JOURNAL OF BACrERIOLOGY, Oct. 1992, p. 6424-6431

Vol. 174, No. 20

0021-9193/92/206424-08$02.00/0 Copyright © 1992, American Society for Microbiology

Development of Thermus-Eschenichia Shuttle Vectors and Their Use for Expression of the Clostridium thermocellum celA Gene in Thermus thermophilus INIGO LASA, M. DE GRADO, M. A. DE PEDRO, AND JOSt BERENGUER* Centro de Biologia Molecular, Universidad Aut6noma de Madrid-Consejo Superior de Investigaciones Cientificas, 28049 Madrid Spain Received 29 April 1992/Accepted 30 July 1992

We describe the self-selection of replication origins of undescribed cryptic plasmids from Thermus aquaticus Y-VH-51B (ATCC 25105) and a Thermus sp. strain (ATCC 27737) by random insertion of a thermostable kanamycin adenyltransferase cartridge. Once selected, these autonomous replication origins were cloned into the Escherichia coli vector pUC9 or pUC19. The bifunctional plasmids were analyzed for their sizes, relationships, and properties as shuttle vectors for Thermnus-Escherichia cloning. Seven different vectors with diverse kanamycin resistance levels, stabilities, transformation efficiencies, and copy numbers were obtained. As a general rule, those from T. aquaticus (pLUl to pLU4) were more stable than those from the Thermus sp. (pMYl to pMY3). To probe their usefulness, we used one of the plasmids (pMYl) to clone in E. coli a modified form of the cellulase gene (ceL4) from Clostridium thermoceflum in which the native signal peptide was replaced in vitro by that from the S-layer gene of T. thermophilus HB8. The hybrid product was expressed and exported byE. coli. When the gene was transferred by transformation into T. thermophilus, the cellulase protein was Also expressed and secreted at 70°C. The production of thermostable enzymes and structural proteins is an economically important field for investigation because of their potential industrial applications (10). Most of these enzymes are presently produced by large-scale fermentation processes, the high cost of which can be justified only for very specific applications (18). For increasing the yield of these processes, thermostable enzymes have been cloned in mesophilic organisms, such as Escherichia coli. However, differences in codon usage or improper folding of the proteins at low temperatures have prevented the expected activities or the desired amounts of the proteins to be obtained. On the other hand, expression of the cloned proteins in thermophilic organisms has been made difficult by the thermal instability of selectable plasmid markers, such as antibiotic resistance genes, at high growth temperatures (7). In 1986, Matsumura et al. demonstrated that point mutations in a kanamycin adenyltransferase gene (kat) increased the thermostability of the enzyme by several degrees (17), thus allowing the selection of cloning vectors for moderate thermophiles, such as Bacillus stearothermophilus. To apply this system to extreme thermophilic eubacteria, we recently described the construction of S-layer (slpA) mutants of Thernus thennophilus HB8 by insertion, through homologous recombination, of a hybrid kat gene controlled by the promoter and ribosome binding site signals of slpA (11). A single copy of the kat gene per chromosome resulted in an MIC of kanamycin of about 20 ,ug/ml in agar plate assays. Similarly, Mather and Fee used this same thermostable kat gene to produce a selectable plasmid for T. thermophilus HB8 by random insertion into its cryptic plasmid, pTT8 (16). We describe the construction of a series of selectable bifunctional E. coli-Thermus plasmids by use of previously undescribed replication origins from two Thermus isolates. *

Corresponding author. 6424

We also demonstrate their usefulness as bifunctional cloning vectors by using one of the plasmids to express a thermostable cellulase gene (ceU) from Clostridium thermocellum in both E. coli and T. thennophilus. The cellulase gene was chosen for its potential properties as a "reporter gene" because of the extracellular nature and thermostability of its product. As a parallel observation from this work, we also demonstrate that the amino-terminal sequence of the T. thennophilus HB8 S-layer gene (5, 6), the sequence of which will appear in the EMBL gene bank under accession number X57333, contains a signal peptide that can direct the secretion of the CelA protein both in T. thermophilus and in E. coli.

MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. T. thermophilus HB8 (ATCC 27634), T. aquaticus Y-VII-51B (ATCC 25105), and a Thermus sp. (ATCC 27737) were obtained from the American Type Culture Collection (Rockville, Md.). T. thernophilus HB27 was generously provided by Y. Koyama. E. coli TG1 [K-12 supE hsdA&5 thi A(lacproAB) F' (traD36 proAB+ laqIq lacZAM15)] and DH5aF' [F' supE44 A(lacZYA-argF)U169 (4)80 lacZAM15) hsdR17 recAl endA41 gyrA96 thi-1 relAl] (Bethesda Research Laboratories, Gaithersburg, Md.) were used as hosts for genetic manipulations of plasmids. Plasmids pUC9 (21) and pUC19 (22) were used for in vitro genetic manipulations. Plasmid pKT1 was constructed in our laboratory (11) and contains a thermostable kanamycin adenyltransferase gene (17) controlled by the transcriptional signals of the slpA gene from T. thennophilus HB8. Plasmid pTC105 (4) was generously provided by P. Beguin and contains the celA gene from C. thermocellum in a 3.2-kbp HindIII-HindIII DNA fragment. Plasmid pSla2.3 is a pUC9 derivative in which the promoter and amino-terminal region of the sipA gene were cloned (5).

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T. thernnophilus HB8 was grown at 70°C under strong aeration in a rich medium containing 8 g of Trypticase (BBL Microbiology Systems, Cockeysville, Md.), 4 g of yeast extract (Okoid, Hampshire, England), and 3 g of NaCl per liter of tap water. The pH of the medium was adjusted to 7.5. For petri plates, 1.5% (wt/vol) agar was added to solidify the medium. When necessary, 100 ,ug of kanamycin per ml was added to plates for selection. Plates were incubated in an inverted position at 70°C in a water-saturated atmosphere. E. coli strains were grown at 37°C in LB medium (12). If a plasmid was present, a selective antibiotic was added (100 ,ug/ml for ampicillin or 30 ,g/ml for kanamycin). Cells were made competent as described previously (13). DNA analysis. Plasmid DNA was purified from E. coli by the alkaline lysis method (2). The same method was used for Thermus spp., but a shorter lysis time was used to limit the degradation of DNA. Total DNA was purified by the method of Marmur (15), but a higher salt concentration was used in the precipitation steps. Most of the DNA techniques were carried out as described by Maniatis et al. (14). Restriction enzyme digestions and ligation reactions were performed as recommended by the manufacturer (Boehringer-Mannheim GmbH, Penzberg, Germany). DNA sequencing was performed by the method of Sanger et al. (20) in accordance with the instructions of the DNA sequencing kit from Pharmacia (Uppsala, Sweden). DNA amplification (19) was performed with the synthetic oligonucleotides described in Results and DNA polymerase from T. aquaticus (Amplitaq; Perkin Elmer-Cetus, Emeryville, Calif.). The amplification reaction mixtures were incubated in a PHC-1-Dry-Block (Techne, Duxford, Cambridge, England). Southern analysis of the chromosomal and plasmid DNAs was performed as described previously (14) with about 5 ,ug of digested DNA per sample. DNA probes were labeled with the Pharmacia kit for random primer labeling. Transformation of T. thermophilus. Essentially the method of Koyama et al. was used (9) for transformation. T. thermophilus HB8 or HB27 was grown at 70°C in the medium described above with 1 mM MgCl2 and 0.5 mM CaCl2. At an optical density at 550 nm of 0.5, samples (0.5 ml) containing approximately 108 CFU were transferred to 5-ml sterile glass tubes, and the desired amount of DNA was added. After 2 h of incubation at 70°C under strong aeration, cells were directly plated onto selective agar plates. Cellulase activity. The production of extracellular cellulase was assayed by a modification of a method described previously (4) to increase the sensitivity. Colonies grown for 24 h on selective plates were transferred to nitrocellulose filters (MSI, Westboro, Mass.), and the filters, with the colonies facing up, were placed on a second set of sterile agar plates. The plates were overlaid with agar (0.7% [wt/vol]) containing 0.5% (wt/vol) carboxymethyl cellulose in 50 mM K2HPO4-12 mM citric acid buffer (pH 6.3). Once the agar was solidified, the plates were incubated at 65°C for 3 h, and the activity was detected by staining of the remaining substrate with Congo red. RESULTS Cloning in E. coli of replication origins from a Thermus sp. and T. aquaticus. As a source of replication origins from Thermus spp., we used T. aquaticus ATCC 25105 and Thermus sp. strain ATCC 27737 because of their biochemical differences and because, to our knowledge, the presence of plasmids within these strains has not been described. As

