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The organization of the leuC, leuD and leuB genes of the extreme thermophile Thermus thermophilus
Gene 222 (1998) 125–132
The organization of the leuC, leuD and leuB genes of the extreme thermophile Thermus thermophilus Masatada Tamakoshi, Akihiko Yamagishi *, Tairo Oshima Department of Molecular Biology, Tokyo University of Pharmacy & Life Science, 1432 Horinouchi, Hachioji, Tokyo 192-0392, Japan Received 27 May 1998; received in revised form 3 September 1998; accepted 3 September 1998
Abstract 3-Isopropylmalate dehydrogenase is encoded by leuB gene while leuC and leuB genes encode the large and small subunits of isopropylmalate isomerase in leucine biosynthetic pathway, respectively. Organization of the leuB, leuC and leuD genes of an extreme thermophile, Thermus thermophilus, was investigated by sequence analysis. Location of the genes was also tested by complementation analysis of leu deficiency of the thermophile and Escherichia coli. The order was the leuC, leuD, and leuB genes and, in contrast to a previous report, they did not overlap with each other. Sequence analysis of the leuC and leuD genes suggested that cysteine residues for iron–sulfur binding and other amino acid residues involved in isomerase activity, which have been inferred from analysis of a related protein, aconitase, were highly conserved. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Complementation analysis; Isopropylmalate dehydrogenase; Isopropylmalate isomerase; Leucine biosynthetic pathway; Sequence analysis
1. Introduction Leucine biosynthetic genes of Escherichia coli (Somers et al., 1973), Salmonella typhimurium (Margolin, 1963), Bacillus subtilis ( Ward and Zahler, 1973), and Lactococcus lactis (Godon et al., 1992) are similarly organized, consisting of four structural genes in a single operon in the order leuABCD containing the regulatory region for the attenuation mechanism (Gemmill et al., 1979; Wessler and Calvo, 1981). These four contiguous structural genes are transcribed as a unit and thus comprise an operon (Burns et al., 1966). The leuA and leuB genes encode 2-isopropylmalate synthetase and 3-isopropylmalate dehydrogenase, respectively, while the leuC and leuD genes encode the large and small subunits * Corresponding author. Tel: +81 426 76 7139; fax: +81 426 76 7145; e-mail: [email protected]. Abbreviations: E. coli, Escherichia coli; IPMDH, 3-isopropylmalate dehydrogenase; IPMI, isopropylmalate isomerase; kb, kilobase(s); leu, leucine biosynthetic operon; leuA, gene encoding 2-isopropylmalate synthase; leuB, gene encoding 3-isopropylmalate dehydrogenase; leuC, gene encoding isopropylmalate isomerase large subunit; leuD, gene encoding isopropylmalate isomerase small subunit; ORF, open reading frame; pyrE, gene encoding orotate phosphoribosyltransferase; rpoD1, gene encoding a sigma factor in a cyanobacterium; T. thermophilus, Thermus thermophilus. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 48 2 - X
of isopropylmalate isomerase (IPMI ), respectively. It is notable that fungal IPMIs are monomeric enzymes (Bigelis and Umbarger, 1975; Reichenbecher and Gross, 1978) whereas a bacterial IPMI is a heterodimer containing products of the leuC and leuD genes ( Fultz and Kemper, 1981). In some organisms, the organization of the leucine biosynthetic genes is different from that described above. For example, the leuB gene is not located near other leu genes in Leptospira interrogans serovar pomona (Ding and Yelton, 1993). In Azotobactor vinelandii, leuC and leuD genes comprise an operon and the leuB gene is near the leuD gene, but there are separate promoters for the leuCD genes and for the leuB gene. The leuA gene was not detected near these genes (Manna and Das, 1997). In Microystis aeruginosa K-81, leuA gene is located in the region upstream of rpoD1 gene, which encodes a principal sigma factor in the cyanobacterium, and there is no stem–loop structure for attenuation upstream of the leuA gene (Asayama et al., 1997). Although the leucine biosynthetic genes except for the leuA gene of Thermus thermophilus have been cloned (Nagahari et al., 1980; Tanaka et al., 1981), the locations of leuC and leuD genes have not been established. Croft et al. have suggested that the order of the leu genes in T. thermophilus is D, C–B, or C, D–B, where the leuB
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and leuC or leuB and leuD genes overlap with each other or are encoded by the same sequence (Croft et al., 1987). However, leuB gene from a closely related thermophile, Thermus aquaticus YT-1, does not overlap with leuD gene ( Kirino and Oshima, 1991). In this report we investigated the location of the leuC and leuD genes of T. thermophilus. The amino acid sequences were also compared with the sequences from other species.
