PLASMID

26, 1-9 ( 199 1)

Insertion of Tn976 into Bacillus pumilus Plasmid pMGD302 and Evidence for Plasmid Transfer by Conjugation CAROLA.HENDRICK,'LAURIEK.JOHNSON,NANCYJ.TOMES, BRENDA K. SMILEY,ANDJOSEPH P. PRICE Microbial

Genetics, Division of Pioneer Hi-Bred International, 7300 Northwest 62nd Avenue, Johnston, Iowa 50131

Inc.,

Received August 20, 1990; revised April 5, 199 1 As part of an effort to develop systems for genetic analysis of strains of Bacilluspumilus which are being used as a microbial hay preservative, we introduced the conjugative Enterococcus faecalis transposon Tn916 into B. pumilus ATCC 1 and two naturally occurring hay isolates of B. pumilus. B. pumilus transconjugants resistant to tetracycline were detected at a frequency of approximately 6.5 X IO-’ per recipient after filter mating with E. faeculis CG110. Southern hybridization confirmed the insertion of Tn916 into several different sites in the B. pumilus chromosome. Transfer of Tn916 also was observed between strains of B. pumilus in filter matings, and one donor strain transferred tetracycline resistance to recipients in broth matings at high frequency (up to 3.4 x 10e5 per recipient). Transfer from this donor strain in broth matings was DNase-resistant and was not mediated by culture filtrates. Transconjugants from these broth matings contained derivatives of a cryptic plasmid (pMGD302. approx 60 kb) from the donor strain with Tn916 inserted at various sites. The plasmids containing Tn916 insertions transferred to a B. pumilus recipient strain at frequencies of approx 5 X 10m6per recipient. This evidence suggests that pMGD302 can transfer by a process resembling conjugation between strains of B. pumilus. 0 1991 Academic Press, Inc.

Hay that is baled at high moisture is susceptible to deterioration from heating (caused by plant respiration and microbial activity) and the growth of spoilage organisms (fungi and thermophilic bacteria). We have been evaluating naturally occurring isolates of Bacillus pumilus for use as microbial hay preservatives (N. Tomes, U.S. Patent 4,820,53 1, April 1989; U.S. Patent 4,863,747, September 1989). While the mode of action of the product is not understood, the hay inoculant allows hay to be baled under high moisture conditions without spoiling (Tomes et aZ., 199 1). It has been observed that Bacillus pumilus and other Bacillus species have antibacterial and antifungal activity in vitro (Korzybski et al., 1978; Loeffler et al., 1986), but whether these activities have any role in the ’ To whom correspondence

efficacy of the microbial product is not known. One of the goals of our group has been to develop genetic techniques for B. pumilus in order to study the role of antibacterial or antifungal compounds in the efficacy of the hay inoculant. Our studies have been hampered by an inability to transform our strains of B. pumilus by conventional techniques and by the lack of transducing phages that will infect these strains. We have identified large cryptic plasmids in hay inoculant strains of B. pumilus and used one of the plasmids as a molecular marker to monitor populations of the inoculant in hay and soil (Hendrick et al., 199 1). We are interested in studying the biology of B. pumilus plasmids because of their potential use in developing gene transfer systems for our isolates. Plasmid transfer by a process resembling conjugation has been observed in vitro for several other species of Bacillus (Bat-

should be addressed.

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0147-619X/91

$3.00

Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form resewed.

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HENDRICK TABLE

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BACTERIALSTRAINSAND PROPERTIES strain

Phenotype

Source or Derivation

Bacillus pumilus 296.5 1 296. I 296.2 296.3 302.41 302. I 302.2 302.3 1.0 1.1 1.2

Enterococcusfaecalis CG110

Escherichia co/i

Sm’ Rf’ Sm’Tc’ Sm’ Rf’ Sm’Tc’ Sm’ Sm’Tc’ Rf’Fus’Tc’ (Tn916)

Ap’Tc’

CG120(PAM120)

Isolate from hay Sm’, 296.5 I Rf’, 296.5 1 CGI 10 X 296.1 Isolate from hay Sm’, 302.4 I Rf’, 302.4 1 CGI 10 x 302. I ATCC No. I Sm’, 1.0 CGllO X 1.1 D. Clewell,

