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Genetic dissection of trehalose biosynthesis in Corynebacterium glutamicum: inactivation of trehalose production leads to impaired growth and an altered cell wall lipid composition

¹ Institut für Mikrobiologie und Genetik, Georg-August-Universität, Grisebachstr. 8, D-37077 Göttingen, Germany
² BASF AG, Ludwigshafen, Germany

Correspondence
Wolfgang Liebl
wliebl{at}gwdg.de

	ABSTRACT

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The analysis of the available Corynebacterium genome sequence data led to the proposal of the presence of all three known pathways for trehalose biosynthesis in bacteria, i.e. trehalose synthesis from UDP-glucose and glucose 6-phosphate (OtsA-OtsB pathway), from malto-oligosaccharides or -1,4-glucans (TreY-TreZ pathway), or from maltose (TreS pathway). Inactivation of only one of the three pathways by chromosomal deletion did not have a severe impact on C. glutamicum growth, while the simultaneous inactivation of the OtsA-OtsB and TreY-TreZ pathway or of all three pathways resulted in the inability of the corresponding mutants to synthesize trehalose and to grow efficiently on various sugar substrates in minimal media. This growth defect was largely reversed by the addition of trehalose to the culture broth. In addition, a possible pathway for glycogen synthesis from ADP-glucose involving glycogen synthase (GlgA) was discovered. C. glutamicum was found to accumulate significant amounts of glycogen when grown under conditions of sugar excess. Insertional inactivation of the chromosomal glgA gene led to the failure of C. glutamicum cells to accumulate glycogen and to the abolition of trehalose production in a otsAB background, demonstrating that trehalose production via the TreY-TreZ pathway is dependent on a functional glycogen biosynthetic route. The trehalose-non-producing mutant with inactivated OtsA-OtsB and TreY-TreZ pathways displayed an altered cell wall lipid composition when grown in minimal broth in the absence of trehalose. Under these conditions, the mutant lacked both major trehalose-containing glycolipids, i.e. trehalose monocorynomycolate and trehalose dicorynomycolate, in its cell wall lipid fraction. The results suggest that a dramatically altered cell wall lipid bilayer of trehalose-less C. glutamicum mutants may be responsible for the observed growth deficiency of such strains in minimal medium. The results of the genetic and physiological dissection of trehalose biosynthesis in C. glutamicum reported here may be of general relevance for the whole phylogenetic group of mycolic-acid-containing coryneform bacteria.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Corynebacterium glutamicum is a Gram-positive soil bacterium that was originally isolated by its ability to produce and excrete glutamic acid (Kinoshita et al., 1957). Today, industrial amino acid production processes using genetically improved strains of this micro-organism are used to satisfy the growing world market for amino acids, in particular L-glutamate and L-lysine (Leuchtenberger, 1996). In the classification system of bacteria, the genus Corynebacterium, together with mycobacteria, nocardia, rhodococci and some other phylogenetically related taxa, belongs the group of mycolic-acid-containing actinomycetes (see Liebl, 2001). Unusually for Gram-positive bacteria, their cell walls contain a characteristic hydrophobic layer (Minnikin & O'Donnell, 1984; Nikaido et al., 1993). It was shown that this layer plays an important role in drug and substrate permeability (Jarlier & Nikaido, 1990; Puech et al., 2000). In contrast to the Gram-negative bacteria, where the outer membrane is composed of phospholipids and lipopolysaccharides, the predominant constituents of the outer lipid layer of corynebacteria and related taxa are the mycolic acid esters. Recently it was shown that the outer hydrophobic barrier of corynebacterial cells represents a lipid bilayer composed of both covalently cell wall-linked mycolates and non-covalently bound glycolipids (Puech et al., 2001). Two trehalose-containing corynomycolic acid esters, i.e. trehalose monocorynomycolate (TMCM) and trehalose dicorynomycolate (TDCM), were shown to be the major free lipid fractions of this lipid bilayer (Puech et al., 2000). The presence of trehalose in C. glutamicum is not restricted only to these two structural components. Significant amounts of free trehalose are observed in C. glutamicum cells as a response to hyperosmotic stress (Skjerdal et al., 1996). Also, it is notable that trehalose was found as one of the by-products excreted into the growth medium during lysine overproduction by C. glutamicum (Vallino & Stephanopoulos, 1993; Wittmann & Heinzle, 2001).

Trehalose (-D-glucopyranosyl -D-glucopyranoside) serves different biological roles in different organisms (for a review, see Argüelles, 2000). In bacteria it can be used as a carbon source (Escherichia coli, Bacillus subtilis), or is synthesized as a compatible solute under osmotic-shock conditions (E. coli), or plays a structural role (Corynebacteriaceae). In yeast and filamentous fungi trehalose is stored intracellularly primarily as a reserve carbohydrate or as a protector against different stress factors.