2. Materials and methods 2.1. Bacterial strains, plasmids and phages The strains of T. thermophilus and E. coli, plasmids and phase DNAs are listed in Table 1. Media for T. thermophilus have been described ( Tanaka et al., 1981) and solidification of the media was performed as described perviously ( Takada et al., 1993). 2.2. DNA manipulations All the routine DNA manipulations, i.e. plasmid preparation, subcloning, agarose gel electrophoresis, were done essentially as described by Sambrook et al. (1989). Restriction endonuclease and DNA modification enzymes were purchased from Toyobo Biochemicals (Osaka, Japan) or Takara Shuzo ( Kyoto, Japan) and used as recommended by the manufacturers. Site-directed mutagenesis with oligonucleotide was performed by the method of Kunkel (1985). Oligonucleotides used for site-directed mutagenesis are listed in Table 2. T. thermophilus MT106 was genetically transformed as described by Koyama et al. (1986). 2.3. Plasmid constructions pJP1232 was constructed as follows. A 0.45 kb BamHI fragment upstream of the leuB gene was cloned in an E. coli vector, pUC119, in the same orientation as pTH4, then the HindIII–BglII fragment in the BamHI fragment was replaced with the HindIII–BglII fragment of pTH4. In order to construct pITB1 and pITB2, pBL5C was partially digested with BamHI restriction endonuclease, followed by the fill-in reaction with Klenow fragment in the presence of the four deoxynucleotides and ligation reaction. 2.4. DNA sequence analysis The DNA fragments digested with appropriate restriction enzymes were subcloned in pUC118 or pUC119 to prepare sequencing templates. The single-stranded DNAs were prepared as described by Messing ( Vieira and Messing, 1987). The DNA sequences were deter-
mined by the dideoxy chain-termination method (Sanger et al., 1977) using an ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit, with an ABI 310 DNA sequencer.
3. Results 3.1. DNA sequencing of T. thermophilus leuC and leuD genes We have previously cloned the HindIII fragment harboring the leuB gene from T. thermophilus ( Tanaka et al., 1981). The DNA fragment cloned in pTH4 was digested with the restriction enzymes indicated in Fig. 1A, and subcloned into E. coli plasmid vectors pUC118 and pUC119. The DNA sequence was determined as described in Section 2.4. Two open reading frames (ORFs) were found between the leuB gene and the small ORF for putative attenuation regulation (Croft et al., 1987). The termination codon TGA of the second ORF overlapped the initiation codon of the leuB gene ( Fig. 1B). Amino acid sequences expected from the two ORFs showed strong similarity to the large and small subunit of isopropylmalate isomerases (IPMIs) from various species (Fig. 2 and Fig. 3). 3.2. Organization of the IPMI genes In order to determine the location of genes encoding the IPMI of T. thermophilus, we examined the ability of the leu operon sequence to complement leuC and leuD mutants of E. coli. It has been shown that the leu operon sequence has a weak promoter which is functional in E. coli and the 5.8 kb HindIII fragment complements the leuB, leuC and leuD mutation of E. coli in an orientationindependent manner (Nagahari et al., 1980; Tanaka et al., 1981; Croft et al., 1987). It was confirmed in the experiment with plasmid pTH4 (Fig. 4). We have previously constructed integration vectors pIW and pINV for T. thermophilus ( Tamakoshi et al., 1997). Both of them contain the leu operon sequence upstream of the leuB gene including the leu promoter sequence. The marker genes are different between these two plasmids; pINV contains pyrE gene as a marker instead of the leuB gene of pIW (Fig. 4). Both plasmids complemented leuC and leuD mutants of E. coli, while only pIW could complement leuB mutant. These results are compatible with the organization of the leu genes shown in Fig. 1. However, pJP1232, which lacked the sequence from the third BamHI site to the start codon of the leuB gene, could not complement any of the mutants tested. The sequence just upstream of the leuB gene seemed to be necessary to complement the leuC or leuD deficiency of E. coli.
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M. Tamakoshi et al. / Gene 222 (1998) 125–132 Table 1 Bacterial strains, plasmids, phages and phage DNAs used in this study Strain or DNA
Description/genotype
Source/reference
the BamHI fragment encoding the leuB gene was completely deleted
Tamakoshi et al. (1995)
F+ leuA371 F+ leuC222 F+ leuD211 supE endA sbcB15 hsdR4 rpsL thi leuB D(lac-proAB) F∞[traD36 proAB lacI q lacZDM15]
Somers et al. (1973) Somers et al. (1973) Somers et al. (1973) this laboratory
Ampr Ampr Ampr Ampr leuB+ leuC+ leuD+ Ampr leuB+ leuC+ leuD+ Ampr leuB+ leuC+ leuD+ Ampr leuB+ Ampr, an integration vector E. coli leuB gene in M13mp18 T. thermophilus leuB gene in M13mp18 Helper phage for single-strand DNA production
Vieira and Messing (1987) Vieira and Messing (1987) Alting-Mees et al. (1992) Tamakoshi et al. (1995) Tamakoshi et al. (1995) Tamakoshi et al. (1995) Tamakoshi et al. (1997) Akanuma et al. (1996) Kirino et al. (1994) Tamakoshi et al. (1997) Vieira and Messing (1987)
Thermus thermophilus MT106 Escherichia coli CV512 CV522 CV524 OM17
Plasmid, phase or phage DNA pUC118 pUC119 pBluescript SKII+ pTH4 pIW pINV pBL5C pIT1 M13D17N NVleuB M13K07
Table 2 Oligonucleotides used in this study Oligonucleotide
Sequence
ELINde
5∞-TAATTCTTCGACATATGACGGTTT CCTTG-3∞ 5∞-TCGATGTACATGTGCGCCAGTT-3∞ 5∞-AACGTCTTAGCCCGGGTTACCCCTTC-3∞ 5∞-GTAAAACGACGGCCAGT-3∞
ELDNde ELTSma Universal primer
Note: Restriction endonuclease recognition sites, NdeI and SmaI, are underlined. ELINde, ELDNde and ELTSma were used for the sitedirected mutagenesis. The universal primer was used for the DNA sequence analysis.
