Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis

Keitarou Kimura1, Lam-Son Phan Tran1,{dagger} and Yoshifumi Itoh1,2

1 Division of Applied Microbiology, National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan
2 Akita Research Institute of Food and Brewing, Sanuki 4-26, Araya-machi, Akita 010-1623, Japan

Correspondence
Yoshifumi Itoh
yosifumi{at}arif.pref.akita.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Many bacteria, including Escherichia coli, have a unique gene that encodes glutamate racemase. This enzyme catalyses the formation of D-glutamate, which is necessary for cell wall peptidoglycan synthesis. However, Bacillus subtilis has two glutamate racemase genes, named racE and yrpC. Since racE appears to be indispensable for growth in rich medium, the role of yrpC in D-amino acid synthesis is vague. Experiments with racE- and yrpC-knockout mutants confirmed that racE is essential for growth in rich medium but showed that this gene was dispensable for growth in minimal medium, where yrpC executes the anaplerotic role of racE. LacZ fusion assays demonstrated that racE was expressed in both types of media but yrpC was expressed only in minimal medium, which accounted for the absence of yrpC function in rich medium. Neither racE nor yrpC was required for B. subtilis cells to synthesize poly-{gamma}-DL-glutamate ({gamma}-PGA), a capsule polypeptide of D- and L-glutamate linked through a {gamma}-carboxylamide bond. Wild-type cells degraded the capsule during the late stationary phase without accumulating the degradation products, D-glutamate and L-glutamate, in the medium. In contrast, racE or yrpC mutant cells accumulated significant amounts of D- but not L-glutamate. Exogenous D-glutamate utilization was somewhat defective in the mutants and the double mutation of race and yrpc severely impaired D-amino acid utilization. Thus, both racemase genes appear necessary to complete the catabolism of exogenous D-glutamate generated from {gamma}-PGA.


Abbreviations: {gamma}-PGA, poly-{gamma}-DL-glutamate

The GenBank/EMBL/DDBJ accession numbers for the sequences of the racE and yrpC genes of strain NAFM5 are AB127053 and AB127054, respectively.

{dagger}Present address: Japan International Research Centre for Agricultural Science, Ohwashi 1-1, Tsukuba, Ibaraki 305-8686, Japan.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
D-Glutamate is an essential component of peptidoglycan (murein) in bacterial cell walls (Osborn, 1969). It also occurs in some non-ribosomal peptide antibiotics (e.g. gramicidin of Bacillus brevis and mycobacillin of Bacillus subtilis) (Zuber et al., 1993) and in capsule poly-{gamma}-DL-glutamate ({gamma}-PGA), a {gamma}-carboxylamide-linked polypeptide of D- or D- and L-glutamate formed by some strains of B. subtilis, Bacillus licheniformis, Bacillus megaterium and Bacillus anthracis (Ashiuchi & Misono, 2002; Thorne, 1993). Two enzymes can catalyse the formation of D-glutamate. D-Amino acid aminotransferase (EC 2.6.1.21, encoded by dat) yields D-glutamate and pyruvate from D-alanine and 2-oxoglutarate (Fotheringham et al., 1998). Glutamate racemase (EC 5.1.1.3) produces D-glutamate in an interconversion reaction between D- and L-glutamate (Doublet et al., 1993). A single gene in many bacteria, including Escherichia coli, encodes the racemase that is the sole source of D-glutamate and hence is essential for cell growth (Doublet et al., 1993). On the other hand, Staphylococcus haemolyticus, Bacillus sphaericus, thermophilic Bacillus sp. YM-1 and perhaps Staphylococcus aureus have both dat and the racemase gene that can functionally complement the D-glutamate-requirement of an E. coli murI mutant. However, whether both genes are required for D-glutamate synthesis by host cells remains unknown (Fotheringham et al., 1998; Pucci et al., 1995). B. subtilis has yheM, which encodes a putative D-amino acid aminotransferase that is similar (65 % amino acid sequence similarity) to S. haemolyticus Dat aminotransferase (Kunst et al., 1997). Moreover, B. subtilis has two glutamate racemase genes, racE and yrpC (Ashiuchi et al., 1998, 1999; Kunst et al., 1997). RacE and YrpC share weak amino acid sequence identity with each other (27 %) and with E. coli MurI (23 and 26 %, respectively), and their enzymic properties differ (Ashiuchi et al., 1998, 1999, 2003).

