Laboratory of Molecular Genetics, School of Agriculture1, and Gene Research Center2, Ibaraki University, Ami, Ibaraki 300-0393, Japan
The United Graduate School, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan3
Author for correspondence: Makoto Shirai. Tel: +81 298 88 8652. Fax: +81 298 88 8653. e-mail: shirai{at}ipc.ibaraki.ac.jp
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ABSTRACT |
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Keywords: amino acid racemase, cyanobacteria, Microcystis, peptide synthetase gene, microcystin biosynthesis
Abbreviations: Adda, 3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4,6-dienoic acid; MCYST, microcystin; Mdha, N-methyldehydroalanine; D-MeAsp, D-erythro-ß-methylaspartic acid; NRPS, non-ribosomal peptide synthetase; PKS, polyketide synthase; UDP-MurNAc-L-Ala-D-Glu, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate
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INTRODUCTION |
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Many D-amino acid racemase genes for peptidoglycan synthesis have been reported in eubacteria (Liu et al., 1997 ; Malathi et al., 1999
; Pucci et al., 1995
). However, no racemase genes have been reported in prokaryotic NRPS. The alanine racemase genes responsible for synthesis of cyclosporin A and HC-toxin were identified in fungal NRPS (Cheng & Walton, 2000
; Hoffmann et al., 1994
). In this study, we showed that the mcyF gene encodes the glutamate racemase involved in microcystin synthesis.
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METHODS |
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DNA manipulation, Southern blotting and sequencing.
Total DNA of Microcystis cells was isolated by the method described previously (Sakamoto et al., 1993 ), and DNA manipulations were performed as described by Sambrook et al. (1989)
. Digested cyanobacterial DNA was separated in 0·8% agarose gels and transferred onto Hybond-NX membranes. DNA fragments (as probes) were labelled using an ECL random prime labelling kit (Amersham Pharmacia Biotech) as described previously (Nishizawa et al., 1999
). Genomic Southern blotting was performed according to the manufacturers instructions. The nucleotide sequence was determined by dideoxy chain termination, using an Applied Biosystems automated sequencer (model 373S) (Nishizawa et al., 1999
).
Construction of a gene-disruption plasmid, and integrative conjugation of Microcystis.
The plasmids for gene disruption of mcyF by homologous recombination were constructed as follows. The 2·5 kb NcoIEcoRI fragment containing mcyF was subcloned from pMCQ3 (Nishizawa et al., 2000 ) by blunt-end ligation into the SmaIBamHI site of pUC119 (ToYoBo), generating pCHI621. The 1·2 kb XbaI fragment containing the CmR gene cassette from pR107XH (Nishizawa et al., 1999
) and the 1·8 kb BamHI fragment containing the mob gene from pSUP5011 (Simon, 1984
) were inserted by blunt-end ligation into the BamHI site (in mcyF) and the HindIII site (in the multi-cloning site) of pCHI621, respectively, generating pJXS10. The plasmids for gene disruption were introduced into Microcystis cells (by conjugation) from E. coli S17-1, and chloramphenicol-resistant (8 µg chloramphenicol ml-1) conjugants were selected as described previously (Nishizawa et al., 1999
).
HPLC analysis of microcystins.
Microcystins were extracted from the dried cells with 5% aqueous acetic acid, purified using BondElute ODS cartridges (Varian) and analysed by HPLC as described previously (Nishizawa et al., 1999 ).
Bacterial complementation test.
E. coli WM335, which requires D-glutamic acid for growth, was grown in LuriaBertani (LB) medium supplemented with 50 µg D-glutamic acid ml-1 and 20 µg thymine ml-1. The racemase expression vector pQE-McyF was constructed as follows (see Fig. 4a). The entire mcyF gene was amplified by PCR with primers 5'-McyF/SphI (5'-CT CGC ATG CAG ACA AAA CTA CCG-3') and 3'-McyF/HindIII (5'-CTC AAG CTT TTT GGG TTT GAA GGC-3') (restriction sites are underlined and modified sequences are shown in italics). PCR was carried out on chromosomal M. aeruginosa K-139 DNA with Taq DNA polymerase (Nippon Gene). PCR products were purified, digested with SphI and HindIII, and cloned into the SphI and HindIII sites of the pQE70 vector (Qiagen). Both the 5'- and the 3'-cloning sites were confirmed by sequencing. The primers (and sequences) used were as follows: QE/F2 primer, 5'-TTG CTT TGT GAG CGG ATA AC-3'; QE/R1 primer, 5'-CAT TAC TGG ATC TAT CAA CAG G-3'. HindIII digestion of pQE70 resulted in removal of the His-tag sequence and appended the amino acid sequence SLIS at the C-terminus of the recombinant protein.
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Computer analysis of DNA.
The DNA sequences were assembled and analysed using GENETYX-MAC software (Software Development).
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RESULTS AND DISCUSSION |
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D-Glutamic acid is fairly widespread in eubacteria, as D-amino acids constitute part of the fundamental tetrapeptide chain of murein in the cell wall (as common components of peptidoglycans) (Fotheringham et al., 1998 ; Liu et al., 1997
; Malathi et al., 1999
; Pucci et al., 1995
). D-Glutamate in a free form is incorporated into the common peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-D-glutamate (UDP-MurNAc-L-Ala-D-Glu), through the action of UDP-MurNAc-L-Ala-D-Glu synthetase (EC 6.3.2.9). In bacteria, enzymes are involved in the biosynthesis of D-glutamic acid through two different pathways: D-glutamate racemase and D-amino acid aminotransferase (Malathi et al., 1999
). Cysteine residues at the active sites of motif 1 [(V/I)x(P/A)CNTA(H/T)] and motif 2 [(I/V)(A/L)GCTH(E/H)x(S/P)], which are thought to form the catalytic centres of these cofactor-independent racemases (Yohda et al., 1996
), were also conserved in McyF (Nishizawa et al., 2000
).
