Folia Microbiol.37 (6), 395-400 (1~)2)

Construction of Shuttle Vectors for Cloning in Escherichia coli and Acetobacterpasteurianus J. GRONF_Sa and J. TURIqAb aDepartment of Biochemistry, bDepartment of Molecular Biology, Faculty of Science, Comenius University 842 15 Bratislava, Czechoslovakia Recefi,ed May 31, 1991 Rea,isedversion June 11, 1992

ABSTRACI'. New cloning vectors were prepared with the aid of a large plasmid isolated from Acetobacter pasteurianus and from plasmids pBR322 and pUC4-KAPA. Of the prepared cloningvectors, pACK5 contains a gene coding for kanamycinresistance, pACI'7 and pACI'71 contain a gene coding for tetracyclineresistance and vector pACG3 with a gene coding for both kanamycin and tetracyclineresistance. The vectors prepared only contained the beginning of replication from the pAC1 plasmid and possessed the ability to replicate within E. coli and A. pasteurianus. The vectors are highly stable in both strains and during the 5-d cultivationunder nonselectiveconditionsare not eliminated.

Acetobacter strains are Gram-negative bacteria that have been widely used in the industry for oxidizing various substrates. Acetic bacteria are characterized by their ability to utilize various saccharides (e.g. glucose, glucitol, ethanol). Their metabolic products are acetic and gluconic acids and others. Recently, the interest in acetic bacteria has increased because they appear to be a suitable source of numerous plasmids (Okomura et al. 1985). One of the plasmids ofAcetobacter pasteurianus appears to be the pAC1 plasmid (Grones et al. 1989). Techniques of gene engineering in cells of the genusAcetobacter have not been adequately developed. Okomura et al. (1985) prepared plasmids pTA5011, pTA5022 possessing tetracycline resistance from the cryptic plasmid pTA5001. Later on, a series of hybrid vectors was prepared employing plasmids from E. coli and A. aceti pAT5001 and pACYC177 (Okomura et al. 1985). The size of the vectors was found to be 26.7-27.2 kb. Chimeric plasmids for cloning into E. coli and Acetobacter sp. were prepared by linking the pMV102 plasmid isolated from Acetobacter sp. with E. coli plasmids (Fukaya et al. 1985). The present paper describes the construction of cloning vectors for cloning into E. coli and A. pasteurianus cells using the initial step of replication from the pAC1 plasmid.

MATERIALS

AND

METHODS

Bacterial strabzs and vectors. Acetobacter pasteurianus strains .~12 and 3614, E. coli DH1 (FrecA1 endA1 gyrA96 thi-1 hsdR17 rm- rm + s u p E 4 4 - ,~-) (Low 1968). The plasmids employed in the

present work are listed in Table I. Cultivation

Table I. Characteristicsof plasmid properties Plasmid

Genetic marker Reference

pAC1 pBR322 pUC4-KAPA pACK5 pACI'7 pACr71 pACG3

Ap, Tc Ap, Km Km Tc Tc Tc, Km

Grones et al. 1989 Bolivaret al. 1977 Barany 1985 thispaper tiffs paper this paper this paper

and

media.

A. pasteurianus

was grown in a YPG cultivation medium containing (g/L): glucose 20, yeast extract 5, peptone 3 (Okomura et al. 1985). E. coli was grown in an LB cultivation medium (Davis et al. 1980). Isolation and purification o f plasmid D N A . Plasmid DNA was isolated from A.pasteurianus using a modified method of Ohmori et al. (1982). The cells were grown

overnight and suspended in 7 mL of TES buffer (Tris-HCi 50 mmol/L, pH 8.0, E D T A disodium salt 20 mmol/L, sucrose 20 mmol/L, W / V ) . The suspension was then supplemented with 4 mL lysozyme (20 g/L) in Tris-HCl (250 mmol/L, pH 8.0) and 4 mL

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Fig. 1, Electron-microscopic visualization of various structural forms of plasmid pAC1 from A. pasteurianus.

