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Keywords:

  • Thermus thermophilus;
  • Gene replacement;
  • Gene deletion;
  • pyrE;
  • leuB

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

A Thermus thermophilus host strain of which the leuB gene was totally deleted was constructed from a ΔpyrE strain by a two step method. First, the leuB gene was replaced with the pyrE gene. Second, the inserted pyrE gene was deleted by using 5-fluoroorotic acid. A plasmid vector with the leuB marker was constructed and the plasmid complemented the leuB deficiency of the host. When the leuB gene from Escherichia coli and its derivative encoding a stabilized enzyme were expressed with the host-vector system, their growth temperature reflected the stability of the enzyme. These results suggest that the gene replacement deletion method using the pyrE gene is useful for the construction of a reliable plasmid vector system and it can be applied to the selection of stabilized enzymes.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Stable enzymes are useful not only for industrial application but also for the research in structural biology. Thermophilic organisms can be used as hosts for the selection of stabilized enzymes. We have previously developed an integration vector system for an extreme thermophile, Thermus thermophilus, and isolated stabilized 3-isopropylmalate dehydrogenases with the system by evolutionary molecular engineering [1–4].

Several plasmid vectors for T. thermophilus have also been constructed by other groups [5–8]. To circumvent the homologous recombination, genes not found in T. thermophilus were used as markers in their systems. However, it is desirable to delete a gene of interest from a T. thermophilus chromosome for a reliable molecular handling although a general method to delete a gene from the organism has not been reported yet.

Sequential disruption of multiple genes has been described with lower eukaryotes by using genes which are involved in the de novo pyrimidine biosynthetic pathway [9–12]. Their deficiency leads to resistance to the bactericidal compound 5-fluoroorotic acid (5-FOA) while the compound is toxic to wild-type cells. We have previously constructed a strain, KT8, in which the pyrE gene (the gene encoding orotate phosphoribosyltransferase) is completely deleted [13]. The pyrE strain shows uracil auxotrophy and 5-FOA resistance. In this study, we replaced a target gene with the pyrE gene in a ΔpyrE strain and regenerated the complete deletion of the pyrE gene. We expressed a gene from a mesophile with a plasmid vector constructed in the host.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Strains and media used

The strains of T. thermophilus and Escherichia coli and plasmids used in this study are listed in Table 1. The rich growth medium and minimum medium for T. thermophilus were described previously [18]. Leucine and isoleucine, 20 μg ml−1 each, were included in the leucine medium [4]. Uracil (20 μg ml−1) and 5-FOA (200 μg ml−1, Sigma) were included in 5-FOA medium [13]. Solidification of media was performed by mixing the double strength media and the 1% Gelrite solution after autoclave sterilization as described previously [18].

Table 1.  Bacterial strains, plasmids and phage DNAs used in this study
Strain or DNADescription/genotypeSource or reference
Strain  
E. coli JM109recA1 endA1 gyrA96 thi supE44[14]
 relA1 hsdR17Δ(lac-proAB) 
 F′(traD36 proAB+lacIqlacZΔM15) 
T. thermophilus MT111ΔpyrEThis study
Plasmids and phage DNA  
pINVAmprpyrE+leuC+leuD+[13]
pITleuBNVAmprleuB+[13]
pIWAmprleuB+leuC+leuD+[13]
pTT8Cryptic[15]
pUC118Ampr[16]
pUC119Ampr[16]
M13D17NVE. coli leuB gene in M13mp18[17]

2.2DNA manipulations

All routine DNA manipulations, plasmid preparations and subcloning in E. coli were done essentially as described by Sambrook et al. [19]. T. thermophilus strains were transformed as described previously [20]. Restriction endonucleases and DNA modification enzymes were used as recommended by the manufacturers. Site-directed mutagenesis was performed as described previously [21]. Oligonucleotides used for site-directed mutagenesis are listed in Table 2.

Table 2.  Oligonucleotides used in this study
TPR-S5′-AGCTTGGTCTTGACAATCCGCCCCTTAGAGTGTATCATGGGGGCAT-3′
  1. Note: TPR-S and TPR-AS were used for the construction of a promoter functional in T. thermophilus. HindIII- and XbaI-termini are formed by annealing of these fragments. IEV and TEV were used for the construction of pTH3ΔEcoRV by site-directed mutagenesis. Restriction endonuclease EcoRV recognition sites are underlined.