The likelihood of three D-glutamate biosynthetic genes in B. subtilis has generated questions as to whether all three are necessary to form the D-glutamate required for peptidoglycan synthesis and if so, whether they cooperate under all growth conditions or play distinct roles under different situations. In addition, whether any of them participate in specific D-glutamate-dependent reactions (e.g. D-glutamate-containing peptide antibiotics and {gamma}-PGA synthesis) other than peptidoglycan synthesis is also intriguing. Along these lines, RacE is thought to play a key role in synthesizing the capsule {gamma}-PGA, which contains D-glutamate (Ashiuchi et al., 1998). On the other hand, YrpC is considered to provide cells with D-glutamate used for cell wall synthesis (Ashiuchi et al., 1999, 2003). However, the complete inactivation of B. subtilis genes has shown that racE is essential for growth in standard laboratory rich medium (Kobayashi et al., 2003). Thus, neither yheM nor yrpC seems to play a role in D-glutamate synthesis, at least in rich medium.

Capsule {gamma}-PGA is specifically synthesized in B. subtilis during the early stationary phase through control of the ComQXPA quorum-sensing mechanism that governs the development of some other stationary-phase phenotypes (Dubnau et al., 2002; Karatas et al., 2003; Lazazzera et al., 1999; Tran et al., 2000). The synthesized capsule is decomposed into its constituent amino acids during the late stationary phase (Kimura & Itoh, 2003; Thorne et al., 1954). B. subtilis can utilize L-glutamate as a nitrogen source (Belitsky, 2002; Belitsky & Sonenshein, 1998; Kane et al., 1981), but whether it can use D-glutamate as a nutrient and the physiological significance of capsule degradation remain unknown.

The present study used a genetic approach to examine the roles of racE and yrpC in growth (as a reflection of D-glutamate formation for peptidoglycan synthesis), in {gamma}-PGA synthesis and in D-glutamate catabolism, particularly in association with {gamma}-PGA degradation. We confirmed that racE is essential for D-glutamate synthesis in rich medium and found that yrpC, as well as racE alone, can direct D-glutamate synthesis to support normal growth in minimal medium. Neither racE nor yrpC is necessary for {gamma}-PGA synthesis, but they significantly contribute to the catabolism of D-glutamate supplied exogenously or from {gamma}-PGA.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, plasmids and media.
Table 1 lists the bacterial strains and plasmids used in this study. Cells were cultured at 37 °C in Luria–Bertani (LB) medium (Sambrook et al., 1989), E9 (Birrer et al., 1994), S7 (Serror & Sonenshein, 1996) or SPII minimal media (Albano et al., 1987). Biotin (0·5 µg ml–1) or amino acids (50 µg ml–1) were added to the minimal medium when required. Escherichia coli and Bacillus subtilis, transformed as described previously (Albano et al., 1987; Inoue et al., 1990), were selected on LB or on SPII agar plates with appropriate supplements and the antibiotics ampicillin (Ap, 100 µg ml–1 for E. coli), chloramphenicol (Cm, 20 µg ml–1 for E. coli and 5 µg ml–1 for B. subtilis), spectinomycin (Spc, 30 µg ml–1 for E. coli and 300 µg ml–1 for B. subtilis) and erythromycin (Erm, 100 µg ml–1 for E. coli and 1 µg ml–1 plus 12·5 µg ml–1 of lincomycin for B. subtilis). Mutants harbouring a Pspac–racE fusion were selected on LB agar containing 1 mM IPTG.


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Table 1. Strains and plasmids

Abbreviations for antibiotics: Ap, ampicillin; Erm, erythromycin; Cm, chloramphenicol; Spc, spectinomycin.

 
Quantification of {gamma}-PGA and D- and L-glutamate.
B. subtilis NAFM5 and its derivatives were cultured in E9 medium without L-glutamate and {gamma}-PGA was purified from the culture supernatants using ethanol (Nagai et al., 1997). The amounts of {gamma}-PGA were determined by HPLC using an Asahipak GAF-7M gel filtration column (7·6x300 cm, Showa Denko) (Nagai et al., 1997) and by comparison with a standard curve generated from a known amount of purified {gamma}-PGA. Chromatography through CROWNPAC CR (+) and CROWNPAC CR (–) HPLC chiral columns (Daicel Chemical Industries) separated D- from L-glutamate in the cultures, and each isomer was quantified by comparing the integrated peak areas with those of the standard glutamates. The polypeptide was hydrolysed in 1 M HCl at 110 °C for 3 h, then the resultant glutamates were purified by passage through a TOYOPAK IC-SP column (Tosoh) and measured by HPLC as above.

DNA isolation and sequencing.
We extracted and purified chromosomal DNA from B. subtilis as described (Bron, 1990). We purified the plasmid DNA used for restriction analysis by alkaline lysis (Bron, 1990) and that for sequencing and plasmid construction using Flex-Prep kits (Amersham Bioscience) or a Midi-Kit (Qiagen). DNA was sequenced using an ABI310 DNA sequencer and Big-Dye terminator sequencing kit (Applied Biosystems). The nucleotide sequences of the racE and yrpC genes of strain NAFM5 are available in the DDJB/EMBL/GenBank databases under accession numbers AB127053 and AB127054, respectively.

