Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany1
Author for correspondence: Alexander Steinbüchel. Tel: +49 251 8339821. Fax: +49 251 8338388. e-mail: steinbu{at}uni-muenster.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: propionyl-CoA, Ralstonia eutropha, 2-methylcitric acid cycle, propionic acid catabolism, methylcitric acid synthase
Abbreviations: DIG, digoxigenin
The GenBank accession numbers for the nucleotide sequences of the prp gene cluster are AF325554 and AF331923.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
During the investigation of Tn5-induced mutants of R. eutropha HF39 with defects in the catabolism of levulinic acid (4-oxopentanoic acid) (Valentin et al., 1992 ; Gorenflo et al., 1998
), we identified a gene cluster, the gene products of which constitute the methylcitric acid cycle in this bacterium. In this study, we present molecular and biochemical data of the methylcitric acid pathway in R. eutropha HF39 and compare the organization of its methylcitric acid cycle genes with those of other bacteria.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Transfer of DNA.
Competent cells of E. coli were prepared and transformed by the CaCl2 procedure as described by Hanahan (1983) . The transduction of genomic DNA of R. eutropha ligated into the cosmid pHC79 to E. coli S17-1 was done after in vitro packaging in
phages as described by Hohn & Murray (1977)
. The packaging extracts were prepared by the method of Scalenghe et al. (1981)
. Electrocompetent cells of S. enterica were prepared and regenerated by the method of Taghavi et al. (1994)
and transformed under the following conditions: 2400 V, 25 µF and 200
.
Tn5 mutagenesis.
Tn5-induced mutants of R. eutropha HF39 impaired in growth on levulinic acid were created by the suicide plasmid technique employing pSUP5011 (Simon et al., 1983a ), which was transferred from E. coli S17-1 to the recipient by conjugation (Friedrich et al., 1981
).
Genotypic characterization of the Tn5-insertion mutants of R. eutropha HF39.
Genomic DNA of the Tn5-insertion mutants was digested with EcoRI, and the genomic EcoRI fragments were ligated to the cosmid pHC79. After in vitro packaging in phages the recombinant cosmids were transduced into E. coli S17-1. Recombinant E. coli clones were selected by their kanamycin resistance conferred by Tn5::mob. The hybrid cosmids were isolated, digested with SalI and ligated into the plasmid pBR325. The recombinant plasmids were transformed into E. coli XL-1 Blue, and clones resistant to kanamycin plus chloramphenicol were selected. The hybrid plasmid of the resulting clones harboured a SalI fragment which included part of Tn5 plus genomic DNA adjacent to the Tn5 insertion. These recombinant plasmids were sequenced using the oligonucleotide 5'-GTTAGGAGGTCACATGG-3', which binds specifically to the IS50L element of Tn5::mob.
DNA sequencing.
The primer-hopping strategy (Strauss et al., 1986 ) was applied to determine the DNA sequence of both strands of DNA. Sequencing was done by using the Sequi Therm EXCEL TM II long-read cycle sequencing kit (Epicentre Technologies) and IRD 800-labelled oligonucleotides (MWG-Biotech). Sequencing was performed in a LI-COR 4000L automatic sequencing apparatus (MWG-Biotech).
The nucleotide sequences of the prp gene cluster were deposited in the GenBank database under the accession numbers AF325554 and AF331923.
Sequence data analysis.
Sequence data were compared with sequences deposited in the GenBank and Prosite databases using the programs BlastSearch 2.0.10 (Altschul et al., 1997 ) and DBGET (Bairoch et al., 1997
).
PCR amplifications.
All PCR amplifications of DNA encoded on plasmids or genomic DNA were carried out as described by Sambrook et al. (1989) . VENTR-DNA polymerase from New England Biolabs was used in all PCR amplifications, which were carried out in an Omnigene HBTR3CM DNA Thermal Cycler (Hybaid).
DNADNA hybridization.
Southern hybridizations were done by the method of Oelmüller et al. (1990) at 68 °C. Colony hybridizations were done by the method of Grunstein & Hogness (1975)
at 68 °C.
Cloning of prpB, prpC and acnM and heterologous expression in E. coli.
Oligonucleotides were constructed to amplify prpB (prpBUP, 5'-AAATCTAGAGGCTTGGCACACCCCTT GCAGTATTG-3'; prpBRP, 5'-TTTTTGTCGACGGCCGCGCGTGAGTCTTGCTTAC-3'), prpC (prpCUP, 5'-AAAGAATTCCCGGCCGCTTGCAG-3'; prpCRP, 5'-TTTTTGTCGACCAACTACCCCTTGTCC-3') and acnM (acnMUP, 5'-AAGAGTCT CGCTGACATGGG ACCACTACC-3'; acnMRP, 5'-AAATCTAGACGGGCCCTTTGTCACAGCTTATGC-3') from genomic DNA of R. eutropha HF39 (restriction sites are underlined). The 984 bp DNA fragment resulting from prpBUP and prpBRP was cloned into pBluescript SK-, resulting in pSK-/prpB. prpCUP contained an EcoRI recognition sequence and prpCRP a SalI recognition sequence to enable forced cloning of prpC downstream of and collinear to the lacZ promoter with EcoRI plus SalI restricted pBluescript SK- DNA, resulting in pSK-/MCSII. acnMUP contained a SacI recognition sequence and acnMRP a XbaI recognition sequence to enable forced cloning of acnM downstream of and collinear to the lacZ promoter of SacI plus XbaI restricted pBluescript SK- DNA; the resulting hybrid plasmid was referred to as pSK-/acnM.