EXPRESSION OF celA IN T. THERMOPHILUS

6425

shown in Fig. 1, total plasmid preparations from 20-ml cultures of both strains were digested with restriction enzyme BamHI or PstI and ligated by use of cohesive ends to a thermostable kanamycin adenyltransferase cartridge (kat). This cartridge was purified from E. coli plasmid pKT1, in which the kat gene is controlled by the promoter region of the S-layer gene (slpA) (11). Ligation mixtures were subsequently used to transform the plasmid-free strain T. thernophilus HB27. After incubation at 70°C for 48 h, a large number of colonies resistant to 20 ,ug of kanamycin per ml were obtained for the PstI-Thennus sp. and BamHI-T. aquaticus ligations. The BamHI-Thermus sp. and PstI-T. aquaticus ligations yielded very few colonies, so they were not used. To clone the putative plasmids from the kanamycinresistant transformant colonies into E. coli, we pooled mixtures of such colonies from both transformations and purified total plasmids. These mixtures were digested with EcoRI, an enzyme with few recognition sites in Thermus spp. and a single recognition site in the kat cartridge, and ligated to the EcoRI site of pUC9 or pUC19. Upon transformation of E. coli, colonies resistant to ampicillin (100 ,ug/ml) and kanamycin (30 pug/ml) were analyzed for the presence of plasmids. This method allowed us to obtain seven Thermus autonomous replicons of different sizes: four from T. aquaticus (pTAQ1 to pTAQ4) and three from the Thermus sp. (pTSP1 to pTSP3). When cloned into pUC vectors, the plasmids were called pLUl to pLU4 and pMY1 to pMY3, respectively (Fig. 1). The plasmids were probed to determine whether they were bifunctional by testing their ability to transform T. thermophilus Hfl27 and to be recovered by transformation into E. coli. The MIC of kanamycin for T. thermophilus cells carrying any one of these plasmids was >200 ,ug/ml. Analysis of pLU and pMY plasmids. The most important properties analyzed in plasmids pLUl to pLU4 and pMY1 to pMY3 are summarized in Table 1. As expected, the restriction patterns of the plasmids revealed the presence of several sites for enzymes that recognize sequences with a high G+C content, such as XhoI. The plasmids either had very few sites with a high A+T content (ClaI and EcoRI) or lacked these sites altogether. Nevertheless, the differences in the restriction patterns between all plasmids but pMY1 and pMY2 suggest that we have cloned distinct replication origins (data not shown). To verify this suggestion, we purified and labeled the corresponding Thermus DNA fragments from all the plasmids and assayed them independently for the ability to hybridize to each other. As Table 1 shows, only plasmids pMY1 and pMY2 had common fragments. Since the procedure for the purification of plasmids from cultures of T. thermophilus HB27 did not yield DNA clean enough to be quantified by spectrophotometry (especially for the pLU plasmids), the transformation efficiencies were assayed with plasmid DNA obtained from E. coli. The efficiencies for the pMY plasmids (Table 2) were about 1/10 those obtained when the same plasmids were purified from T. thermophilus (data not shown). The stability of the plasmids was analyzed by probing the kanamycin resistance of colonies plated after 32 h of growth in nonselective medium, with an intermediate reinoculation during this period. The results (Table 2) demonstrate the relative instability of plasmids pMY1 and pMY2 compared with that of plasmids pMY3 or pLUl to pLU4. A possible explanation for this result could be that plasmids pMY1 and pMY2 have lower copy numbers. To test this possibility, we purified total DNA of T. thermophilus HB27 cells trans-

HCHPSBXN .P X B Sc

Cryptic plasmids of

t

Thermus aquaticus

Cryptic plasmids of Thermus sp m

B

B4

LI Ligation

Ligation

Transformation of Thermus thermophilus HB27 Selection on Kanamycin (20 gg/mI) BX N

PSBX

E PXB

N

pTAQ1 pTAQ2 pTAQ3 pTAQ4

IE 4 *

E E

EP

pTSP1

pTSP2 pTSP3

pUC19

pUC9

Ligation

E Ligation

Transformation of E. coli DH5aF

Selection on Ampicillin (100,g/mI) and Kanamycin (30,g/mI) PSBX I

H

pT)

pTSP pUC9

PEXB

PEKXBSH

FIG. 1. Selection of bifunctional Thennus-E. coli plasmids. Low-scale preparations of plasmids from T. aquaticus Y-VII-51B and the Thermus sp. were digested with the indicated restriction enzymes and ligated to the kat cartridge (KAT). Plasmid mixtures (pTAQ and pTSP) of kanamycin-resistant T. thermophilus HB27 colonies, obtained after transformation with the ligations mixtures, were digested with EcoRI and ligated to pUC9 or pUC19. Both ligation mixtures were then used to transform E. coli cells, and plasmids from individual colonies were analyzed as described in Results. Abbreviations for restriction enzymes: B, BamHI; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; N, NdeI; P, PstI; S, SalI; Sc, SacI; X, XmaI. 6426

EXPRESSION OF celA IN T. THERMOPHILUS

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6427

TABLE 1. Restriction analysis and relationships between plasmids Plasmid

Insert size (kbp)

BamHI

pLUl pLU2 pLU3 pLU4

3.5 8.3 6.8 6.2 4.7 4 9.4

1 2 2 2 2 1 9

pMYl pMY2 pMY3

No. following sites: PstIof theHindIII KpnI

0 2 1 1 1 1 1

0 3 0 0 0 0 3

3 2 2 2 3 1 1

XhoI

pLUl

5 6 4 7 4 4 7

+

formed with each plasmid from kanamycin-resistant, stationary-phase cultures and digested it with EcoRI and BamHI. The DNA fragments were separated by electrophoresis on agarose gels, transferred to nitrocellulose filters, and hybridized with the labeled promoter region of sipA. These digestions generated two labeled DNA fragments, of 1 and 5.8 kbp, corresponding to the kat cartridge of the plasmid and to the S-layer gene of this strain, respectively. The levels of labeling of the DNA bands were related to the copy numbers of the corresponding plasmids (Table 2). Surprisingly, stable plasmids pLU1, pLU2, and pLU4 had copy numbers similar to those of unstable plasmids pMYl and pMY2, making this explanation for the instability improbable. A second explanation for the instability of plasmids pMY1 and pMY2 could be their origin. Southern blots demonstrated that both plasmids hybridized to a 16-kbp PstI band from plasmid preparations of the Thermus sp. (data not shown). This result means that these plasmids were deletion products derived from a larger plasmid, an explanation that could justify their instability. Construction of a fusion between the signal peptide region of sipA and the celA gene of C. thermocelum. The celA gene of C. thermocellum, with its own promoter and signal peptide, can be expressed and exported through the cell envelope of E. coli (4). Preliminary attempts to clone and express this gene in T. thennophilus HB27 by direct insertion of selectable replication origins into plasmid pMY1 were unsuccessful. Therefore, we decided to produce a fusion between this gene and a segment containing the promoter and the aminoterminal region (33 amino acids) of the S-layer gene (slpA) of T. thernophilus HB8 (5). The method used (Fig. 2) was developed in three steps. First, we designed the oligonucleotide shown at the top left of the figure to include a BamHI restriction site after the hypothetical signal peptide of sipA. This oligonucleotide also included an extra adenine to adjust the reading frame. The TABLE 2. Properties of pLU and pMY plasmids as vectors Plasmid

Transformation efficiency (no. of colonies/4Lg)'

pLUl

ix102

pLU2 pLU3 pLU4 pMY1 pMY2 pMY3

1x102 1 x 104 6 x 103 1 x 104 1 x 103 3 x 103

Stability (%)b

Copy no.C

100 100 96 92 14 28 100

4 4 15 2 3 3 40

DNA was purified from m- r- E. coli. b Determined as described in the text. c DNA was purified from stationary-phase cells. a

pLU2

with: Cross-hybridization pLU3 pLU4 pMY1

pMY2

pMY3

+

+ +

+

+ + +

other oligonucleotide used for the amplification step was complementary to the pUC9 vector (reverse primer), into which a fragment of sipA containing the promoter and the amino-terminal region had been cloned. The amplified fragment was digested with XmaI and BamHI and cloned into pUC9 to produce plasmid pPSl. In this plasmid, the promoter and the amino-terminal region (amino acids 1 to 33) of slpA are followed by a polylinker region containing restriction sites for BamHI, Sall, PstI, and HindIII. In the second step, we amplified a celA fragment that extended from its codon for amino acid 29 to the end of the gene. The synthetic oligonucleotide included a SalI restriction site to enable the production of a fusion with the sipA fragment that had been amplified and cloned. The second oligonucleotide (direct primer) was complementary to the region of the pUC9 vector in which the 3.2-kbp HindIIIHindIII fragment was cloned (5). Once amplified, the projected fusion was obtained by cloning the celA fragment between the Sall and HindIII sites of pPS1. Surprisingly, the resultant construction, pPSC1, was positive for cellulase activity in E. coli (Fig. 3C), thus demonstrating the functionality of the signal peptide of the S-layer gene from T. thermophilus HB8 in this mesophile. Finally, to express the fusion protein in T. thermophilus, we inserted into pPSC1 the 5.7-kbp EcoRI fragment from pMY1.1, a derivative of pMYl in which various restriction sites were deleted from the polylinker regions (Fig. 2). The resultant construction, called pTCM1, was then used to transform competent cells of T. thennophilus HB27. Transformants were positive for cellulase activity (Fig. 3B), whereas in a parallel experiment, colonies transformed with pTC105-pTSP1 were negative (Fig. 3A). Figure 3 shows the results of an experiment in which E. coli (Fig. 3C) and T. thermophilus HB8 and HB27 (Fig. 3D) cells transformed with plasmid pMY1.1 (1), pTC105-pTSP1 (2), or pTCM1 (3 and 3') were assayed for the production of extracellular cellulase. When the transcription and secretion signals of the S-layer gene of T. thermophilus HB8 were present, cellulase was synthesized and apparently exported from both the mesophile and the thermophile. Furthermore, the usefulness of the novel bifunctional plasmids was clearly demonstrated. DISCUSSION The use of positive criteria for the selection of plasmids in extreme thermophiles was limited until recently to genes that complement mutants in the synthesis of amino acids (8). Although this system allowed the development of bifunctional Thermus-E. coli vectors (7), the high recombination

frequency in the thermophile (9) produced, after transforma-

6428

J. BACTERIOL.

LASA ET AL. Sal I 36 E

H A K W G A 5'.GCC GGG CAC TGG GCC AAG GAG.3' 3'CC QTQ ACC CGG TTC CT AGM 5' A

AC ACT GTM TCA Gac CC 3' 5' .G GC AAC ACT GTG TCA GCO GCL GOT.3 ' A N T V S A 27

Bam Hi

~~~P

B

H I

RP

:-,II.,.,.,.,.,.,.,.,.,.,""

Bam HI

:"

H

e,

....

DP

Sal I

|X/B S/H

pUC9 X/-B--* EX

N BS B

H

P

0-

.:-.:

- - -

w-

S/H BamHI 5

TGG

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Sa GTC

GALC A?

....ACC CdG GTT CCT AGG CAG CTG TGC Q T W Q G R V S

(GM TCZL...3 CAC

V

AaT.... S

pPSC1 PE

E

E

CELA

P

B

~~pLac

BS

PE

K-'

pTCM1 10.7 Kbp.

E KAT

>.f.e

PBX

FIG. 2. Construction of plasmids used for the expression of celA in T. thermophilus. A DNA fragment containing the promoter and the first coding region of slpA was amplified by the polymerase chain reaction with the oligonucleotides indicated. The fragment (including a new BamHI site) was then cloned into restriction sites XmaI and BamHI of pUC9, and plasmid pPS1 was obtained. The celA gene was amplified and cloned into pPS1 by use of SalI (included in the polymerase chain reaction) and HindIII, and plasmid pPSC1 was obtained. The EcoRI site of this plasmid was further used to clone a replication origin from the Thennus sp. (pMY1.1) containing the selectable kat marker, and the bifunctional plasmid pTCM1 was obtained. Symbols for restriction enzymes are identical to those in Fig. 1. DP, direct primer; RP, reverse primer.

EXPRESSION OF ceL4 IN T. THERMOPHILUS

VOL. 174, 1992

A

6429

B

D

C

a

4

2

.3

2

FIG. 3. Expression of celA in E. coli and T. thermophilus. The assay for the detection of cellulase activity was done on kanamycinresistant colonies of T. thermophilus HB27 transformed with pTC105-pTSP1 (A), kanamycin-resistant colonies of the same strain transformed with pTCM1 (B), E. coli DH5aF' cells transformed with pMYl.l (1), pTC105-pTSP1 (2), pTCM1 (3), and pPSC1 (4) (C), and T. thernophilus HB27 cells transformed with pMY1.1 (1), pTC105-pTSP1 (2), and pTCM1 (3) and T. thermophilus HB8 cells transformed with pTCM1 (3')

(D).

tion, a significant background of wild-type colonies without plasmids. For this reason, the recent adaptation to T. thernophilus of a thermostable kanamycin adenyltransferase gene with very low or no homology to its chromosomic DNA (11, 16) represents an important step in the development of cloning vectors for these extreme thermophiles. In this study, we took advantage of the development (11) of a cartridge in which this selectable resistance marker (17), expressed from

a strong Thermus promoter, can be ligated to the restriction site for PstI, BamHI, or XmaI. The ligation of this cartridge to BamHI- and PstI-digested samples of total plasmids from T. aquaticus and the Thermus sp., respectively, and the subsequent transformation of the plasmid-free strain T. thermophilus HB27 yielded a large number of kanamycinresistant colonies that, hypothetically, should have contained plasmids. As the cartridge contains an internal EcoRI

restriction site, rarely found within the chromosome of

6430

LASA ET AL.

Thermus spp., we used it to clone a mixture of the putative plasmids into pUC9 and pUC19. Analysis of the E. coli colonies that were resistant to both ampicillin and kanamycin yielded seven different (by size) plasmids. These plasmids were demonstrated to be bifunctional, as they could transform T. thennophilus HB27. Furthermore, they also could be purified from transformed cells of the thermophile and recovered by transformation in E. coli. The transformation efficiencies for T. thermophilus HB27 were not directly related to the size of the plasmid (Table 2), as large plasmids, such as pLU3 (10 kbp) and pMY3 (13 kbp), produced more colonies per microgram than did smaller plasmids (pLU1, pLU2, and pMY2). As expected from the presence of a restriction system, transformation efficiencies for plasmids pMYl to pMY3 purified from Thermus spp. were at least 1 order of magnitude higher than those for the same plasmids purified from E. coli. The same could be suggested for the pLU plasmids, although difficulties in the purification of these plasmids from Thermus spp. made it impossible to quantitate the amount of DNA used in the transformation experiments. This effect seems to be related to the intrinsic nature of the replication origins of these plasmids, because it was also extremely difficult to detect clear bands in plasmid preparations of T. aquaticus. The results from the Southern blotting are summarized in Table 1 and clearly demonstrated that only pMY1 and pMY2 had common fragments. This result was in good agreement with those of other experiments, in which both plasmids hybridized to a PstI-generated band of 16 kbp that was observed in plasmids prepared from the Thermus sp. (data not shown). Nevertheless, as minipreparations of plasmid DNA from Thermus spp. frequently appeared to be partially degraded, we believe that pMY1 and pMY2 could represent the replication origin and the PstI site within rearranged fragments of this 16 kbp. The origin of pMY3 is much more clear, as it hybridized to a second PstI band of 9.3 kbp in minipreparations of plasmid DNA from the Thermus sp. The presence of four different replication origins in T. aquaticus was striking and much more difficult to explain, because minipreparations of plasmid DNA from this species often revealed a degradation trail. However, the exochromosomal nature of the cloned fragments was demonstrated by Southern hybridization with a labeled mixture of plasmids pLUl to pLU4. This mixture recognized only the minipreparation