TEXT

Top Abstract Text References

Bacteria normally synthesize D-glutamate and D-alanine as essential components of cell wall peptidoglycan (24). In addition, certain species such as the bacilli synthesize a number of other D-amino acids, including D-phenylalanine, D-asparagine, and D-ornithine, as intermediates in the production of secondary metabolites such as peptide-based antibiotics (2, 13, 36). D-Alanine biosynthesis from L-alanine by alanine racemase appears to be ubiquitous in bacteria (1, 34), but two distinct enzymatic routes have been identified for bacterial D-glutamate biosynthesis. In organisms such as Escherichia coli, Lactobacillus spp., and Pediococcus spp., specific racemases are responsible for the direct biosynthesis of D-glutamate and D-alanine from their L-amino acids (6, 9, 20). Conversely, Bacillus spp., such as Bacillus sphaericus and Bacillus licheniformis, possess a D-amino acid transaminase capable of synthesizing D-glutamate among a broad range of D-amino acids from keto acid precursors, using D-alanine as the amino donor (13, 35). The D-amino acid transaminases of B. sphaericus ATCC 10208 and Bacillus sp. strain YM1 have been particularly well studied (18, 27, 31, 35). The bacilli have generally been considered to synthesize only D-alanine through the action of a racemase, with D-glutamate biosynthesis attributed to the D-amino acid transaminase using -ketoglutarate and D-alanine as substrates (5, 20), although a strain of Bacillus pumilus which lacks D-amino acid transaminase does possess glutamate racemase activity (14). Only one organism, Staphylococcus haemolyticus, has been shown to possess both D-amino acid transaminase and glutamate racemase activities (22).

We have cloned and characterized genes encoding two distinct D-amino acid biosynthetic enzymes from B. sphaericus ATCC 10208 by using a complementation screen with E. coli WM335 (15). This mutant, dependent on exogenously supplied D-glutamate has been used previously to isolate genes encoding D-amino acid transaminase and glutamate racemase (14, 22, 32). Enzymatic assays indicate that the genes isolated in this work encode the D-amino acid transaminase purified from this strain earlier and a previously undetected glutamate racemase. The presence of the two enzymes in B. sphaericus suggests that the bacilli, like Staphylococcus spp., possess two biosynthetic routes to D-glutamate.

Cloning of D-glutamate biosynthetic genes from B. sphaericus. Liquid cultures were grown with aeration at 37°C in Lennox broth (LB) (Gibco/BRL, Gaithersburg, Md.). Plate cultures were grown at 37°C on LB agar or M9 (23) minimal salts-1.5% agar supplemented with 0.2% glucose. E. coli WM335 cultures were supplemented with D-glutamate and thymine, both at 50 µg/ml. Where appropriate, chloramphenicol (10 µg/ml) and ampicillin (200 µg/ml) were added. pBR322 DNA was obtained from New England Biolabs (Beverly, Mass.). Partially MboI-digested chromosomal DNA (16) of B. sphaericus ATCC 10208 was size fractionated on a 0.8% agarose gel, and 2- to 10-kb fragments were ligated to BamHI- and BglII-cleaved pIF306. Plasmid pIF306 is a pBR322 (3) derivative containing a unique BglII site inserted between the vector BamHI and SphI sites. Genes inserted between the BamHI and BglII sites are transcribed from a strong constitutive promoter (21) derived from the pheA (10) promoter of E. coli K-12, located immediately upstream between the unique HindIII and BamHI sites of the vector. The plasmid library was used to transform the restriction-deficient strain XL1 Blue (Stratagene), and approximately 20,000 colonies were obtained. Plasmid DNA prepared from pooled isolates was then introduced into E. coli WM335 and plated on LB-thymine medium in the absence of supplemental D-glutamic acid. Approximately 50 clones which were able to complement the D-glutamic acid deficiency of the host were identified. Restriction analysis of these clones identified two classes of clones represented by the individual plasmid isolates pIF1001 and pIF1002. Each of these plasmids was used to retransform fresh cells of WM335 which were cultured and assayed for D-amino acid transaminase and glutamate racemase activity.