Cells of E. coli harbouring pSK-/prpB, pSK-/MCSII, pSK-/acnM or pBluescript SK- were grown at 30 °C for 16 h in 50 ml LB medium containing 75 µg ml-1 ampicillin plus 0·5 mM IPTG. The cells were harvested (10 min, 4500 r.p.m. at 4 °C), washed with 100 mM Tris/HCl (pH 8·0), resuspended in 5 ml 100 mM Tris/HCl and disrupted by twofold passage through a French press.
Inactivation of the prpB gene of R. eutropha HF39 by insertion of the omega element Km.
For inactivation of prpB by insertion of Km, the 820 bp EcoRI fragment (EE0·82) was used, which contained the incomplete prpB gene (Fig. 1
, A
). This fragment was ligated to EcoRI-digested vector pSKSym, and E. coli XL-1 Blue was transformed with the ligation mixture. Transformants harbouring the hybrid plasmid pSymprpB were obtained. Plasmid pSymprpB was then digested with EcoRV and ligated with
Km, which was recovered by SmaI digestion of plasmid pSKsym
Km (Overhage et al., 1999
). The hybrid plasmid thus obtained was designated pSymprpB
Km. The disrupted prpB gene was isolated from pSymprpB
Km by EcoRI digestion and ligated with EcoRI-digested pSUP202 DNA. E. coli S17-1 was transformed with the ligation mixture, and transformants harbouring the hybrid plasmid pSUPprpB
Km, which conferred resistance to tetracycline and kanamycin, were obtained. Subsequently, pSUPprpB
Km was transferred to R. eutropha HF39 by conjugation, and the transconjugants were selected on NB agar plates containing 160 µg kanamycin ml-1. The transconjugants were tested for tetracycline resistance (encoded by a vector-borne gene) to distinguish between the integration of the whole hybrid plasmid into the chromosome by a single crossover (heterogenotes) and the exchange of the functional prpB gene with the disrupted gene by a double crossover (homogenotes), which resulted in a tetracycline-sensitive phenotype. To confirm the disruption of the prpB gene in the tetracycline-sensitive mutant HF39
prpB
Km, a PCR was performed using the primer prpBUP and prpBRP, which resulted in a single PCR product with the expected size of 1·9 kbp. The sequence of the PCR product was determined, revealing the sequence of prpB
Km.
|
Preparation of crude extracts.
Cells from 20 ml cultures were washed in 10 mM Tris/HCl (pH 7·4) and resuspended in 1 ml of this buffer containing 10 µg DNase I. The cells were disrupted by sonification in a Sonopuls GM 200 (Bandelin, Berlin, Germany) with an amplitude of 16 µm (1 min ml-1). During ultrasonification the samples were cooled in a NaCl/ice bath. Soluble protein fractions of the crude extracts were obtained by 5 min centrifugation at 13000 r.p.m. and 4 °C.
Cultivation of R. eutropha HF39, SK7286 and HF39prpB
Km to determine the enzyme activity of 2-methylcitrate synthase and 2-methylisocitrate lyase.
The specific enzyme activities of 2-methylcitrate synthase and 2-methylisocitrate lyase were measured in R. eutropha HF39, the prpC mutant SK7286 and HF39prpB
Km to confirm the inactivation of prpC or prpB in these mutants. Cells of these strains were grown in 50 ml MM containing 1·0% (w/v) sodium gluconate for 30 h at 30 °C. The cells were harvested, washed twice with sterile saline and transferred to fresh MM containing 0·2% (w/v) sodium levulinate, sodium propionate or sodium gluconate. After incubation for 24 h at 30 °C the cells were harvested, and the enzyme activities were measured in the crude extracts.
Determination of the enzyme activity of 2-methylcitrate synthase, 2-methylisocitrate lyase and 2-methyl-cis-aconitic acid hydratase.
Activity of 2-methylcitrate synthase (EC 4.1.3.31) was measured by the method of Srere (1966) . The cuvette (d=1 cm) contained, in a total volume of 1 ml, 2 mM oxaloacetate, 250 µM propionyl-CoA and 2 mM 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) in 10 mM Tris/HCl (pH 7·4). After addition of the crude extract, the increase of the absorbance at 412 nm (
=13·6 cm2 µmol-1) was measured with an Ultrospec 2000 photometer (Pharmacia).
Activity of 2-methylisocitrate lyase was assayed in a total volume of 0·5 ml containing 66·7 mM potassium phosphate buffer (pH 6·9), 3·3 mM phenylhydrazine, 2·5 mM cysteine, 5 mM MgCl2 and cell extract. The reaction was started by addition of 1·25 mM tripotassium methylisocitrate, which was obtained by alkaline cleavage of the methylisocitric acid lactone (Brock et al., 2001 ; W. Buckel, personal communication). The increase of the absorbance at 324 nm was measured. The molar absorption coefficient of pyruvate phenylhydrazone was 12 mM-1 cm-1 (Luttik et al., 2000
).
Activity of the putative 2-methyl-cis-aconitic acid hydratase was measured in a quartz cuvette (d=1 cm) in 1 ml 100 mM Tris/HCl buffer (pH 8·0) containing 0·1 mM cis-aconitic acid or 1·5 mM tricalcium salt of 2-methylcitric acid plus crude extract. After addition of the substrate, the decrease (cis-aconitic acid; =4·1 cm2 µmol-1) or the increase (tricalcium salt of 2-methylcitric acid;
=4·5 cm2 µmol-1) of the absorbance at 240 nm were measured.
One unit of enzyme activity was defined as the conversion of 1 µmol substrate min-1. The amount of soluble protein was determined by the method of Bradford (1976) , using crystalline bovine serum albumin as standard.
Preparation of the tricalcium salt of 2-methylcitric acid.