Several possible pathways for trehalose biosynthesis are known. The most abundant pathway, i.e. trehalose synthesis from UDP-glucose and glucose 6-phosphate (OtsA-OtsB pathway; Fig. 1a), is widely represented in the prokaryotes and the only one known in the eukaryotes. The first step of this pathway is the condensation of glucose 6-phosphate with UDP-glucose, resulting in the formation of trehalose 6-phosphate and release of UDP. Trehalose is then formed by dephosphorylation of trehalose 6-phosphate. This biosynthetic reaction mechanism has been found in bacteria like E. coli (Kaasen et al., 1994) and in yeast (De Virgilio et al., 1993; Londesborough & Vuorio, 1993). In E. coli, the reactions are catalysed by the enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB). The transcription of both enzymes is induced by osmotic shock or upon entry into the stationary growth phase (Kaasen et al., 1994). In Saccharomyces cerevisiae, both reactions are catalysed by an enzyme complex which consists of two catalytic polypeptides, TPS1 and TPS2, and one regulatory subunit responsible for activation of the complex under stress conditions (Reinders et al., 1997). Coding regions for corresponding enzymes were also identified in the genomes of higher eukaryotes (Arabidopsis Genome Initiative, 2000; Adams et al., 2000). An alternative pathway for trehalose synthesis that uses glycogen as the initial substrate (TreY-TreZ pathway; Fig. 1b) was discovered in some bacteria (Maruta et al., 1996a, b) and archaea (Maruta et al., 1996c). In this case, first the terminal (14) glycosidic bond at the reducing end of the -glucan polymer is transformed into an (11) glycosidic bond via transglycosylation, resulting in the formation of a terminal trehalosyl unit. Subsequently, trehalose is released from the polymer's end via hydrolysis. The enzymes involved in this pathway are maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose hydrolase (TreZ). An additional pathway for trehalose synthesis, which is based on trehalose production from maltose, was discovered in some bacteria (Tsusaki et al., 1996, 1997). In this case, trehalose is synthesized by a single reaction catalysed by trehalose synthase (TreS), which converts the (14) glycosidic bond of maltose into an (11) bond to form trehalose (TreS pathway; Fig. 1c). It was shown (Nishimoto et al., 1996; Nakada et al., 1995) that, although close in their intramolecular transglycosylation activity, TreY and TreS cannot substitute for each other in vivo because of the differences in their substrate specificities.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Trehalose biosynthesis pathways found in bacteria. The three known pathways leading to trehalose from the substrates glucose 6-phosphate and UDP-glucose (a), malto-oligosaccharides or -1,4-glucan polysaccharides (b) and maltose (c) are shown. The C. glutamicum ORFs showing high similarity to trehalose synthesis genes are shown in parentheses.

	METHODS

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Strains, media and cultivation.
The C. glutamicum strains and plasmids which were used in this study are listed in Table 1. Additionally, the E. coli strains XL-1 Blue (Bullock et al., 1987) and S17-1 (Simon et al., 1983) were used for plasmid construction and mobilization of integration vectors into C. glutamicum, respectively. The restriction-deficient C. glutamicum strain R163 (Liebl et al., 1989a) was used for preparation of plasmid constructs before their electroporation into the C. glutamicum type strain. The strains were maintained on LB plates supplemented with antibiotics as required.

Recombinant DNA techniques.
Basic methods such as plasmid isolation, DNA restriction and ligation were performed according to Sambrook et al. (1989). C. glutamicum plasmid DNA was isolated by alkaline extraction (Birnboim & Doly, 1979) after previous treatment of the cells with 10 µg lysozyme ml^-1 for 30 min at 37 °C. Genomic DNA from C. glutamicum was isolated as described by Lewington et al. (1987). PCR reactions were carried out using Pfu polymerase (Promega). Some of the PCR products were cloned directly into the vector pCR4 using the TOPO Cloning Kit (Invitrogen).

The following primers were used in this study (regions that are not homologous to the original gene sequences are in italics; regions that are present only in the original sequence but not in the primer are in parentheses; restriction sites used for cloning purposes are underlined): tre351_f, GGG GAT CCA AAA GAC CAC CGC AAA GAA GAC; tre351_r, CCT CTA GAG CAG TAA AGC AAG CGG AAG AA; otsAB_f, GGG CAT GC(A) GTA TGC GGA AAG CGT GCG ATT G; otsAB_r, GGA AGC TTG CCC CAA ATA ACC GCA AAG CCA; treZ_f, GGT CTA GAG CGT TGG TGT AGG CAT TAA C; treZ_r, GGT CTA GAC GCA AAA GCC TGG TCA GTT G; treS_f, GGT CTA GAT GAG GCG AAA GTG GTG AAA GT; treS_r, GGT CTA GAC ATT CGC GGG ACA ACA CAA T; glg_f, GGG TCT AGA GTA TCC ACC AGA GGT TTA CG; glg_r, GGG TCT AGA TTA AAT CTT CCG CGT CAT CGA AAG; otsB_f, GGG GAT CCA AGG TGC CAG GGC TTT AAA G; otsB_r, GGG GAT CCG GAA CCA GAA GTG GAA TTG G; treZ_f2, GGG GAT CCC GGG TGA CTT GCA AAA CCT C; treZ_r2, GGG GAT CCG CAA AAG CCT GGT CAG TTG; treS_f3, GGG TCG ACA TGA GGC GAA AGT GGT GAA AG; treS_r3, GGG TCG ACA CAT TCG CGG GAC AAC ACA A.

Construction of otsAB, treZ, treS and glgA : : Km mutants of C. glutamicum DSM 20300.
The two-step recombination system (Schäfer et al., 1994), based on the inability of C. glutamicum carrying the sacB gene to grow in media with high sucrose concentrations, was used for the chromosomal inactivation of the trehalose and glycogen biosynthesis genes of C. glutamicum. For each planned inactivation experiment, a mobilizable C. glutamicum integration vector was constructed which contained the gene of interest but with an internal deletion, thus providing two homology regions for recombination.