Characterization of the B. sphaericus glr gene. Plasmid DNA isolation, restriction analysis, and ligation were performed by standard methodology as previously described (16). Plasmid pIF1001 carried a 2.34-kb MboI fragment of B. sphaericus chromosome. Sequence analysis of this fragment (25) revealed a single large open reading frame of 684 bp encoding a 28,876-Da protein (GenBank accession no. U26733). Strong primary sequence identity was observed between this protein sequence and the glutamate racemases of B. pumilus (50%), S. haemolyticus (50%), Pediococcus pentosaceus (43%), Lactobacillus fermentus (37%), Lactobacillus brevis (40%), and to a lesser extent E. coli (26%). Strong sequence identity (50 and 30%, respectively) was also observed between B. sphaericus glutamate racemase and the putative products of the racE (GenBank accession no. Z75208) and yrpC (GenBank accession no. U93875) genes identified in the B. subtilis genomic sequence. The sequence alignment is shown in Fig. 1. The peptide also displayed a low level of primary sequence identity (21%) to the aspartate racemases of Streptococcus thermophilus and Desulfurococcus sp., but no significant identity was observed with alanine racemase of B. subtilis or Bacillus stearothermophilus or with B. subtilis amino acid racemase (GenBank accession no. Z94043). Identification of the ATG start codon of the protein was strongly supported by the sequence homology between the protein's N terminus and the other known glutamate racemase sequences, the presence of a consensus Bacillus ribosome binding site GAGG 7 bp upstream of the ATG codon, and the absence of upstream ATG, TTG, and GTG start codons capable of initiating the same open reading frame. Downstream of the coding sequence, there is a region of strong dyad symmetry typical of a bacterial transcription terminator.

View larger version (100K): [in this window] [in a new window]   FIG. 1.   Primary sequence homology between glutamate racemase of B. sphaericus, glutamate racemases from other bacteria, and the putative gene products of the B. subtilis racE and yrpC genes. The consensus sequence shows the amino acids that are conserved in four or more sequences. Sequence identities (percentages) between B. sphaericus glutamate racemase and the individual enzymes are as follows: B. pumilis, 50%; S. haemolyticus, 50%; P. pentosaceus, 43%; L. brevis, 40%; L. fermentus, 37%; E. coli, 26%; B. subtilis RacE, 50%; and B. subtilis YrpC, 30%.

Glutamate racemase activity of pIF1001. Cultures of strains to be assayed were inoculated from single colonies into 10 ml of LB and grown with shaking at 37°C. These were used to inoculate 50-ml LB cultures which were grown for 8 h in a 1-liter shake flask from an initial optical density at 600 nm of 0.05. Cells for glutamate racemase assay were washed, resuspended in 50 mM Tris HCl buffer (pH 8.0), and lysed in a French pressure cell at 1,000 lb/in². Supernatant fluid was recovered for the assay following centrifugation at 14,000 × g for 30 min at 4°C. Glutamate racemase activity was assayed by an L-glutamate dehydrogenase-coupled assay as described previously (22). Assay results were corrected for nonspecific reduction of NADP, which was determined from control assays carried out in the absence of D-glutamate. One unit is defined as 1 µmol of NADP reduced per min at 37°C. The results are summarized in Table 1. Measurements of glutamate racemase activity in pIF1001-bearing cells were consistently three to four times higher than those of the control cells (WM335 carrying pIF306), while the activity of WM335 carrying pIF1002 was not higher than that of the control. The protein encoded on pIF1001 was concluded to be a glutamate racemase of B. sphaericus ATCC 10208, and the gene was designated glr. The glr gene does not appear to be part of an operon, as there are no significant open reading frames upstream or downstream of the gene. Moreover, glr is oriented on pIF1001 such that expression is convergent with the upstream pheA-derived promoter region. This suggests that the native glr promoter is present on the fragment cloned in pIF1001, although we have not yet identified the transcription start site in B. sphaericus. Analysis of the DNA sequence upstream of the coding region did not reveal sequences with significant homology to the consensus Bacillus ^a 35 (TTGACA) and 10 (TATAAT) regions reported for several B. sphaericus genes expressed during vegetative growth (4, 33). The codon usage of glr is generally consistent with that of moderately expressed E. coli genes (7).

Characterization of the B. sphaericus dat gene. Plasmid pIF1002 contains a 2.5-kb insert, and sequence analysis revealed an open reading frame of 848 bp encoding a protein of 31,392 Da (GenBank accession no. U26732). The N-terminal coding sequence was subsequently determined by using additional primers and was found to agree entirely with that of the purified enzyme (data not shown). This confirmed that the gene cloned in pIF1002 encodes D-amino acid transaminase, and we have named it dat. The C-terminal amino acid sequence of the B. sphaericus ATCC 10208 D-amino acid transaminase has been previously reported to consist of the peptide sequence VI (FY)LAL-COOH (5). The VI(FY)LAL peptide does not occur anywhere in the peptide sequence, and the origin of this stretch of sequence is unknown. The coding sequence is preceded by a strong Bacillus ribosome binding site (AAAGGA) located 9 bp upstream of the ATG start codon. The gene is followed by a region of extensive dyad symmetry typical of bacterial transcription terminators.

