germany

Correspondence
Helmut Görisch
Goerisch{at}lb.TU-Berlin.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The response regulator AgmR was identified to be involved in the regulation of the quinoprotein ethanol oxidation system of Pseudomonas aeruginosa ATCC 17933. Interruption of the agmR gene by insertion of a kanamycin-resistance cassette resulted in mutant NG3, unable to grow on ethanol. After complementation with the intact agmR gene, growth on ethanol was restored. Transcriptional lacZ fusions were used to identify four operons which are regulated by the AgmR protein: the exaA operon encodes the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the exaBC operon encodes a soluble cytochrome c550 and an aldehyde dehydrogenase, the pqqABCDE operon carries the PQQ biosynthetic genes, and operon exaDE encodes a two-component regulatory system which controls transcription of the exaA operon. Transcription of exaA was restored by transformation of NG3 with a pUCP20T derivative carrying the exaDE genes under lac-promoter control. These data indicate that the AgmR response regulator and the exaDE two-component regulatory system are organized in a hierarchical manner. Gene PA1977, which appears to form an operon with the agmR gene, was found to be non-essential for growth on ethanol.


Abbreviations: PQQ, pyrroloquinoline quinone; QEDH, quinoprotein ethanol dehydrogenase

{dagger}Present address: Institut für Mikrobiologie der Technischen Universität Braunschweig, Spielmannstraße 7, D-38106 Braunschweig, Germany.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Pseudomonas aeruginosa ATCC 17933 grows aerobically on ethanol. The substrate is oxidized to acetaldehyde by a periplasmic quinoprotein ethanol dehydrogenase (QEDH) that contains pyrroloquinoline quinone (PQQ) as cofactor (Rupp & Görisch, 1988). The QEDH transfers electrons to an endoxidase via a soluble cytochrome c550 (Reichmann & Görisch, 1993). To study the ethanol oxidation system of P. aeruginosa, chemical mutants unable to grow on ethanol were isolated (Schobert & Görisch, 1999). Several genes were identified to be involved in ethanol oxidation. The exaA gene encodes the QEDH (Diehl et al., 1998), and cytochrome c550 is encoded by the exaB gene (Schobert & Görisch, 1999). An NAD+-dependent acetaldehyde dehydrogenase is encoded by the exaC gene, which is cotranscribed with exaB (Schobert & Görisch, 1999). The PQQ biosynthetic genes are clustered in the pqqABCDE operon, while a pqqF gene is located 14 kb upstream of pqqABCDE. The acsA gene encodes an acetyl-CoA synthetase which converts acetate to acetyl-CoA (Kretzschmar et al., 2001). A malate : quinone oxidoreductase encoded by the mqo gene is also essential for growth on ethanol (Kretzschmar et al., 2002).

After phenotypic characterization and complementation of different regulatory mutants, it was concluded that six or seven different genes might be involved in regulation of the ethanol oxidation system (Schobert & Görisch, 2001; Görisch, 2003). Until now, only the two-component regulatory system ExaDE that controls the transcription of exaA, but not of exaBC, has been identified (Schobert & Görisch, 2001).

The previously isolated mutant MS15 did not produce PQQ or the apoprotein of QEDH (Schobert & Görisch, 1999). From this information, together with promoter-probe studies, it was assumed that MS15 had a defect in a regulatory gene which controls not only expression of QEDH but also expression of cytochrome c550 and the PQQ biosynthetic enzymes. Mutant MS15 was complemented by cosmid pTB3001, which contains a 25 kb insert (Schobert & Görisch, 1999). In this study, we identify and characterize the defective regulatory function of mutant MS15.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
The strains and plasmids used in this work are listed in Table 1.


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Table 1. Strains and plasmids used in this study

 
Escherichia coli was cultivated in Luria-Bertani (LB) medium, and P. aeruginosa was cultivated in LB or minimal medium (Rupp & Görisch, 1988) containing different carbon sources. Alcohols were added at 0·5 % (v/v). Glucose and succinate were used at 40 mM, and acetate at 20 mM.

Antibiotics were added at the following concentrations: tetracycline, 20 µg ml–1; carbenicillin, 100 µg ml–1; kanamycin, 50 µg ml–1.

General genetic techniques and PCR.
Routine recombinant DNA work was performed according to the protocols described by Sambrook et al. (1989) and Ausubel et al. (2002). Triparental matings were performed as described by Kretzschmar et al. (2001).