For inactivation of the otsA-otsB genes, a fragment of 1·5 kb carrying the entire otsA ORF was amplified using the primers tre351_f and tre351_r and cloned into the EcoRV restriction site of pBluescript KS, resulting in pBlueKS : : otsA. Then, a 0·65 kb region carrying part of otsB was amplified with the primers otsAB_f and otsAB_r. The PCR product, cut with HindIII and SphI, served to replace a 0·90 kb HindIII–SphI fragment of the otsA-carrying plasmid, resulting in the in-frame fusion of the 5'-part of otsA with the 3'-part of otsB. Using XbaI, the resulting otsAB ORF was cloned into the mobilizable integration vector pCLiK8.2. A mobilizable treZ inactivation plasmid was constructed as follows. A 2·5 kb treZ fragment was amplified with the primers treZ_f and treZ_r. The PCR product was cut with XbaI and cloned into pCLiK3, before introduction of an internal 0·65 kb in-frame deletion into treZ with SalI. The treZ gene was cloned via XbaI into pCLiK8.2. For chromosomal inactivation of treS, the gene was cloned in pBluescriptKS after amplification with the PCR primers treS_f and treS_r. Upon digestion of the resulting plasmid with EcoRV and StyI, and treatment with Klenow enzyme, the plasmid was religated, resulting in an 0·65 kb in-frame deletion in the cloned treS ORF. The truncated gene was cloned into the mobilizable plasmid pK18mobsac (Schäfer et al., 1994) using XbaI.

The three final constructs for inactivation of the OtsA-OtsB, TreY-TreZ and TreS pathways, designated pCLiK8.2 : : otsAB, pCLiK8.2 : : treZ and pK18ms : : treS, respectively, were transformed into E. coli S17-1 and mobilized into heat-stressed C. glutamicum according to the procedure described by Schäfer et al. (1990). Successful first recombinants (chromosomal integration mutants) were selected by plating on LB plates with 20 µg kanamycin ml^-1. For selection of the second recombination event, the integration mutants were plated on agar containing 5–10 % (w/v) sucrose. In some cases, trehalose was added at 2 % (w/v).

A putative glycogen synthase gene (glgA) was inactivated by single-step chromosomal integration. For this purpose, a 0·6 kb internal fragment of glgA was amplified using glg_f and glg_r as the PCR primers. The PCR product was cloned into the integration vector pCLiK6 using its unique XbaI site. The resulting plasmid was mobilized using E. coli S17-1 as described above. The integration mutants were selected on LB medium supplemented with kanamycin.

The genotype of the mutants obtained was verified by Southern blot analysis and with specific PCR reactions. During the preparation of this work, mutants in the genes otsA, treY and treS (but not otsB, treZ and glgA) were reported by Wolf et al. (2002).

Construction of pWLQ2 : : otsAB, pWLQ2 : : otsA, pWLQ2 : : treZ and pWLQ2 : : treS.
Expression plasmids carrying the various trehalose biosynthesis genes were constructed using the C. glutamicum–E. coli shuttle expression vector pWLQ2 (Liebl et al., 1992). A 1·6 kb BamHI–SalI fragment of pBlueKS : : otsA (see above) was ligated with pWLQ2 opened with the same enzymes. In the resulting plasmid (pWLQ2 : : otsA) the otsA gene is under the control of the P_tac promoter. For construction of pWLQ2 : : otsAB, the otsB gene was amplified from the C. glutamicum chromosome using the primers otsB_f and otsB_r. After cloning the PCR product in pCR4-TOPO, the 1 kb BamHI fragment was excised and inserted into the BamHI site of pWLQ2 : : otsA, yielding pWLQ2 : : otsAB with both ots genes under regulation of the P_tac promoter.

For construction of pWLQ2 : : treZ, a 2·5 kb PCR product generated with the primers treZ_f2 and treZ_r2 was cloned into pCR4-TOPO. The treZ gene was excised with BamHI and recloned in the BamHI site downstream of the P_tac promoter of pWLQ2. For the construction of pWLQ2 : : treS, the chromosomal C. glutamicum treS gene was amplified as a 2 kb fragment using the primers treS_f3 and treS_r3. After initial cloning into pCR4-TOPO, the treS gene was excised and recloned into pWLQ2 using artificially added SalI sites. The plasmid pWLQ2 : : treS was isolated, in which treS is orientated collinearly to the P_tac promoter. All plasmids were transformed into C. glutamicum strains by electroporation (Liebl et al., 1989a). The strains were grown with kanamycin selection at 20 µg ml^-1. P_tac-driven gene expression was induced by addition of IPTG at a final concentration of 1 mM.

Isolation and analysis of lipids.
Cell lipids were isolated as described by Puech et al. (2000). The cells were harvested and washed after approximately 10 h incubation (growth at 210 r.p.m. at 30 °C) as described below (see Sample preparation). For lipid extraction the wet cells were suspended in CHCl₃/CH₃OH [1 : 1 (v/v)] and shaken at room temperature for 16 h. Remaining bacterial residues were re-extracted twice with CHCl₃/CH₃OH [2 : 1 (v/v)] and the organic phases were pooled and concentrated in a vacuum centrifuge. Water-soluble contaminants were removed by additional extraction with water [2 : 1 (v/v)] and the organic phases were freeze-dried, yielding the crude lipid extracts. Lipid extracts were dissolved in chloroform at a final concentration of 50 µg µl^-1 and analysed by TLC. Samples were applied to silica-gel-coated aluminium plates (type G-60, 5x10 cm, Merck) and developed with CHCl₃/CH₃OH/H₂O [30 : 8 : 1 (by vol.)] in a tightly sealed chamber at 4 °C. Glycolipids were visualized by spraying with a 0·2 % (w/v) anthrone solution in conc. H₂SO₄ followed by heating (at 100 °C for 10–15 min).

