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  • J. Microbiol. Biotechnol. (2012), 22(9), 12791287http://dx.doi.org/10.4014/jmb.1203.03023First published online May 19, 2012pISSN 1017-7825 eISSN 1738-8872

    Genetic Transformation of Geobacillus kaustophilus HTA426 by ConjugativeTransfer of Host-Mimicking Plasmids

    Suzuki, Hirokazu1* and Ken-ichi Yoshida

    2

    1Organization of Advanced Science and Technology, Kobe University, Hyogo 657-8501, Japan2Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Hyogo 657-8501, Japan

    Received: March 12, 2012 / Revised: May 3, 2012 / Accepted: May 10, 2012

    We established an efficient transformation method for

    thermophile Geobacillus kaustophilus HTA426 using

    conjugative transfer from Escherichia coli of host-mimicking

    plasmids that imitate DNA methylation of strain HTA426

    to circumvent its DNA restriction barriers. Two conjugative

    plasmids, pSTE33T and pUCG18T, capable of shuttling

    between E. coli and Geobacillus spp., were constructed.

    The plasmids were first introduced into E. coli BR408,

    which expressed one inherent DNA methylase gene (dam)

    and two heterologous methylase genes from strain

    HTA426 (GK1380-GK1381 and GK0343-GK0344). The

    plasmids were then directly transferred from E. coli cells

    to strain HTA426 by conjugative transfer using pUB307

    or pRK2013 as a helper plasmid. pUCG18T was introduced

    very efficiently (transfer efficiency, 10-5-10

    -3 recipient

    -1).

    pSTE33T showed lower efficiency (10-7-10

    -6 recipient

    -1)

    but had a high copy number and high segregational stability.

    Methylase genes in the donor substantially affected the

    transfer efficiency, demonstrating that the host-mimicking

    strategy contributes to efficient transformation. The

    transformation method, along with the two distinguishing

    plasmids, increases the potential of G. kaustophilus HTA426

    as a thermophilic host to be used in various applications

    and as a model for biological studies of this genus. Our

    results also demonstrate that conjugative transfer is a

    promising approach for introducing exogenous DNA into

    thermophiles.

    Keywords: Geobacillus kaustophilus, transformation, mimicking,

    conjugative transfer, restriction-modification

    The genus Geobacillus comprises aerobic or facultatively

    anaerobic, Gram-positive, thermophilic bacilli that were

    reclassified from the genus Bacillus in 2001 [20].

    Members of this genus have been isolated from various

    land and marine hot environments and also from cool soil

    environments, thereby demonstrating their wide distribution

    [14]. These bacteria are able to grow at temperatures above

    45oC and have historically served as important sources of a

    wide variety of thermostable enzymes [14]. Moreover,

    Geobacillus spp. have attracted considerable interest in the

    field of microbial bioprocessing. Bioprocessing at elevated

    temperatures has many practical advantages [30]; the

    Geobacillus genus includes several promising species,

    such as those showing ethanol tolerance [5] or arsenate

    resistance [6] and these capable of long-chain alkane

    degradation [9, 13] or metabolizing herbicide [14]. Genetic

    engineering plays important roles in the study and exploitation

    of these properties, as demonstrated in ethanol production

    by genetically engineered Geobacillus thermoglucosidasius

    [5]. However, the use of genetic engineering in this genus,

    even for essential plasmid transformation, has been

    reported only in Geobacillus stearothermophilus [7, 17-

    19, 31] and G. thermoglucosidasius [5, 29].

    Previously, we reported an efficient transformation

    method for Streptomyces griseus IFO 13350 using host-

    mimicking plasmids that imitate DNA methylation of this

    bacterium to circumvent the restriction barriers of its

    restriction-modification (R-M) systems [25]. R-M systems

    defend the host bacterium against invasion by exogenous

    DNA, such as bacteriophage DNA, and thus hamper

    genetic transformation of bacteria. All four types (types I-

    IV) of R-M systems digest exogenous DNA selectively by

    differentiating from host-endogenous DNA on the basis of

    host-specific DNA methylation [21]. Thus, host-mimicking

    plasmids circumvent restriction barriers, allowing efficient

    transformation.

    *Corresponding authorPhone: +81 92 642 7609; Fax: +81 92 642 3053;E-mail: hirokap@xpost.plala.or.jpPresent address: Hirokazu Suzuki, Department of Bioscience andBiotechnology, Faculty of Agriculture, Graduate School, KyushuUniversity, Fukuoka 812-8581, Japan

  • 1280 Suzuki and Yoshida

    In this study, the host-mimicking strategy was applied to

    establish a plasmid transformation method for G. kaustophilus

    HTA426. This thermophile, isolated from a deep-sea

    sediment of the Mariana Trench [26], is able to grow under

    aerobic conditions between 42oC and 74oC (optimally at

    60oC) [27, 28]. Bacterial growth is observed even in media

    containing 3% (w/v) NaCl [26, 27]. The publication of the

    entire genome sequence [28] has also provided excellent

    opportunities for widening biological understanding and

    practical applications of this strain. Here we report the

    efficient transformation of G. kaustophilus HTA426 using

    conjugative transfer of host-mimicking plasmids from

    Escherichia coli. The successful implementation of our

    method increases the potential of strain HTA426 as a

    thermophilic host in various applications and a model in

    biological studies of this genus. It also demonstrates the

    benefits of the host-mimicking strategy and conjugative

    transfer as a promising approach for introducing exogenous

    DNA into thermophiles.