Construction of lacZ fusions.
The racE gene is co-transcribed with upstream ysmB (encoding a transcriptional regulator of the MarR family that is thought to control the ysmB racE operon) from nucleotide (nt) –47 relative to the ysmB translation initiation codon (+1) (Kada et al., 2004). The 349 bp intergenetic region between yrpC and yrpB (a putative 2-nitropropane dioxygenase gene) contains a potential {rho}-independent transcription terminator (9 bp loop and 12 bp stem) with a polyT sequence between nt –329 and –293 of the yrpC translation initiation codon (Kunst et al., 1997). A 1511 bp fragment carrying the promoter region of the ysmB-racE operon was amplified by PCR using chromosomal DNA from strain BD630 or NAFM5 and primers having an EcoRI or a BamHI site (underlined) at 5'-GGGAATTCACCTCTTCCGCATGGGAGAGGTT-3' (corresponding to nt –1078 to –1056 of ysmB) and 5'-CCGGATCCTCTTCATTTCATGCTGCAGTTTCA-3' (complementary to nt –39 to –16 of racE), respectively. We also amplified 284 bp of the yrpC promoter region using the primer set 5'-CGTAATTTCATCCACTGACTCTGGA-3' (nt –292 to –268 of yrpC) and 5'-CTCGGATCCACGCTTTCTGTCTCATTCTT-3' (complementary to nt –28 to –9 of yrpC). The amplified DNA fragments were cloned into plasmid pUC119 (Vieira & Messing, 1987) at the HincII site and their nucleotides were verified by sequencing. The EcoRI and BamHI fragment of the racE promoter was excised from the plasmid and inserted between the corresponding sites of the lacZ transcription fusion vector, pDG1661 (Guérout-Fleury et al., 1996). Using the EcoRI site in the multi-cloning sequence on the plasmid, the yrpC promoter sequence obtained as an EcoRI–BamHI fragment was cloned into the fusion vector as above. The resultant fusions were introduced into the amyE locus of strains BD630 and NAFM5 by double crossover recombination, to create strains LS37 [amyE : : (racElacZ)], LS33 [amyE : : (yrpClacZ)], NAFM9 [amyE : : (racElacZ)] and NAFM11 [amyE : : (yrpClacZ)].

Construction of mutants.
The DNA regions of yrpC and racE were amplified by PCR using B. subtilis BD630 chromosomal DNA as the template and the following primer pairs: 5'-CGTAATTTCATCCACTGACTCTGGA-3' (corresponding to nt –292 to –268 of yrpC) and 5'-TTCTTAGTTCATGTTTCTCTTCAGGAG-3' (complementary to nt +819 to +892 of yrpC), 5'-AATGAGCTTGTTGAGCGGGTCAAGG-3' (nt –213 to –198 of racE) and 5'-TAGACCGAAGAATTCCGGCAAAACAG-3' (complementary to nt +914 to +939 of racE). The amplified yrpC and racE fragments were cloned into the plasmid pBluescript II KS+ (Stratagene) at the EcoRV site and between the PstI and EcoRV sites (the racE fragment had been cut with PstI at –34 bp of racE) respectively, to verify the nucleotides by sequencing. Spectinomycin-resistant (Spcr) and erythromycin-resistant (Ermr) cassettes (Guérout-Fleury et al., 1995) were then inserted as BamHI fragments into the unique BclI site of yrpC and into the unique Bst1107I site of racE, respectively, on the plasmids (the BamHI fragment of racE : : Ermr had been blunted using a DNA blunting kit; Takara-Bio, http://www.takara-bio.co.jp). After linearization with ScaI (the yrpC : : Spcr plasmid) or BamHI (the racE : : Ermr plasmid), the plasmid DNAs were transformed into strains BD630 and NAFM5 to replace the relevant genes by double crossover recombination, yielding strains LS27 (yrpC : : Spcr), NAFM12 (yrpC : : Spcr), LS32 (racE : : Ermr) and NAFM21 (racE : : Ermr). The racE segment was recloned as a NotI–BclI fragment (nt –34 to +733 of racE) between the NotI and BamHI sites downstream of the Pspac promoter on plasmid pMUTIN2 (Vagner et al., 1998), resulting in plasmid pMUTIN2–racE. The integration of pMUTIN2–racE into the racE locus on the BD630 and NAFM5 chromosomes via a single crossover resulted in strains LS29 [racE : : (Pspac–racE)] and NAFM19 [racE : : (Pspac–racE)], respectively. Likewise, replacement of the yrpC gene with yrpC : : Spcr in strains LS29 and NAFM19 using the linearized yrpC : : Spcr plasmid DNA yielded strains LS30 [racE : : (Pspac–racE) yrpC : : Spcr] and NAFM20 [racE : : (Pspac–racE) yrpC : : Spcr]. We deleted codY of strains LS33 [amyE : : (yrpClacZ)] and LS37 [amyE : : (racElacZ)] by transformation with the chromosomal DNA of strain PS37 (unkU : : Spcr {Delta}codY), to generate strains LS44 [amyE : : (yrpClacZ) unkU : : Spcr {Delta}codY] and LS42 [amyE : : (racElacZ) unkU : : Spcr {Delta}codY] respectively.

