1 Institute for Biotechnology, Research Centre Juelich, D-52425 Juelich, Germany
2 School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Correspondence
Lothar Eggeling
l.eggeling{at}fz-juelich.de
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ABSTRACT |
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INTRODUCTION |
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We are particularly interested in lipid synthesis of Corynebacterium glutamicum. This organism is used for the large-scale production of L-glutamate and L-lysine, currently produced in amounts of 1·2x106 and 6x105 tonnes per year, respectively (Eggeling & Sahm, 1999). It is known that the lipid content of C. glutamicum influences the amino acid excretion properties of this organism (Hoischen & Krämer, 1990
). For instance, an altered phospholipid composition of C. glutamicum, as achieved by overexpression of the cardiolipin synthetase gene cls, results in enhanced L-glutamate excretion (Nampoothiri et al., 2002
). It is also known from studies with Mycobacterium species that the outer lipid layer, consisting largely of mycolic acids bound to trehalose and the arabinogalactan polymer, contributes significantly to the flux properties of the cell envelope (Jarlier & Nikaido, 1994
). Accordingly, mutants of Mycobacterium smegmatis with decreased mycolic acid content exhibit an increased permeability (Liu & Nikaido, 1999
), and an increased content of the porin MspA decreases the diffusion barrier of Mycobacterium bovis for glucose, which becomes manifest by an accelerated growth rate (Mailaender et al., 2004
). However, whereas the major interest relates to the influx of solutes (antibiotics) in the case of Mycobacterium, for C. glutamicum it rather concerns the efflux of solutes (amino acids).
In this regard, we are interested in cell wall and lipid synthesis of C. glutamicum (Eggeling & Sahm, 2001), which is closely related to Brevibacterium ammoniagenes. This latter organism possesses two FAS-I proteins (Stuible et al., 1997
). This is surprising since Corynebacterium diphtheriae, and even Mycobacterium tuberculosis, have only one FAS-I enzyme (Cole et al., 1998
). However, as the recent establishment of the genome sequence revealed, C. glutamicum also possesses two FAS-I genes (Kalinowski et al., 2003
). Since duplicated versions of genes with similar or related functional activities frequently occur in M. tuberculosis rather than in C. glutamicum, here, we address the following questions: are the two FAS-I enzymes functionally active, what is their function in vivo, is regulation of both genes apparent, and what is their relation to mycolic acid synthesis?
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METHODS |
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Plasmid and mutant construction.
To enable deletion of fasB crossover, PCR was applied. The primers used were pfasB5'out (5'-CGGAATTCCGCGCGTTCAGCCCCCGTATTA-3'), pfasB5'in (5'-CCCATCCACTAAACTTAAACGCGAGCTCAGCCCAGTGTCC-3'), pfasB3'out (5'-CGGAATTCCGGTAGCCACCGACGTAGGACTG-3') and pfasB3'in (5'-TGTTTAAGTTTAGTGGATGGGGTACGGCATGATCGACAACTTT-3'). For the second PCR, the primer pairs pfasB5'out and pfasB3'out were used again. The resulting fragment was ligated with EcoRI cleaved pK19mobsacB (Schäfer et al., 1994) to generate pK19mobsacB
fasB. The primers used to construct the inactivation plasmid pK18mobfasAint were pfasA5' (5'-GTGCTTACGTGAACATTCCAGG-3') and pfasA3' (5'-ACCGCCGGAGGACATGGAGA-3'). The PCR fragment obtained was ligated with SmaI-cleaved pK18mob. The inserts in all final constructs used in this study were confirmed by sequencing.
The chromosomal fasB gene was deleted by using plasmid pK19mobsacBfasB in a similar procedure as described for the Cg-pks deletion (Gande et al., 2004
). fasA was inactivated using pK18mobfasAint as described by Schäfer et al. (1994)
. The strain C. glutamicum
fasB was used for the inactivation of fasA by use of pK18mobfasAint. All recombinants with chromosomal genes disrupted or deleted were confirmed by absence of free plasmid, and at least two independent PCR analyses using different primer pairs.
Lipid analysis.
Fatty acid methyl esters were prepared as described by Klatte et al. (1994), and identified by gas chromatography with model 5898A microbial identification system (Microbial ID). Trimethylsilylated derivatives of mycolic acids were analysed by high-temperature gas chromatography on an HP 5790A gas chromatograph (Hewlett Packard), equipped with a flame-ionization detector on a 12 m high throughput screening (HTS) column with hydrogen gas as the carrier. Derivatives were identified by comparing their retention times to those of standards and by gas chromatography mass spectrometry analysis on a KRATOS MS50 spectrometer (ion source temperature set to 200 °C and ionization energy to 70 eV), respectively.
Preparation of 13C-labelled mycolic acids.