The trehalose content of the lipid extracts was quantified after saponification of the crude lipid extract according to Liu & Nikaido (1999), with modifications: aliquots of the samples were taken before the water extraction, freeze-dried and dissolved in 5 % (w/v) potassium hydroxide. The samples were incubated for 1 h at 100 °C, cooled, and aliquots were directly used for trehalose determination by high-pH HPLC (see below).

Sample preparation for trehalose and glycogen determination.
Samples of cultures (1·5 ml) were rapidly cooled on ice and centrifuged (13 000 r.p.m., 4 °C, 15 min). All subsequent manipulations were done at 4 °C. The supernatant was collected and frozen at -20 °C for subsequent extracellular trehalose determination. The cells were washed with BMC medium and also stored as a pellet at -20 °C. In order to minimize changes in the extracellular osmotic conditions, ice-cold medium with the same salt and sugar composition as the growth medium was used for washing. Aliquots of the washed cells were used for determination of cell dry weight.

Cells were opened by sonication (40 % amplitude, 0·5 s cycle) in 500 µl 10 mM sodium/potassium phosphate buffer pH 6. Cellular debris was removed by centrifugation (13 000 r.p.m., 4 °C, 15 min) and the supernatant was used for trehalose and/or glycogen determination.

Trehalose determination.
An enzymic trehalose determination assay was used which was based on the quantitative hydrolysis of trehalose to two molecules of glucose, using recombinant trehalase from E. coli. For this purpose, the E. coli trehalase TreA was overexpressed and partially purified as described by De Smet et al. (2000). Samples of 5–20 µl were incubated with or without recombinant trehalase (5 U) in 90 µl 10 mM sodium/potassium phosphate buffer pH 6·0 for 1 h at 37 °C. The glucose liberated was assayed by the addition of 900 µl freshly prepared enzyme-colour reagent solution from an oxidase/peroxidase glucose detection kit (Sigma 510-DA). Trehalose was calculated from the difference of the glucose amounts in the samples with and without trehalase treatment. A significant background was observed during the measurement of extracellular trehalose at a high concentration of maltose, i.e. in culture supernatants of 10 % (w/v) maltose-containing BMC broth, which was caused either by contamination of the maltose with trehalose or by non-specific interference of maltose with the enzymic trehalose assay. The background was determined by the enzymic assay of samples of sterile maltose BMC and subtracted from the values obtained from culture supernatants.

For more complex samples such as crude cell extracts where a high background of glucose was observed, trehalose was measured with high-pH ion chromatography (HPIC) at room temperature using a Carbo-Pak PA1 column installed in a DX500-HPLC system (DIONEX) supplied with a pulsed amperometric detector ED40. Samples of 25 µl of 10-fold diluted crude extracts were applied to the column and eluted with a linear gradient from 0 to 80 mM sodium acetate in 150 mM sodium hydroxide. The column was regenerated by a 10 min wash with 500 mM sodium acetate followed by 10 min equilibration with 150 mM sodium hydroxide. Trehalose was detected as a single peak with a retention time of approximately 3·3 min. Quantification was based on calibration with defined amounts of a trehalose standard solution.

Glycogen determination.
The amount of intracellular glycogen in C. glutamicum was assayed by hydrolysis with amyloglucosidase (Brana et al., 1982). For this purpose, samples (200 µl) of crude cell extracts (prepared as described above) were mixed with 2 vols 97 % (v/v) ethanol, pelleted and redissolved with heating in the same volume of 10 mM sodium/potassium phosphate buffer pH 6·0. Samples of 5–50 µl were incubated with amyloglucosidase (60 mU; Boehringer Mannheim) in 90 µl 100 mM sodium acetate buffer pH 4·5 for 1 h at 37 °C. The amount of glucose liberated was determined enzymically as described above. The amount of glycogen was calculated from the difference in glucose concentration between the amyloglucosidase-treated samples and control samples without amyloglucosidase.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Analysis of C. glutamicum genome sequence data
The available sequences from the raw C. glutamicum genome data (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html; accession no. NC_003450) were screened for the presence of ORFs with similarity to genes known to be involved in trehalose metabolism. For the initial identification of potential candidates the suggested genome annotations were used. In addition, a BLAST search was made that was based on the enzymes for trehalose synthesis of Mycobacterium tuberculosis, a human pathogen phylogenetically related to Corynebacterium bacteria, which possesses all three known pathways for trehalose biosynthesis (De Smet et al., 2000). ORFs with high similarity to all five genes involved in the different pathways were also found in C. glutamicum (Fig. 1).

The ORFs NCgl2535 and NCgl2537 were designated as otsA and otsB, respectively, because they putatively encode polypeptides with significant similarity to the enzymes trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of the OtsA-OtsB pathway (52 % and 28 % identity with M. tuberculosis OtsA and OtsB, respectively). The two genes are separated by an additional ORF (NCgl2536) with the same orientation as otsA and otsB but unknown function (Fig. 2a). One of the ORFs upstream of otsA (NCgl2533) encodes a transmembrane threonine exporter (Simic et al., 2001). An oppositely oriented ORF downstream of otsB is predicted to encode a LacI-family-type transcription regulator which might be involved in the regulation of the otsA and otsB genes.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Organization of the trehalose (a, b, c) and glycogen (d) biosynthesis genes on the C. glutamicum chromosome. The genes directly involved in biosynthesis of trehalose [otsA/otsB (a), treY/treZ (b) and treS (c)] and of glycogen [glgA/glgC (d)] are drawn in black. The deletions made during chromosomal gene inactivation are marked with dashed boxes (a, b, c). The scheme of integrational disruption of glgA is shown in (d). The fragments amplified by PCR and used for cloning and expression, or for inactivation of biosynthesis genes, are shown as black lines below the genes. For details of cloning and gene inactivation see the text.