EDTA disodium salt (25 mmol/L). The mixture was incubated for i h at 37 ~ following incubation 3 mL of 10 % SDS was added and the mixture was incubated again for 30 min at 30 ~ After incubation NaCI was added to the final concentration of i mol/L. The lyzate was centrifuged for 30 min at 16 000 g. Pancreatic RNAase (10 rag/L) was added to the supernatant, followed by a 30-min incubation at 37 ~ After incubation PEG 6000 was added to the final concentration of 10 %. The sediment obtained by centrifugation was dissolved in TE buffer containing i g CsC1 per i mL of nucleic acid solution and 80/.LL ethidium bromide (100 g/L) and centrifuged at 830 Hz for 18 h in the VTi65 rotor of a Beckman centrifuge. Plasmid DNA was isolated from E. coli according to Birnboim and Doly (1979). Transformation. Transformation of A. pasteurianus 3614 cells by the plasmid was carried out using the following procedure: 25 mL of YPG cultivation medium was inoculated with 0.1 mL of an overnight cell culture and incubated with shaking overnight at 28 ~ After cultivation the cells were cooled and centrifuged for 10 min at 10 000 g at 4 ~ The sediment was suspended in 12 mL of sterile

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CONSTRUCrlON OF SltU"ITLE VECrORS 397

cold solution A (in mmol/L: NaCI 100, MgCI2 5, Tris-HC1 10; pH 7.6). After stirring, the suspension was centrifuged and the sediment was suspended in 12 mL of sterile cold solution B (in mmol/L: CaCI2 100, KCI 250, MgCI2 5, Tris-HC1 10; pH 7.6) and placed into a cold bath for i h. After centrifugation the cells were resuspended in 4 mL of solution B. To 2 mL aliquots of competent cells 1 - 20/xg of plasmid DNA was added and, after mixing, the cells were left for 1 h on ice. The YPG cultivation medium (3.8 mL) was then added to the cell suspension followed by shaking at 28 ~ in the presence of the appropriate antibiotics. After transformation, the cells were seeded in 50, 100, 200 and 300/zL aliquots on YPG agar plates containing either kanamycin (30 mg/L) or tetracycline (15 mg/L). E. coli cells were transformed according to Mandel and Higa (1971). For selecting transformants kanamycin (final concentration 30 mg/L) and tetracycline (final concentration 15 mg/L) were added into the cultivation medium. Enzymes. Restriction endonucleases EcoRI, EcoRV, PvulI and Sinai were obtained from the Development Center, Comenius University, Bratislava; restriction endonucleases BgllI, HindllI, PstI were obtained from the Institute of Sera and Vaccines, Prague, and restriction endonuclease Ball and T4 DNA ligase were from the Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava. Electron microscopy. Preparations for electron microscopy were prepared according to Kleinschmidt (1968) using a protein technique. The preparations were rotary-shadowed with an P t - P d alloy (4:1) and examined under the electron microscope BS 540 (Tesla, Brno, Czechoslovakia). Gel electrophoresis. Elcctrophoresis of nucleic acids was done in 1 % agarose gel in TAE buffer according to Thomas and Davis (1975). Stability of vectors. The stability of prepared vectors was examined after transforming into E. coli and A. pasteurianus. E. coli cells transformed by plasmids pACK5, pACT7, pACT71 and pACG3 were cultivated in the LB medium and A. pasteurianus in YPG medium with the appropriate antibiotic. Flasks containing liquid LB and YPG media without antibiotics were inoculated with 1/100 volumes of cultures grown overnight in the presence of the appropriate antibiotic. Cells were grown under vigorous shaking at 28 ~ (A. pasteurianus) and at 37 ~ (E. coli) for 24 h. From the cultivation medium 1/100 aliquots were withdrawn and another flask containing the cultivation medium without antibiotic was inoculated. The described procedure was repeated five times at one-day intervals. After each one-day cultivation the cells were plated on solid agar without antibiotic and 100 grown colonies were plated on agar plates containing the antibiotic. The grown colonies were counted and plasmid DNA was isolated from every individual colony.