3.3. Complementation analysis in T. thermophilus The requirement of the correct sequence around the BamHI site for the leucine biosynthesis in T. thermophilus was examined with integration vectors pITB1 and pITB2. pITB1 and pITB2 have the same sequence except that the nearest upstream or downstream BamHI restriction site was disrupted in pITB1 or pITB2, respectively, to cause a frameshift. pITB2 could transform MT106, in which the BamHI fragment containing the leuB gene was totally deleted (Tamakoshi et al., 1995), into Leu+ whereas pITB1 could not ( Fig. 5). The result suggests that the ORF beyond the nearest upstream BamHI site is responsible for leucine biosynthesis also in T. thermophilus.
3.4. Expression of the leuB gene of E. coli in T. thermophilus 3-Isopropylmalate dehydrogenase (IPMDH ) and IPMI are encoded by different genes in E. coli. IPMDH is encoded by leuB gene and leuC and leuD genes encode large and small subunits of IMPI, respectively. To express the leuB gene of E. coli in place of the leuB gene of T. thermophilus in the thermophile without influencing the other genes, we constructed the integration vector pITEleuB in which the T. thermophilus leuB gene was replaced with the E. coli leuB gene from the initiation codon to a site close to the termination codon as described in Fig. 6. Then the DleuB strain MT106 was transformed with pITEleuB. The transformants could grow on a minimal plate at 53°C without leucine. This result clearly indicates that the leuB gene does not overlap with the leuC or leuD genes in T. thermophilus.
4. Discussion In this paper we have investigated the location of the leuC and leuD genes of T. thermophilus by sequence analysis and complementation analysis of the thermophile as well as E. coli. The complementation analysis of T. thermophilus with pITB1 and pITB2 indicated that there is a gene involved in the leucine biosynthetic pathway beyond the nearest BamHI site upstream of the leuB gene (Fig. 1). DNA sequencing analysis
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Fig. 1. (A) Sequencing strategy and physical map of pTH4. Restriction endonuclease sites: H, HindIII: Sp, SphI: Sm, SmaI: B, BamHI: Xh, XhoI: Bg, BglII. Open circle indicates the promoter and small ORF for putative attenuation regulation (Croft et al., 1987). (B) The nucleotide sequence between the initiation codon of the leuB gene and the nearest 5∞ BamHI site (Suzuki et al., 1997) and its deduced amino acid sequence. The BamHI site is underlined. An asterisk indicates a termination codon. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB017135.
Fig. 2. Multiple alignment of the deduced amino acid sequences of the large subunit of bacterial IPMI of selected species. Species and accession numbers of sequences are Thermus thermophilus ( T.the), Salmonella typhimurium (S.typ, X51476), Escherichia coli ( E.coli, AE000117), Bacillus subtilis (B.sub, Z75208), Lactococcus lactis (L.lac, M90761), Actinoplanes teichomyceticus (A.tei, X94647), Azotobacter vinelandii (A.vin, Y11280). Conserved amino acid residues (*) and relatively conserved residues (+) are indicated. Active site residues are dotted.
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Fig. 5. Complementation analysis of T. thermophilus MT106. T. thermophilus MT106 was transformed as described in Section 2.2. Thick lines represent sequences originated from T. thermophilus and a broken line indicates deletion. The + and − signs represent growth and non-growth, respectively, in the absence of leucine. B and (B) indicate BamHI site and the BamHI site filled-in to cause a frameshift, respectively.
Fig. 3. Multiple alignment of the deduced amino acid sequences of the small subunit of bacterial IPMI of selected species. Species and accession numbers of sequences are Thermus thermophilus ( T.the), Salmonella typhimurium (S.typ. X02528), Escherichia coli (E.col, AE000117), Bacillus subtilis (B.sub, Z75208), Lactococcus lactis (L.lac M90761), Azotobacter vinelandii (A.vin, Y11280). Conserved amino acid residues (*) and relatively conserved residues (+) are indicated. Active site residues are dotted.