Enzyme assays.
{beta}-Galactosidase activities determined in cell extracts using o-nitrophenyl {beta}-galactoside as the substrate are expressed as Miller units (Nicholson & Setlow, 1990).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Growth phenotypes of the racE and yrpC mutants
We first inactivated yrpC in B. subtilis BD630 (a derivative of {gamma}-PGA non-producing strain 168) and NAFM5 (a {gamma}-PGA-producing strain of non-168 lineage) by inserting a spectinomycin-resistance (Spcr) cassette (Guérout-Fleury et al., 1995). The resultant mutants, strains LS27 (yrpC : : Spcr) and NAFM12 (yrpC : : Spcr), proliferated normally in S7 minimal media as well as in LB medium without D-glutamate supplement (Fig. 1a, b; data for NAFM12 not shown). The mutants also proliferated normally in E9 and SPII minimal media (data not shown), indicating that yrpC is dispensable for growth at least in these media. We then knocked out racE in strains BD630 and NAFM5 by adding an erythromycin-resistance (Ermr) cassette (Guérout-Fleury et al., 1995), to create strains LS32 (racE : : Ermr) and NAFM21 (racE : : Ermr), respectively. These insertional mutants could be selected on S7 minimal plates without D-glutamate, but not on LB plates, confirming the indispensability of racE for growth in rich medium (Kobayashi et al., 2003) and indicating the dispensability of this gene in minimal medium. We then investigated growth properties of the mutants in some detail. Strain LS32 grew comparably to the wild-type and the yrpC mutant strains in S7 medium without D-glutamate (Fig. 1a, b). In contrast, the racE mutant failed to grow in LB medium (Fig. 1b). Exogenous D-glutamate did not support the growth of the racE mutants in LB medium at any concentration up to 8 mM, because yrpC is not expressed in this medium (see below) and L-glutamate in lb medium probably antagonizes D-glutamate uptake, a situation analogous to that of E. coli murI mutants (Doublet et al., 1993). The growth properties of strain NAFM21, a racE null mutant of strain NAFM5, were identical to those of strain LS32 (data not shown). To examine whether yheM is involved in D-glutamate synthesis in minimal medium, we replaced racE with a Pspac–racE fusion, an IPTG-inducible racE gene, in strains NAFM5, NAFM12 (yrpC : : Spcr), BD630 and LS27 (yrpC : : Spcr), to generate strains NAFM19 [racE : : (Pspac–racE)], NAFM20 [yrpC : : Spcr racE : : (Pspac–racE)], LS29 [racE : : (Pspac–racE)] and LS30 [yrpC : : Spc racE : : (Pspac–racE)], respectively. Strains NAFM19 and LS29 proliferated normally in S7 medium without D-glutamate or IPTG, confirming that yrpC alone can direct the synthesis of a sufficient amount of D-glutamate for growth in minimal medium. The racE : : (Pspac–racE) mutants did not grow in LB medium; however, when 1 mM IPTG was added, the growth returned to normal. Double mutants of yrpC : : Spcr and racE : : (Pspac–racE), strains NAFM20 and LS30, did not grow in either S7 or LB medium, unless 0·3 mM D-glutamate (in S7 medium) or 1 mM IPTG (in S7 and LB media) was added (data not shown). Furthermore, in LB medium without IPTG, the racE : : (Pspac–racE) mutant proliferated only for 2 h (about 3 generations) probably due to exhausting the cellular D-glutamate pools then losing viability during further incubation (Fig. 1c). We thus concluded that while racE is essential for growth in rich medium, racE and yrpC can individually complete D-glutamate synthesis in minimal medium, and yheM plays no significant role in D-glutamate synthesis at least under our growth conditions.