Cells were grown as before, but since only the 13C-U-labelled oleic acid was available, this was added filter-sterilized, since autoclaving the medium together with the acid resulted in poor growth. The 13C-labelled mycolic acid methyl esters (MAMEs) were then extracted from the defatted cells as described previously (Gande et al., 2004). Briefly, mycolic acids were released from the defatted cells by treatment with 1 ml 5 % tetrabutylammonium hydroxide at 100 °C, overnight. The samples were then allowed to cool and CH2Cl2 (4 ml), water (3 ml) and CH3I (100 µl) were added, and the entire contents mixed for 30 min. The upper aqueous layer was discarded, and the lower organic layer was washed twice with water (4 ml). The lower organic layer containing the MAMEs was dried under reduced pressure, resuspended in CH2Cl2 and analysed by electrospray mass spectrometry (ES-MS). The measurements were carried out in the positive-ion mode on a triple quadropole LCT instrument (Micromass) fitted with an atmospheric pressure electrospray source. The samples were injected directly by using a Rheodyne injector (Rheodyne Europe) with methanol as the mobile phase. The machine was run at a flow rate of 200 µl min1 with the cone voltage at 35 V and the spraying needle voltage set at 3 kV. The scan rate was 1 s and the mass range was from 200 to 2000.
Transcript quantification.
Total mRNA was prepared from either glucose, acetate or propionate-grown cultures harvested at mid-exponential phase (Wendisch et al., 2001), using the RNeasy Mini kit (Qiagen). Samples were treated with DNase I and extracted with phenol/chloroform isoamyl alcohol. After further purification by precipitation and solubilization in water, the RNA amount and quality was judged and quantified in a denaturing formamide gel and by photometric analysis, respectively. Equal amounts of 500 ng RNA were transcribed into cDNA using random hexamer primers (Invitrogen). The product was used in real-time PCR using a LightCycler instrument with SYBR Green I as the fluorescence dye following the instructions of the supplier (Roche Diagnostics).
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RESULTS |
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The parallel attempted deletion of fasA with the appropriate construct was unsuccessful. We therefore constructed plasmid pk18mobfasAint carrying an internal fragment of fasA to enable disruption of this gene. Three kanamycin-resistant clones were obtained on complex medium plates (brain heart infusion) containing 0·2 g oleic acid l1 plus 0·4 g butter hydrolysate l1 after 4 days. Further analyses by PCR confirmed integration of the vector into fasA, and one mutant was termed C. glutamicum fasA. The same plasmid pk18mobfasAint was also used to disrupt fasA in C. glutamicum fasB to yield the double mutant C. glutamicum fasAB.
fasB is non-essential
As shown in Fig. 1, growth of the fasB mutant in liquid minimal medium CGXII-glucose was almost indistinguishable from that of the wild-type, whereas growth of the fasA mutant and that of the double mutant was not apparent. This may indicate that fasB plays a subordinate role, whereas the fasA-encoded protein is apparently the major fatty acid synthase. Interestingly, FAS-IA exhibits the highest identity to the single fatty acid synthase of M. tuberculosis (Cole et al., 1998
). Supplementation with 0·03 % oleate restored growth of both the fasA and fasAB mutants. Interestingly, in the fasA background the fasB mutation causes a discernible effect in that the final growth yield was slightly reduced (Fig. 1
).
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Lipid composition of fas mutants
To analyse the lipid composition, the wild-type and mutants were grown to an OD600 of 59, and lipids were extracted, derivatized and analysed (Table 1). The fatty acids present in C. glutamicum were dominated by 16 : 0 (palmitate) and 18 : 1
9c (oleate), thus confirming earlier analyses (Hoischen & Krämer, 1990
). Also tuberculostearic acid (10Me18 : 0) was present, which is characteristic for a number of Corynebacterianeae (Collins et al., 1982
). As expected, upon addition of oleate to wild-type cultures, this fatty acid increased in abundance in the extractable lipids, as did the content of the 36 : 2 mycolic acid, which is made up of two oleate molecules (Table 2
). Interestingly, the results showed that in the absence of fasB the relative content of palmitate is strongly reduced, suggesting that palmitate is primarily synthesized by the fasB-encoded fatty acid synthase. This is in full agreement with the absence of 32 : 0 and 32 : 1 mycolic acids and the reduced content of the mycolic acids of type 34 : 1 and 34 : 0. The consequences of the fasB mutation are even more pronounced when comparing the fasA with the fasAB mutant. Thus, fasB encodes a fatty acid synthase mostly synthesizing 16 : 0, whereas the fasA-encoded enzyme synthesizes 18 : 1 together with 18 : 0, with a small amount of 16 : 0.