revealed that it is the leuD gene encoding the small subunit of IPMI ( Fig. 3). Previously, Croft et al. (1987) have suggested that the order of the leu genes in T. thermophilus is D, C–B, or C, D–B, where the leuB and leuC or leuB and leuD genes overlap with each other or are encoded by the same sequence. These conclusions have been derived from their complementation experiments, especially the inability of the plasmid pNZ1232 to complement both leuD and leuC mutants of E. coli and the sequence data
Fig. 4. Complementation of Escherichia coli leuB, leuC and leuD mutants. E. coli leuB, leuC and leuD mutants were transformed with the plasmids listed. Plasmid pTH4 was described previously ( Tamakoshi et al., 1995). pIW and pINV are integration vectors constructed for the thermophile ( Tamakoshi et al., 1997). pJP1232 was constructed as described in Section 2.3. The + and − signs represent growth and non-growth, respectively, in the absence of leucine. Short arrows indicate the direction of transcription from a promoter sequence. Restriction sites: H, HindIII; B, BamHI; Bg, BglII.
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Fig. 6. Construction of an integration vector pITEleuB for the integration of E. coli leuB gene into T. thermophilus. NdeI and a SmaI sites were introduced at the initiation codon and at a site immediately downstream of the termination codon of the E. coli leuB gene ( Kirino et al., 1994), with the primers ELINde and ELTSma, respectively. The NdeI site within the leuB gene was eliminated with the primer ELDNde and ELTSma, respectively. The NdeI site within the leuB gene was eliminated with the primer ELDNde without changing the amino acid sequence (M13D17NV ). Phage NVleuB has been constructed previously ( Tamakoshi et al., 1997). The NdeI–EcoRV fragment including the leuB gene of T. thermophilus in NVleuB was replaced with the NVEleuB and was cloned in the BamHI site of pIT1. pITEleuB was used for the integration of the E. coli leuB gene into the chromosome of T. thermophilus. Restriction sites: N, NdeI: Sm, SmaI; B, BamHI; V, EcoRV. Sm/V means the site of ligation between SmaI and EcoRV sites.
of the 0.45 kb BamHI fragment reported by Kagawa et al. (1984). The inability of the plasmid pJP1232 harboring the same fragment as pNZ1232 to complement the leuC and leuD mutants was confirmed in our experiment. Although, the sequence of the 0.45 kb BamHI fragment has been revised (Suzuki et al., 1997), and the sequence analyzed in this report revealed the leuC and leuD genes at 3∞ region of the leuB gene. Especially the leuD gene continues beyond the BamHI site in front of the leuB gene and its termination codon overlaps with the initiation codon of the leuB gene to form an ATGA motif. Overlaps have been found in some T. thermophilus genes (Hoshino et al., 1993; Kosuge et al., 1994; Koyama and Furukawa, 1990) and are related to the possible translation coupling (Oppenheim and Yanofsky, 1980). The presence of the ORF beyond the 0.45 kb BamHI fragment is further confirmed in our complementation experiments with a T. thermophilus mutant. As shown in Section 3.2, the plasmid pJ1232 could not support the growth of leuC mutant of E. coli. The result suggests that the T. thermophilus leuC gene product cannot associate with the E. coli leuD gene product efficiently or the complex of them cannot catalyze the isomerization between 2- and 3-isopropylmalate, because the leuC gene of T. thermophilus in pJP1232 is expected to be expressed in E. coli. This result indicates the limitation of the heterologous complementation analysis.
It seems that there are some variations in the arrangement of leucine biosynthetic genes. The organization of the genes leuCDB in T. thermophilus was different from that in well-characterized organisms, such as E. coli, in which the order is leuBCD (Somers et al., 1973). Although, it seems that the genes of subunits of the enzyme IPMI are transcribed sequentially in the same operon in any bacteria. The deduced amino acid sequences of the leuC and leuD gene products showed significant homology to the equivalent polypeptides from other species ( Figs. 2 and 3). Aconitase and iron responsive protein ( IRP) are structurally related to IPMI (Hentze and Argos, 1991; Prodromou et al., 1992). The leuC and leuD gene products correspond to the three N-terminal domains and the C-terminal domain of aconitase, respectively. Active site residues have been predicted from the sequence alignment, and most of the active site residues are conserved in IPMIs, suggesting the similar reaction mechanism. They are indicated by dots in Fig. 2 and Fig. 3. Three cysteine residues involved in the ligand cysteine residues Cys-347, -407 and -410 were also found in T. thermophilus leuC product, although the presence of the ligand in IPMIs has not been confirmed yet. Additional cysteine residues Cys-127 and -221 are also conserved in all IPMIs as can be seen in Fig. 2. No or few cysteine residues have been found in some thermophilic enzymes, in contrast to the counterparts of mesophiles ( Kagawa et al., 1984; Koyama and Furukawa,
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1990), except for the case of metal binding (Mather et al., 1991; Nureki et al., 1991). The absence of cysteine residue is expected to contribute to increase the stability of thermophile proteins since cysteine residue is unstable at higher temperatures in the presence of free oxygen ( Volkin and Klibanov, 1987). Accordingly, these two conserved cysteine residues may have some structural or functional roles in IPMIs.
Acknowledgement This work was supported by Grants-in-Aids for Scientific Research from the Ministry of Education, Science, and Culture of Japan (No. 09780552).