Cells of the Tn5-mutant VG17 of R. eutropha HF39 were grown for 24 h at 30 °C in 1 l MM containing 1·0% (w/v) disodium succinate plus 160 µg kanamycin ml-1. The cells were harvested (20 min, 4000 r.p.m.), washed with sterile saline and transferred to 500 ml fresh MM containing 0·2% (w/v) sodium propionate. After incubation for 24 h at 30 °C, 0·6% (w/v) disodium succinate and 0·4% (w/v) sodium propionate were added. After further incubation for 48 h at 30 °C, the cells were sedimented by centrifugation, and the supernatant was lyophilized. The supernatant was resuspended in 0·1 vol. double-distilled water, and 2-methylcitric acid was extracted by the method of Brock et al. (2000) . To purify this extract, 2 vols double-distilled water was added, and the solution was neutralized with CaOH2. Addition of 1 vol. acetone or 2-propanol resulted in the precipitation of a slightly yellowish powder, which could be purified to a white powder by repeating this precipitation step. GC/MS analysis (see below) of the white powder identified this substance as 2-methylcitric acid.
Analysis of the supernatant of resting cells.
Cells of the Tn5-insertion mutants VG17, P2 and SK7287, the null-allele mutants HF39prpB
Km and HF39prpD
Km, and R. eutropha HF39 were grown in 50 ml MM containing 0·5% (w/v) disodium succinate as carbon source for 24 h at 30 °C. The cells were harvested, washed twice with sterile saline and transferred to fresh MM containing 0·1% (w/v) sodium propionate. After 24 h incubation at 30 °C 0·1% (w/v) sodium propionate and 0·25% (w/v) disodium succinate were added. Aliquots of 1 ml were withdrawn at different times and centrifugated for 5 min at 13000 r.p.m. The clear supernatants were lyophilized, subsequently esterified and used for GC/MS analysis.
GC/MS analysis.
The white powder and cell-free lyophilized supernatants were subjected to methanolysis in the presence of sulfuric acid (5 h at 100 °C), and the resulting methyl esters of the organic acids were characterized by coupled gas chromatography/mass spectrometry (GC/MS) using an HP 6890 gas chromatograph equipped with a model 5973 mass selective detector (Hewlett Packard).
Electrophoretic methods.
Proteins were separated under denaturing conditions in polyacrylamide gels, which were stained with Coomassie brilliant blue R as described by Osborn & Weber (1969) .
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular characterization of Tn5-induced mutants defective in the catabolism of levulinic acid
The insertion of Tn5 into the genomes of these mutants was confirmed by Southern hybridization using EcoRI-digested DNA isolated from the mutants and the central digoxigenin (DIG)-labelled 5·1 kbp HindIII fragment of Tn5 as probe.
To map the insertions of Tn5 in seven of these mutants, SalI restriction fragments conferring kanamycin resistance were cloned. Sequencing of these SalI fragments, employing an oligonucleotide hybridizing to the terminal IS50L region of Tn5, revealed strong homologies to structural genes involved in the catabolism of propionate. In the mutants SK7286 and SK7290 Tn5 mapped at an identical position, and the amino acid sequence of the putative translational product revealed 78·0% similarity to the prpC gene products of S. enterica serovar Typhimurium and E. coli (Horswill & Escalante-Semerena, 1997 ; Textor et al., 1997
), which encodes a 2-methylcitrate synthase. The sequence of the Tn5 insertion locus in the mutants VG17 and P2 revealed strong similarities to aconitate hydratase genes (Prodromou et al., 1992
) and genes encoding iron-responsive element binding proteins (Yu et al., 1992
). The Tn5-insertion locus in the mutant SK7287 mapped 38 bp downstream of the SalI recognition sequence and showed no similarities to other genes. However, the insertion had probably occurred in proximity to those in VG17 and P2 as indicated by the identical sizes of the Tn5-containing EcoRI and SalI fragments. In the mutants VG3, 3-29, 3-27 and SK4507, Tn5 mapped in a gene whose putative translational product showed strong similarities to the prpR gene product of S. enterica and E. coli (Horswill & Escalante-Semerena, 1997
; Textor et al., 1997
).
Cloning and sequencing of native genomic fragments
A DIG-labelled Tn5::mob-harbouring EcoRI restriction fragment cloned from mutant VG17 was used to identify the native 3·7 kbp EcoRI fragment referred to as VG17EE3·7 in a genomic library in E. coli S17-1 prepared from EcoRI-digested DNA in the cosmid pHC79 (Fig. 1, A). It was subsequently ligated to linearized pBluescriptSK- and transferred to E. coli XL-1 Blue. Another gene library was prepared from HindIII-digested genomic DNA of strain HF39 and cosmid pHC79 in E. coli S17-1. Colony hybridization with DIG-labelled EE3·7 DNA identified a clone which harboured a 8·5 kbp HindIII fragment (HH8·5) containing a 3·2 kbp HindIIIEcoRI fragment as part of the native 3·7 kbp EcoRI fragment (Fig. 1, A)
. With DIG-labelled EE10·1 Tn5::mob-containing DNA of the mutant SK4507, an 8·4 kbp HindIII fragment was identified. When it was digested with EcoRI, five fragments, of 2·8, 2·6, 0·82, 0·372 and 0·218 kbp, were obtained. With the DIG-labelled prpC gene as a probe a 2·9 kbp HindIII restriction fragment (HH2·9) was identified in the hybrid plasmid of the positive E. coli clone (Fig. 1, A)
. The restriction fragments shown in Fig. 1(A)
were cloned into the vectors pBluescript SK- or pBCSK+ and fully sequenced. Six ORFs were identified, five of which showed similarities to structural genes (Fig. 1, B)
.