TPR-AS5′-CTAGATGCCCCCATGATACACTCTAAGGGGCGGATTGTCAAGACCA-3′
IEV5′-ACGGCCACCTTGATATCGTCCTCCTGGGG-3′
TEV5′-CCCCATCTTAGGATATCTGGCGGAGGAC-3′

Southern blotting was performed according to the protocol described by Sambrook et al. [19]. DNA was capillary-transferred to a Nylon membrane (Hybond N+, Amersham) using 0.4 N NaOH as a transfer buffer, overnight. The preparation of labelled probes and detection were performed using the ECL random prime labelling and detection system (Amersham).

2.3Plasmid constructions

pITDpyrE, a plasmid used for the replacement of the leuB gene encoding 3-isopropylmalate dehydrogenase in T. thermophilus, was constructed as shown in Fig. 1. The NdeI-EcoRV fragment containing the leuB gene was replaced with the NdeI-EcoRV pyrE gene fragment of pINV. The resulted 0.7-kbp BamHI fragment containing the pyrE gene was cloned in the BamHI site of the leu operon in the same direction as the construct of pITDpyrE.

image

Figure 1. Construction of the plasmid pITDpyrE used for replacement of the leuB gene with the pyrE gene. The thin lines and thick lines represent E. coli cloning vectors and sequences originated from T. thermophilus, respectively. Solid and open arrows indicate the leuB and pyrE genes of T. thermophilus, respectively. Restriction sites: B, BamHI; N, NdeI; V, EcoRV.

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The plasmid pTH3ΔEcoRV used to delete the pyrE gene from the leuB locus was constructed as shown in Fig. 2. Two EcoRV sites were introduced around the initiation and the termination codons of the leuB gene with the primers IEV and TEV in the fragment that encodes the leu operon cloned from T. thermophilus HB8 by site-directed mutagenesis. The resulting plasmid was digested with EcoRV and self-ligated.

image

Figure 2. Construction of a ΔleuB plasmid, pTH3ΔEcoRV, for gene targeting of the integrated pyrE gene. It was used to transform T. thermophilus MT115 to construct a ΔleuBΔpyrE strain, TTY1. Sequences of the nucleotides around the termination codon of the leuD gene and the initiation codon of the leuB gene are shown. Sequences of the amino acids encoded by them are also shown in A and C. Thin and thick lines represent E. coli vectors and fragments of T. thermophilus DNA, respectively. The EcoRV sites are underlined. An asterisk indicates a termination codon. Restriction sites: H, HindIII; B, BamHI; V, EcoRV.

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An E. coli-T. thermophilus shuttle vector, pT8leuB, was constructed by cloning three fragments sequentially into pUC118 (Fig. 3): (i) a synthetic promoter which has HindIII- and XbaI-termini described in Fig. 3 (B), (ii) the NdeI-EcoRI fragment containing the T. thermophilus leuB gene with a NdeI-XbaI linker of which the sequence is shown in Fig. 3B and (iii) a cryptic plasmid, pTT8, from T. thermophilus HB8 after digestion with BglII and cloning in the BamHI site of pUC119.

image

Figure 3. Construction of an autonomously replicating plasmid vector for T. thermophilus. (A) Structure of pT8leuB. Thin and thick lines represent an E. coli cloning vector and a fragment of T. thermophilus plasmid pTT8, respectively. An open circle indicates the synthetic promoter. Restriction sites: X′ and X, XbaI; K, KpnI; P, PstI; Bc, BclI; N, NdeI; V, EcoRV; E, EcoRI. X′ is regenerated by the ligation of blunt-ended HindIII of pUC118 and XbaI of pUC119. SaKSm indicates SacI-KpnI-SmaI sites from the multiple cloning site of pUC119. (B) The DNA sequence around the synthetic promoter region. The sequences upstream of the −35 region, between the −35 and −10 regions and downstream of the −10 region were designed based on three T. thermophilus promoters, P43, P214 and P31, reported [23]. The corresponding sequences are underlined. Two synthetic oligonucleotides, TPR-S and TPR-AS, were used for a T. thermophilus plasmid vector construction. The −35 and −10 regions are shown by dotted letters.

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pT8EleuB, a plasmid for expression of the wild-type E. coli leuB gene in T. thermophilus, was constructed by replacing the NdeI-EcoRV fragment containing the T. thermophilus leuB gene of pT8leuB with a NdeI-SmaI fragment containing the E. coli leuB gene reported previously [17]. pT8ELMV, a plasmid for the expression of a mutant E. coli leuB gene, was constructed essentially in the same way after the introduction of mutations Glu-256Leu and Met-259Val into the E. coli leuB gene as described previously [22].

