Fachgebiet Technische Biochemie, Institut für Biotechnologie der Technischen Universität Berlin, Seestraße 13, D-13353 Berlin, Germany1
Author for correspondence: Helmut Görisch. Tel: +49 30 31427582. Fax: +49 30 31427581. e-mail: Goerisch{at}lb.TU-Berlin.De
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ABSTRACT |
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Keywords: exaAB, regulation, sensor kinase, response regulator, divergent promoter
Abbreviations: Cb, carbenicillin; MM, minimal medium; PQQ, pyrroloquinoline quinone; QEDH, quinoprotein ethanol dehydrogenase; QMDH, quinoprotein methanol dehydrogenase; Tc, tetracycline
The GenBank accession number for the sequence reported in this work is AJ009858.2 (=CAB 95009.1).
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INTRODUCTION |
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METHODS |
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Database searches were made using the programs BLAST or gapped BLAST (Altschul et al., 1997 ). For DNA editing and protein-sequence alignment, the program DNAMAN (Lynnon Biosoft) was used. Protein sequence alignment was done using CLUSTAL W (Thompson et al., 1994
). Searches for transmembrane helices were done using the SOSUI system described by Hirokawa et al. (1998)
. Prosite profiles were searched using the ProfileScan server at the Swiss Institute for Experimental Cancer Research (http://www.isrec.isb-sib.ch/software/PFSCAN_form.html).
ß-Galactosidase assay.
ß-Galactosidase activity was determined with cells treated with toluene, according to the procedure of Miller (1992) . P. aeruginosa containing promoter-probe vectors was grown on different media containing Tc. Cultures were harvested at an OD620 of 0·5 and cooled on ice for 5 min. Cells were washed and resuspended in the same volume of 20 mM potassium phosphate buffer (pH 7·2) containing 1 mM EDTA (buffer A). Prior to the assay, cells were diluted in Z-buffer (Miller, 1992
).
Determination of ß-galactosidase activity in P. aeruginosa mutants unable to grow on ethanol was performed after induction on ethanol: the constructs containing pTB3138 or pTB3139 were grown overnight in LB-Tc medium. An aliquot of 0·5 ml was washed and diluted fourfold in minimal medium (MM)-ethanol-Tc medium. After shaking of the cultures for 6 h at 37 °C, the ß-galactosidase activity induced was determined. The OD620 was measured at the beginning and the end of the induction period, to detect any growth of revertants.
Specific activity of QEDH.
The QEDH activity was measured as described by Rupp & Görisch (1988) . The protein concentration was determined according to Bradford (1976)
.
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RESULTS |
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To determine if exaB and exaC are both under the control of the exaB promoter, the lacZ promoter-probe vectors pTB3140 and pTB3141 were constructed. First, a 1·7 kb HindIIIAgeI fragment of pTB3070 was cloned into a HindIII/SmaI-digested pBluescript vector. In this vector (pTB3134), the partial exaC gene ends at the former AgeI site, which is followed by a XbaI site. This vector is fused to pEDY305 via the XbaI site. To generate a promoter-probe vector containing a transcriptional lacZ fusion with the exaC gene, including the complete exaB promoter region, the pBluescript vector was removed by digestion with PstI. The resulting promoter-probe vector was religated and named pTB3141 (Fig. 2).
To generate a promoter-probe vector for the exaC gene, without the exaB promoter region, the fusion plasmid between pTB3134 and pEDY305 was digested with XhoI to eliminate the pBluescript vector together with a 1 kb fragment carrying the exaAexaB promoter region. The resulting promoter-probe vector, pTB3140, contains the partial exaC gene with a 0·4 kb upstream sequence to the XhoI site within the exaB gene, but no exaB promoter (Fig. 2).
Establishing the ß-galactosidase assay
The vectors pTB3138, pTB3139, pTB3140, pTB3141 and pEDY305 were transferred into wild-type P. aeruginosa ATCC 17933 by triparental mating. To obtain reproducible ß-galactosidase transcription levels, it was essential, in the expression experiments, to inoculate with precultures grown on MM-succinate. To determine if the specific ß-galactosidase activity during growth on ethanol depends on the growth phase, P. aeruginosa pTB3138 was grown on MM-ethanol-Tc medium and then ß-galactosidase activity was measured as a function of growth. The activity (Miller units) was constant between OD620 values of 0·2 and 0·7. In all subsequent experiments, ß-galactosidase activity was measured with cultures at an OD620 of 0·5.