For PCR reactions, genomic DNA isolated from P. aeruginosa ATCC 17933 was used as template and Pfu DNA polymerase (Promega) was used for amplification. For primer design, the sequence of P. aeruginosa strain PAO1 could be used, since the nucleotide identity between PAO1 and ATCC 17933 is 99 % (Schobert, 1999). Primers were designed using the primer3 internet tool (Rozen & Skaletsky, 1998). For amplification, oligonucleotides with restriction sites (indicated in bold, below) for BamHI and PstI were used. The forward primer for amplification of the agmR gene was 5'-ACAGGATCCCGTCCAGCCCCTGGCAGTAG-3' and the reverse primer was 5'-AGACTGCAGCAGGGCGGTGAAACTGAC-3'. The 1·67 kb PCR product was cloned between the BamHI–PstI sites of pUCP20T and pUC18, resulting in plasmids pTB7060 and pTB7062 (Fig. 1). For amplification of gene PA1977, the forward primer was 5'-CAGGATCCGAACAAGCAGATCGCCTAC-3' and the reverse primer was 5'-AGACTGCAGTCATGCTTCGCCATCGAGAAC-3'. The 1·5 kb PCR product was cloned between the BamHI–PstI sites of pUCP20T and pUC18, resulting in plasmids pTB7061 and pTB7059 (Fig. 1).



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Fig. 1. Physical and restriction map of the cloned DNA fragments. Cosmid pTB3001 and plasmids pTB3070, pTB3112, pTB3143 and pTB3144 are subclones of pTB3001; fragments were cloned into pUCP20T. Inserts of pTB7067, pTB7060 and pTB7061 were amplified by PCR and cloned into pUCP20T. Inserts of pTB7059 and pTB7062 were amplified by PCR and cloned into pUC18. B, BamHI; M, MluI; P, PstI; S, SacI. Positions of the inserted kanamycin-resistance genes are indicated.

 
Electrotransformation of P. aeruginosa ATCC 17933 was performed as described by Smith & Iglewski (1989).

Site-directed mutagenesis.
For site-directed inactivation of genes of P. aeruginosa ATCC 17933 by a kanamycin-resistance cassette, the sacB-based strategy with the suicide vector pEX18Ap (Hoang et al., 1998) was employed. Sucrose-resistant colonies were obtained by streaking P. aeruginosa merodiploids on LB plates supplemented with 5 % sucrose. For inactivation of the agmR gene, the SmaI-digested kanamycin-resistance cassette was ligated into the blunted BstXI site of pTB7062, resulting in pTB7063, and the complete insert was cloned in pEX18Ap, resulting in pTB7064. The Kmr gene is transcribed in the same orientation as the agmR gene. For inactivation of the gene PA1977, the SmaI-digested kanamycin-resistance cassette was ligated into the BsaAI site of pTB7059, resulting in pTB7065, and the complete insert was cloned in pEX18Ap, resulting in pTB7068. The Kmr gene is transcribed in the same orientation as PA1977.

Construction of promoter-probe vectors.
A promoter-probe vector was constructed to study the transcriptional regulation of the pqqABCDE operon. A 2·5 kb XhoI–BamHI fragment of pTB3070 containing the pqqAB promoter region was cloned between the XhoI–BglII sites of vector pEDY305 (Schwartz et al., 1998) to construct a transcriptional pqqABlacZ fusion, resulting in plasmid pTB7023 (Fig. 2). To study transcriptional regulation of the two-component regulatory system exaDE, the corresponding promoter region was amplified by PCR. The forward primer was 5'-TCAGATCTGTTCATCAGGCCGTTGAGG-3' and the reverse primer was 5'-AGTCTAGAGATGCCCGTCAGGTACTGG-3'; BglII and XbaI restriction sites are indicated in bold. To construct a transcriptional exaDlacZ fusion, the 1·01 kb PCR product was cloned between the BglII–XbaI sites of the promoter-probe vector pQF50 (Farinha & Kropinski, 1990), resulting in plasmid pTB7074 (Fig. 2).



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Fig. 2. Physical and restriction map of promoter-probe vectors used in this study. Promoter regions are indicated with arrows. pTB3138, pTB3139 and pTB7023 are derivatives of pEDY305; pTB7074 is a derivative of pQF50. B, BamHI; P, PstI; S, SalI; X, XhoI. *Vectors constructed by Schobert & Görisch (1999).

 
{beta}-Galactosidase assay.
Activity of {beta}-galactosidase was determined with cells treated with toluene, according to the procedure of Miller (1992).

Determination of {beta}-galactosidase activity in P. aeruginosa mutants unable to grow on ethanol was performed after induction on ethanol, as described by Schobert & Görisch (2001).