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Fig. 1. Growth properties of wild-type (a) and mutants of racE and yrpC (b) in rich and minimal media, and indispensability of racE for growth in rich medium (c). (a, b) Overnight cultures of test strains shaken at 37 °C in S7 minimal medium (Serror & Sonenshein, 1996) were inoculated into fresh LB medium or S7 minimal medium at 1 % of volume. The cultures were incubated as above and cell growth was measured using a spectrophotometer at 600 nm (OD600). {square}, Wild-type (BD630) in LB medium; {blacksquare}, wild-type (BD630) in S7 medium; {triangleup}, strain LS27 (yrpC : : Spcr) in LB medium; {circ}, strain LS32 (racE : : Ermr) in LB medium; {blacktriangleup}, strain LS27 (yrpC : : Spcr) in S7 medium; {bullet}, strain LS32 (racE : : Ermr) in S7 medium. (c) Strain LS29 [racE : : (Pspac–racE)] cells growing exponentially in LB medium containing 1 mM IPTG were harvested by centrifugation, washed twice with sterilized saline and suspended in fresh LB medium with ({bullet}) or without ({circ}) 1 mM IPTG. Viable cells in cultures shaken at 37 °C were counted on LB plates containing 1 mM IPTG at 30 min intervals.

 
Regulation of racE and yrpC expression
The distinct growth phenotypes of the racE and yrpC mutants implied that these genes are differently expressed in rich and minimal media. We accordingly constructed BD630 and NAFM5 derivatives harbouring a racElacZ or a yrpClacZ fusion at the amyE locus and measured {beta}-galactosidase activities in these strains. In the BD630 derivatives, RacE–LacZ synthesis (in strain LS37) increased as the culture proliferated and reached maximal levels at the mid-exponential phase, followed by a gradual decrease after the culture entered the stationary phase (Fig. 2). The YrpC–LacZ fusion (in strain LS33) was expressed with similar kinetics but at lower levels (Fig. 2). The fusions were also expressed in the NAFM5 derivatives with similar kinetics and at essentially the same levels (data not shown). The {beta}-galactosidase activities directed by yrpClacZ in both strains growing in LB medium were negligible (Table 2). Casamino acids [0·1 % (w/v)] reduced racElacZ and yrpClacZ expression in S7 medium by 30 % and 50 %, respectively (Table 2). The global regulator CodY controls the expression of nutrition-responsive genes in B. subtilis (Molle et al., 2003; Ratnayake-Lecamwasam et al., 2001). However, a codY mutation did not relieve repressed expression of the fusions in LB medium (12 units from racE–lacZ and <1 unit from yrpClacZ), suggesting that the nutrient-dependent expression of neither racE nor yrpC is regulated by codY.



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Fig. 2. Expression of yrpClacZ and racElacZ fusions in S7 minimal medium. Strains LS33 [amyE : : (yrpClacZ), circles] and LS37 [amyE : : (racElacZ), triangles] were shaken in S7 medium (20 ml) at 37 °C. Growth at 600 nm (filled symbols) and {beta}-galactosidase activities (open symbols) were determined in portions (1 ml) removed every hour.

 

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Table 2. Expression of racElacZ and yrpClacZ fusions in B. subtilis under various growth conditions

{beta}-Galactosidase activities in B. subtilis BD630 derivatives LS37 [amyE : : (racElacZ)] and LS33 [amyE : : (yrpClacZ)], and NAFM5 derivatives NAFM9 [amyE : : (racElacZ)] and NAFM11 [amyE : : (yrpClacZ)], measured in exponentially growing cells (OD600 1·0) in the media indicated are expressed as Miller units (Nicholson & Setlow, 1990). Values are means of three measurements; standard deviations were below 5 % of the corresponding values (not shown).

 
Effects of racE and yrpC mutations on the production and the D-glutamate content of {gamma}-PGA
To investigate whether racE or yrpC is required for {gamma}-PGA synthesis in vivo, we determined the amounts and D-glutamate contents of the capsule polypeptides produced by racE (strain NAFM19) and yrpC (strain NAFM12) knockout mutants and compared them with those of the wild-type strain, NAFM5. The racE and yrpC mutants produced 0·9 and 0·7 mg {gamma}-PGA ml–1 and the polypeptides contained 86 % and 87 % D-glutamate, respectively. These values were comparable to those of the polypeptide produced by the wild-type strain (1·0 mg ml–1 and 86 % D-glutamate). Thus, neither racE nor yrpC is necessary for {gamma}-PGA synthesis.

Effects of exogenous D- and L-glutamate on the synthesis and the D-glutamate content of {gamma}-PGA
Since strain NAFM20 [racE : : (Pspac–racE) yrpC : : Spcr] absolutely requires exogenous D-glutamate for growth, we could not exclude the possibility that D-glutamate is a precursor of {gamma}-PGA synthesis. Assuming that D-glutamate is the precursor, adding D-glutamate to the growth medium should promote {gamma}-pga synthesis and increase the D-amino acid content as a consequence of elevated cellular pools of the precursor. Wild-type strain NAFM5 produced 1·0 mg {gamma}-PGA ml–1 in the absence of exogenous D-glutamate, but 0·7, 0·2 and 0·1 mg ml–1 at exogenous D-glutamate concentrations of 2, 3 and 5 mM, respectively, without influencing the D-glutamate contents of the {gamma}-PGAs. Since D-glutamate did not affect the growth of strain NAFM5 even at 5 mM (see below), it might prevent a process involved in {gamma}-PGA synthesis rather than in cell growth. In contrast to D-glutamate, L-glutamate (e.g. 5 mM) stimulated {gamma}-PGA synthesis 2·5-fold, but without influencing the D-glutamate content. These results rule out the involvement of racE and yrpC in {gamma}-PGA synthesis and suggest that D-glutamate competes with L-glutamate, the likely precursor of the polypeptide (Urushibata et al., 2002), during its synthesis in vivo.