A positive factor is needed for transcription of exaA
To investigate the effect of an additional exaA promoter copy on the specific enzymic activity of QEDH, P. aeruginosa strains containing the following plasmids were grown on MM-ethanol-Tc until an OD620 of 0·5 was reached: pTB3138, containing the 0·89 kb PstISalI fragment of pTB3070 with the complete exaAexaB promoter region but with a truncated exaA gene; pTB3001, a pLAFR3 derivative containing a 20 kb genomic DNA fragment with the exaAexaB promoter region and the complete exaA gene; and pLAFR3, the empty vector, which served as a control. All three vectors are derivatives of pRK290, Tc resistant and larger than 20 kb. As an additional control, the P. aeruginosa wild-type was grown on MM-ethanol medium. The specific activity of QEDH was the same in the P. aeruginosa wild-type without any plasmid as it was in P. aeruginosa containing pLAFR3 or pTB3001. Thus, neither the presence of Tc nor a second copy of the exaA gene and its promoter influences the specific activity of QEDH. In P. aeruginosa containing pTB3138, with the exaA promoter followed by a truncated exaA gene, the specific QEDH activity dropped to 50% of that of the wild-type. Since the native form of QEDH is a homodimer, the polypeptide product produced by the truncated exaA gene might interfere with subunit association, thereby reducing the enzyme activity. This, however, is unlikely, because it is known from the QEDH X-ray structure that none of the 18 N-terminal amino acids of the truncated exaA gene product is involved in subunit association (Keitel et al., 2000 ). The results suggest that a positive factor, needed for starting transcription at the exaA promoter, is limiting.
Transcription levels of exaA, exaB and exaC
ß-Galactosidase activities were determined with P. aeruginosa strains containing the various lacZ fusion vectors, after growth on different carbon sources (Table 2). The promoter activities of exaA and exaB were monitored with wild-type P. aeruginosa containing pTB3138 or pTB3139. Induction of both promoters was observed with the four alcohols tested. The ß-galactosidase activities during growth on MM-succinate-Tc and MM-acetate-Tc were low. The highest induction of both promoters was observed after growth on ethanol.
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Identification of regulatory mutants in P. aeruginosa
Recently, we isolated and characterized 21 mutants of P. aeruginosa unable to grow on ethanol. These mutants were grouped into four classes by biochemical characterization (Schobert & Görisch, 1999 ). Plasmids pTB3138 (exaA::lacZ fusion) and pTB3139 (exaB::lacZ fusion) were used to identify regulatory mutants with a diminished transcription rate for exaA and/or exaB by transferring the plasmids separately into all mutants by triparental mating. Since the mutants are unable to grow on ethanol, ß-galactosidase activity was determined after induction on MM-ethanol-Tc medium.
Six mutants (MS9, MS11, MS12, MS13, MS16 and MS21) showed significantly reduced transcription rates for the exaA promoter, which amounted to about 10% of the wild-type level (Fig. 3). These mutants were divided into two groups. Group A contained the four mutants (MS9, MS11, MS16 and MS21) that showed low transcription rates for both the exaA promoter and the exaB promoter. Group B contained the mutants (MS12 and MS13) in which only the transcription rate of the exaA promoter is significantly reduced (the transcription rate of the exaB promoter being even higher than that in the wild-type) (Fig. 3
).
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The second ORF present on the pTB3144-insert DNA corresponds to a previously sequenced gene of a putative regulatory-response protein (Diehl et al., 1998 ) and was named exaE. The previously reported sequence (Diehl et al., 1998
) was corrected and is now available under accession number AJ009858.2. It starts at position 1195 and ends at position 1872. The exaE gene encodes a protein of 225 aa (Fig. 6
), and a ProfileScan search showed the presence of two motifs, i.e. a histidine-receiving module in bacterial sensor systems at positions 2118, and a helixturnhelix profile at positions 148198, belonging to the luxR family (Henikoff et al., 1990
). Sequence comparison showed that the putative receiving aspartate residue is located at position 53. Other highly conserved residues in the histidine-receiving module of response regulators described by Parkinson (1993)
are also found, i.e. aspartate 7+8, and lysine 103.
