Fachgebiet Technische Biochemie, Institut für Biotechnologie der Technischen Universität Berlin, Seestraße 13, D-13353 Berlin, Germany1
Author for correspondence: Helmut Görisch. Tel: +49 30 314 27582. Fax: +49 30 314 27581. e-mail: Goerisch{at}lb.TU-Berlin.De
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ABSTRACT |
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Keywords: ethanol oxidation, acetate metabolism, acetate kinase, phosphotransacetylase, Pseudomonas aeruginosa
Abbreviations: ACK, acetate kinase; ACS, acetyl-CoA synthetase; PQQ, pyrroloquinoline quinone; PTA, phosphotransacetylase; QEDH, quinoprotein ethanol dehydrogenase
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INTRODUCTION |
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Recently, we isolated mutants of P. aeruginosa unable to grow on ethanol. Mutants MS1 and MS8 have a defect in a structural or regulatory gene of either the electron transport chain specific for the quinoprotein ethanol oxidation system or the metabolism of acetaldehyde and acetate (Schobert & Görisch, 1999 ). The present communication describes the identification of the gene acsA, which encodes ACS, essential in P. aeruginosa ATCC 17933 for growth on ethanol.
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METHODS |
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Triparental matings between E. coli JM109 and P. aeruginosa mutants were carried out by mixing aliquots of overnight cultures on LB agar. E. coli HB101 carrying pRK2013 was used as helper strain. After 6 h at 37 °C, cells were resuspended and spread on both succinate and selective ethanol minimal medium.
For PCR amplification of the acsA gene, the 4·8 kb fragment of pTB4102 was used as template. Pfu DNA polymerase (Stratagene) was used according to the instructions of the manufacturer. As primers the oligonucleotide 5'-AGT GGA TCC GTT GAT CTC GCT GTG G-3' complementary to a sequence 205 bp upstream of the start codon and the oligonucleotide 5'-AGT GGA TCC GAA GTG TTA CCG CGC C-3' 109 bp downstream of the stop codon TGA of ORF2 were used.
Construction of an acsA::Kmr mutant.
A pUC19 derivative, pTB4108, containing a 5·8 kb fragment from pTB4107 with the kanamycin-resistance gene of transposon Tn5 in the acsA gene, was used to transform P. aeruginosa by electroporation (Smith & Iglewski, 1989 ). Potential site-directed double-crossover mutants with a Kmr phenotype were selected for loss of ampicillin resistance.
Induction of enzyme activity.
Strains were grown in minimal medium with succinate as carbon source. At an OD620 of 0·8, cells were collected by centrifugation, washed twice and resuspended in twice the amount of minimal medium with 0·5% (v/v) ethanol to induce the ethanol oxidation system. After incubation for 5 h at 37 °C, cells were harvested by centrifugation.
Preparation of cell-free extracts.
Bacteria were grown to late-exponential phase, harvested, and washed twice with 20 mM potassium phosphate buffer (pH 7·0). The wet cell paste (12 g) was resuspended in 10 ml 10 mM Tris/HCl buffer (pH 7·9). After cell disruption by sonication, the cell homogenate was centrifuged for 30 min at 6000 g. The supernatant contained 1·54 mg protein ml-1 and was stored at -80 °C as cell-free extract. Under these conditions, ACS and ACK activities were stable for several days. For the detection of low enzyme activity, cell-free extracts with about 10 mg protein ml-1 were prepared.
Enzyme assays.
Enzyme activities were determined with cell-free extracts. For standard tests, 0·51·3 mg protein was used in a total test volume of 1 ml. For the determination of low enzyme activity, about 6 mg protein was used per test. ACS activity was assayed in 0·1 M Tris/HCl buffer, pH 8·5, by monitoring the formation of acetyl-CoA from acetate, CoA and ATP (Jones & Lipman, 1955 ; Berg, 1962
). ACK activity was determined by measuring the formation of acetyl-P from acetate and ATP (Aceti & Ferry, 1988
; Eggen et al., 1991
). Acetyl-CoA and acetyl-P were determined as the trivalent iron complex of acetylhydroxamate at 540 nm. Enzyme activities below 0·025 U per assay mixture cannot be detected. One unit (U) is defined as 1 µmol product formed in 20 min. Using the same cell extract, standard deviations of the enzymic test were calculated as ±9% for ACS and ±3% for ACK activity. PTA activity was determined with acetyl phosphate and CoA as substrates. Formation of acetyl-CoA was followed by the absorption increase of the thioester linkage at 233 nm (Thompson & Chen, 1990
). Protein concentrations were determined using the method of Groves et al. (1968)
.
Internet tools.
The following internet services were used: BLAST for DNA or protein database searches (Altschul et al., 1997 ), PROSITE for searching protein sequence motifs (Bairoch et al., 1997
), and Pseudomonas genome project version from 2.2.2000 (http://pseudomonas.bit.uq.edu.au/) for obtaining the DNA sequence of the acsA gene.
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RESULTS AND DISCUSSION |
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Wild-type P. aeruginosa expressed both ACS and ACK activity, but no PTA was found under any growth condition used (data not shown). In control experiments with cell-free extracts prepared from E. coli K-12 grown on glucose, PTA activity was easily demonstrated. ACS activity was induced on malonate, 2,3-butanediol, ethanol and acetate, while no activity could be detected on succinate and glucose (Table 3). In contrast, ACK activity is almost independent of the carbon source used.
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The activity of ACS was also determined in the wild-type and mutant MS1 after induction on ethanol. Whilst wild-type cells displayed measurable ACS activity, the mutant did not. The ACK activity was the same (Table 3), but no PTA activity was found.