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Deletion of the leuB gene from the chromosome

A strategy to construct a ΔleuBΔpyrE strain of T. thermophilus consisted of two steps (Fig. 4A): (1) replacement of the leuB gene of a ΔpyrE strain with the pyrE gene and (2) deletion of the pyrE gene.

image

Figure 4. Th construction of mutant strains of T. thermophilus. (A) Physical maps of T. thermophilus strains. MT111 has the deletion of the pyrE gene. MT111 was derived from KT8 [13] by repairing the mutation in the leu operon with the wild-type leu operon sequence. MT115 was one of the transformants of MT111 with pITDpyrE. TTY1 is one of the transformants of MT115 with pTH3ΔEcoRV. Broken lines and a double slash indicate deletion and separation of these two loci on the chromosome, respectively. Black and open arrows indicate the leu and pyrE genes, respectively. Restriction sites: Bg, BglII; B, BamHI; K, KpnI; N, NdeI; V, EcoRV. (B) Southern blot analysis of chromosomal DNA of T. thermophilus strains with the NdeI-EcoRV pyrE 0.55-kbp fragment as a probe. Chromosomal DNA was digested with BamHI, electrophoresed on a 1% agarose gel and then transferred to a nylon membrane. Lane 1, MT111 (ΔpyrE); lane 2, MT115 (ΔleuB); lane 3, TTY1 (ΔleuBΔpyrE). The positions of size markers are shown by horizontal lines and their sizes are given in kbp. (C) Southern blot analysis with the BglII-BglII leuB 1.9-kbp fragment as a probe. DNA was digested with BglII, electrophoresed on a 1.5% agarose gel and then transferred to a nylon membrane. Lanes and positions of size markers are the same as in (B). Physical maps of the strains are shown in (A).

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For the gene replacement, pITDpyrE (Fig. 1), in which the leuB gene was replaced with the pyrE gene in the leu operon, was used to transform the ΔpyrE strain MT111. Some of the Ura+ transformants were randomly selected and all of them showed leucine auxotrophy. The physical map of one of the strains, MT115, was analyzed by Southern blotting (Fig. 4B and C). When a BamHI digest of chromosomal DNA was probed with the pyrE gene (B), a 0.65-kbp band could be seen in MT115 (lane 2), which was not seen in the parental strain MT111 (lane 1). When the leuB locus was analyzed (C), the 1.9-kbp leuB BglII fragment in MT111 (lane 1) was shortened to 1.4 kbp in MT115 (lane 2). These results show the replacement of the leuB gene with the pyrE gene in MT115.

To delete the pyrE gene inserted at the leuB locus, MT115 was transformed with pTH3ΔEcoRV (Fig. 2) and the 5-FOA resistant clones were isolated. When the pyrE gene was used as a probe, the 0.65-kbp band disappeared in one of the 5-FOAr transformants TTY1 (B, lane 3). The 1.4-kbp BglII leuB gene fragment in MT115 was shortened to 0.85 kbp in TTY1 (C, lane 3). The difference corresponds to the deletion of the pyrE gene. These results show that TTY1 lacks the leuB gene in addition to regeneration of the complete deletion of the pyrE gene.

Theoretically, any cloned T. thermophilus gene can be deleted from MT111 or TTY1 as a parental strain as long as the disruption is not lethal.

3.2Construction of a T. thermophilus-E. coli shuttle plasmid vector

An autonomously replicating plasmid vector for the thermophile was constructed (Fig. 3). pT8leuB consists of a replication origin of a cryptic plasmid, pTT8, from T. thermophilus HB8, T. thermophilus leuB gene as a marker, a synthetic promoter designed for the gene expression in the thermophile and an E. coli plasmid vector. The synthetic promoter was designed to drive a strong transcription in T. thermophilus and to have minimum homology with the host chromosomal DNA. For these purposes, a sequence around the −35 and −10 regions was designed based on three different strong promoters reported by Maseda and Hoshino [23](Fig. 3B). The plasmid pT8leuB could transform TTY1 into Leu+.

3.3Expression of wild-type and mutant E. coli leuB genes in T. thermophilus

To show the applicability of the host-vector system constructed above, we tried to express a gene from a mesophile and a mutant of it. TTY1 was transformed with a plasmid, pT8EleuB, which was constructed by replacing the T. thermophilus leuB gene of pT8leuB with the E. coli wild-type leuB gene. All transformants could grow at 60°C without leucine but not at 65°C. TTY1 was also transformed with another plasmid, pT8ELMV, which directs the expression of an in vitro mutagenized leuB gene encoding a stabilized E. coli 3-isopropylmalate dehydrogenase [22]. The transformants could grow at a temperature of 65°C. These results show that the system described here is useful to compare the stabilities of enzymes in the thermophile and can be used for selecting stabilized mutant enzymes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

This work was supported by Grants-in-Aids for Scientific Research from the Ministry of Education, Science and Culture of Japan (Number 09780552).

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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