Function of the putative gene product of ORF1
The deduced amino acid sequence of this 1962 bp ORF, which started with an ATG at position 1962 (see Fig. I, included as supplementary data with the on-line version of this paper at http://mic.sgmjournals.org), showed 4043% identity to the prpR gene product of E. coli and S. enterica serovar Typhimurium and to various other transcriptional activators belonging to the 54-(rpoN) family (Palacios & Escalante-Semerena, 2000
). The prpR gene product is probably a transcriptional activator of the prpBCAOD operon in R. eutropha HF39 and belongs to the
54 family of transcriptional activators. A feature of
54-dependent transcription, in addition to a -24/-12 promoter sequence, is its dependency on an activator protein. Most of these activator proteins bind to conservative sequences with a twofold rotational symmetry, which are designated upstream activator sequences (UAS) and are located 80120 bp upstream of the
54-dependent promoter (Kustu et al., 1989
). At 83 bp upstream of the putative
54-dependent promoter of the prpBCAOD operon in R. eutropha HF39 a putative UAS was identified (TGT-N12-ACA), which strongly resembled the UAS of nifA (TGT-N10-ACA) of bacteria (Alvarez-Morales et al., 1986
) and the putative UAS of acoD of R. eutropha (TGT-N11-ACA) (Priefert & Steinbüchel, 1992
). A second putative UAS was located 102 bp upstream of the putative
54-dependent promoter (TTGAATTNCAAA-N8-TTTGNAATTCAA). This imperfect palindromic sequence overlapped with the putative
70-dependent promoter of the prpR gene. The binding of an activator protein to this putative UAS could repress the
70-dependent transcription of prpR. The propionate-negative phenotype of the rpoN-negative mutant HF149 of R. eutropha (not shown in detail) provided further evidence for
54-dependent transcription of prp genes.
Function of the putative gene product of ORF2
The amino acid sequence deduced from the 909 bp nucleotide sequence of ORF2 exhibited identities of 76·7% and 75·9% and similarities of 87·7% and 86·2% to the prpB gene products of E. coli (Textor et al., 1997 ) and S. enterica (Horswill & Escalante-Semerena, 1997
), respectively. Furthermore, the prpB gene product also showed significant identities to carboxyphosphonoenolpyruvate phosphonomutases (3240%) and isocitrate lyases (2531%) of several organisms. The function of the prpB gene product as a 2-methylisocitric acid lyase was suggested for E. coli (Textor et al., 1997
).
From the amino acid alignment (see Fig. II, included as supplementary data with the on-line version of this paper at http://mic.sgmjournals.org) and from the tentative ribosome-binding site, which preceded the ATG at position 2243, it was concluded that this codon is probably the putative translational start codon of prpB of R. eutropha. PrpB consisted of 302 amino acids with a calculated molecular mass of 32314 Da and a pI of 5·15. Thirty-six nucleotides upstream of the 5'-terminal region of prpB a consensus 54 promoter sequence was identified as in S. enterica serovar Typhimurium LT2 (Palacios & Escalante-Semerena, 2000
).
prpB was heterologously expressed in E. coli XL-1 Blue (pSK-/prpB), and a protein of approximately 32±1 kDa was synthesized from the recombinant E. coli strain, which corresponded well with the molecular mass calculated for the prpB gene product (see Fig. IIIA, included as supplementary data with the on-line version of this paper at http://mic.sgmjournals.org).
Inactivation of the prpB gene of R. eutropha HF39 by insertion of the omega element Km. Since no Tn5 insertion mapped in prpB, the phenotype of a mutant with defective prpB was unknown. For inactivation of prpB, pSUPprpB
Km was transferred to R. eutropha HF39 by conjugation and the null-allele mutants were selected as described in Methods. The homogenote HF39
prpB
Km could not grow on MM agar plates containing sodium propionate as a sole carbon source.
Determination of 2-methylisocitrate lyase activity. The activity of 2-methylisocitrate lyase was measured in crude extracts of propionate-induced [0·140 U (mg protein)-1] and uninduced cells [0·0024 U (mg protein)-1] of R. eutropha HF39 and propionate-induced cells of HF39prpB
Km [no activity measurable]. The results revealed that the formation of 2-methylisocitrate lyase is induced by propionic acid in R. eutropha HF39. Furthermore, 2-methylisocitrate lyase activity of 0·191 U (mg protein)-1 was determined in the recombinant E. coli (pSK-/prpB) in comparison to only 0·0036 U (mg protein)-1 in an E. coli strain harbouring only pBluescript SK-.