Universityof Michigan D. Clewell, University

of Michigan

tisti et al., 1985; Gonzalez et al., 1982; Koehler and Thorne, 1987) and these mating systems have been useful in the genetic analysis of plasmid-borne genesin B. anthrucis and B. thuringiensis. As an initial step in developing genetic systems for hay inoculant strains, we introduced the 16.4-kb conjugative Enterococcusfuecalis transposon Tn916 (Gawron-Burke and Clewell, 1982; Clewell and Gawron-Burke, 1986; Murphy, 1989) into B. pumilus. Tn916 or related elements have been introduced by conjugation into B. thuringiensis (Naglich and Andrews, 1988), B. subtilis (Christie et al., 1987, Scott et al., 1988) B. unthrucis (Ivins et al., 1988) and many other grampositive species (Murphy, 1989). In this paper we describe the insertion of Tn916 into a cryptic B. pumilus plasmid which appears to have the ability to transfer between B. pumilus isolates by a process resembling conjugation. MATERIALS

AND METHODS

Bacterial strains, media, and growth conditions. Bacterial strains used in this study are shown in Table 1. E. coli cultures were grown at 37°C on LB medium (Maniatis et al.,

ET AL.

1982) containing 50 pg ampicillin/ml. Bucillus isolates were grown at 28 “C in tryptic soy broth (TSB)2 (Difco Laboratories, Detroit, MI), TSB containing 1.5% agar (TSA), brain heart infusion broth (BHI) (Difco), or BHI containing 1.5% agar; stock cultures were maintained at -70°C in TSB plus 20% glycerol. Selective antibiotic concentrations were: streptomycin sulfate (Sm) (Sigma Chemical Co., St. Louis, MO), 1000 pg/ml; rifampin (Rf) (Sigma), 100 pug/ml; tetracycline (Tc) (Sigma), 20 pg/ml. Minimal medium contained the following: KH,PO,, 0.72 g/liter; K,HPO, . 3H20, 1 g/liter; NaCl, 1 g/ liter; sodium citrate, 0.5 g/liter; NH,S04, 4 g/liter; MgSO, . 7H20, 0.7 g/liter; glycerol, 10 ml/liter; agar, 15 g/liter. Muting conditions. For filter matings between donor and recipient strains, cells were grown in 16-mm culture tubes on a roller drum in 5 ml BHI broth containing the appropriate antibiotic. After overnight growth at 28°C the cells were subcultured into 45 ml of BHI in 250-ml sidearm flasks at 28°C. When the cultures reached early log phase (Aho = 0.6), cells were pelleted by centrifugation ( 10 min, 16OOg),washed once with potassium phosphate buffer (0.0125 M at pH 7. I), and resuspended in an equal volume of buffer. The donor (Tc’) and recipient (Sm’ or RP) cells were mixed at ratios of 1:9 or 1:1 and filtered onto a 0.45~pm nitrocellulose filter (25 mm diameter) (Millipore Corp., Bedford, MA). The filters were placed cellside up on BHI agar and were incubated for 4 h at 28°C. The cells were washed from the filter with 2 ml of phosphate buffer, vortexed vigorously for 30 s, diluted, and plated on BHI containing both selective antibiotics. Donor and recipient populations after mating also were determined independently after 4 h at 28°C. Transfer frequency was expressedas the number of Tc’ transconjugants per milliliter divided by the viable count of recipient ‘Abbreviations used: TSB, tryptic soy broth; TSA, TSB containing 1.5%agarose;BHI, brain heart infusion broth; SDS, sodium dodecyl sulfate; D:R, donor:recipient ratio.

PLASMID

TRANSFER

IN Bacillus pumilus

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tures by the method of Kado and Liu (198 1) as modified by Koehler and Thome (1987) for B. subtilis. The E. coli pAM120 plasmid used for hybridization experiments was extracted from 500-ml LB cultures by a modified alkaline lysis method (Maniatis et al., 1982) and purified further by chromatography on pZ523 columns (5 Prime-3 Prime, Inc., Paoli, PA) according to the manufacturer’s instructions. Chromosomal DNA from B. pumilus was purified from 5-ml overnight BHI cultures by the method of Engwall and Atherly (1986), except that 10 mg of lysozyme/ml was used for cell lysis. DNA was cut with restriction endonucleases (Promega, Inc., Madison, WI) according to manufacturer’s directions, and fragment sizes were determined by agarosegel electrophoresis (0.7 to 1.O%SeaKem GTG agarose; FMC BioProducts, Rockland, ME) in Tris borate buffer (Meyers et al., 1976), using DNA fragment size standards obtained from Bethesda Research Laboratories, Inc. (Gaithersburg, MD) ( 1-kb DNA ladder and high-molecular-weight DNA markers). Labeling probe. Plasmid PAM 120 (1 pg) was linearized by restriction endonuclease digestion and labeled with 50 PCi of [a-32P]dCTP by the random priming method of Screening transconjugantsfor mutations. Feinberg and Vogelstein (1983). A library of Tn916-containing B. pumilus Southern hybridization. Plasmid DNA transconjugants from matings of E. faecalis (300 to 400 ng) or total bacterial DNA (2.0 CGl 10 and B. pumilus 296.1 was made by pg) was cut with restriction enzymes, sepapicking individual colonies from selection rated by electrophoresis on a 1.0% agarose plates to master grids on TSA containing Sm gel, and transferred to positively charged and Tc. At the sametime colonies were inocu- nylon membranes (BioTrace RP, Gelman lated into wells of microtiter plates contain- Sciences, Inc., Ann Arbor, MI) by standard ing 100 ~1TSA + 20% glycerol for permanent methods (Maniatis et al., 1982). Hybridizastorage at -70°C. Screening for auxotrophic tion was carried out in 50% formamidedX mutants was performed by replica-plating SSC (1X SSC is 0.15 M NaCl plus 0.015 M master grids to minimal medium. Auxo- sodium citrate)-0.5% sodium dodecyl sulfate trophs were identified by failure of colonies (SDS)-5X Denhardt solution- 100 Kg of salto grow on minimal medium; they were puri- mon sperm DNA/ml-25 mM NaPO,, pH fied by streaking twice for single colonies on 6.8. Filters were hybridized overnight at TSA + Tc, and then diagnosis of auxotrophic 42°C with 32P-labeled PAM 120 DNA ( lo5 requirements was carried out according to cpm/ml for filters containing plasmid DNA; Davis et al. (1980). lo6 cpm/ml for filters containing chromoDNA techniques.B. pumilus plasmids were somal DNA). They were washed at room temisolated from 5-ml overnight BHI broth cul- perature with 2X SSC-0.1% SDS, at 42°C (Sm’ or Rp) cells in the mating mixture after 4 h. Controls consisting of donor and recipient cells alone were treated similarly to determine the frequency of spontaneous resistant mutants in the population. For broth matings, donor and recipient cultures were prepared as described above, except that the cells were resuspended in an equal volume of BHI broth, then mixed at 1:1 and 1:9 ratios, incubated statically for 4 h at 28°C and then diluted and plated on selective medium. To determine that transfer of resistance was not due to either transformation or transduction, the following experiments were performed. Filter and broth matings in the presence of DNase were carried out as described above, except that cell mixtures contained 10 pg of DNase (Sigma; type I)/ml and 10 mM MgS04, and the filters were incubated on BHI containing 100 pg of DNase/ml and 10 mM MgS04. To determine the possibility of transduction, the donor and recipient were grown as described above, then the donor culture was centrifuged ( 10 min, 16OOg),and the supematant was filtered through a 0.22pm filter (Millipore). Equal volumes of the cell-free donor filtrate and the recipient culture were mixed and treated as described above for filter and broth matings.

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HENDRICK TABLE 2 FREQUENCYOF TRANSFEROF Tn916 FROM Enterococcusfaecalis CGl 10 TO Bacillus pumilus ISOLATES’

Recipient 296.1

296.1 302.1 302.1

1.1

Mating conditions”

Transfer frequency (per recipient)

Filter Broth Filter Broth Filter ( 16 h)

6.2 x lO-7 *

nd’ 6.5 x 10-7d

nd 1.2 x 10-s

n 4 h at 37’C, except as noted; donor:recipient ratio, 1:9. * Mean of seven experiments. ’ nd, transfer not detected (frequency,

Insertion of Tn916 into Bacillus pumilus plasmid pMGD302 and evidence for plasmid transfer by conjugation.

As part of an effort to develop systems for genetic analysis of strains of Bacillus pumilus which are being used as a microbial hay preservative, we i...
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