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The organization of the leuC, leuD and leuB genes of the extreme thermophile Thermus thermophilus Masatada Tamakoshi, Akihiko Yamagishi *, Tairo Oshima Department of Molecular Biology, Tokyo University of Pharmacy & Life Science, 1432 Horinouchi, Hachioji, Tokyo 192-0392, Japan Received 27 May 1998; received in revised form 3 September 1998; accepted 3 September 1998
Abstract 3-Isopropylmalate dehydrogenase is encoded by leuB gene while leuC and leuB genes encode the large and small subunits of isopropylmalate isomerase in leucine biosynthetic pathway, respectively. Organization of the leuB, leuC and leuD genes of an extreme thermophile, Thermus thermophilus, was investigated by sequence analysis. Location of the genes was also tested by complementation analysis of leu deficiency of the thermophile and Escherichia coli. The order was the leuC, leuD, and leuB genes and, in contrast to a previous report, they did not overlap with each other. Sequence analysis of the leuC and leuD genes suggested that cysteine residues for iron–sulfur binding and other amino acid residues involved in isomerase activity, which have been inferred from analysis of a related protein, aconitase, were highly conserved. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Complementation analysis; Isopropylmalate dehydrogenase; Isopropylmalate isomerase; Leucine biosynthetic pathway; Sequence analysis
1. Introduction Leucine biosynthetic genes of Escherichia coli (Somers et al., 1973), Salmonella typhimurium (Margolin, 1963), Bacillus subtilis ( Ward and Zahler, 1973), and Lactococcus lactis (Godon et al., 1992) are similarly organized, consisting of four structural genes in a single operon in the order leuABCD containing the regulatory region for the attenuation mechanism (Gemmill et al., 1979; Wessler and Calvo, 1981). These four contiguous structural genes are transcribed as a unit and thus comprise an operon (Burns et al., 1966). The leuA and leuB genes encode 2-isopropylmalate synthetase and 3-isopropylmalate dehydrogenase, respectively, while the leuC and leuD genes encode the large and small subunits * Corresponding author. Tel: +81 426 76 7139; fax: +81 426 76 7145; e-mail: [email protected]. Abbreviations: E. coli, Escherichia coli; IPMDH, 3-isopropylmalate dehydrogenase; IPMI, isopropylmalate isomerase; kb, kilobase(s); leu, leucine biosynthetic operon; leuA, gene encoding 2-isopropylmalate synthase; leuB, gene encoding 3-isopropylmalate dehydrogenase; leuC, gene encoding isopropylmalate isomerase large subunit; leuD, gene encoding isopropylmalate isomerase small subunit; ORF, open reading frame; pyrE, gene encoding orotate phosphoribosyltransferase; rpoD1, gene encoding a sigma factor in a cyanobacterium; T. thermophilus, Thermus thermophilus. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 48 2 - X
of isopropylmalate isomerase (IPMI ), respectively. It is notable that fungal IPMIs are monomeric enzymes (Bigelis and Umbarger, 1975; Reichenbecher and Gross, 1978) whereas a bacterial IPMI is a heterodimer containing products of the leuC and leuD genes ( Fultz and Kemper, 1981). In some organisms, the organization of the leucine biosynthetic genes is different from that described above. For example, the leuB gene is not located near other leu genes in Leptospira interrogans serovar pomona (Ding and Yelton, 1993). In Azotobactor vinelandii, leuC and leuD genes comprise an operon and the leuB gene is near the leuD gene, but there are separate promoters for the leuCD genes and for the leuB gene. The leuA gene was not detected near these genes (Manna and Das, 1997). In Microystis aeruginosa K-81, leuA gene is located in the region upstream of rpoD1 gene, which encodes a principal sigma factor in the cyanobacterium, and there is no stem–loop structure for attenuation upstream of the leuA gene (Asayama et al., 1997). Although the leucine biosynthetic genes except for the leuA gene of Thermus thermophilus have been cloned (Nagahari et al., 1980; Tanaka et al., 1981), the locations of leuC and leuD genes have not been established. Croft et al. have suggested that the order of the leu genes in T. thermophilus is D, C–B, or C, D–B, where the leuB
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and leuC or leuB and leuD genes overlap with each other or are encoded by the same sequence (Croft et al., 1987). However, leuB gene from a closely related thermophile, Thermus aquaticus YT-1, does not overlap with leuD gene ( Kirino and Oshima, 1991). In this report we investigated the location of the leuC and leuD genes of T. thermophilus. The amino acid sequences were also compared with the sequences from other species.