RESULTS A N D DISCUSSION Plasmid DNA was isolated from A. pasteurianus. Plasmid pAC1 was visualized by electron microscopy (Fig. 1). On the basis of this, we determined the size of plasmid DNA to be 18.5 kb (Grones et al. 1989). Purified plasmid DNA was cleaved by restriction endonuclcases. Enzymes EcoRI, PstI and PvulI split the plasmid into three fragments, HindlII into five different fragments. Plasmid pAC1 isolated from A. pasteurianus was not cleaved with restriction endonucleases, such as Sau3AI, BamHI, Sinai, PvuI and others. Because the plasmid does not bear any selection marker for a simple detection in A. pasteurianus cells we decided to incorporate into it a gene encoding kanamycin and tetracycline resistance. The plasmid was partially cleaved with restriction endonuclease EcoRI. A gene for kanamycin resistance was selected from a casette vector pUC4-KAPA by cleaving EcoRI and the gene coding for tetracycline resistance was selected from the plasmid by cleaving with EcoRI and PvulI. After ligating, E. coli DH1 cells were transformed using a ligation mixture and transformants were selected on LB agar plates containing kanamycin and tetracycline. After analyzing the recombinants we obtained by cloning vectors pACK5 and its derivatives (Grones et al. 1991) and pACq'7 and its derivatives. A scheme for constructing the individual vectors is shown in Fig. 2. The vectors prepared contained fragments from the pAC1 plasmid and an appropriate selection marker. Vector pACK5 possesses as a selection marker a gene coding for kanamycin resistance while for cloning of a foreign gene restriction sites for SmaI and XhoI can be utilized. The cloning vector pACT7 contains a 2066-bp-long fragment of plasmid pBR322 bearing a gene coding for tetracycline resistance and possesses two cleavage sites for EcoRI and PvulI. The vector contains a segment coding for a nonessential portion of plasmid pBR322 located between restriction sites for Ball and PvulI. The fragment was cleaved using

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EcoRI

EcoRI ~eomHI

EcoRI~

EcoRI EcoRI

PvuII EcoRI ~'Pvull

RI

Klenow

portinl

Er

~EcoRl EcoRI%, v_ ~

~BomHI ~'3"9kb ;Sa/I

.XhoI ~ " Sinai

H;ndllI

Pvul[ ~'------.'J'Boll EcoRZ

RI

T Eco

EcoRI XhoI

EcoRl ~- HindIII

Stool

o RI

1 Bali+Pvull

x'~..~/ Sa/[ Eco

RI/

EcoRI~ Fig. 2. Schematic representation of constructing cloning vectors from plasmid pAC1 from

A.pasteurianus.

the above restriction endonucleases; thus the size of the vector decreased approximately by 600 bp and a new smaller derivative p A C ~ I was prepared. The vector contains a gene coding for tetracycline resistance and possesses one site for EcoRI only. The vector contains all restriction sites that are components of the tetracycline gene, such as EcoRI, HindlII, EcoRV, BamHI, SalI and others. pACT71 (containing only one site for EcoRI) was employed for constructing another vector. Applying the restriction endonuclease EcoRI to plasmid pUC4-KAPA we selected a gene coding for kanamycin resistance which was then inserted into the EcoRI site of vector pAC'I~I. After transforming E. coli DH1 cells and selecting transformants a vector pACG3 containing genes for kanamycin and tetracycline resistance and a segment from plasmid pAC1 was obtained. On the basis of partial cleavage the size of the plasmid was determined to be 4.8 kb. The vector possesses two sites for EcoRI that mark the gene coding for kanamycin resistance and two HindlII and Sail in genes coding for resistance

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C O N S T R U C T I O N OF S H U ' I T L E VECTORS

399

to antibiotics. Sites for the restriction endonucleases SrnaI and XhoI and in the gene for tetracycline resistance EcoRV, BamHI and others can be employed for cloning into the gene for kanamycin resistance. The above-described cloning vectors contain the beginning of replication from plasmid pAC1. All steps of vector preparation were carried out in E. coli cells. Vectors were retransformed into A. pasteurianus 3614. Comparison of transformation to A. pasteurianus and E. coli revealed that the efficacy of transformation inA. pasteunanus is only 10 % of that in E. coli.

105

I

l

I

I

I

% I00

O!