Catabolic functions of racE and yrpC
B. subtilis 168 and its derivatives (e.g. strain BD630) can utilize L-glutamate as a source of nitrogen, but not of carbon (Belitsky & Sonenshein, 1998), because the gudB gene [encoding the catabolic (NAD+-dependent) glutamate dehydrogenase responsible for L-glutamate utilization] of this strain specifies an inactive enzyme due to a 9 bp duplication in the coding region. Removal of the duplication confers GudB activity and consequently enables cells to utilize L-glutamate as a carbon source and even more efficiently as nitrogen source (Belitsky & Sonenshein, 1998; Kane et al., 1981). Strain NAFM5 (non-168 lineage) could not utilize L-glutamate as a carbon source, suggesting that the GudB of this strain is also inactive, but this strain can utilize the amino acid as a nitrogen source. Strain BD630 initiated slow growth (doubling time of 90 min) after a lag-time of about 8 h in minimal medium containing 1 mM D-glutamate as the sole nitrogen source (Fig. 3a). The racE and yrpC mutations alone and in combination did not cause further defects in D-glutamate utilization by strain BD630 (data not shown). This strain could not grow in minimal medium containing high concentrations of D-glutamate as a nitrogen source (Fig. 3a), perhaps due to D-glutamate cytotoxicity (see below). On the other hand, strain NAFM5 proliferated faster (doubling time of 75 min) in minimal medium supplemented with 1 mM D-glutamate as the sole nitrogen source (Fig. 3b). Growth rate and cell yield improved as the D-glutamate concentration increased, but without reaching the levels achieved at the corresponding concentrations of L-glutamate (doubling time of 40 min at 8 mM, Fig. 3b). A block of racE or yrpC did not influence the utilization of D-glutamate below 5 mm and the growth rate was reduced but by only 10 (yrpc mutant) and 20 % (race mutant) at 8 mm D-glutamate (data not shown). The double mutant, NAFM20 [racE : : (Pspac–racE) yrpC : : Spcr], continued to utilize D-glutamate at concentrations below 2 mM. Increasing D-glutamate concentrations further retarded the growth rate of this double mutant (doubling time of 200 min, cf. 65 min of the wild-type strain) at 5 mM and almost abolished it at 8 mM (Fig. 3b).



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Fig. 3. Utilization of D-glutamate by strains BD630 and NAFM5 as nitrogen source. Strains BD630 (a) and NAFM5 (b) were grown in E9 medium containing 2 % (w/v) glucose and various concentrations of D- or L-glutamate as the sole nitrogen source as follows: 8 mM L-glutamate ({bullet}); 1 mm D-glutamate ({triangleup}); 5 mm D-glutamate ({blacktriangleup}); and 8 mm D-glutamate ({circ}). (b) Growth of strain NAFM20 (racE : : Pspac–racE Ermr, yrpC : : Spcr) in E9 medium containing 8 mM D-glutamate as the sole nitrogen source ({blacksquare}). Cell growth was measured at 600 nm (OD600).

 
Toxicity of D-glutamate for strains bd630 and nafm5
since strains bd630 and nafm20 can utilize low concentrations of D-glutamate, the defective growth of these strains at high concentrations might be due to the cytotoxicity of D-glutamate rather than an inability to catabolize it. We accordingly examined the toxic effect of D-glutamate on strains BD630, NAFM5 and their racE or yrpC mutants by measuring their growth in minimal medium containing NH4Cl as a nitrogen source and various concentrations of D-glutamate. Fig. 4 shows that the D-amino acid significantly decreased and abolished the growth of strain BD630 at 1 and 5 mM, respectively. On the other hand, the D-amino acid had no and minimal effects on the growth of strain NAFM5 below 5 mM and at 8 mM, respectively. The racE or yrpC mutants of strain NAFM5 were more sensitive to D-glutamate than the wild-type. The D-amino acid had no effect on the growth of strain NAFM12 (yrpC : : Spcr) at 1 mM but inhibited growth at higher concentrations. Strain NAFM19 (racE : : Pspac–racE) was more sensitive to D-glutamate than strain NAFM12, probably because NAFM19 lacks RacE, which is expressed more abundantly than YrpC (Ashiuchi et al., 1998, 1999). To examine whether strain BD630 is specifically sensitive to D-glutamate or also to other D-amino acids, we incubated strains BD630 and NAFM5 (for comparison) in E9 minimal medium containing NH4Cl as the nitrogen source and various concentrations of D-tyrosine, D-serine or D-alanine. At concentrations above 0·1 mM and 5 mM, respectively, D-tyrosine and D-serine prevented the growth of strain BD630. In contrast, these D-amino acids had no effect on the growth of strain NAFM5 at the same concentrations. D-Alanine (8 mM) moderately suppressed the growth of strain BD630 but had little effect on that of strain NAFM5. Thus, strain BD630 appears more sensitive than strain NAFM5 to different D-amino acids. We consider a possible cause for the sensitivity of strain BD630 to D-amino acids in the Discussion.