Internet tools.
BLAST was used for DNA or protein database searches (Altschul et al., 1997), and the Pseudomonas aeruginosa Genome Database (Stover et al., 2000) was used to obtain DNA sequences of PAO1. For prediction of transmembrane helices in proteins, the TMHMM program was used (Krogh et al., 2001).


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Complementation of MS15
The previously isolated chemical mutant MS15 is unable to grow on ethanol, but growth is restored after complementation with cosmid pTB3001 (Schobert & Görisch, 1999). To identify the defective gene, MS15 was transformed by subclones of cosmid pTB3001. Transformation by pTB3112 and pTB3143 restored growth on ethanol, albeit with longer generation times than the wild-type containing the empty pUCP20T vector (Table 2). The overlapping parts of pTB3112 and pTB3143 contained four complete genes: exaD, exaE, PA1977 and agmR (Fig. 1). The two-component regulatory system exaDE (PA1979/1980) had been characterized previously, and strains with mutations in these genes showed properties different from MS15 (Schobert & Görisch, 2001). Accordingly, the genes PA1977 and agmR (PA1978) were amplified with specific primers by PCR and cloned into pUCP20T (Fig. 1). The plasmids pTB7060, containing the agmR gene (PA1978), and pTB7067, containing both agmR and PA1977, restored growth on ethanol of MS15, but the complemented mutant showed a prolonged lag phase and grew more slowly than the wild-type (Table 2). We assume that the defective AgmR protein produced by the chemical mutant MS15 is still able to bind to regulatory DNA sequences, and thereby interferes with the positive function of the native AgmR protein. Plasmid pTB7061, containing the gene PA1977, did not restore growth (Table 2). These results indicate that the agmR gene is defective in mutant MS15. The agmR gene encoded a protein of 221 amino acids which showed a high similarity to response regulators of two-component regulatory systems of the LuxR family (Henikoff et al., 1990; Schweizer, 1991).


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Table 2. Growth of P. aeruginosa mutants on various substrates

Results represent the mean of two independent growth tests; generation times (h) are shown in parentheses. –, No growth; +, growth with longer generation times than wild-type; ++, growth like wild-type; (+), growth as + but with prolonged lag phase; ND, not determined.

 
Inactivation of the agmR gene and characterization of mutant NG3
To investigate the function of the agmR gene, it was inactivated by insertion of the kanamycin-resistance cassette of transposon Tn5, using pTB7064. An agmR : : Kmr mutant, NG 3, was isolated. The position of the Kmr cassette inserted in the chromosome is indicated in Fig. 1. The insertion of the 1 kb kanamycin-resistance cassette was confirmed by PCR analysis using the agmR-specific primers.

Growth of mutant NG3 on various carbon sources was compared with the wild-type (Table 2). Mutant NG3 was unable to grow on ethanol and 1,2-propanediol. It grew on butanol, with a longer generation time than the wild-type, while on glycerol, acetate, glucose and succinate no differences in growth rates were found. Growth of NG3 on ethanol was restored after complementation with pTB7060 and pTB7067. In contrast to the complemented chemical mutant MS15, the agmR : : Kmr mutant NG3 after complementation was able to grow on ethanol like the wild-type (Table 2).

The agmR gene was initially supposed to be an activator for glycerol metabolism (Schweizer, 1991). However, generation of an agmR : : Tcr mutant of P. aeruginosa PAO1 shows no detectable glp phenotype (Schweizer, 1992; Schweizer & Po, 1996). In contrast, a Pseudomonas putida agmR : : Kmr mutant (AVP2) was described to be unable to grow on decanol, ethanol and glycerol (Vrionis et al., 2002). The specific activity of QEDH in mutant AVP2 was also reduced. The agmR gene of P. putida has 86 % nucleotide identity to the agmR gene of P. aeruginosa. Even though the P. putida agmR : : Kmr AVP2 mutant was unable to grow on glycerol, the P. aeruginosa agmR : : Kmr mutant NG3 grows on glycerol like the wild-type. In P. aeruginosa ATCC 17933, the glycerol metabolism appears not to be regulated by AgmR.

Promoter activities of the exaA, exaBC, pqqABCDE and exaDE operons
The different promoter-probe vectors pTB3138, pTB3139 (Schobert & Görisch, 2001), pTB7023 and pTB7074 (Fig. 2) were transferred into P. aeruginosa wild-type and mutant NG3 by triparental mating or electrotransformation. After induction on minimal medium with ethanol, the {beta}-galactosidase activity was determined (Table 3).


