Introduction of Tn916 and Pamp1 Into Streptococcus Bovis JB1 by Conjugation ROBERT B

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1991, p. 2710-2713 Vol. 57, No. 9 0099-2240/91/092710-04$02.00/0

Introduction of Tn916 and pAMP1 into Streptococcus bovis JB1 by Conjugation ROBERT B. HESPELL AND TERENCE R. WHITEHEAD* Fermentation Biochemistry Research, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department ofAgriculture, 1815 N. University Street, Peoria, Illinois 61604 Received 12 March 1991/Accepted 25 June 1991

The transposon Tn916 and self-mobilizing plasmid pAM,l1 were conjugated from Enterococcus faecalis to the ruminal bacterium Streptococcus bovis JB1. Transconjugants were identified by resistance to tetracycline (Tn916) or erythromycin (pAMfr1) and by Southern hybridization analyses. Transfer frequencies were 7.0 x 10-6 and 1.0 x 10-6 per recipient cell for Tn916 and pAMfi1, respectively. The transconjugants JB1/Tn916 and JB1/pAMII1 were used as donors for matings with E. faecalis, Bacillus subtilis, and the ruminal bacterium Butyrivibrio fibrisolvens. While pAMj31 was successfully transferred to all three organisms, Tn916 was transferred only into B. subtilis and B. fibrisolvens at very low frequencies. This is the first report of conjugal DNA transfers between two ruminal organisms.