Also, an ORF (NCgl2221) was identified in the C. glutamicum genome which is significantly related (up to 64 % identity) to the trehalose synthase genes of other bacteria. This gene was designated treS. The start of the ORF located immediately downstream of treS (NCgl2222; Fig. 2c) overlaps the 3'- end of the treS ORF by 4 bp. ORFs with high similarity to NCgl2222 are found also directly downstream of treS in Streptomyces coelicolor and M. tuberculosis. In other bacteria like Ralstonia solanacearum, Pseudomonas aeruginosa and Chlorobium tepidum, the treS and NCgl2222 homologues are fused in one ORF. Although nothing is known about the properties and physiological role of these putative NCgl2222-similar proteins, the genome data suggest a close functional connection with trehalose synthase.

To check the possibility of glycogen serving as a substrate for trehalose biosynthesis via the TreY-TreZ pathway, the C. glutamicum genome was scoured for putative genes for enzymes that may be involved in glycogen synthesis (Preiss & Greenberg, 1965). Two ORFs, NCgl1073 and NCgl1072, were found whose translation products are highly similar to the (putative) enzymes ADP-glucose pyrophosphorylase (GlgC) and glycogen synthase (GlgA). The deduced C. glutamicum GlgC and GlgA amino acid sequences are related to the corresponding M. tuberculosis homologues at 61 % and 59 % identity, respectively. The two ORFs are situated next to each other but are oriented divergently, with their start codons separated by 51 bp (Fig. 2d). An additional ORF (NCgl0389) with significant similarity to (putative) glycogen synthase enzymes was found. However, due to the genetic surroundings of NCgl1072 this gene and not NCgl0389 was preferred for investigation of its role in glycogen synthesis.

In summary, exploration of the C. glutamicum genome data indicated the presence of all three pathways for trehalose biosynthesis observed in bacteria, thus suggesting a similar gene outfit for this purpose as in the related M. tuberculosis. In addition, the genome data suggested the presence of the pathway for glycogen synthesis in C. glutamicum.

Accumulation of free trehalose by C. glutamicum
As described by Vallino & Stephanopoulos (1993) and Wittmann & Heinzle (2001), lysine-overproducing mutants of C. glutamicum accumulate up to 6 g trehalose l^-1 in the culture broth under conditions close to those used for industrial lysine production. Attempts to connect this trehalose accumulation with changes in the osmolarity of the growth medium, using the type strain of C. glutamicum and NaCl addition to increase the osmolarity, were not successful (data not shown). On the other hand, when sucrose was used instead of NaCl for adjustment of the medium's osmolarity, a significant long-term increase of the extracellular trehalose was observed.

The growth and trehalose accumulation by the type strain of C. glutamicum in minimal BMC medium with two different sugar concentrations, i.e. 0·5 % (w/v) sucrose (Fig. 3a) and 10 % (w/v) sucrose (Fig. 3b), was followed. In the case of the low-sugar medium C. glutamicum stopped its growth at an OD₆₀₀ of about 12, due to substrate limitation. In this case the trehalose accumulated in the culture broth did not exceed 0·1 g l^-1. In contrast, when grown with an excess of sucrose the bacteria reached a final OD₆₀₀ of more than 16. Under these conditions, the type strain accumulated up to 0·9 g trehalose l^-1 during the late exponential and the stationary phase. Monitoring of the intracellular trehalose level showed that in the case of high sucrose supply, intracellular levels of about 20 µg trehalose per mg dry cell weight were reached, which is about four times the maximum intracellular trehalose level detected in the case of low-sucrose supplementation. Under low- as well as high-sucrose conditions, the intracellular trehalose concentration dropped to extremely low values in stationary-phase cells (Fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 3. Dependence of trehalose and glycogen accumulation by C. glutamicum on the sucrose concentration in the medium. The type strain of C. glutamicum was grown in minimal BMC broth supplemented with 0·5 % sucrose (a) or 10 % sucrose (b). Growth curves were recorded by monitoring the OD600 of the cultures (). Intracellular (hatched bars) and extracellular (black bars) trehalose, and intracellular glycogen (white bars), were measured after 12, 24, 48 and 92 h of growth. The results shown are from at least three replicate experiments. DCW, dry cell weight.

Inactivation of the C. glutamicum trehalose biosynthesis pathways
In order to determine the role of the different pathways proposed from the genome data analysis in C. glutamicum trehalose biosynthesis in vivo, three mutants were constructed by chromosomal inactivation of at least one gene of each pathway (Fig. 1; see Methods). For inactivation of the OtsA-OtsB pathway a 2·4 kb chromosomal fragment was removed, resulting in the in-frame fusion of truncated otsA and otsB genes. In this mutant, designated C. glutamicum otsAB, more than 70 % of the otsA gene, the entire ORF NCgl2536, and more than 95 % of otsB were deleted (Fig. 2a). Inactivation of the TreY-TreZ pathway was achieved by in-frame deletion of a 645 bp fragment of the treZ gene (Fig. 2b). Preceding efforts to inactivate the first gene of the pathway (treY) were unsuccessful, perhaps due to polar effects of such deletions on the NCgl2038 ORF. The third proposed pathway for trehalose synthesis in C. glutamicum, i. e. the TreS pathway, which uses maltose as a precursor (Fig. 2c), was inactivated by the in-frame deletion of a 459 bp internal fragment of treS, resulting in a truncated gene which no longer encoded a functional trehalose synthase (data not shown). Thus, three C. glutamicum DSM 20300 single mutants were obtained and named otsAB, treZ and treS, according to the pathway targeted for inactivation in each case.

