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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences of the racE and yrpC genes of strain NAFM5 are AB127053 and AB127054, respectively.
Present address: Japan International Research Centre for Agricultural Science, Ohwashi 1-1, Tsukuba, Ibaraki 305-8686, Japan.
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INTRODUCTION |
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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 -PGA synthesis) other than peptidoglycan synthesis is also intriguing. Along these lines, RacE is thought to play a key role in synthesizing the capsule
-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 -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 -PGA synthesis and in D-glutamate catabolism, particularly in association with
-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
-PGA synthesis, but they significantly contribute to the catabolism of D-glutamate supplied exogenously or from
-PGA.
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METHODS |
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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
-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 EcoRIBamHI 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 NotIBclI 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 pMUTIN2racE. The integration of pMUTIN2racE into the racE locus on the BD630 and NAFM5 chromosomes via a single crossover resulted in strains LS29 [racE : : (PspacracE)] and NAFM19 [racE : : (PspacracE)], 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 : : (PspacracE) yrpC : : Spcr] and NAFM20 [racE : : (PspacracE) 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
codY), to generate strains LS44 [amyE : : (yrpClacZ) unkU : : Spcr
codY] and LS42 [amyE : : (racElacZ) unkU : : Spcr
codY] respectively.
Enzyme assays.
-Galactosidase activities determined in cell extracts using o-nitrophenyl
-galactoside as the substrate are expressed as Miller units (Nicholson & Setlow, 1990
).
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RESULTS |
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Effects of exogenous D- and L-glutamate on the synthesis and the D-glutamate content of -PGA
Since strain NAFM20 [racE : : (PspacracE) yrpC : : Spcr] absolutely requires exogenous D-glutamate for growth, we could not exclude the possibility that D-glutamate is a precursor of -PGA synthesis. Assuming that D-glutamate is the precursor, adding D-glutamate to the growth medium should promote
-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
-PGA ml1 in the absence of exogenous D-glutamate, but 0·7, 0·2 and 0·1 mg ml1 at exogenous D-glutamate concentrations of 2, 3 and 5 mM, respectively, without influencing the D-glutamate contents of the
-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
-PGA synthesis rather than in cell growth. In contrast to D-glutamate, L-glutamate (e.g. 5 mM) stimulated
-PGA synthesis 2·5-fold, but without influencing the D-glutamate content. These results rule out the involvement of racE and yrpC in
-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 : : (PspacracE) 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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DISCUSSION |
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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 -DL-glutamyl hydrolase encoded by ywtD, which lies immediately downstream of the
-PGA synthetic operon, can hydrolyse
-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.
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ACKNOWLEDGEMENTS |
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Received 21 January 2004;
revised 25 May 2004;
accepted 7 June 2004.
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