2. Materials and methods 2.1. Bacterial strains, plasmids and phages The strains of T. thermophilus and E. coli, plasmids and phase DNAs are listed in Table 1. Media for T. thermophilus have been described ( Tanaka et al., 1981) and solidification of the media was performed as described perviously ( Takada et al., 1993). 2.2. DNA manipulations All the routine DNA manipulations, i.e. plasmid preparation, subcloning, agarose gel electrophoresis, were done essentially as described by Sambrook et al. (1989). Restriction endonuclease and DNA modification enzymes were purchased from Toyobo Biochemicals (Osaka, Japan) or Takara Shuzo ( Kyoto, Japan) and used as recommended by the manufacturers. Site-directed mutagenesis with oligonucleotide was performed by the method of Kunkel (1985). Oligonucleotides used for site-directed mutagenesis are listed in Table 2. T. thermophilus MT106 was genetically transformed as described by Koyama et al. (1986). 2.3. Plasmid constructions pJP1232 was constructed as follows. A 0.45 kb BamHI fragment upstream of the leuB gene was cloned in an E. coli vector, pUC119, in the same orientation as pTH4, then the HindIII–BglII fragment in the BamHI fragment was replaced with the HindIII–BglII fragment of pTH4. In order to construct pITB1 and pITB2, pBL5C was partially digested with BamHI restriction endonuclease, followed by the fill-in reaction with Klenow fragment in the presence of the four deoxynucleotides and ligation reaction. 2.4. DNA sequence analysis The DNA fragments digested with appropriate restriction enzymes were subcloned in pUC118 or pUC119 to prepare sequencing templates. The single-stranded DNAs were prepared as described by Messing ( Vieira and Messing, 1987). The DNA sequences were deter-
mined by the dideoxy chain-termination method (Sanger et al., 1977) using an ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit, with an ABI 310 DNA sequencer.
3. Results 3.1. DNA sequencing of T. thermophilus leuC and leuD genes We have previously cloned the HindIII fragment harboring the leuB gene from T. thermophilus ( Tanaka et al., 1981). The DNA fragment cloned in pTH4 was digested with the restriction enzymes indicated in Fig. 1A, and subcloned into E. coli plasmid vectors pUC118 and pUC119. The DNA sequence was determined as described in Section 2.4. Two open reading frames (ORFs) were found between the leuB gene and the small ORF for putative attenuation regulation (Croft et al., 1987). The termination codon TGA of the second ORF overlapped the initiation codon of the leuB gene ( Fig. 1B). Amino acid sequences expected from the two ORFs showed strong similarity to the large and small subunit of isopropylmalate isomerases (IPMIs) from various species (Fig. 2 and Fig. 3). 3.2. Organization of the IPMI genes In order to determine the location of genes encoding the IPMI of T. thermophilus, we examined the ability of the leu operon sequence to complement leuC and leuD mutants of E. coli. It has been shown that the leu operon sequence has a weak promoter which is functional in E. coli and the 5.8 kb HindIII fragment complements the leuB, leuC and leuD mutation of E. coli in an orientationindependent manner (Nagahari et al., 1980; Tanaka et al., 1981; Croft et al., 1987). It was confirmed in the experiment with plasmid pTH4 (Fig. 4). We have previously constructed integration vectors pIW and pINV for T. thermophilus ( Tamakoshi et al., 1997). Both of them contain the leu operon sequence upstream of the leuB gene including the leu promoter sequence. The marker genes are different between these two plasmids; pINV contains pyrE gene as a marker instead of the leuB gene of pIW (Fig. 4). Both plasmids complemented leuC and leuD mutants of E. coli, while only pIW could complement leuB mutant. These results are compatible with the organization of the leu genes shown in Fig. 1. However, pJP1232, which lacked the sequence from the third BamHI site to the start codon of the leuB gene, could not complement any of the mutants tested. The sequence just upstream of the leuB gene seemed to be necessary to complement the leuC or leuD deficiency of E. coli.
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M. Tamakoshi et al. / Gene 222 (1998) 125–132 Table 1 Bacterial strains, plasmids, phages and phage DNAs used in this study Strain or DNA
Description/genotype
Source/reference
the BamHI fragment encoding the leuB gene was completely deleted
Tamakoshi et al. (1995)
F+ leuA371 F+ leuC222 F+ leuD211 supE endA sbcB15 hsdR4 rpsL thi leuB D(lac-proAB) F∞[traD36 proAB lacI q lacZDM15]
Somers et al. (1973) Somers et al. (1973) Somers et al. (1973) this laboratory
Ampr Ampr Ampr Ampr leuB+ leuC+ leuD+ Ampr leuB+ leuC+ leuD+ Ampr leuB+ leuC+ leuD+ Ampr leuB+ Ampr, an integration vector E. coli leuB gene in M13mp18 T. thermophilus leuB gene in M13mp18 Helper phage for single-strand DNA production
Vieira and Messing (1987) Vieira and Messing (1987) Alting-Mees et al. (1992) Tamakoshi et al. (1995) Tamakoshi et al. (1995) Tamakoshi et al. (1995) Tamakoshi et al. (1997) Akanuma et al. (1996) Kirino et al. (1994) Tamakoshi et al. (1997) Vieira and Messing (1987)
Thermus thermophilus MT106 Escherichia coli CV512 CV522 CV524 OM17
Plasmid, phase or phage DNA pUC118 pUC119 pBluescript SKII+ pTH4 pIW pINV pBL5C pIT1 M13D17N NVleuB M13K07
Table 2 Oligonucleotides used in this study Oligonucleotide
Sequence
ELINde
5∞-TAATTCTTCGACATATGACGGTTT CCTTG-3∞ 5∞-TCGATGTACATGTGCGCCAGTT-3∞ 5∞-AACGTCTTAGCCCGGGTTACCCCTTC-3∞ 5∞-GTAAAACGACGGCCAGT-3∞
ELDNde ELTSma Universal primer
Note: Restriction endonuclease recognition sites, NdeI and SmaI, are underlined. ELINde, ELDNde and ELTSma were used for the sitedirected mutagenesis. The universal primer was used for the DNA sequence analysis.