Physiological investigation of the prpB null-allele mutant. To get a hint of the physiological function of the prpB translational product in the methylcitric acid cycle of R. eutropha, three-stage growth experiments were carried out as described in Methods with the null-allele mutant HF39prp
Km and strain R. eutropha HF39 as control. In the mutant HF39
prpB
Km six components accumulated in the supernatant (Fig. 2b
). The mass spectrum of the compound with a retention time of 35·35 min in the gas chromatogram revealed an identity of 92·8% to the trimethyl ester of 2-methylcitric acid (NIST database). Comparison of the peak at 36·00 min with the mass spectrum of the trimethylester of isocitric acid (NIST database) revealed that nearly all key fragments showed a 1415 higher m/z (Fig. 2c
), which corresponds to the mass of the additional methyl group in 2-methylisocitric acid (not contained in the database). This suggests that this component of the supernatant is 2-methylisocitric acid. The component with a retention time of 39·91 min showed an identical mass spectrum as the component with a retention time of 36·00 min, except that two fragments with m/z 146 and m/z 189 were missing. Esterification of the trisodium salt of isocitric acid and GC/MS analysis revealed a retention time for the methyl ester of this substance of 40·85 min and the key fragments had an m/z which was 1415 lower than the m/z of the peaks in the 39·91 min peak mass spectrum. The lactone of 2-methylisocitric acid and isocitric acid might be formed as a side product of esterification, as described for esterification of these substances in the supernatant of Yarrowia lipolytica R-2 (Tabuchi & Serizawa, 1975
), whereas the dimethyl ester of the lactone of 2-methylisocitric acid gave a single sharp peak with a later retention time than the trimethyl ester of 2-methylcitric acid (Tabuchi & Serizawa, 1975
). These properties of the dimethyl ester of the methylisocitric acid lactone correspond well with the properties of the dimethyl ester of the putative 2-methylisocitric acid lactone in the supernatant of the mutant HF39
prpB
Km. Furthermore, 2-methylisocitric acid and 2-methylisocitrate lactone, which was a gift from W. Buckel (Phillips-Universität Marburg, Germany), were used as GC/MS standards and they revealed identical retention times and mass spectra as the substances with retention times of 36·00 and 39·91 min in the supernatant of HF39
prpB
Km. In the supernatant of Y. lipolytica R-2, which produced 2-methylisocitric acid from odd-carbon n-alkanes, citric acid, 2-methylcitric acid and 2-methyl-cis-aconitic acid were additionally identified (Tabuchi & Serizawa, 1975
). The disruption of prpB caused the accumulation of 2-methylisocitric acid and probably as a consequence of the equilibrium 2-methylcitric acid was accumulated. This result provides clear evidence that the prpB gene product is involved in the cleavage of 2-methylisocitric acid into succinate and pyruvate in the methylcitric acid cycle of R. eutropha HF39 (Fig. 3
) as shown for the translational product of ICL2 in Sacch. cerevisiae (Luttik et al., 2000
).
|
|
Determination of 2-methylcitrate synthase activity and expression of prpC in E. coli. In crude extracts, R. eutropha HF39 exhibited significantly higher specific activity of 2-methylcitrate synthase [0·354 U (mg protein)-1] than SK7286 [0·01 U (mg protein)-1]. In addition, the specific 2-methylcitrate synthase activity of HF39 was approximately twofold higher in cells cultivated on propionate [0·354 U (mg protein)-1] than in those cultivated on levulinate [0·145 U (mg protein)-1].
The Tn5-containing SalI fragment of mutant SK7286 was subcloned in pBluescript SK- and sequenced. Analysis of the nucleotide sequence made it possible to design oligonucleotides for the amplification of prpC from genomic DNA of R. eutropha by PCR. prpC was cloned into pBluescript SK-, resulting in pSK-/MCSII. The recombinant E. coli strain harbouring pSK-/MCSII synthesized a protein of approximately 42±1 kDa, which corresponded well with the calculated molecular mass of 42720 Da for the prpC gene product. Crude extracts of E. coli (pSK-/MCSII) exhibited a 2-methylcitrate synthase specific activity of 2·86 U (mg protein)-1, if propionyl-CoA was used as substrate, whereas with acetyl-CoA the specific activity was only approximately 10% of this value. Crude extracts of an E. coli strain harbouring only pBluescript SK- exhibited no 2-methylcitrate synthase activity using propionyl-CoA as substrate and 0·009 U (mg protein)-1 with acetyl-CoA as substrate.
Phenotypic complementation of the Tn10-induced null-allele prpC mutant JE3907 of S. enterica. When pSK-/MCSII was transferred to the Tn10-induced null-allele prpC mutant JE3907 of S. enterica by electroporation, phenotypic complementation of this mutant occurred, as revealed by growth of the recombinant strain in no-carbon E medium containing 0·5% (w/v) sodium propionate. However, the growth of the complemented mutant exhibited a lag phase of about 180 h, whereas the lag phase was only 50 h in a recombinant mutant strain JE4176, harbouring prpC of S. enterica in pBAD30. In contrast, JE3907 harbouring only the vector did not grow at all, even after a prolonged incubation period.
Function of the putative gene product of ORF4
Downstream and at a distance of 118 bp from ORF3, ORF4 started with an ATG at position 4525, and it was preceded by a putative ribosome-binding site (see Fig. I, included as supplementary data with the online version of this paper at http://mic.sgmjournals.org). The putative translational product of ORF4 has a calculated size of 94726 Da and a pI of 5·36. The comparison of the deduced amino acid sequence of ORF4 with the primary structures of other proteins exhibited the highest similarity, of 85% and 80·3%, to the aconitate hydratases of Pseudomonas putida KT2440 and Neisseria meningitidis serogroup B (see Fig. V, included as supplementary data with the online version of this paper at http://mic.sgmjournals.org), whose genes are located downstream of the prpC gene in a cluster (Fig. 4). In contrast, the identity of the gene product of ORF4 to Aco1 of E. coli (Prodromou et al., 1992
) and to AcoN of L. pneumophila (Mengaud & Horwitz, 1993
), which catalyse the isomerization step in the citric acid cycle, was only 45%. A dendrogram of the amino acid sequences of several aconitate hydratases revealed that the putative acnM translational products form a cluster, which separates them from aconitases acting in the citric acid cycle (Fig. 5
). The amino acid sequences of aconitate hydratases include three highly conserved cysteine residues, which represent the ligands for the 4Fe4S cluster. These three cysteine residues also occurred in the acnM gene product of R. eutropha (C477, C480 and C491). These similarities of ORF4 to aconitate hydratases gave a hint that the encoded enzyme catalyses a hydration/dehydration reaction (2-methylcitric acid
2-methyl-cis-aconitic acid
2-methylisocitric acid) as part of the methylcitric acid cycle in R. eutropha HF39; it was therefore referred to as acnM.