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Expression of fasA and fasB
The physiological data clearly point to expression of both fas genes. We performed transcript quantifications to verify this and to assay for regulation. For this purpose, cDNA was synthesized from total mRNA prepared from either glucose-, acetate- or propionate-grown cultures. This was amplified via real-time PCR with fasA-specific primer pairs. Using three independent experiments and calibration curves from known amounts of fasA DNA, these quantifications gave transcript ratios of 0·18±0·01 for acetate/glucose, 1·62±0·22 for propionate/glucose and 9·03±1·54 for propionate/acetate (mean±SD, n=3), showing, in particular, reduced fasA steady-state transcript level during growth on acetate. Additional quantifications made with a different set of fasA primer pairs yielded almost identical results (data not shown). Since the growth substrates, in particular propionate, alter growth rates this may also influence gene transcript levels. Therefore, the mRNAs of gyrA, fbp and ddh were quantified as reference genes. Subsequently, the fasA transcripts were normalized to these reference genes and the mean value of relative ratios calculated, giving 0·12±0·05 for acetate/glucose, 2·01±0·55 for propionate/glucose and 16·77±2·55 for propionate/acetate. This confirms that in acetate-grown cells, relative to glucose, the fasA transcript is reduced by about eightfold, whereas that of propionate-grown cells is increased by about twofold (Fig. 3).
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In a separate experiment fasA and fasB transcript quantifications were run in parallel to assess for their presence relative to each other. This revealed that on glucose the fasA transcript accounted for 68·0 % of both transcripts, whereas it was 46·9 % on acetate and 80·3 % on propionate, indicating a relative adjustment of the two transcript steady-state levels to each other (Fig. 3).
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DISCUSSION |
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As evident from the mutational studies, FAS-IB is clearly the less important fatty acid synthase, whose absence does not result in a rigorous phenotype, but has rather subtle consequences. Growth is virtually unaltered under the conditions used and with the substrates assayed when fasB is deleted. In addition, the fasB mutant reveals that C. glutamicum tolerates to some degree changes in fatty acid composition as known for a number of organisms, including Escherichia coli (Cronan & Rock, 1996). The fact that FAS-IB cannot serve to replace FAS-IA agrees with the observation that an oleic acid auxotrophic mutant of B. ammoniagenes could only be complemented by fasA, but not fasB (Stuible et al., 1996
). Nevertheless, the fasB mutation has some effect on C. glutamicum, which is apparent by the altered lipid composition, and the increased L-glutamate accumulation in the fasA background. Since it is assumed that the L-glutamate exporter present in the cytoplasmic membrane is influenced by membrane tension and lipid composition in its activity (Nampoothiri et al., 2002
), the interplay of the appropriate lipid environment with the carrier protein at the molecular level might be one reason for increased L-glutamate accumulation with the fasAB double mutant. Interestingly, some classically derived L-glutamate producers of C. glutamicum are oleic acid auxotrophs (Okazaki et al., 1967
). According to the present study these mutants are most likely fasA mutants. However, since the fasAB mutation is advantageous over the fasA mutation with respect to L-glutamate accumulation, the development of appropriate double mutants is an option to further improve L-glutamate formation. In this respect it is interesting to mention that an increase in temperature is one classical means to trigger mutants of C. glutamicum from non-excretion to L-glutamate excretion, and that the ratio of oleate, stearate and palmitate synthesized by the isolated fatty acid synthase of B. ammoniagenes varies considerably with temperature (Kawaguchi et al., 1981
).
Although about 85 % of the cellular fatty acids during growth on propionate are still even-chain-length fatty acids, growth on this substrate is characterized by the presence of a number of uneven fatty acids. C. glutamicum utilizes the methylcitrate pathway with propionyl-CoA as an intermediate (Claes et al., 2002), and it is therefore most likely that unspecificity of the
-ketoacyl ACP-synthase activity of FAS-I leads to utilization of propionyl-CoA, instead of acetyl-CoA, as the priming unit in fatty acid synthesis. This deviant fatty acid composition of C. glutamicum on propionate could well be the reason that utilization of this substrate is associated with poor growth. The results obtained with the 13C-labelled oleic acid demonstrate that the short mycolic acids synthesized in C. glutamicum originate from the FAS-I-synthesized fatty acids. The three FAS-II modules present in C. glutamicum are not involved. This agrees with the current model of a Claisen-type condensation of two pre-made fatty acid molecules with the recent demonstration of the involvement of a carboxylated intermediate (Gande et al., 2004
; Portevin et al., 2004
).
Both fas genes are clearly subject to substrate-dependent regulation. The strong reduction of the fasA mRNA level on acetate is also reflected by the reduced contribution of fasA to the total fas transcripts (Fig. 3). Surprisingly, the downregulation, when the bacteria are exclusively grown on acetate, is not reflected in an altered fatty acid or mycolic acid composition. One reason for this might be that during growth on acetate the intracellular acetyl-CoA concentration is increased to 145 µM in C. glutamicum instead of the 24 µM present during growth on glucose (Wendisch et al., 1997
). Together with the appropriate kinetic properties of the acetyl-CoA carboxylase AccD1AccBC (Gande et al., 2004
) this might ensure a sufficiently high supply of the product malonyl-CoA for the FAS-I enzymes. It might be expected that in C. glutamicum a regulatory system is present to adapt the lipid composition of the cell and the lipid environment of transporters in response to the growth conditions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 7 March 2005;
revised 26 April 2005;
accepted 26 April 2005.
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