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Table 3. Activities of the exaA, exaB, exaDE and pqqAB promoters in wild-type and mutant NG3 after induction on ethanol

 
The promoter activities of the exaA and exaBC operons in mutant NG3 were reduced, relative to the wild-type, by a factor of about 200. The promoter activity of the pqqABCDE operon was reduced by a factor of about 25 (Table 3). These results indicate that the agmR gene product might be a positive regulator of the quinoprotein ethanol regulon of P. aeruginosa. The {beta}-galactosidase activity of the exaDE promoter in the wild-type was 406±141 U. In the mutant NG3 the activity of the exaDE promoter was again abolished (5·2±3·6 U), indicating that the AgmR protein also controls transcription of the exaDE operon (Table 3). As reported previously, the two-component system ExaDE controls transcription of the exaA gene encoding the QEDH (Schobert & Görisch, 2001). In mutants with defects in the exaD or exaE gene, transcription of the exaA gene is drastically reduced, and no active enzyme or apo-QEDH are found. Transcription of the exaBC and pqqABCDE operons, however, is not impaired (Görisch, 2003). In mutant NG3, with an inactivated agmR gene, transcription of the exaDE operon is prevented, which abolishes transcription of the exaA gene, and it appears that there is a hierarchical organization of the two regulatory functions AgmR and ExaDE. To demonstrate that AgmR is not directly involved in the expression of the exaA gene, mutant NG3, containing the promoter-probe vector pTB3138, was transformed by the pUCP20T derivative pTB3144 carrying the exaDE genes under control of the lac promoter. As expected, the transcription of the exaDE system resulted in restored expression of the exaA gene (Table 3). In mutant NG3 containing empty vectors, no activity was determined. The data prove that the regulatory factors AgmR and ExaDE in P. aeruginosa are organized in a hierarchical way. Fig. 3 summarizes the transcriptional regulation of the different components of the quinoprotein ethanol oxidation system. The AgmR protein controls transcription of a regulon consisting of three operons (exaBC, pqqABCDE and exaDE), which are essential for ethanol oxidation in P. aeruginosa ATCC 17933. The two-component regulatory system exaDE then controls transcription of the exaA operon encoding the QEDH.



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Fig. 3. Model of the regulation of the quinoprotein ethanol oxidation system of P. aeruginosa controlling the expression of QEDH, cytochrome c550 and PQQ biosynthetic enzymes. p, Promoter; +, positive interaction.

 
Inactivation of the gene PA1977 and characterization of mutant NG4
Downstream of the gene agmR, gene PA1977 is located, the function of which is unknown. Gene PA1977 and the agmR gene apparently form an operon, because they are separated by only 10 bp and no sequence resembling a transcriptional terminator has been found downstream of agmR (Schweizer, 1991). A database search for conserved domains with the PA1977 sequence did not reveal similarities to known sensor kinases, but the protein was predicted to contain 10 transmembrane helices (Krogh et al., 2001). Gene PA1977 was inactivated by insertion of a kanamycin-resistance cassette of transposon Tn5, using pTB7068. A PA1977 : : Kmr mutant (NG4) was isolated. The position of the Kmr cassette inserted in the chromosome is indicated in Fig. 1. The presence of the kanamycin-resistance cassette in PA1977 was confirmed by PCR analysis, using the PA1977-specific primers.

Mutant NG4 was able to grow on ethanol, 1,2-propanediol, butanol, glucose, succinate and acetate, albeit with a longer generation time than the wild-type, but complementation of mutant NG4 with pTB7061 or pTB7067 did not restore wild-type behaviour. Growth on glycerol was not impaired (Table 2). The function of gene PA1977 remains unknown, and apparently it is not involved in the control of the quinoprotein ethanol oxidation system of P. aeruginosa.

Concluding remarks
In the present work, the product of the agmR gene was identified as a general regulator of the quinoprotein ethanol oxidation system of P. aeruginosa ATCC 17933. The agmR : : Kmr mutant NG3 was unable to grow on ethanol and 1,2-propanediol, but complementation with the agmR gene restored growth on ethanol with wild-type behaviour.

The AgmR response regulator controls transcription of a regulon consisting of the three operons exaBC, exaDE and pqqABCDE, which are essential for ethanol oxidation in P. aeruginosa. The regulatory factors AgmR and ExaDE are organized in a hierarchical way. So far, the corresponding sensor kinase to the AgmR regulator is not known. Experiments are under way in our laboratory to identify such a sensor kinase.


   ACKNOWLEDGEMENTS
 
We thank Dr E. Schwartz, Humboldt Universität, Berlin, for the gift of plasmid pEDY305. This work was supported by the Deutsche Forschungsgemeinschaft.


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Received 3 November 2003; revised 7 January 2004; accepted 3 February 2004.



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