|
|
The crude extract of E. coli(pSK-/acnM) was used immediately after preparation, because FeS clusters of aconitases are known to be sensitive to oxygen (Kennedy et al., 1983 ). The enzyme activity in the crude extracts was determined as described in Methods using the tricalcium salt of 2-methylcitric acid or cis-aconitic acid (Sigma) as substrate. With tricalcium 2-methylcitrate no enzyme activity was measurable in crude extracts of either strain. A reason for this could be that the tricalcium 2-methylcitrate preparation contained an inhibitor for the acnM gene product. As 2-methyl-cis-aconitic acid was not available, the analogous substrate cis-aconitic acid was used at a concentration of 0·1 mM. The specific enzyme activity in crude extracts of E. coli carrying pSK-/acnM was 21·5-fold higher [2·84 U (mg protein)-1] than that in crude extracts of E. coli harbouring pBluescript SK- [0·132 U (mg protein)-1]. This result indicates that the acnM gene product might catalyse the hydration of 2-methyl-cis-aconitic acid to 2-methylisocitric acid, and it was therefore designated 2-methyl-cis-aconitic acid hydratase.
Function of the putative gene product of ORF5 and identification of the Tn5-insertion locus in the mutant SK7287
The Tn5-insertion locus of the mutant SK7287 has been identified in the 3'-terminal region of the 1191 bp ORF5 (Fig. 1, B; see also Fig. I, included as supplementary data with the online version of this paper at http://mic.sgmjournals.org), which started with an ATG at position 7195. The deduced amino acid sequence (396 aa) exhibited the highest similarity, of 71·5%, to a conserved hypothetical 40·9 kDa protein in the prp locus of N. meningitidis serogroup B (see Fig. VI, included as supplementary data with the online version of this paper at http://mic.sgmjournals.org). Furthermore, it showed 33% identity to the hypothetical 39·5 kDa yraM gene product of Bacillus subtilis (Parro et al., 1997
) and a similarity of 36% to a hypothetical 37·1 kDa protein in the modCbioA intergenic region in the genome of E. coli (Blattner et al., 1997
). The amino acid alignment of the hypothetical 39·5 kDa protein of B. subtilis revealed weak similarities (24%) to the pduG gene product of S. enterica (Bobik et al., 1997
), which catalyses the reactivation of a diol dehydratase.
Based on computer analysis with the program PSORT (Klein et al., 1985 ) the product of ORF5 may be associated with the membrane, but there are no data available to support this suggestion.
Physiological investigations of null-allele mutants of acnM and ORF5. To analyse the physiological functions of the acnM and ORF5 translational products in the methylcitric acid cycle of R. eutropha HF39, three-stage growth experiments were carried out as described in Methods, with the Tn5 mutants VG17, P2 and SK7287 and R. eutropha HF39 as control. All the Tn5 mutants converted succinate into fumarate, malate and 2-methylcitrate. After a period of 48 h, fumarate and malate had completely disappeared in the supernatants of the mutants VG17, P2 and SK7287 and were completely converted into 2-methylcitrate (Fig. 2a). These results showed clearly that both the acnM and the ORF5 mutant are impaired in the conversion of 2-methylcitric acid into 2-methylisocitric acid. Polar effects in the mutants VG17, P2 and SK7287 can be excluded, because a defect in the gene located downstream (prpD) caused no accumulation of 2-methylcitric acid. As ORF5 is always localized together with acnM (Fig. 4
), it is conceivable that both gene products are required for the conversion of 2-methylcitric acid into 2-methylisocitric acid.
Function of the putative gene product of ORF6
ORF6 (1455 bp), which started with the ATG at position 8412 (see Fig. I, included as supplementary data with the online version of this paper at http://mic.sgmjournals.org), encoded a protein with a theoretical molecular mass of 53001 Da and a pI of 6·64. The deduced amino acid sequence showed 78·9% and 80·2% similarity to the prpD gene products of E. coli (Textor et al., 1997 ) and S. enterica (Horswill & Escalante-Semerena, 1997
), respectively (see Fig. VII, included as supplementary data with the online version of this paper at http://mic.sgmjournals.org), and 60·4% similarity to the ypo2 gene product of Sacch. cerevisiae, a hypothetical 57·7 kDa membrane protein in the cit3hal1 intergenic region (Q12428).
Inactivation of the prpD gene of R. eutropha HF39 by insertion of the omega element Km. Since no Tn5 insertion mapped in ORF6, the phenotype of a mutant with defective ORF6 was unknown. For inactivation of prpD, pSUPprpD
Km was transferred to R. eutropha HF39 by conjugation and the null-allele mutants were selected as described in Methods. The homogenote HF39prpB
Km was not impaired in growth on MM agar plates containing sodium propionate, sodium valerate or sodium levulinate and kanamycin.
The function of prpD in S. enterica as 2-methylcitric acid dehydratase was proposed by Horswill & Escalante-Semerena (1999b ); however, analysis of the prpD null alleles showed that a mutation in prpD did not block the ability of R. eutropha to use propionate as sole carbon source. This ORF is obviously not required for a functional methylcitric acid cycle, or the function of the prpD gene product can be taken over by another enzyme or a non-enzymic hydration of the methylaconitate occurs.
Physiological investigation of the prpD null-allele mutant. To get a hint of the physiological functions of the prpD translational products, three-stage growth experiments were carried out as described in Methods with HF39prpDKm and R. eutropha HF39 as control. In the supernatant of HF39prpD
Km no 2-methylcitric acid or any other organic acid was detectable after a period of 48 h.