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Fig. 4. Toxicity of D-glutamate to strains BD630 (a), NAFM5 (b), NAFM12 (c) and NAFM19 (d). Strain BD630 (wild-type), NAFM5 (wild-type), NAFM12 (yrpC : : Spcr) and NAFM19 (racE : : Ermr) were grown in E9 medium containing 2 % (w/v) glucose (as carbon source), 0·7 % (v/v) NH4Cl (as nitrogen source) and various amounts of D-glutamate ({bullet}, 0 mM; {circ}, 1 mM; {blacktriangleup}, 5 mM; {triangleup}, 8 mM). Cell growth was measured at 600 nm (OD600).

 
Accumulation of D-glutamate derived from {gamma}-PGA in racE and yrpC mutant cultures
Like other B. subtilis and B. licheniformis strains that produce {gamma}-PGA (Kambourova et al., 2001; Ko & Gross, 1998; Thorne et al., 1954), strain NAFM5 actively produces capsule {gamma}-PGA during the early stationary phase (between 2 and 3 days of incubation in E9 minimal medium) and then degrades it during the late stationary phase (between 4 and 7 days of incubation). The amounts of {gamma}-PGA produced by strain NAFM5 reached about 1 mg ml–1, which approximately corresponded to 8 mM of glutamate. However, we did not detect either L- or D-glutamate (<0·03 mM) in NAFM5 cultures even after 7 days when over 90 % of the polypeptide had been degraded, implying that the cells had internalized and utilized the degradation products. To examine the participation of racE and yrpC in the utilization of D-glutamate derived from {gamma}-PGA, we measured the amounts of D- and L-glutamate in the cultures of NAFM5 (wild-type), NAFM12 (yrpC : : Spcr) and NAFM19 [racE : : (Pspac–racE)]. Little L-glutamate (<0·03 mM) was detected in either wild-type or mutant cultures throughout the stationary phase (up to 7 days). In contrast, D-glutamate became detectable after 3 days in both NAFM12 and NAFM19, but not in the wild-type, and increased to maximal levels after 7 days (Fig. 5). Thus, NAFM5 cells appear to take up the D- and L-glutamate degraded from {gamma}-pga and both race and yrpc are probably involved in the catabolic conversion of D-glutamate to the L-form.



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Fig. 5. D-Glutamate accumulation in culture medium by racE and yrpC mutants. Strains NAFM5 (wild-type; {circ}), NAFM12 (yrpC : : Spcr; {triangleup}) and NAFM19 (racE : : Pspac–racE Ermr; {bullet}) were cultured in E9 minimal medium. Amounts of D- and L-glutamate were determined by HPLC in aliquots taken at the indicated times (see Methods).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Whole-genome sequencing has identified two putative glutamate racemase genes in B. anthracis (racE-1 and racE-2), B. cereus (BC0866 and BC4496) and in B. subtilis (racE and yrpC), as well as three in Thermoanaerobacter tengcongensis (murI-1, murI-2 and murI-3) (Bao et al., 2002; Ivanova et al., 2003; Kunst et al., 1997; Read et al., 2003). However, not all Bacillus species have two glutamate racemase genes. For example, Bacillus halodurans has a single racemase gene (Takami et al., 2000). B. subtilis RacE, B. anthracis RacE-1, -2 and B. cereus BC4496, BC0866 are closely related and constitute the glutamate racemase group of Bacillus (Fig. 6). In contrast, YrpC is distantly related to this group and resembles Clostridium glutamate racemase (Fig. 6). Furthermore, racE, racE-2 and BC4496 all lie in the conserved racE (racE-2 or BC4496)-gerM-rph-synA-synB cluster. The neighbouring genes of racE-1 and BC0866 are also perfectly conserved in the B. anthracis and B. cereus chromosomes but are totally distinct from those of the yrpC locus on the B. subtilis chromosome. The origin of yrpC appears to be distinct from that of the other Bacillus racemase genes but close to that of the clostridial genes.



