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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Institut für Mikrobiologie der Technischen Universität Braunschweig, Spielmannstraße 7, D-38106 Braunschweig, Germany.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Antibiotics were added at the following concentrations: tetracycline, 20 µg ml1; carbenicillin, 100 µg ml1; kanamycin, 50 µg ml1.
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 BamHIPstI 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 BamHIPstI sites of pUCP20T and pUC18, resulting in plasmids pTB7061 and pTB7059 (Fig. 1
).
|
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 XhoIBamHI fragment of pTB3070 containing the pqqAB promoter region was cloned between the XhoIBglII 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 BglIIXbaI sites of the promoter-probe vector pQF50 (Farinha & Kropinski, 1990
), resulting in plasmid pTB7074 (Fig. 2
).
|
Determination of -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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
-galactosidase activity was determined (Table 3
).
|
|
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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ausubel, F. A., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (editors) (2002). Current Protocols in Molecular Biology. New York: Wiley.
Boyer, H. W. & Roulland-Dussoix, D. (1969). A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 14, 459472.
Cetin, E. T., Töreci, K. & Ang, Ö. (1965). Encapsulated Pseudomonas aeruginosa (Pseudomonas mucosus) strains. J Bacteriol 89, 14321433.[Medline]
Diehl, A., von Wintzingerode, F. & Görisch, H. (1998). Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer sequence of the gene and deduced structural properties of the enzyme. Eur J Biochem 257, 409419.[Abstract]
Farinha, M. A. & Kropinski, A. M. (1990). Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters. J Bacteriol 172, 34963499.[Medline]
Figurski, D. H. & Helinski, D. R. (1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 16481652.[Abstract]
Görisch, H. (2003). The ethanol oxidation system and its regulation in Pseudomonas aeruginosa. Biochim Biophys Acta 1647, 98102.[Medline]
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557580.[Medline]
Henikoff, S., Wallace, J. C. & Brown, J. P. (1990). Finding protein similarities with nucleotide sequence databases. Methods Enzymol 183, 111132.[Medline]
Hoang, T. T., Karkhoff-Schweizer, R. R., Kutchma, A. J. & Schweizer, H. P. (1998). A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212, 7786.[CrossRef][Medline]
Kretzschmar, U., Schobert, M. & Görisch, H. (2001). The Pseudomonas aeruginosa acsA gene, encoding an acetyl-CoA synthetase, is essential for growth on ethanol. Microbiology 147, 26712677.
Kretzschmar, U., Rückert, A., Jeoung, J. & Görisch, H. (2002). Malate : quinone oxidoreductase is essential for growth on ethanol or acetate in Pseudomonas aeruginosa. Microbiology 148, 38393847.
Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305, 567580.[CrossRef][Medline]
Miller, J. M. (1992). A Short Course in Bacterial Genetics, a Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Reichmann, P. & Görisch, H. (1993). Cytochrome c550 from Pseudomonas aeruginosa. Biochem J 289, 173178.[Medline]
Rozen, S. & Skaletsky, H. J. (1998). Primer3. Code available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html.
Rupp, M. & Görisch, H. (1988). Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa. Biol Chem Hoppe-Seyler 369, 431439.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schobert, M. (1999). Molekulargenetische Untersuchungen zum Ethanol-oxidierenden System in Pseudomonas aeruginosa. PhD thesis, Technische Universität Berlin, Germany.
Schobert, M. & Görisch, H. (1999). Cytochrome c550 is an essential component of the quinoprotein ethanol oxidation system in Pseudomonas aeruginosa: cloning and sequencing of the genes encoding cytochrome c550 and an adjacent acetaldehyde dehydrogenase. Microbiology 145, 471481.[Abstract]
Schobert, M. & Görisch, H. (2001). A soluble two-component regulatory system controls expression of quinoprotein ethanol dehydrogenase (QEDH) but not expression of cytochrome c550 of the ethanol-oxidation system in Pseudomonas aeruginosa. Microbiology 147, 363372.
Schwartz, E., Gerischer, U. & Friedrich, B. (1998). Transcriptional regulation of Alcaligenes eutrophus hydrogenase genes. J Bacteriol 180, 31973204.[Abstract]
Schweizer, H. P. (1991). The agmR gene, an environmentally responsive gene, complements defective glpR, which encodes the putative activator for glycerol metabolism in Pseudomonas aeruginosa. J Bacteriol 173, 67986806.[Medline]
Schweizer, H. P. (1992). Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 6, 11951204.[Medline]
Schweizer, H. P., Klassen, T. R. & Hoang, T. (1996). Improved methods for gene analysis in Pseudomonas. In Molecular Biology of Pseudomonads, pp. 229237. Edited by T. Nakazawa, K. Furukawa, D. Haas & S. Silver. Washington, DC: American Society for Microbiology.
Schweizer, H. P. & Po, C. (1996). Regulation of glycerol metabolism in Pseudomonas aeruginosa: characterization of the glpR repressor gene. J Bacteriol 178, 52155221.[Abstract]
Smith, A. W. & Iglewski, B. H. (1989). Transformation of Pseudomonas aeruginosa by electroporation. Nucleic Acids Res 17, 10509.[Medline]
Staskawicz, B., Dahlbeck, D., Keen, N. & Napoli, C. (1987). Molecular characterization of cloned avirulence genes from race 0 to race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol 169, 57895794.[Medline]
Stover, C. K., Pham, X. Q., Erwin, A. L. & 28 other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959964.[CrossRef][Medline]
Vrionis, H. A., Daugulis, A. J. & Kropinski, A. M. (2002). Identification and characterization of the AgmR regulator of Pseudomonas putida: role in alcohol utilization. Appl Microbiol Biotechnol 58, 469475.[CrossRef][Medline]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33, 103119.[CrossRef][Medline]
Received 3 November 2003;
revised 7 January 2004;
accepted 3 February 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |