Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan1
Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma 370-1292, Japan2
Author for correspondence: Hiromi Nishida. Tel: +81 3 5841 7828. Fax: +81 3 5841 8490. e-mail: hnishida{at}iam.u-tokyo.ac.jp
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ABSTRACT |
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Keywords: Deinococcus radiodurans, Thermus thermophilus, lysine biosynthesis, gene disruption, evolution
Abbreviations: DAP, diaminopimelate
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INTRODUCTION |
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Lysine biosynthesis via the aminoadipate pathway has been suggested for an extremely thermophilic anaerobic member of the Archaea, Thermoproteus neutrophilus; the presence of this pathway in this organism was inferred from acetate-assimilation patterns (Schäfer et al., 1989 ). In addition, another extremely thermophilic anaerobic member of the Archaea, Pyrococcus, has a gene cluster similar to that responsible for lysine biosynthesis in Thermus thermophilus (Nishida et al., 1999
). At the present time, no bacterium other than T. thermophilus is known to synthesize lysine via the aminoadipate pathway, and no organism is known to synthesize lysine through pathways other than the aminoadipate or DAP pathways.
The complete nucleotide sequence of the Deinococcus radiodurans genome has been determined (White et al., 1999 ). Genome analysis has revealed that approximately half of all of the ORFs identified in D. radiodurans encode proteins of unknown function. It has been remarked that the absence of key enzymes for lysine biosynthesis via the DAP pathway in D. radiodurans is a puzzling feature, since this organism does not require lysine for growth (Makarova et al., 2001
). No gene cluster similar to the T. thermophilus lys gene cluster is seen within the D. radiodurans genome sequence, although genes homologous to the lys cluster are found in an unco-ordinated fashion within the genome (Nishida, 2001a
; Miyazaki et al., 2001
). The existence of this set of homologous genes suggests that D. radiodurans synthesizes lysine in the same way as T. thermophilus. However, D. radiodurans also possesses a homologue of lysA, DR1758. In almost all bacteria, the product of lysA expression catalyses the decarboxylation of DAP in the last step of the typical DAP pathway for lysine biosynthesis (Miyazaki et al., 2001
).
The deduced amino-acid sequence of another D. radiodurans gene, DR1420, shows 68% identity to that of T. thermophilus lysZ, whose product is assumed to function as an N-acetylaminoadipate kinase based on sequence similarity with other proteins and the auxotrophic nature of strains mutated in this gene (Nishida et al., 1999 ). The deduced amino-acid sequence of DR1758 shows 32% identity to the DAP decarboxylase (LysA) of Escherichia coli.
The aim of this study was to disrupt DR1420 (the lysZ homologue) and/or DR1758 (the lysA homologue) in D. radiodurans and to test the requirement of lysine for growth in mutants carrying the disrupted gene(s).
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METHODS |
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Disruption of DR1420 in D. radiodurans.
This was performed by using the direct insertional mutagenesis technique described by Funayama et al. (1999) with slight modifications. First, a 1·4 kb DNA fragment containing DR1420 was amplified by PCR using the primers 5'-CGAGGCCGGGGCAACGCGGGAAGG-3' and 5'-ATGCGGTCCTGCGCGAATTCGAGG-3'. Since DR1420 has a single Eco47III site (AGC
GCT), we designed the PCR primers so that the Eco47III site was at the middle of the amplified fragment. The amplification was carried out using PCR Beads (Amersham Pharmacia Biotech) and the following protocol: denaturation at 95 °C for 5 min, followed by 30 cycles at 96 °C for 1 min and 60 °C for 1 min, with a final extension at 60 °C for 10 min. The PCR product was then cloned into the pDrive Cloning Vector (Qiagen). Plasmid pDrive was digested with Eco47III; the linearized plasmid was then ligated to a 0·9 kb HincII fragment containing the chloramphenicol-resistance gene (cat) from pKatCAT (Funayama et al., 1999
). We then performed a PCR using the resulting plasmid as the template and the same primers as described above. The PCR product was purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech). It was then used to transform D. radiodurans.
D. radiodurans was cultured in TGY broth (2 ml) until an OD660 value of 0·6 was reached. The cells were collected by centrifugation (7000 r.p.m. for 5 min), washed in 2 ml of TGY broth, resuspended in 200 µl of TGY broth and then mixed with 80 µl of 0·3 M CaCl2. Aliquots (30 µl) of the suspension were added to 10 µl (0·5 µg) of the PCR product prepared above, and the mixture was incubated at 30 °C. After 90 min incubation, 2 ml of TGY broth was added to the sample and this was incubated for an additional 24 h. Finally, 200 µl of the suspension was spread onto a TGY plate containing chloramphenicol. Colonies that grew on the plate were picked up as candidate strains with a knockout in the DR1420 gene. Disruption of DR1420 was confirmed by PCR using the primers 5'-ACGCCATAACCGCCATGATA-3' and 5'-TCTAGGAAACCACAGTCC-3', the positions of which in the genomic sequence of D. radiodurans were just outside those of the primers used for constructing the disruption.
Disruption of DR1758 in D. radiodurans.
The DR1758 gene has a single HincII [GT(C,T)(A,G)AC] site. We performed the PCR amplification so that a 1·2 kb DNA fragment containing the HincII site at the middle of the fragment would be generated. The PCR primers used were 5'-AAGGTAAGTGCTGCTCAT-3' and 5'-GACATGGCGCACAGCACC-3'; amplification was carried out using the same protocol as described for the disruption of DR1420. The PCR product was cloned into the pCR2.1 Vector (Invitrogen); the plasmid was then digested with HincII. The linearized plasmid was ligated with a 1·0 kb HincII fragment from pKatAPH2, a kanamycin-resistant version of pKatCAT. Using the resulting plasmid as the template, PCR was performed with the primers described above. The amplified DNA fragment was introduced into D. radiodurans ATCC 13939T and the DR1420 disruptant to generate a DR1758 disruptant and a DR1420/DR1758 double-disruptant, respectively. Disruption was confirmed by PCR, using primers 5'-GGGCCCAGCTTAAAGCGC-3' and 5'-ATCATCGCGCCGATGACC-3'.
Auxotrophic complementation test.
The mutants were cultured in TGY broth for 24 h. After harvesting of the cultures by centrifugation, the cell pellets were washed three times with a minimal medium developed for D. radiodurans (Venkateswaran et al., 2000 ) that does not contain lysine. The cells were then cultured in this minimal medium at 27 °C for 2 days. The optical density of the cultures was determined at 660 nm, using a spectrophotometer.
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RESULTS AND DISCUSSION |
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It was also shown that the lysA homologue DR1758 was not essential for lysine biosynthesis in D. radiodurans (Table 1). This result was expected, because other genes encoding key enzymes for lysine biosynthesis via the DAP pathway are absent from the D. radiodurans genome. It was also found that DR1758 was not related to genes involved in the aminoadipate pathway for lysine biosynthesis. Thus, at present, it is difficult to infer the reason as to why D. radiodurans has a lysA homologue. The DR1420/DR1758 double disruptant could also grow in the minimal medium used in this study (Table 1
).
We have shown that the system for lysine biosynthesis in D. radiodurans is not identical to that of T. thermophilus, even though the former has homologues of the genes involved in the lysine biosynthetic pathway of the latter. Through further studies, including a combination of gene-disruption and biochemical analyses, we hope to elucidate the nature of the lysine biosynthetic pathway in D. radiodurans and to elucidate the evolutionary relationship between the aminoadipate pathway and the DAP pathway.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 5 February 2002;
revised 8 April 2002;
accepted 17 May 2002.
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