1 Institute for Biotechnology, Research Centre Jülich, D-52425 Jülich, Germany
2 School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
3 Pathology, University Hospital, D-52074 Aachen, Germany
Correspondence
Lothar Eggeling
l.eggeling{at}fz-juelich.de
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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Our interest in C. glutamicum stems from the use of this bacterium for amino acid production and amino acid export. Currently, more than 1·8x106 t of amino acids are produced annually with this bacterium, and it is clear that specific mechanisms must exist for amino acids to pass through the cell wall and its lipid layers. We succeeded in identifying specific exporters for L-lysine (Vrljic et al., 1996), L-threonine (Simic et al., 2001
) and L-isoleucine (Kennerknecht et al., 2002
), each representing a new functional and structural type of transporter (Vrljic et al., 1999
; Eggeling & Sahm, 2003
). The L-lysine exporter and its subfamilies are widely distributed in a large number of bacteria. The native physiological function of the L-lysine export system of C. glutamicum is the sensing of intracellular concentrations of either L-lysine or L-arginine by the regulator LysG, with subsequent induction of lysE, the exporter gene, at an elevated intracellular concentration of about 2030 mM to prevent toxic accumulation of these basic amino acids (Bellmann et al., 2001
). L-Glutamate has been manufactured for almost 50 years using C. glutamicum, and, with an annual production of 1·2x106 t, this amino acid represents by far the greatest fraction produced with this bacterium. Nevertheless, the efflux of L-glutamate remains the least understood. The reason for this is that specific treatments are necessary for L-glutamate excretion, e.g. addition of detergents, biotin limitation or a temperature upshift (Uy et al., 2003
). Consequently, a number of diverse hypotheses exist to explain L-glutamate excretion by C. glutamicum (Hoischen & Krämer, 1990
; Eggeling et al., 2001
; Kimura, 2003
). In addition, no exporter for L-glutamate export has been identified to date.
We report here that EMB treatment results in L-glutamate efflux of C. glutamicum, and we also report on genome-wide consequences of gene expression caused by the addition of EMB, which is discussed in the context of the mode of action of EMB in M. tuberculosis.
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METHODS |
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Genomic mutations.
To replace Pemb with Ptet, the plasmid pK19mobsacB-Ptet-emb was introduced into C. glutamicum by electroporation. Recombinants with the integrated vector used 15 µg kanamycin ml1. Positive selection for the loss of vector in a second round of homologous recombination was by growth in the presence of 10 % sucrose. Finally, clones were analysed for successful exchange using PCR and two different sets of primer pairs. The resulting strain was transformed with pJC1-Pgap-tetR (in the presence of 200 ng anhydrotetracycline ml1) to provide the Tet repressor, and to yield 13032 : : Ptet-emb(pJC1-Pgap-tetR), in which emb expression is under control of TetR.
Cultivation conditions.
Pre-cultures of C. glutamicum were grown on complex medium CGIII, and growth experiments were done using salt medium CGXII containing 4 % (w/v) glucose and 30 mg protocatechuic acid l1 in 50 ml Erlenmeyer baffled flasks with shaking at 120 r.p.m. (Keilhauer et al., 1993). EMB was added after sterilization as an aqueous solution. Where appropriate, C. glutamicum was cultured with kanamycin at a concentration of 25 µg ml1. After transformation, a lower concentration of kanaymcin (15 µg ml1) was used. Growth was followed as optical density (OD600) of appropriately diluted culture samples.
To cultivate 13032 : : Ptet-emb(pJC1-Pgap-tetR), a pre-culture was grown in BHI (brain heart infusion; Difco) containing 200 ng anhydrotetracycline ml1. After 24 h, aliquots were harvested, washed with 0·9 % NaCl, resuspended in CGXII, and used for the inoculation of a CGXII culture to an OD600 of 1·0. After 24 h, this culture, without anhydrotetracycline, was used to inoculate a second CGXII culture without washing. After 24 h, this procedure was repeated. Only in the third GXII culture was the anhydrotetracycline-dependent growth clearly detected.
Strain 13032(pEKEx2emb), and the control strain containing the empty plasmid, were pre-cultivated in CGIII without induction. After washing with 0·9 % NaCl, the CGXII culture (containing 0·1 mM IPTG) was inoculated to give a starting OD600 of 0·5.
Electron microscopy.
Cells were grown on the salt medium CGXII, either with or without 100 mg EMB l1. At an OD600 of about 10, 1 ml cells were pelleted, and resuspended in 0·1 M sodium phosphate buffer, pH 7·2, containing 3 % glutardialdehyde. The cells were embedded in 3 % agarose, followed by fixation with 1 % OsO4, water removal, and embedding in epoxy resin. Ultrathin sections were placed on a copper grid, stained with uranyl acetate and lead citrate, and inspected with a Philips EM 400 transmission electron microscope.