Organization of the prp locus in Gram-negative bacteria
Analysis of data from genome sequencing projects of various organisms showed that genes homologous to the prp genes of R. eutropha HF39 are widespread in Gram-negative bacteria. A comparison of these putative prp genes revealed two distinct classes of the organization of the prp clusters (Fig. 4). In the enterobacteria E. coli and S. enterica (Blattner et al., 1997
; Horswill & Escalante-Semerena, 1997
) these loci consist of the structural genes prpR, prpB, prpC, prpD and prpE, in which prpBCDE are organized in an operon (Fig. 4
). In the prp loci of R. eutropha HF39, Pseudomonas putida KT2440, P. aeruginosa, N. meningitidis serogroup B and Burkholderia sacchari IPT101T (data not shown) prpE is missing and in Ralstonia metallidurans CH34, N. meningitidis serogroup B, Vibrio cholerae and B. sacchari IPT101T (data not shown) prpD is missing (Fig. 4
). However, the prp loci of these bacteria additionally include acnM and ORF5. The prpE gene product is not required for the functionality of the methylcitric acid cycle during growth of R. eutropha on levulinic acid, as the cycle is inducible during the degradation of levulinic acid. The levulinic acid degradation pathway is not known in detail yet. Furthermore, the function of PrpE can be taken over by the product of the acoE gene of R. eutropha (Priefert & Steinbüchel, 1992
). In P. putida KT2440 a second putative aconitate hydratase gene is divergently transcribed from the prp locus. A putative regulator gene, which showed identities to regulators of the gntR family, is localized collinear to the other genes of the prp cluster in the pseudomonads. The prp locus of the enterobacterium V. cholerae is particularly interesting because it represents a mixture of both classes of prp loci: a regulator gene of the Pseudomonas type, acnM, ORF5 and prpE occur in the cluster, but prpD is missing.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alvarez-Morales, A., Betancourt-Alvarez, M., Kaluza, K. & Henneke, H. (1986). Activation of the Bradyrhizobium japonicum nifH and nifDK operons is dependent on promotor-upstream DNA sequences. Nucleic Acids Res 14, 4207-4227.[Abstract]
Aoki, H., Uchiyama, H., Umetsu, H. & Tabuchi, T. (1995). Isolation of 2-methylisocitrate dehydratase, a new enzyme serving in the methylcitric acid cycle for propionate metabolism, from Yallowia lipolytica. Biosci Biotechnol Biochem 59, 1825-1828.
Bairoch, A., Bucher, P. & Hoffmann, K. (1997). The PROSITE database, its status in 1997. Nucleic Acids Res 25, 217-221.
Birnboim, H. C. & Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-1523.[Abstract]
Blattner, F. R., Plunkett, G. I. I. I., Bloch, C. A. & 14 other authors (1997). The complete genome sequence of Escherichia coli K-12. Science 277, 14531474.
Bobik, T. A., Xu, Y., Jeter, R. M., Otto, K. E. & Roth, J. R. (1997). Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase. J Bacteriol 179, 6633-6639.[Abstract]
Bolivar, F. (1978). Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique EcoRI sites for selection of EcoRI generated recombinant DNA molecules. Gene 4, 121-136.[Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.[Medline]
Brock, M., Fischer, R., Linder, D. & Buckel, W. (2000). Methylcitrate synthase from Aspergillus nidulans: implications for propionate as an antifungal agent. Mol Microbiol 35, 961-973.[Medline]
Brock, M., Darley, D., Textor, S. & Buckel, W. (2001). 2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans: characterization and comparison of both enzymes. Eur J Biochem (in press).
Bullock, W. O., Fernandez, J. M. & Stuart, J. M. (1987). XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with ß-galactosidase selection. BioTechniques 5, 376-379.
Friedrich, B., Hogrefe, C. & Schlegel, H. G. (1981). Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus. J Bacteriol 147, 198-205.[Medline]
Gorenflo, V., Schmack, G. & Steinbüchel, A. (1998). Biotechnological production and characterization of polyesters containing 4-hydroxyvaleric acid and medium-chain-length hydroxyalkanoic acids. Macromolecules 31, 644-649.
Grunstein, M. & Hogness, D. S. (1975). Colony hybridization: a method for the isolation of cloned DNA that contains a specific gene. Proc Natl Acad Sci USA 72, 3961-3965.[Abstract]
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.[Medline]
Hogrefe, C., Römermann, D. & Friedrich, B. (1984). Alcaligenes eutrophus hydrogenase genes (hox). J Bacteriol 158, 43-48.[Medline]
Hohn, B. & Collins, J. (1980). A small cosmid for efficient cloning of large DNA fragments. Gene 11, 291-298.[Medline]
Hohn, B. & Murray, K. (1977). Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proc Natl Acad Sci USA 74, 3259-3263.[Abstract]
Horswill, A. R. & Escalante-Semerena, J. C. (1997). Propionate catabolism in Salmonella typhimurium LT2: two divergently transcripted units comprise the prp locus at 8·5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon. J Bacteriol 179, 928-940.[Abstract]
Horswill, A. R. & Escalante-Semerena, J. C. (1999a). The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase. Microbiology 145, 1381-1388.[Abstract]
Horswill, A. R. & Escalante-Semerena, J. C. (1999b). Salmonella typhimurium LT2 catabolizes propionate via the 2-methylcitric acid cycle. J Bacteriol 181, 5615-5623.
Kennedy, M. C., Emptage, M. H., Dreyer, J.-L. & Beimert, H. (1983). The role of iron in the activation-inactivation of aconitase. J Biol Chem 258, 11098-11105.
Klein, P., Kanehisa, M. & DeLisi, C. (1985). The detection and classification of membrane-spanning proteins. Biochim Biophys Acta 815, 468-476.[Medline]
Kustu, S., Santero, E., Keener, J., Opham, D. & Weiss, D. (1989). Expression of 54(ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev 53, 367-376.