Based on the single mutants just described, all possible combinations of double mutants (otsAB/treZ, otsAB/treS, treZ/treS), as well as a triple mutant inactivated in all three trehalose synthesis pathways (otsAB/treZ/treS), were constructed. During the construction of the otsAB/treZ and the otsAB/treZ/treS mutants we faced difficulties in obtaining the second-step (vector excision) recombinants carrying the desired deletion. Instead of obtaining nearly equal numbers of the desired deletion variants and clones resulting from reversion of the vector integration event (Schäfer et al., 1994), only the latter type of second-step recombinants were obtained. The problem was overcome after addition of 2 % (w/v) trehalose to the medium used for the sacB-based selection of clones carrying the second recombination event. This interesting observation was a first indication that these two mutant strains had severe difficulties in growing without trehalose in the medium.

During incubation of the otsAB/treZ and otsAB/treZ/treS mutant strains in liquid minimal medium without trehalose with moderate shaking (about 150 r.p.m.) aggregates of cells were observed which rapidly sedimented at the bottom of the culture tubes. Although the increase of culture agitation to 210 r.p.m. resulted in the improvement of growth, the strains carrying mutations in both the OtsA-OtsB and the TreY-TreZ pathways were significantly impaired in their growth in minimal media compared to the other trehalose synthesis mutants and the type strain (Fig. 4a).

View larger version (31K): [in this window] [in a new window]   Fig. 4. Phenotypic characterization of mutants with inactivated trehalose biosynthesis pathways. Growth curves in BMC medium containing 1 % sucrose in comparison with the wild-type are shown in (a). In addition, all mutants and the type strain were grown in 10 % sucrose BMC medium. The intracellular trehalose concentration was determined after 48 h (b), and extracellular trehalose was measured after 72 h (c). The bars in (b) and (c) correspond to the following strains: wild-type (1), otsAB (2), treZ (3), treS (4), otsAB/treZ (5), otsAB/treS (6), treZ/treS (7), otsAB/treZ/treS (8). All data shown are mean values from at least three replicate experiments. DCW, dry cell weight.

Growth of the mutants otsAB/treZ and otsAB/treZ/treS on different substrates known to be utilized by C. glutamicum was investigated by cultivation at 30 °C at 150 r.p.m. in 5 ml BMC medium supplemented with different carbon sources at 1 % (w/v) (Table 2). It is noteworthy in this context that C. glutamicum DSM 20300 is unable to grow on trehalose as the sole source of carbon and energy. On most of the sugar substrates tested the wild-type strain reached a maximum OD₆₀₀ of above 15, while the mutant strains displayed significantly impaired growth. In contrast, growth of the mutants on acetate or pyruvate was not as severely affected as growth on sugar substrates. Trehalose addition to sucrose cultures largely relieved the growth defect of the mutants. This phenomenon of complementation of the mutants by trehalose addition was investigated in more detail by recording growth curves (see below, Fig. 5).

View this table: [in this window] [in a new window]   Table 2. Comparison of the growth of the double mutant otsAB/treZ and the triple mutant otsAB/treZ/treS with that of the type strain The strains were grown at 30 °C, 150 r.p.m., in tubes containing 5 ml BMC broth supplemented with different substrates as specified, at a final concentration of 1 % (w/v) (unless noted otherwise). ±, Weak growth.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Growth characteristics of trehalose-non-producing mutants in 1 % sucrose BMC in the absence or presence of 2 % trehalose. Addition of trehalose led to the nearly complete restoration of growth of the mutants otsAB/treZ and otsAB/treZ/treS. The data shown are mean values from at least three replicate experiments.

Complementation of otsAB/treZ and otsAB/treZ/treS by addition of trehalose

Growth of otsAB/treZ and otsAB/treZ/treS on maltose
The fact that the double mutant otsAB/treZ and the triple mutant otsAB/treZ/treS displayed similar growth behaviour in minimal medium with most of the substrates tested (Table 2) indicates that the presence of an intact treS gene had no significant effect on growth under these conditions. Taking into account that trehalose synthase (TreS) catalyses trehalose production from maltose we investigated the growth phenotype of both mutants on BMC minimal media supplemented with 1 % (w/v) maltose as the sole carbon source (Table 2; Fig. 6a). While growth of the triple mutant otsAB/treZ/treS was significantly impaired in this medium, the otsAB/treZ strain with an intact treS gene displayed a similar growth rate to the wild-type.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Phenotypic differences between otsAB/treZ, otsAB/treZ/treS and the wild-type grown in BMC minimal medium supplemented with maltose as the carbon source. Bacterial growth was determined on minimal medium with 1 % maltose (a); the accumulation of intracellular (b) and extracellular (c) trehalose was measured after growth for 48 h and 72 h, respectively, in BMC medium with 10 % maltose. The values for extracellular trehalose were corrected by subtraction of the assay background as described in Methods. All data shown are mean values from at least three replicate experiments. DCW, dry cell weight.