fraction and did not hybridize to the chromosomal DNA. With the double intent of probing the functionality of these plasmids and of developing secretion vectors for Thermus spp., we decided to prepare a fusion between a thermostable and easy-to-assay enzyme (CeUA) and the signal peptide of the S-layer gene of T. thermophilus HB8 (5). Since the S-layer is the outermost envelope of T. thermophilus (3), this construction should have been exported and, because of the absence of other hydrophobic sequences along the structural celA gene (1), secreted into the medium. The expression of this fusion in E. coli (Fig. 3) revealed an important collateral observation: the amino-terminal sequence of slpA acts as an effective export signal in E. coli (Fig. 3C). However, because of the low level of expression of the fusion, we could not sequence the amino terminus. Therefore, we could not determine whether the signal peptide was actually processed in the mesophile. The insertion of a replication origin from the Thermus sp. with the kat cartridge into the EcoRI site of pPSC1 allowed the cloning of the fusion gene into T. thermophilus. The expression of cellulase was detected in the surrounding

J. BAcrERIOL.

medium of T. thermophilus cells transformed with plasmid pTCM1 (Fig. 3B and D), whereas in cells transformed with pTC105-pTSP1, it was not detected. This result strongly suggests that the cellulase expressed from plasmid pTCM1 is actively exported in T. thermophilus although the possibility exists that part of the observed activity was due to a small amount of cell lysis. However, in experiments not described here, cellulase activity was detected in the supernatant of liquid cultures, in which the protein concentration was too low to be detected by Coomassie blue staining. Therefore, the bulk of the activity probably did not originate from cell lysis. The cellulase activity observed in T. thermophilus was apparently lower than that detected when the same plasmid was cloned in E. coli. This effect could have been related in part to the high temperature at which the thermophile was grown (70°C); such high-temperature growth makes the expressed product unstable. To facilitate the use of the celA gene as a reporter in the future, investigators should isolate ceLA mutants with increased thermal stability by direct selection of colonies transformed with mutagenized pTCM1. The results presented in this paper clearly demonstrate that genes cloned in E. coli can be expressed in T. thennophilus by use of our bifunctional plasmids. Nevertheless, before these plasmids can be generally used, it will be important to analyze the regulatory mechanisms of promoters from Therinus spp. and to apply this information to the control of the expression of cloned genes. ACKNOWLEDGMENTS We are very grateful to M. Matsumura for giving us the thermostable kanamycin adenyltransferase gene and to P. B6guin for providing the celA gene. The comments and suggestions of A. Ottolenghi are also acknowledged. This work was supported by grant BI091-0523 from the CICYT and by an institutional grant from the Fundaci6n Ram6n Areces. I. Lasa is the recipient of a fellowship from F.P.I. REFERENCES 1. Beguin, P., P. Cornet, and J.-P. Aubert. 1985. Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum. J. Bacteriol. 162:102-105. 2. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 3. Cast6n, J. R., J. L. Carrascosa, M. A. de Pedro, and J. Berenguer. 1988. Identification of a crystalline surface layer on the cell envelope of the thermophilic eubacterium Thermus thermophilus. FEMS Microbiol. Lett. 51:225-230. 4. Cornet, P., J. Millet, P. BEguin, and J.-P. Aubert. 1983. Characterization of two cel (cellulose degradation) genes of Clostridium thermocellum coding for endoglucanases. Bio/Technology 1:589-594. 5. Faraldo, M. M. 1990. Ph.D. thesis. Universidad Aut6noma de Madrid, Madrid, Spain. 6. Faraldo, M. M., M. A. de Pedro, and J. Berenguer. 1991. Cloning and expression in E. coli of the structural gene coding for the monomeric protein of the S-layer of Thermus thermophilus HB8. J. Bacteriol. 173:5346-5351. 7. Koyama, Y., Y. Arikawa, and K. Furukawa. 1990. A plasmid vector for an extreme thermophile, Thermus thennophilus. FEMS Microbiol. Lett. 72:97-102. 8. Koyama, Y., and K. Furukawa. 1990. Cloning and sequence analysis of tryptophan synthetase genes of an extreme thermophile, Thermus thermophilus HB27: plasmid transfer from replica-plated Escherichia coli recombinant colonies to competent T. thermophilus cells. J. Bacteriol. 172:3490-3495. 9. Koyama, Y., T. Hoshino, N. Tomizuka, and K. Furukawa. 1986. Genetic transformation of the extreme thermophile Thermus

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thermophilus and other Thermus spp. J. Bacteriol. 166:338-340. 10. Kristjansson, J. K. 1989. Thermophilic organisms as sources of thermostable enzymes. Trends Biotechnol. 7:349-353. 11. Lasa, I., J. R. Cast6n, L. A. Fernandez-Herrero, M. A. de Pedro, and J. Berenguer. 1992. Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8. Mol. Microbiol. 6:1555-1564. 12. Lennox, E. X. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190-206. 13. Mandel, M., and A. Higa. 1970. Calcium dependent bacteriophage DNA infection. J. Mol. Biol. 53:159-162. 14. Maniatis, T., E. F. Fritsch, and J. Sambrook 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208-218. 16. Mather, M. W., and J. A. Fee. 1992. Development of plasmid cloning vectors for Thennus thermophilus HB8: expression of a heterologous, plasmid-borne kanamycin nucleotidyltransferase gene. Appl. Environ. Microbiol. 58:421-425.

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