Luttik, M. A. H., Kötter, P., Salomons, F. A., van der Klei, I. J., van Dijken, J. P. & Pronk, J. T. (2000). The Saccharomyces cerevisiae ICL2 gene encodes a mitochondrial 2-methylisocitrate lyase involved in propionyl-CoA metabolism. J Bacteriol 182, 7007-7013.
Marmur, J. (1961). A procedure for the isolation of desoxyribonucleic acids from microorganisms. J Mol Biol 3, 208-218.
Mengaud, J. M. & Horwitz, M. A. (1993). The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive element-binding protein. J Bacteriol 175, 5666-5676.[Abstract]
Miyakoshi, S., Uchiyama, H., Someya, T., Satoh, T. & Tabuchi, T. (1987). Distribution of the methylcitric acid cycle and ß-oxidation for propionate in fungi. Agric Biol Chem 51, 2381-2387.
Oelmüller, U., Krüger, N., Steinbüchel, A. & Friedrich, C. G. (1990). Isolation of prokaryotic RNA and detection of specific mRNA with biotinylated probes. J Microbiol Methods 11, 73-84.
Osborn, M. & Weber, K. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244, 4406-4412.
Overhage, J., Priefert, H., Rabenhorst, J. & Steinbüchel, A. (1999). Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene. Appl Microbiol Biotechnol 52, 820-828.[Medline]
Palacios, S. & Escalante-Semerena, J. C. (2000). prpR, ntrA and ihf functions are required for expression of the prpBCDE operon, encoding enzymes that catabolize propionate in Salmonella enterica serovar Typhimurium LT2. J Bacteriol 182, 905-910.
Parro, V., San Román, H., Galindo, I., Purnelle, B., Bolotin, A., Sorokin, A. & Mellado, R. P. (1997). A 23911 bp region of the Bacillus subtilis genome comprising genes located upstream and downstream of the lev operon. Microbiology 143, 1321-1326.[Abstract]
Priefert, H. & Steinbüchel, A. (1992). Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus. J Bacteriol 174, 6590-6599.[Abstract]
Prodromou, C., Artymuik, P. J. & Guest, J. R. (1992). The aconitase of Escherichia coli. Nucleotide sequence of the aconitase gene and amino acid sequence similarity with mitochondrial aconitases, the iron-responsive-element-binding protein and isopropylmalate isomerases. Eur J Biochem 204, 599-609.[Abstract]
Pronk, J. T., van der Linden-Beuman, A., Verduyn, C., Scheffers, W. A. & van Dijken, J. P. (1994). Propionate metabolism in Saccharomyces cerevisiae: implications for the metabolon hypothesis. Microbiology 140, 717-722.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Scalenghe, F., Turco, E., Edström, J. E., Pirotta, V. & Melli, M. (1981). Microdissection and cloning of DNA from specific region of Drosophila melanogaster polytene chromosomes. Chromosoma 82, 205-216.[Medline]
Schlegel, H. G., Kaltwasser, H. & Gottschalk, G. (1961). Ein Submersverfahren zur Kultur wasserstoffoxidierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38, 209-222.
Simon, R. (1984). High frequency mobilization of Gram-negative bacterial replicons by the in vitro Tn5-mob transposon. Mol Gen Genet 196, 413-420.[Medline]
Simon, R., Priefer, U. & Pühler, A. (1983a). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1, 784-791.
Simon, R., Priefer, U. & Pühler, A. (1983b). Vector plasmids for in vivo and in vitro manipulations of gram-negative bacteria. In Molecular Genetics of the BacteriaPlant Interaction, pp. 98106. Edited by A. Pühler. Berlin/Heidelberg/New York: Springer.
Srere, P. A. (1966). Citrate-condensing enzymeoxaloacetate binary complex. J Biol Chem 241, 2157-2165.
Srivastava, S., Urban, M. & Friedrich, B. (1982). Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5. Arch Microbiol 131, 203-207.[Medline]
Strauss, E. C., Kobori, J. A., Siu, G. & Hood, L. E. (1986). Specific-primer-directed DNA sequencing. Anal Biochem 154, 353-360.[Medline]
Tabuchi, T. & Serizawa, N. (1975). The production of 2-methylcitric acid from odd-carbon n-alkanes by a mutant of Candida lipolytica. Agric Biol Chem 39, 1049-1054.
Taghavi, S., van der Lelie, D. & Mergeay, M. (1994). Electoporation of Alcaligenes eutrophus with (mega) plasmids and genomic DNA fragments. Appl Environ Microbiol 60, 3585-3591.[Abstract]
Textor, S., Wendisch, V. F., De Graaf, A. A., Müller, U., Linder, M. I., Linder, D. & Buckel, W. (1997). Propionate oxidation in Escherichia coli: evidence for operation of a methylcitrate cycle in bacteria. Arch Microbiol 168, 428-436.[Medline]
Vagelos, P. R. (1959). Propionic acid metabolism. IV. Synthesis of malonyl coenzyme A. J Biol Chem 234, 490-497.
Valentin, H. E., Schönebaum, A. & Steinbüchel, A. (1992). Identification of 4-hydroxyvaleric acid as a constituent of biosynthetic polyhydroxyalkanoic acids from bacteria. Appl Microbiol Biotechnol 36, 507-514.
Yu, Y., Radisky, E. S. & Leibold, E. A. (1992). The iron-responsive element binding protein: purification, cloning and regulation in rat liver. J Biol Chem 267, 19005-19010.
Received 22 January 2001;
revised 29 March 2001;
accepted 23 April 2001.