Plasmid complementation of otsAB mutations
C. glutamicum otsAB/treZ strains carrying expression plasmids with the otsA gene (pWLQ2 : : otsA) and both ots genes (pWLQ2 : : otsAB) were constructed and checked for their ability to grow in 1 % (w/v) sucrose-containing BMC medium in the absence of trehalose (Fig. 7). The plasmid carrying both otsA and otsB efficiently complemented the mutant's growth deficiency under these conditions. This observation excludes the possibility that the mutant's growth phenotype is a result of polar effects that could have been caused by the deletion introduced into the chromosome, and also shows that ORF NCgl2536, the ORF located between otsA and otsB on the chromosome (see Fig. 2) which was not supplied on the plasmid, is not essential for trehalose production and normal growth in minimal medium. Transformation of the otsAB/treZ double mutant with pWLQ2 : : otsA led to a significant improvement of growth in 1 % (w/v) sucrose BMC broth, but did not result in the complete complementation of the mutant's growth deficiency (Fig. 7). An explanation for this could be the in vivo substitution of the function of trehalose phosphate phosphatase (OtsB) by a different, perhaps non-specific, phosphatase, or the assumption that the presence of trehalose 6-phosphate instead of trehalose in the C. glutamicum cell is sufficient for a partial restoration of bacterial growth.

View larger version (12K): [in this window] [in a new window]   Fig. 7. Complementation of the C. glutamicum otsAB/treZ mutant using plasmid copies of the otsA and otsB genes. Plasmids carrying only the otsA gene or both otsA and otsB were transformed into the otsAB/treZ mutant. The transformants were grown in 1 % sucrose BMC broth containing 20 µg kanamycin ml-1 and 1 mM IPTG for induction of gene expression. The data shown are mean values from at least three replicate experiments.

Lipid composition of the trehalose-non-producing mutant C. glutamicum otsAB/treZ

The otsAB/treZ mutant was grown in 30 ml 1 % (w/v) sucrose-containing BMC broth with or without the addition of 2 % (w/v) trehalose. The cells were harvested after 10 h and equal amounts of wet cells were used for cell wall lipid isolation. The lipids were separated using silica-gel TLC plates developed with a chloroform/methanol/water solvent system and compared with the lipids isolated from the type strain grown under the same conditions (Fig. 8). The spots detected after anthrone staining were identified based on the C. glutamicum glycolipid profile described by Puech et al. (2000). When grown in the absence of trehalose, the mutant strain lacked both major trehalose-containing glycolipids in its cell wall lipid fraction. The missing trehalose-corynomycolates were not substituted by other, trehalose-less corynomycolates (such as glucose monocorynomycolate, GMCM, which was observed to be accumulated in a csp1-inactivated C. glutamicum mutant; Puech et al., 2000). In the presence of trehalose in the culture broth, the otsAB/treZ mutant is able to produce trehalose corynomycolates. However, in contrast to the wild-type strain, the trehalose-supplemented mutant contained TMCM as the predominant glycolipid while TDCM was missing. Based on the proposed trehalose corynomycolate biosynthetic pathway (Shimakata & Minatogawa, 2000), it may be possible that a high concentration of trehalose present in the medium results in a shift of the equilibrium in the TDCM synthesis reaction in favour of TMCM. Analysis of the lipids from the type strain DSM 20300 grown in medium with 2 % trehalose supports this hypothesis (authors' unpublished data).

View larger version (23K): [in this window] [in a new window]   Fig. 8. TLC analysis of cellular lipid extracts. Equal amounts (100 µg each) of cell lipid extracts from the type strain grown in 1 % sucrose BMC medium (lane 1), and the mutant otsAB/treZ grown in 1 % sucrose BMC medium with addition of 2 % trehalose (lane 2) and without trehalose (lane 3), were separated on TLC silica-gel plates and developed with CHCl3/CH3OH/H2O (30 : 8 : 1, by vol.) in a tightly sealed chamber at 4 °C. The spots were visualized by spraying with 0·2 % anthrone dissolved in conc. H2SO4 followed by heating at 100 °C. NPL, non-polar lipids; TDCM, trehalose dicorynomycolate; TMCM, trehalose monocorynomycolate.

A mutant designated as glgA : : Km was obtained after site-specific integration of pCLiK6 : : glgA' into the chromosome of C. glutamicum, resulting in disruption of the NCgl1072 ORF (see Fig. 2d). The mutant was unable to accumulate glycogen under conditions of excess sucrose (see legend to Fig. 9). Two additional mutants were made by disruption of the NCgl1072 ORF in the chromosome of the otsAB and otsAB/treS mutants. The mutants were designated as otsAB/glgA : : Km and otsAB/treS/glgA : : Km, respectively. The phenotypic comparison of the C. glutamicum otsAB/treZ and otsAB/treZ/treS mutants with the two isogenic mutants lacking glycogen synthase (GlgA) instead of TreZ did not reveal differences between the four mutant strains with respect to their ability to grow in minimal media without trehalose (Fig. 9) and their inability to produce and accumulate trehalose (see legend to Fig. 9). The fact that the glgA : : Km and treZ mutants showed identical phenotypes in the otsAB as well as the otsAB/treS background strongly supports the idea that TreZ and GlgA are involved in one and the same pathway for trehalose biosynthesis. Also, these results provide evidence for the importance of trehalose synthesis from glycogen in C. glutamicum.