95

90

85 105

I

I

I

I

I

i

100

95

4

-

90

85i

0

I

I

1

I

I

1

2

3

~;

5 d

Fig. 3. Stability of cloning vectors (resistant cells, %) during the 5-d cultivation in E. coli (top) andA. pasteurianus (bottom); 1 - pACI71, 2 - pACq'7, 3 - pACKS, 4 - pACG3.

Plasmid pMV102 was employed for preparing the cloning shuttle vectors pMV302 and pMV331 for A. aceti; Fukaya et al. (1985) used plasmid pMV102 for the same purpose. The prepared shuttle vectors contain the beginning of replication from plasmid pMV102 as well as the ColE1 replicon from plasmids pBR322, pBR325 and pACYC177. Both replicons secure replication in E. coli and A. aceti. On the other hand, vectors pACK5, pACT71 and pACG3 contain the beginning of replication from plasmid pAC1 only and possess a similar ability to replicate within E. coli and A. pasteurianus as vectors of the pMV series do. StabilitY of vectors. This was examined after transformation into E. coli and A. pasteurianus. Of the strains tested a higher stability of the cloned vectors was found in A. pasteurianus where it reached almost 100 %; E. coli was a little less stable (about 85 %) during the 5-d cultivation in the absence of antibiotics (Fig. 3). Vectors with kanamycin resistance are less stable than vectors with tetracycline resistance in E. coli DH1 cells as compared with A. pasteurianus 3614.

REFERENCES BARArCr F.: Single stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering. Gene 37, 111-119 (1985). BZRNaOIM H.C., DOLY J.: A rapid alkaline extraction procedure for screening of recombinant plasmid DNA. NucL4cids Res. 7,

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BOLIVAR F., RODRIGUEZ R.I,., GREENE P.J., BELACtl N.C., IIEYNECKER II.L., BOYER H.W., CROSA J.It., FOLKOW S.: Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95 - 106 (1977). DAVIS R.W., Boa~'Elrq I)., Roall J.R.: Advance Bacterial Genetics. Cold Spring Ilarbor Laboratory, Cold Spring Harbor, New York 1980. (]RONF.S .]., SKERI'3qOVA M., BEI)ERKOV.~ K., TERRA J.: Isolation and characterization of plasmid pAC1 from Acetobacterpasteurianus. Bioldgia (Bratislava ) 44, 1191-1196 (1989). GRO~ES J., .Sm,:RE~OVA M., TURgrA J.: Preparation of recombinant plasmids with kanamycin resistance in plasmid pAC1 from Acetobacterpasteurianus. Biolrgia (Bratislava) 46, 673- 678 (1991) FUKAYA M., OKUMURA T., MASAI II., UOZUMI T., BEPPU T.: Construction of new shuttle vectors for Acetobacter. Agric.Biol.Chem. 49, 2083- 2090 (1985). KLEINSCHMIDT A.K.: Monolayer techniques in electron microscopy of nucleic acid molecules. Methods Enzymol. 128, 361-377 (1968). l~ow B.: Formation of merodiploids in matings with a class of Rec recipient strains ofEscherichia coli K12. Proc.Nat.Acad.Sci. USA 60, 160 - 168 (1968). MANDEL M., HIGA A.: Calcium dendrit bacteriophage I)NA infection. J.Mol.Biol. 53,154-174 (1970). MANIATIS T., FRrrSCH E.F., SAMBROOK J.: Molecular Cloning - a Laboratory Manual. (.'old Spring Ilarbor Laboratory, Cold Spring Ilarbor, New York 1982. OItMORI S., UOT~ONI T., BEPPU T.: Loss of acetic acid resistance and ethanol oxidizing ability in an Acetobacter strain. Agric.BioLChem. 46, 381 - 389 (1982). OKOMt;RA II., UOZI;MI W., BEPPU T.: Construction of plasmid vectors and genetic transformation system forAcetobacter aceti. Agric.Biol.Chem. 49. 1011-1017 (1985). "I'IlOMAS P., DAVIS R.W.: Studies on the cleavage of bacteriophge lambda DNA with EcoRl restriction endunuclease. J.MoLBiol. 91, 315- 382 (1975).