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Fig. 6. Phylogenetic tree of bacterial glutamate racemases. Amino acid sequences of enzymes from GenBank (http://www.ncbi.nlm.nih.gov) were aligned using CLUSTAL W software and a phylogenetic tree was calculated using the neighbour-joining (NJ) method (www.ddbj.nig.ac.jp). The bar indicates amino acid substitution per residue and numbers at branches are bootstrap estimates. B. subtilis RacE and the YrpC are boxed.

 
In accordance with its genetic features, racE alone can complete D-glutamate synthesis in both rich and in minimal media, whereas yrpC can do so only in minimal medium because this gene is not expressed in rich medium (Fig. 1b, Table 2). The primary structures of racE and yrpC are diverse, yet their expression is controlled in a growth-phase- and nutrient-dependent manner (Fig. 2, Table 2). However, yrpC is more tightly regulated than racE by nutrients (Fig. 2, Table 2). The turnover rates of cell wall peptidoglycan synthesis correlate with growth rates (Cheung et al., 1983). Cells growing rapidly in rich medium would require more precursors for peptidoglycan synthesis than slowly growing cells in minimal medium. In fact some, if not all peptidoglycan synthetic genes, including gcaS (encoding UDP-N-acetylglucosamine pyrophosphorylase), dltABCD (responsible for D-alanine-D-alanine carrier protein synthesis), mbl (MreB-like protein) and murE-mraY-murD (UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate and phospho-N-acetylmuramoyl-pentapeptide synthesis), are repressed 3–7-fold under amino acid starvation (Eymann et al., 2002). In addition to D-glutamate biosynthesis, both racE and yrpC participate in D-glutamate catabolism (Fig. 3b). In accordance with their roles in both D-glutamate synthesis and catabolism, high levels of racE and yrpC are expressed during the mid-exponential phase when cells actively proliferate (regulation as peptidoglycan synthetic genes), and these levels are partially suppressed by amino acids (regulation as catabolic genes) (Table 2).

The expression of racE and yrpC is neither induced by D-glutamate nor repressed by ammonia (data not shown). However, these genes are abundantly expressed in minimal medium when compared with the glutamate racemase genes of other bacteria (Ashiuchi et al., 1998) and are therefore capable of participating in D-glutamate catabolism. B. subtilis appears to possess at least two D-glutamate catabolic pathways. While the less efficient pathway directly catabolizes D-glutamate perhaps via an oxidase or a transaminase, the other is more efficient and involves RacE and YrpC. When the latter pathway was blocked by the racE and yrpC double mutation, B. subtilis NAFM5 could not complete D-glutamate catabolism and consequently the cellular pools of D-glutamate increased to toxic levels, preventing cell growth (Fig. 3b). Strain BD630 cannot utilize high concentrations (>=5 mM) of D-glutamate, although it has levels of both RacE and YrpC that are comparable to those of NAFM5 (data not shown) and it can utilize D-glutamate at low concentrations (Fig. 3a). Strain BD630 appears to be so susceptible to D-glutamate cytotoxicity that it cannot thrive in the presence of high D-glutamate concentrations (Fig. 3a). Strain BD630 but not strain NAFM5 exhibits hyper-susceptibility to D-amino acids such as D-tyrosine and D-serine. D-aminoacyl-trna deacylase degrades aminoacyl-trnas charged with D-amino acids to prevent mis-incorporation of D-amino acids into proteins (Yang et al., 2003) and a mutant devoid of this enzyme becomes sensitive to D-amino acids (Soutourina et al., 2000). A possible explanation for the apparent sensitivity of B. subtilis 168 to different D-amino acids is that this strain lacks D-amino acid deacylase activity. Experiments are required to substantiate this hypothesis.

The {gamma}-DL-glutamyl hydrolase encoded by ywtD, which lies immediately downstream of the {gamma}-PGA synthetic operon, can hydrolyse {gamma}-PGA in vitro into 470 kDa and 11 kDa fragments (Suzuki & Tahara, 2003). These degradation intermediates appear to be further degraded into D- and L-glutamate (Fig. 5). The second degradation enzyme should be identified to elucidate the mechanism underlying capsule degradation. Furthermore, the physiological effects expressed by mutants lacking the degradation enzymes must be demonstrated to prove the function of the polypeptide capsule as a nutrient.


   ACKNOWLEDGEMENTS
 
We are grateful to A. L. Sonenshein for the gift of B. subtilis PS29 (unk : : Spcr) and PS37 (unk : : Spcr {Delta}codY). This work was supported in part by a grant from the Ministry of Agriculture, Forestry and Fisheries.


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Received 21 January 2004; revised 25 May 2004; accepted 7 June 2004.



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