3.3. Complementation analysis in T. thermophilus The requirement of the correct sequence around the BamHI site for the leucine biosynthesis in T. thermophilus was examined with integration vectors pITB1 and pITB2. pITB1 and pITB2 have the same sequence except that the nearest upstream or downstream BamHI restriction site was disrupted in pITB1 or pITB2, respectively, to cause a frameshift. pITB2 could transform MT106, in which the BamHI fragment containing the leuB gene was totally deleted (Tamakoshi et al., 1995), into Leu+ whereas pITB1 could not ( Fig. 5). The result suggests that the ORF beyond the nearest upstream BamHI site is responsible for leucine biosynthesis also in T. thermophilus.
3.4. Expression of the leuB gene of E. coli in T. thermophilus 3-Isopropylmalate dehydrogenase (IPMDH ) and IPMI are encoded by different genes in E. coli. IPMDH is encoded by leuB gene and leuC and leuD genes encode large and small subunits of IMPI, respectively. To express the leuB gene of E. coli in place of the leuB gene of T. thermophilus in the thermophile without influencing the other genes, we constructed the integration vector pITEleuB in which the T. thermophilus leuB gene was replaced with the E. coli leuB gene from the initiation codon to a site close to the termination codon as described in Fig. 6. Then the DleuB strain MT106 was transformed with pITEleuB. The transformants could grow on a minimal plate at 53°C without leucine. This result clearly indicates that the leuB gene does not overlap with the leuC or leuD genes in T. thermophilus.
4. Discussion In this paper we have investigated the location of the leuC and leuD genes of T. thermophilus by sequence analysis and complementation analysis of the thermophile as well as E. coli. The complementation analysis of T. thermophilus with pITB1 and pITB2 indicated that there is a gene involved in the leucine biosynthetic pathway beyond the nearest BamHI site upstream of the leuB gene (Fig. 1). DNA sequencing analysis
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Fig. 1. (A) Sequencing strategy and physical map of pTH4. Restriction endonuclease sites: H, HindIII: Sp, SphI: Sm, SmaI: B, BamHI: Xh, XhoI: Bg, BglII. Open circle indicates the promoter and small ORF for putative attenuation regulation (Croft et al., 1987). (B) The nucleotide sequence between the initiation codon of the leuB gene and the nearest 5∞ BamHI site (Suzuki et al., 1997) and its deduced amino acid sequence. The BamHI site is underlined. An asterisk indicates a termination codon. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB017135.
Fig. 2. Multiple alignment of the deduced amino acid sequences of the large subunit of bacterial IPMI of selected species. Species and accession numbers of sequences are Thermus thermophilus ( T.the), Salmonella typhimurium (S.typ, X51476), Escherichia coli ( E.coli, AE000117), Bacillus subtilis (B.sub, Z75208), Lactococcus lactis (L.lac, M90761), Actinoplanes teichomyceticus (A.tei, X94647), Azotobacter vinelandii (A.vin, Y11280). Conserved amino acid residues (*) and relatively conserved residues (+) are indicated. Active site residues are dotted.
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Fig. 5. Complementation analysis of T. thermophilus MT106. T. thermophilus MT106 was transformed as described in Section 2.2. Thick lines represent sequences originated from T. thermophilus and a broken line indicates deletion. The + and − signs represent growth and non-growth, respectively, in the absence of leucine. B and (B) indicate BamHI site and the BamHI site filled-in to cause a frameshift, respectively.
Fig. 3. Multiple alignment of the deduced amino acid sequences of the small subunit of bacterial IPMI of selected species. Species and accession numbers of sequences are Thermus thermophilus ( T.the), Salmonella typhimurium (S.typ. X02528), Escherichia coli (E.col, AE000117), Bacillus subtilis (B.sub, Z75208), Lactococcus lactis (L.lac M90761), Azotobacter vinelandii (A.vin, Y11280). Conserved amino acid residues (*) and relatively conserved residues (+) are indicated. Active site residues are dotted.
revealed that it is the leuD gene encoding the small subunit of IPMI ( Fig. 3). Previously, Croft et al. (1987) have suggested that the order of the leu genes in T. thermophilus is D, C–B, or C, D–B, where the leuB and leuC or leuB and leuD genes overlap with each other or are encoded by the same sequence. These conclusions have been derived from their complementation experiments, especially the inability of the plasmid pNZ1232 to complement both leuD and leuC mutants of E. coli and the sequence data
Fig. 4. Complementation of Escherichia coli leuB, leuC and leuD mutants. E. coli leuB, leuC and leuD mutants were transformed with the plasmids listed. Plasmid pTH4 was described previously ( Tamakoshi et al., 1995). pIW and pINV are integration vectors constructed for the thermophile ( Tamakoshi et al., 1997). pJP1232 was constructed as described in Section 2.3. The + and − signs represent growth and non-growth, respectively, in the absence of leucine. Short arrows indicate the direction of transcription from a promoter sequence. Restriction sites: H, HindIII; B, BamHI; Bg, BglII.