Biochemical analysis.
To quantify arabinose and galactose in cell wall arabinogalactan, cultures with and without EMB (50 µg ml1) were grown for 4 h, then 1 µCi ml1 (37 kBq ml1) [14C]glucose (230 mCi mmol1; 9·25 GBq mmol1; AP Biotech) was added, and after 4 h cultivation, the cells were harvested. They were immediately disrupted by sonication, followed by preparation of the insoluble cell wall mycolyl-arabinogalactan-peptidoglycan complex as described by Westphal & Jann (1965). Briefly, the disrupted cell extract was harvested by centrifugation and treated with 2 % SDS in PBS (0·1 M K2HPO4, 0·01 M NaCl, pH 7·4) at 95 °C for 1 h. After centrifugation, the pellet was washed with water, acetone/water (80 : 20, v/v) and acetone, and allowed to dry. An aliquot was resuspended in 400 µl 2 M trifluoroacetic acid, and incubated for 3 h at 120 °C. After drying under nitrogen, the hydrolysed residue was resuspended in water (1 ml) and chloroform (1 ml), sedimented by centrifugation, and the upper aqueous phase was recovered, dried, and finally resuspended in 100 µl water. An aliquot (50 000 c.p.m.) from each strain was subjected to TLC using silica gel plates (5735 silica gel 60F254; Merck), developed in pyridine/ethyl acetate/glacial acetic acid/water (5 : 5 : 1 : 3, by vol.). Autoradiograms were produced by 23 days' exposure to Kodak X-Omat AR film to reveal 14C-labelled sugars, and they were compared with known standards.
For lipid analysis, cells grown on CGXII plus 5 µCi (185 kBq) [14C]acetate ml1 (62 mCi mmol1; 2·3 GBq mmol1; Amersham) were used, which were harvested, washed and freeze-dried. Free lipids were extracted as described by Gande et al. (2004), and the de-lipidated extracts were used to release and isolate the bound lipids. Aliquots from each strain were subjected to TLC using silica gel plates (5735 silica gel 60F254; Merck), either developed in CHCl3/CH3OH/H2O (60 : 16 : 2, by vol.) for the free lipids, or in petroleum ether/acetone (95 : 5, v/v) for the bound lipids. Autoradiograms were produced by 23 days' exposure to Kodak X-Omat AR film to reveal 14C-labelled lipids, 14C-labelled fatty acid methyl esters (FAMES) and 14C-labelled mycolic acid methyl esters (MAMES), which were compared with known standards (Puech et al., 2000
, 2001
; Gibson et al., 2003
).
Preparation of total RNA.
Total RNA was prepared as described previously (Wendisch et al., 2001) from control and EMB cultures (25 ml) treated at OD600 1·0, which were harvested in the exponential and early-stationary growth phases. The quality of RNA was analysed by UV spectrophotometry and denaturing formaldehyde agarose gel electrophoresis. The prepared RNA was stored at 70 °C until use.
DNA microarray analyses.
The generation of whole-genome DNA microarrays, synthesis of fluorescently labelled cDNA from total RNA, microarray hybridization, washing and data analysis were performed as described previously (Ishige et al., 2003; Polen & Wendisch, 2004
). Genes that exhibited significantly changed mRNA levels (P<0·05 in a Student's t-test) by a factor of at least three were determined in two series of DNA microarray experiments. Series one consisted of six comparisons of the wild-type cultivated in CGXII with or without 500 µg EMB ml1 harvested in the exponential growth phase after 56·5 h at fixed OD600, whereas series two consisted of 14 comparisons of the wild-type cultivated either with or without EMB, but harvested at fixed times (0, 2, 4, 6, 8, 10, 15 and 25 h).
Genes that were differentially expressed in at least one of these experiments were tabulated. If one of the genes identified belonged to a putative operon, the operon genes were also added to a table consisting of a of total 107 genes. For those 76 genes that exhibited detectable signals in more than 3/4 of the experiments, a hierarchical cluster analysis was made as described (Eisen et al., 1998; Polen & Wendisch, 2004
).