View larger version (14K): [in this window] [in a new window]   Fig. 9. Phenotypic comparison of the glgA : : Km and treZ mutants. The single recombination mutant glgA : : Km and the mutants otsAB/glgA : : Km and otsAB/treS/glgA : : Km were compared with otsAB/treZ, otsAB/treZ/treS, and the wild-type. Growth was monitored in 1 % sucrose BMC broth supplemented with 20 µg kanamycin ml-1 to maintain the marker insertion in glgA : : Km mutants. The data shown are mean values from at least three replicate experiments. While the wild-type produced about 68·1±14·3 µg glycogen [mg dry cell weight (DCW)]-1 after 24 h growth, no glycogen was found in the glgA : : Km mutant. The level of intracellular trehalose after 24 h growth was 18·6±3·6 µg (mg DCW)-1 and 10·5±1·9 µg (mg DCW)-1, respectively, for the wild-type and the glgA : : Km mutant, while the mutants otsAB/glgA : : Km, otsAB/treS/glgA : : Km, otsAB/treZ and otsAB/treZ/treS did not reveal significant amounts of intracellular trehalose [all assay values below 0·4 µg (mg DCW)-1]. Extracellular trehalose accumulation, which was measured after 72 h growth in 10 % sucrose BMC broth supplemented with 20 µg kanamycin ml-1 where appropriate, was 0·43±0·12 µg ml-1 and 0·52±0·09 µg ml-1, respectively, for the wild-type and the glgA : : Km mutant, while the mutants otsAB/glgA : : Km, otsAB/treS/glgA : : Km, otsAB/treZ and otsAB/treZ/treS did not reveal significant amounts of extracellular trehalose (all assay values below 0·03 µg ml-1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

It is not known if the OtsA-OtsB and TreY-TreZ pathways are used simultaneously in wild-type cells and, if so, if the quantitative contribution of both pathways to trehalose production is similar. From the energetic point of view, the OtsA-OtsB pathway is more efficient than the TreY-TreZ pathway. The synthesis of 1 mol trehalose via the OtsA-OtsB pathway is achieved from 1 mol glucose 6-phosphate and 1 mol UDP-glucose, while 1 mol trehalose produced via the TreY-TreZ pathway consumes 2 mol ADP-glucose (for glycogen synthesis). If one assumes that trehalose is produced mainly for synthesis of the cell wall lipids TDCM and TMCM, and that trehalose phosphate and not free trehalose is needed as a precursor for this purpose (also see below; Shikimakata & Minatogawa, 2000), the energy balance is even more in favour of the OtsA-OtsB pathway, because phosphorylated trehalose is an intermediate of the OtsA-OtsB but not of the TreY-TreZ pathway. Therefore it seems reasonable to speculate that only under energy- and substrate-excess conditions could the TreY-TreZ pathway be preferred over the OtsA-OtsB pathway. On the other hand, our results show that glycogen, which can serve as a substrate for the TreY-TreZ pathway, is present in C. glutamicum cells also under conditions of low sugar supply, although not in the same amounts as under sugar-excess conditions. Also, we observed that the TreY-TreZ pathway alone is sufficient to support C. glutamicum growth not only under sugar excess (Fig. 4a) but also under low-sugar conditions (0·5 %, w/v, sucrose; data not shown). Further experiments are needed to determine the individual contribution of each of the OtsA-OtsB and TreY-TreZ pathways to trehalose biosynthesis in wild-type C. glutamicum cells under different growth conditions.

Our data suggest that the TreS pathway plays only a supporting role in trehalose synthesis. Analysis of the growth and trehalose accumulation characteristics of the otsAB/treZ and otsAB/treZ/treS mutants (Fig. 6) demonstrated that this pathway is involved in trehalose synthesis during growth on maltose-containing medium. It is interesting to note that while the wild-type strain and the otsAB/treZ mutant revealed similar levels of intracellular trehalose, the otsAB/treZ mutant accumulated much less extracellular trehalose than the wild-type (Fig. 6b, c), whose extracellular trehalose level after growth on maltose was about the same as on sucrose (Fig. 4c). At present it is not known if the wild-type strain, which contains all three functional trehalose biosynthesis pathways, preferentially utilizes the TreS pathway during growth on maltose. However, the difference in extracellular trehalose accumulation between the wild-type strain and the mutant retaining the TreS pathway as the only trehalose biosynthesis pathway after growth on maltose suggests that in the wild-type both other pathways have a dominant role for trehalose synthesis also when the bacteria are grown on an excess of maltose.

Our results show that C. glutamicum accumulates glycogen when grown under conditions of sugar excess. A glycogen synthesis pathway using ADP-glucose as precursor, similar to that in other bacteria (Preiss & Greenberg, 1965), was predicted from the genome data. Using chromosomal insertion mutagenesis, we showed that the ORF NCgl1072 (together with its neighbour NCgl1073) is involved in glycogen synthesis in C. glutamicum. We were able to connect glycogen synthesis with trehalose synthesis, showing that otsAB mutants simultaneously impaired in glycogen synthesis (otsAB/glgA : : Km and otsAB/treS/glgA : : Km) displayed an identical growth and trehalose synthesis phenotype as the otsAB mutants with an inactivated TreY-TreZ pathway (otsAB/treZ and otsAB/treZ/treS) (see Fig. 9). The growth deficiency of the mutant blocked simultaneously in glycogen synthesis and in the OtsA-OtsB pathway was observed under most growth conditions, including low (1 %) sucrose (Fig. 9), which confirms the important role of trehalose synthesis from glycogen not only under sugar-excess growth conditions.

Impact of trehalose biosynthesis on the growth physiology and cell wall lipid composition of C. glutamicum
The trehalose dependence of growth of the otsAB/treZ and the otsAB/treZ/treS mutants on the majority of the substrates tested indicates the importance of this disaccharide for these bacteria. This is in accordance with the fact that C. glutamicum, just like the related mycobacteria (De Smet et al., 2000), has established three independent pathways for trehalose biosynthesis. One