D-Amino acid transaminase activity of pIF1002. Cells for D-amino acid transaminase assay were prepared as described above for the glutamate racemase assay but were washed and resuspended in 1 ml of 50 mM potassium phosphate buffer (pH 8.5). Extracts of E. coli WM335 carrying pIF1002 were assayed for D-amino acid transaminase activity by the lactate dehydrogenase-coupled assay as described previously (11). Assay results were corrected for nonspecific oxidation of NADH, which was determined by assaying in the absence of D-alanine. One unit of activity is defined as that which produces 1 µmol of pyruvate (NAD) per min at 37°C. The results shown in Table 1 indicated levels of D-alanine-dependent NADH oxidation approximately 1,000-fold higher than those of control cultures carrying only the vector pIF306. Similar assays conducted with WM335 transformed with pIF1001 had shown no increase in D-alanine-dependent NADH oxidation over the levels in the controls. The specific activity of the purified B. sphaericus D-amino acid transaminase has been reported to be 116 to 160 U/mg, indicating that the WM335/pIF1002 cells express Dat to approximately 5% of releasable cell protein. The high level of dat expression may be due in part to the colinear orientation of the dat coding sequence with the constitutive pheA promoter located upstream on the vector. To determine if transcription occurred in the absence of a heterologous promoter, the dat gene and 400 bp of upstream untranslated sequence were isolated by PCR and subcloned onto the low-copy-number vector pLG338 (28) between the unique SalI and EcoRI sites. Oligonucleotides used to isolate the B. sphaericus dat gene by PCR had the sequences 5'-GATGTCGACGTTAATCCAAACGTTAGC-3' and 5'-GACGAATTCTTTTAGGTAGCTCTTTTTAATC-3'. The resulting plasmid, pIF372, was able to confer wild-type growth upon WM335 in the absence of exogenous D-glutamate and showed D-amino acid transaminase activity of 0.3 U/mg. These results indicated that dat expression could originate from the 400-bp upstream sequence. However, neither this transcription start site nor that used in B. sphaericus has been identified.

Primary sequence conservation among known D-amino acid transaminase enzymes. Genes encoding D-amino acid transaminases have previously been isolated from the thermophilic Bacillus sp. strain YM1 (30) and from S. haemolyticus (22). In addition, we have previously identified and characterized the dat gene of B. licheniformis (32) (GenBank accession no. U26947). The sequence identity between the enzymes encoded by these genes and the D-amino acid transaminase of B. sphaericus was examined as shown in Fig. 2. The B. sphaericus D-amino acid transaminase shows 67% identity to that of the thermophilic Bacillus sp. strain YM1, 48% identity to the D-amino acid transaminase of S. haemolyticus, and 42% identity to the B. licheniformis D-amino acid transaminase. The sequence also shows 42% identity to the putative D-amino acid transaminase encoded by the B. subtilis yheM gene (GenBank accession no. Y14082) identified by the sequencing of the genome. The crystal structure of the YM1 enzyme has been determined to 1.9-Å resolution, identifying a novel enzyme fold for a transaminase and enabling the assignment of residues believed to be important to the catalytic mechanism (29). Alignment of the primary sequence of B. sphaericus D-amino acid transaminase with those of the YM1 enzyme and the other D-amino acid transaminases not surprisingly shows complete conservation of K145 (important for substrate-cofactor proton transfer) and residues Y31, R50, E177, I204, and T205, which are all involved in cofactor ion pairing. There is also partial conservation of the serine residues S179 to S181, which contribute to cofactor positioning. Interestingly, the region from Ser240 to Ser243, which has been suggested to be important in substrate specificity through side chain discrimination, shows sequence variations in the known D-amino acid transaminase enzymes.

View larger version (61K): [in this window] [in a new window]   FIG. 2.   Primary sequence homology between the D-amino acid transaminase, other known microbial D-amino acid transaminases, and the gene product of the B. subtilis yheM gene. The consensus sequence shows the amino acids that are conserved in three or more sequences. Individual percentages for sequence conservation with B. sphaericus D-amino acid transaminase are as follows: Bacillus sp. strain YM1, 67%; S. haemolyticus, 48%; B. licheniformis, 42%; and B. subtilis YheM, 42%. Asterisks indicate residues assigned active functions in the YM1 enzyme (29): substrate-cofactor proton transfer (K145), cofactor ion pairing (Y31, R50, E177, S179 to S181, I204, and T205), and substrate side chain recognition (S240 to S243).

Conclusions. In this report we describe the isolation from B. sphaericus ATCC 10208 of the dat gene encoding the D-amino acid transaminase and a second gene we have designated glr, which encodes a hitherto unknown glutamate racemase. This is the first clear report of a Bacillus strain possessing both D-glutamate biosynthetic enzymes, although the genomic sequence of B. subtilis indicates that it may also possess both enzymes. The B. sphaericus enzymes display high primary sequence identity to other known D-amino acid transaminases and glutamate racemases.