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DISCUSSION |
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The physiological characterization of six mutants and the results of transformation experiments restoring growth by different cosmids and various subclones derived from cosmid pTB3001 allowed us to estimate the number of regulatory genes involved. Mutants MS16 and MS21 acquired wild-type behaviour after transformation by the same two cosmids and were unable to grow on acetate, but they differ in their ability to produce PQQ. Mutants MS9 and MS11 show identical phenotypes. They produce PQQ, grow poorly on succinate and not on acetate or glucose, but different cosmids restore growth on ethanol. In all four mutants (MS9, MS11, MS16 and MS21), no activity of exaA and exaB promoters was found (Table 3). The properties of these four mutants indicate that four different regulatory genes might be involved. However, in mutants MS12 and MS13, only induction of the exaA promoter was impaired, indicating that the two divergent promoters are regulated differently. The two mutants show identical phenotypes and both are converted to wild-type behaviour by cosmid pTB3001 and by different subclones thereof. Subclone pTB3144 restores growth on ethanol with both mutants, whereas pTB3145 restores growth on ethanol only for mutant MS12.
Plasmid pTB3145 carries a gene encoding a previously sequenced response regulator (Diehl et al., 1998 ). At that time, it was unknown if this gene was involved in the regulation of the ethanol-oxidation system. Sequencing of the part of pTB3144 that also restores the growth of MS13 led to the identification of a gene encoding a histidine sensor kinase. This gene ends only 15 bp upstream of the response-regulator gene and shares the same orientation. Both genes form a typical bacterial two-component system and were named exaDE. The sensor kinase shares 3035% identity with other bacterial sensor kinases. Although the typical histidine kinase motif is present, no transmembrane helices were found, indicating that ExaD is a soluble, cytoplasmic sensor kinase.
The cytoplasmic localization of the soluble ExaD sensor kinase raises the question of the nature of the signal molecule activating this enzyme. So far, there is no indication that ExaD might be activated in a manner similar to CheA, a soluble kinase of E. coli involved in chemotaxis, the autophosphorylation of which is controlled by methyl-accepting chemotaxis proteins. These methyl-accepting chemotaxis proteins are membrane-bound chemoreceptors that detect chemical stimuli in the periplasm.
In contrast, in methylotrophic bacteria, both sensor kinases essential for growth on methanol (MxbD, MxcQ) are membrane bound, with a periplasmic loop that is assumed to be the receptor domain. In M. extorquens, a hierarchy of the different regulatory genes is presumed (Springer et al., 1997 ): the two-component system mxcQE controls the expression of mxbDM, which regulates expression of the mxaF promoter and the pqq biosynthesis genes. There is also a single response-regulator gene, mxaB, without an adjacent sensor kinase; mxaB is essential for mxaF induction and is also involved in induction of the pqq biosynthesis genes (Morris & Lidstrom, 1992
; Ramamoorthi & Lidstrom, 1995
).
Our data clearly show that the response regulator ExaE is necessary to induce transcription from the exaA promoter. The divergent promoter region between exaA and exaB does not contain sequences that can be unabigiously identified as known 70,
54 or other consensus sequences known for Pseudomonas (Ronald et al., 1992
). However, we found a 23 bp sequence motif with dyad symmetry (CGTCCGGGAA-N3-TTCCCGGACG) between exaA and exaB. It is located 113 bp upstream of exaA and 177 bp upstream of exaB and might constitute a binding motif for the response regulator ExaE.
Concluding remarks
We have investigated the regulatory network controlling the ethanol-oxidation system in P. aeruginosa. Our results with the six regulatory mutants studied show that six different genes might be involved. In one group of regulatory mutants, the induction of both exaA and exaB is impaired, whereas in another group only induction of exaA is impaired. The observed regulatory pattern presumably allows P. aeruginosa to use cytochrome c550 also as an electron acceptor with enzymes for other carbon sources besides ethanol. Experiments designed to identify the signal molecule of the exaD sensor kinase and to verify binding of the exaE response regulator in the exaA promoter region are under way. In addition, cloning and characterization of the other regulatory genes involved will be performed to establish whether or not there is a hierarchical organization of the different regulatory factors.
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ACKNOWLEDGEMENTS |
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Received 20 July 2000;
revised 20 October 2000;
accepted 2 November 2000.