Disruption of the mcyF gene
To determine whether the cloned racemase gene, mcyF, in the mcy operon is required for microcystin biosynthesis, gene disruption of mcyF in M. aeruginosa K-139 and Microcystis sp. S-70 was carried out. However, no disruptants were isolated for strain K-139. The restriction barrier of strain K-139 may interfere with recombination (Takahashi et al., 1996 ). Genomic Southern hybridization demonstrated the presence of mcyF in Microcystis sp. S-70, which produces MCYST-LR, -RR and -YR (Nishizawa et al., 2000
). Therefore, insertional mutagenesis of mcyF in strain S-70 was performed, and the chloramphenicol-resistant conjugant Microcystis sp. S-70JX1 was isolated.
To demonstrate integration of the CmR cassette into the mcyF gene on the chromosome, genomic Southern hybridization was carried out. Total DNA from mutant S-70JX1 was digested with either HincII or HindIII and then probed with the 0·75 kb BglII fragment containing a part of the racemase gene (Fig. 2a). A 2·9 kb signal and a 5·0 kb signal, obtained with HincII and HindIII, respectively, were detected in the control (Microcystis sp. S-70). A 0·8 kb signal and a 6·2 kb signal, however, were detected in mutant JX1 (Fig. 2b
). In the case of HindIII digestion, the position of this signal coincided with that of a signal obtained with the probe of the CmR cassette fragment (data not shown). Furthermore, to confirm mcyF gene disruption and gene organization in strain S-70JX1, the 6·2 kb HindIII fragment, including mcyEmcyFmcyG (Fig. 2b
), was cloned from this strain. Sequence analysis showed that the gene organization in strain S-70 was the same as that in strain K-139, and nucleotide sequence identity between both strains was 95·5%.
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Complementation of a bacterial mutant deficient in D-Glu synthesis
To confirm that the mcyF gene encodes the glutamate racemase, the McyF racemase was expressed in D-glutamic-acid-requiring E. coli WM335, using the T5 promoter transcriptiontranslation system of pQE vectors (Fig. 4a). E. coli WM335 transformed with pQE-McyF expressed a protein of about 28 kDa (Fig. 4b
), the molecular mass of which coincided well with the value predicted for McyF on the basis of its nucleotide sequence. The pQE-McyF-transformed E. coli grew exponentially and survived on agarose plates and in LB liquid medium (data not shown) without D-glutamic acid as well as in those supplemented with 50 µg D-glutamic acid ml-1. However, E. coli WM335 carrying the expression vector pQE70 or pCHI621 showed no growth on agarose plates without D-glutamic acid, and a rapid decrease in OD660 was observed shortly after inoculation into liquid medium without D-glutamic acid (data not shown). These observations showed that the mcyF gene was capable of complementation of the D-glutamic-acid-requiring phenotype in E. coli. Gene disruption and complementation analysis demonstrated that D-Glu in the cyclic heptapeptide microcystin is synthesized from the primary metabolite L-Glu by a glutamate racemase (McyF). These results indicated that McyF is involved in D-glutamic acid synthesis. Moreover, this is the first report showing the occurrence of a glutamate racemase gene in prokaryotic non-ribosomal peptide synthesis.
Phylogenetic analysis of the D-amino acid racemase
The microcystin molecule is composed of the following three D-amino acid residues: D-Ala, D-MeAsp and D-Glu (Fig. 1). The microcystin synthetase was shown previously to contain one epimerization domain in McyA and one racemase, McyF (Nishizawa et al., 2000
). In NRPS, D-amino acid synthesis is generally catalysed by an epimerase, which is encoded by an epimerization domain at the carboxy-terminal end of the thiolation domain (Stein et al., 1995
). Our results indicated that a glutamic acid racemase, McyF, is responsible for the production of D-Glu.
On the other hand, a sequence similarity search of D-amino acid racemases from bacteria and fungi, which are available in the GenBank, EMBL and DDBJ databases, was carried out using the program in GENETYX-MAC (see Methods). Our results revealed that McyF has 22·430·1% sequence similarity to aspartate racemase, 16·923·7% similarity to glutamate racemase, and 15·121·2% similarity to alanine racemase. The phylogenetic relationships between these racemase genes were analysed by the neighbour-joining method, using GENETYX-MAC, and the resulting dendrogram is shown in Fig. 5. These racemase genes were tightly clustered, except for the glutamate racemase of Haemophilus influenzae. D-Amino acid racemases can be roughly divided into two groups, namely the Ala type and the Glu type. Aspartate racemases belong to the glutamate racemase cluster. Two cysteine residues, which are thought to form the catalytic centre, were highly conserved among the aspartate and glutamate racemases, as well as among their surrounding amino acid sequences (Nishizawa et al., 2000
).
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ACKNOWLEDGEMENTS |
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Received 3 October 2000;
revised 6 December 2000;
accepted 29 January 2001.