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Fig. 6. Construction of an integration vector pITEleuB for the integration of E. coli leuB gene into T. thermophilus. NdeI and a SmaI sites were introduced at the initiation codon and at a site immediately downstream of the termination codon of the E. coli leuB gene ( Kirino et al., 1994), with the primers ELINde and ELTSma, respectively. The NdeI site within the leuB gene was eliminated with the primer ELDNde and ELTSma, respectively. The NdeI site within the leuB gene was eliminated with the primer ELDNde without changing the amino acid sequence (M13D17NV ). Phage NVleuB has been constructed previously ( Tamakoshi et al., 1997). The NdeI–EcoRV fragment including the leuB gene of T. thermophilus in NVleuB was replaced with the NVEleuB and was cloned in the BamHI site of pIT1. pITEleuB was used for the integration of the E. coli leuB gene into the chromosome of T. thermophilus. Restriction sites: N, NdeI: Sm, SmaI; B, BamHI; V, EcoRV. Sm/V means the site of ligation between SmaI and EcoRV sites.
of the 0.45 kb BamHI fragment reported by Kagawa et al. (1984). The inability of the plasmid pJP1232 harboring the same fragment as pNZ1232 to complement the leuC and leuD mutants was confirmed in our experiment. Although, the sequence of the 0.45 kb BamHI fragment has been revised (Suzuki et al., 1997), and the sequence analyzed in this report revealed the leuC and leuD genes at 3∞ region of the leuB gene. Especially the leuD gene continues beyond the BamHI site in front of the leuB gene and its termination codon overlaps with the initiation codon of the leuB gene to form an ATGA motif. Overlaps have been found in some T. thermophilus genes (Hoshino et al., 1993; Kosuge et al., 1994; Koyama and Furukawa, 1990) and are related to the possible translation coupling (Oppenheim and Yanofsky, 1980). The presence of the ORF beyond the 0.45 kb BamHI fragment is further confirmed in our complementation experiments with a T. thermophilus mutant. As shown in Section 3.2, the plasmid pJ1232 could not support the growth of leuC mutant of E. coli. The result suggests that the T. thermophilus leuC gene product cannot associate with the E. coli leuD gene product efficiently or the complex of them cannot catalyze the isomerization between 2- and 3-isopropylmalate, because the leuC gene of T. thermophilus in pJP1232 is expected to be expressed in E. coli. This result indicates the limitation of the heterologous complementation analysis.
It seems that there are some variations in the arrangement of leucine biosynthetic genes. The organization of the genes leuCDB in T. thermophilus was different from that in well-characterized organisms, such as E. coli, in which the order is leuBCD (Somers et al., 1973). Although, it seems that the genes of subunits of the enzyme IPMI are transcribed sequentially in the same operon in any bacteria. The deduced amino acid sequences of the leuC and leuD gene products showed significant homology to the equivalent polypeptides from other species ( Figs. 2 and 3). Aconitase and iron responsive protein ( IRP) are structurally related to IPMI (Hentze and Argos, 1991; Prodromou et al., 1992). The leuC and leuD gene products correspond to the three N-terminal domains and the C-terminal domain of aconitase, respectively. Active site residues have been predicted from the sequence alignment, and most of the active site residues are conserved in IPMIs, suggesting the similar reaction mechanism. They are indicated by dots in Fig. 2 and Fig. 3. Three cysteine residues involved in the ligand cysteine residues Cys-347, -407 and -410 were also found in T. thermophilus leuC product, although the presence of the ligand in IPMIs has not been confirmed yet. Additional cysteine residues Cys-127 and -221 are also conserved in all IPMIs as can be seen in Fig. 2. No or few cysteine residues have been found in some thermophilic enzymes, in contrast to the counterparts of mesophiles ( Kagawa et al., 1984; Koyama and Furukawa,
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1990), except for the case of metal binding (Mather et al., 1991; Nureki et al., 1991). The absence of cysteine residue is expected to contribute to increase the stability of thermophile proteins since cysteine residue is unstable at higher temperatures in the presence of free oxygen ( Volkin and Klibanov, 1987). Accordingly, these two conserved cysteine residues may have some structural or functional roles in IPMIs.
Acknowledgement This work was supported by Grants-in-Aids for Scientific Research from the Ministry of Education, Science, and Culture of Japan (No. 09780552).
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