Two functional FAS-I type fatty acid synthases in Corynebacterium glutamicum

Eva Radmacher1, Luke J. Alderwick2, Gurdyal S. Besra2, Alistair K. Brown2, Kevin J. C. Gibson2, Hermann Sahm1 and Lothar Eggeling1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The lipid-rich Corynebacterianeae, to which Corynebacterium glutamicum and Mycobacterium species belong, produce both fatty acids and mycolic acids. Compared with most other bacteria, C. glutamicum possesses two fatty acid synthases, encoded by fasA (8907 kb; FAS-IA) and fasB (8988 kb; FAS-IB). Here, it was shown by mutational analyses that fasA is essential but fasB is not. However, in a fasA background, the fasB mutation results in a slightly reduced growth yield, L-glutamate production is increased, and comparative lipid analysis suggests that in vivo FAS-IB is active primarily to supply palmitate. Transcript quantifications revealed that the fasB transcript contributes 32 % to both fas transcripts during growth on glucose, affirmative for fasB expression, and that fasB is subordinate to fasA. The fasA transcript is downregulated by 8·3-fold during growth on acetate as compared with glucose. The lipid analyses also demonstrate that cells grown on propionate produce a number of uneven fatty acids (e.g. 15 : 0, 17 : 0, 17 : 1), which are not present in cells grown on glucose or acetate, suggesting that fatty acid synthase in vivo may also use propionyl-CoA as the priming unit in fatty acid synthesis. The fatty acid auxotrophic fasAB double mutant was used to determine the suggested incorporation of fatty acids into mycolic acids. Supplementation of this mutant with uniformly labelled [13C]oleate and analysis of isolated mycolic acids confirmed that mature mycolic acids in the mutant consist exclusively of two fused [13C]oleate molecules. In addition to an altered phospholipid profile, the fasB mutant also exhibits differences in its mycolic acid profile. Taken together, the results show that although FAS-IA is the most relevant fatty acid synthase of C. glutamicum and FAS-IB is supplementary, both synthases are necessary to produce the characteristic lipid environment of this organism.


Abbreviations: ES-MS, electrospray mass spectrometry; MAMEs, mycolic acid methyl esters


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fatty acid synthases (FASs) are large multienzyme complexes of more than 2 MDa, which catalyse successive cycles of a multistep reaction to generate mature fatty acids (Cronan & Rock, 1996). At least seven discrete functional domains are involved in this reaction sequence, which are either fused to form one polypeptide as is the case with FAS-I or occur as individual polypeptides, referred to as FAS-II. FAS-I is characteristically found in eukaryotes, whereas bacteria usually possess FAS-II. A remarkable exception to this rule are members of the Corynebacterianeae, which possess FAS-I. Constituent members of this suborder are Mycobacterium and Corynebacterium species (Stackebrandt et al., 1997), and Mycobacterium possesses FAS-II in addition to FAS-I. The FAS-I synthase supplies the fatty acids for phospholipids and as precursors for mycolic acid synthesis. These latter lipids are {alpha}-branched, {beta}-hydroxylated fatty acids, which form an outer lipid layer in the Corynebacterianeae.

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?


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Growth conditions.
The wild-type C. glutamicum (ATCC 13032) and mutants derived from this strain were used throughout this study. Cells were grown on salt medium CGXII (Eggeling & Bott, 2005), which was inoculated to an initial OD600 of 1 from a culture grown overnight on complex medium CGIII. When cultures were grown with acetate or propionate as the substrate, an additional CGXII culture for adaptation was used prior to inoculation into CGXII to follow growth. To grow fas mutants, cultures contained additionally 0·03 % (w/v) sodium oleate plus 1 % (w/v) Brij35, autoclaved together. The mutants were inoculated to an initial OD600 of 2.