    MATERIALS AND METHODS

    Bacterial Strains, Culture Conditions, Plasmids, and Primers

    Table 1 lists the bacterial strains and plasmids used in this study.

    Table 2 lists the primers used. E. coli strains were grown in Luria-

    Bertani (LB) medium at 37oC. Ampicillin (50 g/ml), kanamycin

    (25 g/ml), chloramphenicol (12.5 g/ml), and tetracycline (6.5 g/ml)

    were used when necessary. E. coli JM109 and plasmid pCR4Blunt-

    TOPO were used for DNA manipulation. G. kaustophilus was

    grown at 60oC in LB medium. Kanamycin (5 g/ml) was added when

    necessary. The solid medium contained 2% (w/v) agar. The E. coli-

    Table 1. Strains and plasmids used in this study.

    Strain or plasmid Relevant description(s)a

    Reference or source

    G. kaustophilus strains

    HTA426 Wild type JCM 12893

    MK24 Strain HTA426 harboring pSTE33T This study

    MK30 Strain HTA426 harboring pUCG18T This study

    E. coli strains

    JM109 Strain used for DNA manipulation in E. coli Takara Bio

    ER1793 F-, e14-(mcrA-), (mrr-hsdRMS-mcrBC)114::IS10, dcm+, dam+ New England Biolabs

    IR27 F-

    , e14-

    (mcrA-

    ), (mrr-hsdRMS-mcrBC)114::IS10, dcm::lacZ, dam::metB [25]

    IR24 F-, e14-(mcrA-), (mrr-hsdRMS-mcrBC)114::IS10, dcm::lacZ, dam+ This study

    BR397 Strain IR27 harboring pUB307 and pIR207 This study

    BR398 Strain IR24 harboring pUB307 and pIR207 This study

    BR408 Strain IR24 harboring pUB307 and pIR408 This study

    BR409 Strain ER1793 harboring pUB307 and pIR408 This study

    KR397 Strain IR27 harboring pRK2013 and pIR207 This study

    KR398 Strain IR24 harboring pRK2013 and pIR207 This study

    KR408 Strain IR24 harboring pRK2013 and pIR408 This study

    Plasmids

    pCR4Blunt-TOPO Cloning vector, ColE1 replicon, AmpR, KmR Invitrogen

    pUB307 Derivative of IncP-1 plasmid RP1, Tra+, oriV, oriT, Km

    R, Tet

    R[3]

    pRK2013 Derivative of IncP-1 plasmid RK2, Tra+, ColE1 replicon, oriT, KmR [10]

    pSTE33 E. coli-Geobacillus shuttle plasmid, pSTK1 replicon, KmR (TK101), AmpR [18]

    pSTE33T pSTE33 carrying oriT This study

    pUCG18 E. coli-Geobacillus shuttle plasmid, pBST1 replicon, KmR (TK101), AmpR [29]

    pUCG18T pUCG18 carrying oriT This study

    pIR200 Derivative of pACYCDuet-1, p15A replicon, CmR

    [25]

    pIR201 Derivative of pACYCDuet-1, lac promoter, p15A replicon, CmR [25]

    pIR207 Derivative of pACYCDuet-1, hsp70 promoter, p15A replicon, CmR This study

    pIR380 pIR207 carrying GKP08 This study

    pIR399 pIR207 carrying GK0343-GK0344 This study

    pIR401 pIR207 carrying GK1380-GK1381 This study

    pIR408 pIR207 carrying GK0343-GK0344 and GK1380-GK1381 This study

    aAmp

    R, Cm

    R, Km

    R, and Tet

    R denote genes coding for resistance to ampicillin, chloramphenicol, kanamycin, and tetracycline, respectively. Km

    R (TK101)

    denotes TK101 encoding thermostable kanamycin nucleotidyltransferase.

  • PLASMID TRANSFORMATION OF G. KAUSTOPHILUS 1281

    Geobacillus shuttle plasmids pSTE33 and pUCG18 were provided

    by Dr. Issay Narumi and Dr. David J. Leak, respectively.

    DNA Methylation Analysis

    Chromosomal DNA (100 g) was purified by ultracentrifugation

    and degraded to deoxynucleosides by sonication followed by enzymatic

    degradation using nuclease P1 (Wako Pure Chemicals) and E. coli

    alkaline phosphatase (Takara Bio) [8, 25]. The resulting deoxynucleosides

    were passed through Microcon YM-3 filters (Millipore) and analyzed

    by reversed-phase high-performance liquid chromatography (HPLC).

    The operating conditions were as follows: column, Pegasil-B

    (4.6 250 mm; Senshu Kagaku); column temperature, 40oC; flow

    rate, 1 ml/min; solvent A, 50 mM sodium acetate, pH 5.0; solvent B,

    methanol; detection wavelengths, 26