Subcloning and characterization of the acsA gene
Cosmid pTB3018 from a cosmid gene library of P. aeruginosa restored growth on ethanol to mutants MS1 and MS8. Plasmid pTB4100, carrying a 6·9 kb fragment derived from cosmid pTB3018, also complemented both mutants. Recently, we showed by sequencing a 6·7 kb fragment carrying the exaABC gene cluster that the DNA sequences of P. aeruginosa ATCC 17933 and P. aeruginosa PAO1 differ only slightly by about 2% (Schobert & Görisch, 1999 ; Diehl et al., 1998
). Therefore, we used the published PAO1 sequence to evaluate the relevant ATCC 17933 sequence. We sequenced 250 bp from the 5'- and 3'-end of the 6·9 kb insert of pTB4100. By comparison to the genome database of P. aeruginosa PAO1, we identified four ORFs. Further subclones were isolated and tested for complementation by triparental mating. Only pTB4102, with an intact ORF2, complemented both mutants MS1 and MS8 (Fig. 2
). Clones carrying only the intact ORF2 were obtained by PCR. In pTB4105, ORF2 is oriented linear with the lac promoter of the vector pUCP20T, whilst in pTB4106, it is integrated in an antilinear orientation. Complementation with both plasmids restored growth on ethanol to mutants MS1 and MS8, indicating that the P. aeruginosa promoter of ORF2 is present in the 205 bp region in front of the start codon. We confirmed that the complementation occurred in trans and was not a result of a homologous recombination. Plasmid DNA was prepared from complemented mutant strains and used to transform E. coli JM109. The resulting transformants were used again in triparental matings to complement successfully the original mutants MS1 and MS8.
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Proteins of the ACS family (Wang et al., 1999 ) share two conserved amino acid sequence regions and both, motif I and motif II, are found in the polypeptide sequence encoded by the acsA gene. Motif I represents the AMP-binding site of proteins that catalyse the reaction of ATP and carboxylic acids to acyl adenylates and the transfer of the acyl residue. The function of the conserved motif II is unknown.
Inactivation of the acsA gene
To demonstrate that the gene product of the acsA gene is essential for growth on ethanol, the gene was inactivated by site-directed mutagenesis using pTB4108 (Fig. 2d). A Kmr mutant, UK1, was obtained as described in Methods. The mutant was unable to grow on ethanol, and PCR with genomic DNA confirmed the presence of the kanamycin-resistance cassette in the acsA gene (acsA::Kmr) (data not shown). The Kmr gene is transcribed in the same orientation as the acsA gene. The acsA::Kmr allele in mutant UK1 does not express a dominant negative variant of ACS, since complementation with pTB4105, carrying only the acsA gene, restored wild-type growth on ethanol and led to a threefold higher expression of ACS activity compared to wild-type cells (Table 2
and Table 3
). Like mutants MS1 and MS8, mutant UK1 showed poor growth on acetate (Table 2
).
Expression of the acsA gene in complemented mutants MS1, MS8 and UK1
The acsA gene in P. aeruginosa indeed encodes an enzyme with ACS activity as demonstrated by the data shown in Table 3. Wild-type extracts showed ACS activity after induction by ethanol. In contrast, extracts of mutants MS1 and UK1 showed no detectable ACS activity under standard test conditions.
Mutants transformed by triparental mating with plasmid pTB4105 or pTB4106 were grown on ethanol. Cell-free extracts of the complemented mutants showed a three- to five-times higher specific activity of ACS compared to wild-type extracts (Table 3). The acsA gene encodes a polypeptide of 72 kDa. With cell-free extracts of induced wild-type cells, a 70 kDa polypeptide band is readily detected by SDS-PAGE, and cell-free extracts from complemented mutants showed an increased intensity of this 70 kDa band (data not shown).
Since mutant UK1, like mutants MS1 and MS8, grows on acetate, albeit poorly, we tried to detect low levels of ACS activity. Cell extract with a high protein concentration was prepared from UK1 cells grown on acetate, and up to 6 mg protein (ml assay mixture)-1 was used. Under these conditions acetyl-CoA formation with a specific activity of about 10% of wild-type level was detected (Table 3). Apparently this low activity is sufficient to support poor growth of UK1 on acetate. The acetyl-CoA-forming enzyme in mutant UK1, however, showed a different substrate specificity to that observed with ACS in wild-type cells. While ACS in wild-type extracts activated acetate and propionate equally well, the acetyl-CoA-forming activity in UK1 extracts showed about twice the activity with propionate as substrate compared to acetate. With caproate, both, mutant and wild-type extracts, showed only about 15% of the activity compared to acetate (data not shown).
Concluding remarks
In the present paper, we demonstrate that the putative acsA gene of P. aeruginosa encodes an ACS activity. This enzyme, which so far has not been described in P. aeruginosa, is essential for growth on ethanol, 2,3-butanediol and malonate. In contrast, mutants with a defect in the acsA gene, however, can still grow on acetate, albeit poorly. By comparison with P. aeruginosa PAO1, where the presence of a second putative acs gene, acsB, was inferred from sequence similarities, a second acs gene may also be present in P. aeruginosa ATCC 17933. In cell extracts of mutant UK1, where the acsA gene is interrupted by the insertion of a kanamycin-resistance cassette, a residual low acetyl-CoA-forming activity was found, which shows a different substrate specificity compared to the wild-type acsA gene product. Whether this activity is caused by an acetyl-CoA-forming enzyme encoded by a putative acsB gene or whether the product of the acsA::Kmr allele shows a residual low activity with an accidentally modified substrate specificity is presently under investigation in our laboratory.
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ACKNOWLEDGEMENTS |
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Received 4 June 2001;
accepted 18 June 2001.