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{Delta}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 pK19mobsacB{Delta}fasB 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 {Delta}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 min–1 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).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Generation of fasA, fasB mutants
The two fas genes of C. glutamicum are very similar in size: 8907 bp for fasA and 8988 bp for fasB. Their polypeptides share identities of 44 % over their entire length, and even at the nucleotide level stretches of identities exist, with the longest one from nt 7559 to 8297 (fasA coordinates), where the DNA sequences are 74 % identical. To delete fasB, the non-replicative plasmid pK19mobsacB{Delta}fasB was made, carrying sequences of the 5'- and 3'-end of fasB, enabling deletion of 6075 nt of chromosomal fasB. Using this plasmid C. glutamicum was transformed to kanamycin resistance, indicating vector integration into the chromosome. For selection of the second recombination and looping out of the vector (Schäfer et al., 1994), one kanamycin-resistant recombinant was chosen and plated on sucrose-containing Luria–Bertani and growing clones were scored for further analysis. From approximately 100 clones analysed, fasB was deleted in six of them, whereas in the others the wild-type situation was restored. Colonies of the fasB deletion mutant were more yellowish than the wild-type, but growth on plates was not altered (see below). One of these C. glutamicum fasB clones was chosen for further analyses.

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 l–1 plus 0·4 g butter hydrolysate l–1 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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Fig. 1. Dependence of C. glutamicum fas mutants on oleic acid supply. Growth on CGXII-glucose of C. glutamicum wild-type ({lozenge}, {blacklozenge}), fasA ({circ}, {bullet}), fasB ({triangleup}, {blacktriangleup}) and the fasAB double mutant ({square}, {blacksquare}) without (open symbols) and with 0·03 % oleate (filled symbols).

 
Mycolic acids stem from pre-made fatty acids
As evident, the double mutant is fully dependent on the exogenous addition of fatty acids. Therefore, it offers the possibility to determine the suggested incorporation of fatty acid chains to yield the mycolic acid core. To this end the mutant was grown with uniformly labelled [13C]oleic acid, harvested after 16 h and the MAMEs prepared were analysed by ES-MS (Fig. 2). The dominant species has a mass of 621·4, which is consistent with a uniformly labelled 36 : 2 mycolic acid. Also a mycolic acid species with a mass of 585·3 is present, where no label is incorporated. This is probably due to mycolic acids originating from the inoculum, since a high initial density derived from an unlabelled pre-culture was used to enable growth of the mutant. In addition, a minor ‘mixed’ species (m/z 603·4) is present where either the mero-chain or the {alpha}-branch is labelled. Therefore, the newly synthesized mycolic acid originated entirely from oleate, which was fully incorporated into the mature species. This fully corroborates the view that two fatty acids are condensed via a Claisen-like reaction, where recent evidence has been obtained of an intermediate carboxylation reaction to activate the {alpha}-carbon of the incoming fatty acid of the nascent mycolic acid (Portevin et al., 2004; Takayama et al., 2005). In addition, the results offer an explanation for the poor growth of the mutant, since its fatty acid together with its mycolic acid composition differ from that of the wild-type (see below), thereby creating conditions strongly deviating from the natural lipid composition.



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Fig. 2. Identification of MAMEs from C. glutamicum by ES-MS. MAMEs were isolated from the [13C]oleate labelled C. glutamicum fasAB double mutant. Only oleate derived C36 : 2 MAMEs are apparent, where m/z 603·6 represents MAMEs with both branches labelled, m/z 585·3 unlabelled MAMEs and m/z 603·4 MAMEs where either the mero-chain or the {alpha}-branch is labelled.

 
fasA mutants enable glutamate excretion
In earlier reports, it was shown that oleic acid auxotrophic mutants of C. glutamicum excrete L-glutamate (Okazaki et al., 1967). We therefore grew the mutants on CGXII-glucose with 0·03 % oleate together with 1 % Brij35. The wild-type and the fasB mutant accumulated 0·3 mM and 0·1 mM L-glutamate, respectively, after 48 h, whereas the fasA mutant produced a high concentration of 46·2 mM L-glutamate. This was even further enhanced to 58·8 mM with the fasAB double mutant, showing that the fasB mutation has an additional positive influence on the final L-glutamate concentration in the fasA background.

Lipid composition of fas mutants
To analyse the lipid composition, the wild-type and mutants were grown to an OD600 of 5–9, 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{omega}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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Table 1. Fatty acid profiles of the wild-type and mutants of C. glutamicum grown under different conditions

Abbreviations: wt, wild-type; fasA, fasB, fasAB, the corresponding mutant analysed; Ol, oleic acid supplied. The substrates were: Gl, glucose; Ac, acetate; Pr, propionate. Fatty acids below the detection limit of 0·25 % are shown as –. The cumulative percentages are not 100 % due to rounding. Determinations were done from two independent cultures, with observed variances of less than 5 %. The data from one culture are given.