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RESULTS |
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Resistance is related to emb expression
Mycobacterium avium and M. tuberculosis have three emb genes, and at least one of these (embB) is suggested to be the target of EMB (Escuyer et al., 2001). However, Corynebacterium species have only one emb gene. This is in accordance with the notion that the genome of Corynebacterium species is considered to represent the archetype of Corynebacterineae, and has a low frequency of structural alterations and gene duplications (Nakamura et al., 2003
). The emb gene of C. glutamicum was cloned into pEKEx2, and the emb-overexpressing recombinant was assayed for EMB sensitivity. As shown in Fig. 2
, this rendered the strain no longer susceptible to EMB at the concentration of 15 mg l1, and no glutamate efflux occurred (data not shown). The MIC (Etest) of EMB for the wild-type was 0·75 µg ml1, and for the overexpressing strain it was 1·5 µg ml1. Direct proof of overexpression was obtained by real-time PCR using emb cDNA as standards, resulting in a 5·2±0·8-fold increased emb transcript in the overexpressing strain. This illustrates that overexpression of the single emb gene of C. glutamicum, which is more related to the mycobacterial embC than to embA or embB, was sufficient to confer EMB resistance.
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Comparative analysis of the effect of EMB on RNA levels
The hierarchical cluster analysis of the global gene expression patterns in response to EMB addition is shown in Fig. 6. The experiments are shown in columns, and the ORFs in rows. As might be expected, the clustering of the experiments is according to the time points where samples were taken, including those experiments where cells were grown to a constant OD600 (which took 56·5 h). The closest clustering was at around 410 h, indicating that at this point in time global gene expression levels remained comparatively unaltered.
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Clusters 3 and 4 contained downregulated genes. The strongest response (0·3-fold decrease) was present for NCgl0511 (a permease in an operon with NCgl050913), and NCgl1131 and NCgl1646, which both encode putative secreted hydrolases. The known genes of the clusters depicted in Fig. 6 all encode membrane proteins. Furthermore, ORFs NCgl191517, proposed to encode components of an oligopeptide uptake system, were also reduced in their expression. Interestingly, in a comparative analysis of global expression changes in E. coli, a high number of membrane proteins exhibited altered expression as a consequence of ampicillin addition (Shaw et al., 2003
). Thus, envelope stress might preferentially affect the expression of membrane proteins, which could also be the case for EMB. The cytochrome supercomplex genes, and that of the F0F1-type ATP synthase, are presumably downregulated due to poor growth of the cultures, as described for other organisms (Morita, 1997
).
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DISCUSSION |
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Of course we inspected the upregulated genes in particular for carriers which could be candidates for the claimed L-glutamate exporter (Hoischen & Krämer, 1990). Cluster 1 includes the gene for the mechanosensitive ion channel YggB, which has been shown to enable betaine efflux, but this is not known to be involved in glutamate export (Nottebrock et al., 2003
). In principle, each permease could be the carrier in demand, requiring inactivation of carrier genes for efflux assays.
With respect to the consequences of EMB addition on the mRNA levels of cytosolic enzymes, it is striking that no genes for enzymes of central metabolism are affected. Only a weak decrease of the odhA and lpd mRNA levels is apparent (Fig. 7); the products of these genes are likely to be constituents of the 2-oxoglutarate dehydrogenase complex competing with glutamate dehydrogenase for the common substrate (Kimura 2003
; Uy et al., 2003
). This, and the fact that sugar conversion to L-glutamate occurs although growth is retarded (Fig. 1a
), means that transcription of genes of central metabolism is rather robust despite a strongly disordered cell envelope. This agrees with similar observations made for M. tuberculosis in response to the addition of antibiotics (Waddell et al., 2004
).
There are a number of genes known whose altered expression facilitates L-glutamate efflux. This holds for overexpression of cma, acp, plsC and fadD15 (Nampoothiri et al., 2002), genes of phospholipid biosynthesis, reduced expression of dtsR1 encoding the
-subunit of acetyl-CoA carboxylase (Kimura et al., 1997
; Gande et al., 2004
), and reduced expression of alr, the alanine racemase gene (Eggeling et al., 2001
), as well as ltsA encoding an amidotransferase (Hirasawa et al., 2000
). Differential expression of these genes might alter lipid or peptidoglycan composition of the cell envelope. The emb gene represents the first description that an alteration of the arabinogalactan within the cell envelope results in L-glutamate efflux. Nevertheless, it appears that there is no single molecular event directly causing efflux. Instead, the common feature is merely a strongly disordered cell envelope (Eggeling & Sahm, 2001
). Since, in addition, a carrier must be present for L-glutamate export (Hoischen & Krämer 1990
), we speculate that as a consequence of the disordered cell envelope a carrier becomes activated due to an increased membrane tension or an altered lipid environment (Tillman & Cascio, 2003
). Alternatively, the carrier gene could be expressed as a consequence of the obviously disordered cell envelope. In the latter case, comparative genome-wide expression profilings using not only EMB addition, but also the other conditions eliciting L-glutamate efflux, might help to further track the export carrier, and to unravel L-glutamate efflux and production by C. glutamicum.
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ACKNOWLEDGEMENTS |
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Received 2 December 2004;
revised 31 January 2005;
accepted 3 February 2005.
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