 

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Table 2. Mycolic acid profiles of the wild-type and mutants of C. glutamicum grown under different conditions

The abbreviations are as for Table 1. Mycolic acids below the detection limit of 1 % are shown as –. The cumulative percentages are not 100 % due to rounding. Determinations were done from two independent cultures, with observed variances of less than 5 %. The data from one culture are given.

 
Growth and lipid composition on acetate and propionate
We also assayed the lipid profiles of C. glutamicum grown on 2 % acetate and 1 % propionate. The growth rates (in h–1) were 0·38 for acetate, 0·40 for glucose and 0·15 for propionate (data not shown). The growth rates of the fasB mutant were indistinguishable from the wild-type, whereas with the fasA mutant (supplemented with oleate) no substantial growth on either acetate or propionate was obtained. Lipids of the wild-type grown on the three different substrates were analysed as before. When cells were grown on acetate more or less the same fatty acid and mycolic acid profiles were present as on glucose, which was not the case with those grown on propionate (Table 1). In particular, fatty acids with odd numbers were apparent (15 : 0, 17 : 1 and 17 : 0), suggesting utilization of the propionate skeleton during fatty acid synthesis. Evidently, these fatty acids are also accepted as substrates by the single pks-encoded polyketide synthase of C. glutamicum (Gande et al., 2004), since the corresponding uneven mycolic acids were identifiable in propionate-grown cells (Table 2).

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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Fig. 3. Quantifications of fas mRNA steady-state levels. The fasA transcript is given in black, the fasB transcript hatched. (a) Transcript levels in cells grown on acetate (Ac) or propionate (Pr) relative to glucose are given. (b) Contribution of the two transcripts to the fas transcripts, the sum set to 100 %, are given, as quantified in cells grown on glucose (Gl), acetate (Ac) or propionate (Pr).

 
The same quantification using two different primer pairs and controls for fasB produced the ratios 0·45±0·02 for acetate/glucose, 0·76±0·02 for propionate/glucose and 1·79±0·11 for propionate/acetate, and as relative ratios 0·28±0·07 for acetate/glucose, 0·95±0·09 for propionate/glucose and 3·44±0·44 for propionate/acetate. Therefore, the fasB transcript level is less stringently controlled than fasA. Relative to glucose, it is decreased about twofold on acetate, but almost unaltered on propionate.

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).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Unlike C. diphtheriae, C. glutamicum and Corynebacterium efficiens are unusual in having two FAS-I genes. C. glutamicum also possesses three FAS-II modules (NCgl0281, NCgl0283 and NCgl0527). In Mycobacterium, FAS-II modules are present in addition to their single FAS-I, to elongate the mero-chain of the mycolic acids (Takayama et al., 2005). However, the mycolic acids of Corynebacterium species are not elongated, and C. diphtheriae and C. efficiens do not possess FAS-II modules, which indicates a subordinate role for these modules in C. glutamicum, if functional at all. In contrast, both FAS-I enzymes are active in C. glutamicum. The fasB deletion, in both the wild-type and the fasA mutant, leads to a strongly reduced content of palmitate. The activity and specificity of FAS-IB agrees completely with the in vitro activity of the FAS-IB enzyme of B. ammoniagenes, which also has two FAS-I enzymes and is closely related to the L-glutamate-excreting coryneform bacteria (Stuible et al., 1996). The isolated FAS-IB synthesizes primarily palmitate, and to a lesser extent stearate, but not oleate (Stuible et al., 1997). In contrast, the isolated FAS-IA of B. ammoniagenes produces stearate and oleate, illustrating the specificity of the enzymes and that chain length and desaturation are inherent properties of the FAS-I enzymes.

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 {beta}-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 AccD1–AccBC (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.


   ACKNOWLEDGEMENTS
 
This work was funded in part by a Degussa grant (Düsseldorf, Germany) to E. R. G. B. acknowledges support as a Lister Institute-Jenner Research Fellow and the Medical Research Council (UK).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 7 March 2005; revised 26 April 2005; accepted 26 April 2005.



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