The microflora of the rumen is responsible for degradation 0.3% glucose as the energy source. E. faecalis CG110 of feed materials ingested by ruminants. There is consider- containing Tn916 in the chromosome (5), CG180 containing able interest in modifying ruminal fermentation to increase Tn9J6 on the plasmid pAM180 (5), JH2-2 containing pAM31 the efficiency of digestion. One approach to achieve this is to (11), and RH110 containing Tn9J6zAE (14) were also grown genetically alter species of ruminal microorganisms to en- on RGM-glucose with 10 jxg of tetracycline per ml or hance degradation of feed materials (for reviews, see refer- erythromycin as necessary. B. subtilis BR151 was grown ences 4 and 6). In the last few years, various genes encoding aerobically on brain heart infusion (BHI) medium (Difco, enzymes involved mainly in cellulose and xylan degradation Detroit, Mich.) at 37°C. Plasmids pDL216, a derivative of have been cloned from several species, but genetic exchange pAMP1 (11), and pAM120 (5), containing Tn916, were used systems for ruminal bacteria have not yet been developed as probes for Southern hybridization analysis of pAMP1 and (7). Tn9O6 transconjugants, respectively. Although the bacterial population of the rumen consists Mating protocols. For S. bovis JB1 and E. faecalis mat- predominantly of gram-negative species, two ecologically ings, the procedures described previously (8) were used with important gram-positive species are Butyrivibriofibrisolvens some modifications. The cells were grown to late-log or and Streptococcus bovis. The major role of B. fibrisolvens is early-stationary phase, and the cells were allowed to filter xylan degradation, but many strains are also amylolytic and mate for 7 to 12 h at 37°C. Each filter was washed with pectinolytic. S. bovis is an active starch-degrading ruminal carbohydrate-free DM, and appropriate dilutions were per- organism and produces an extracellular amylase (2). A gene formed with DM. S. bovis JB1 transconjugants were selected encoding amylase activity has recently been cloned from S. on DM agar plates overlaid with 4 ml of DM agar containing bovis in our laboratory (15). The development of genetic 0.2% sterile insoluble cornstarch (U.S. Biochemicals, Cleve- transfer systems for this organism would allow us to intro- land, Ohio) and 10 jig of tetracycline or erythromycin. The duce the cloned amylase gene or other genes into S. bovis cornstarch was added to the DM overlay as a dry, auto- and to create useful mutants for genetic study. Recently, we claved powder. S. bovis colonies produced large clearing have shown conjugal transfer of the transposon Tn916 (1) zones on the starch-agar. E. faecalis strains were unable to and the self-mobilizing plasmid pAM31 from Enterococcus grow on DM containing starch or other carbohydrates. faecalis into a variety of B. fibrisolvens strains which repre- For S. bovis and B. fibrisolvens matings, strains were sent different DNA relatedness groups (8). We now report on grown on DM containing 0.3% cellobiose to mid-log (B. the conjugal transfer of Tn9J6 and pAMP1 from E. faecalis fibrisolvens) or late-log (S. bovis) phase. Mating procedures into S. bovis JB1. Interestingly, unlike B. fibrisolvens trans- similar to those described above were used, except that the conjugants, S. bovis JB1 transconjugants containing Tn9J6 DM overlay contained 0.3% L-arabinose and 0.1% remazol or pAM,B1 could conjugally transfer these elements to E. brilliant blue xylan (Sigma Chemical Co., St. Louis, Mo.). faecalis, B. fibrisolvens, or Bacillus subtilis. Unlike B. fibrisolvens, S. bovis can neither grow on this (A preliminary report of this work has been presented [9].) medium nor produce colonies which have clearing zones on the remazol brilliant blue xylan. MATERIALS AND METHODS For S. bovis and B. subtilis matings, both organisms were Bacterial strains, plasmids, and media. S. bovis JB1 (13) grown on BHI medium until mid-log phase, and a procedure and B. fibrisolvens strains were grown anaerobically either similar to that described above was used except that BHI on a defined medium (DM[3]) or on RGM medium, a medium was substituted for RGM medium and matings were complex yeast extract-Trypticase-salts medium (10), with carried out aerobically. B. subtilis transconjugants were selected on a minimal salts agar medium [K2HPO4, 14.0; KH2PO4, 6.0; (NH4)2SO4, 2.0; sodium citrate, 1.0; MgSO4; * Corresponding author. Casamino Acids, 0.2; tryptophan, 0.05 (pH 7.0); each in 2710 VOL. 57, 1991 INTRODUCTION OF Tn9l6 AND pAM,Bl INTO S. BOVIS JB1 2711 grams per liter], containing 0.2% L-arabinose as the carbon A. B. source and 10 ,ug of tetracycline or erythromycin per ml. S. 1 2 34 5 1 2 3 45 bovis JB1 cannot use arabinose and was unable to grow on this medium. Conjugation frequencies are expressed as transconjugants per donor or recipient cell at the time of mating. DNA analyses. Total DNA was isolated from JB1 trans- Kb conjugants by using a modified miniprep procedure sug- gested by David Odelson, Central Michigan University, Mt. Pleasant. Briefly, each isolate was inoculated into 3 ml of 23.1- RGM-glucose containing the appropriate antibiotic. After overnight growth at 37°C, 6 ml of RGM-glucose containing 20 mM DL-threonine was added and the culture was incu- 9.4- bated at 37°C for 1 h. Nonsterile powdered glycine was then 6.6- added to a 5% (wt/vol) final concentration, and the culture was incubated at 37°C for 1 h. Cells were pelleted by 4.4- centrifugation and washed once at room temperature with 1.5 ml of TE buffer (10 mM Tris HCl, 1 mM EDTA) containing 50 mM NaCl. The cells were pelleted by centrifugation and suspended in 0.3 ml of TE buffer containing 25% 2.3- glucose. The following additions were made to the cell 2.0- suspension: 3 ,ul of a 10-mg/ml concentration of DNase-free RNase A, 70 RIu of 1.5% lysozyme in TE buffer, and 10 ,ul of 0.5 M EDTA. The cells were allowed to incubate at room temperature for 1 h, and then 40 ,ul of 10% sodium dodecyl sulfate was added to lyse the cells. After incubation at room temperature for 30 min, 65 pI of 5 M potassium acetate was added and the mixture was incubated on ice for 30 min. The precipitate was removed by centrifugation. The supernatant FIG. 1. Southern hybridization analysis of total DNA from S. phenol-chloro- bovis JB1 and Tn916 transconjugants. DNA was probed with fluid was recovered and extracted once with biotinylated DNA synthesized from pAM120. (A) Photograph of form (1:1, vol/vol). The DNA in the aqueous phase was 0.8% agarose gel stained with ethidium bromide. (B) Detection of precipitated with ethanol and pelleted by centrifugation. The hybridizing DNA with alkaline phosphatase-strepavidin conjugate DNA pellet was dissolved in TE buffer for further analysis. after transfer to nitrocellulose. Lanes: 1, biotinylated lambda- The DNA was digested with restriction endonucleases ac- HindlIl molecular size markers; 2 to 5, HindIll-digested DNA (2, cording to instructions of the manufacturer, electrophoresed JB1; 3, JB1-T1; 4, JB1-T2; 5, JB1-T3). through agarose gels in 89 mM Tris-68 mM phosphoric acid-2 mM EDTA, and stained with ethidium bromide. DNA hy- bridizations were done with biotinylated probes as described previously (8, 12). tetracycline resistance gene replaced by an erythromycin Materials. Restriction enzymes, [biotin-14]dATP, and bi- resistance gene (14). It is not clear why we were unable to otinylated lambda-HindIII molecular size markers were pur- isolate Tn9J6AE transconjugants. We are currently attempt- chased from Bethesda Research Laboratories (Gaithersburg, ing to isolate different strains of E. faecalis containing Md.). Nitrocellulose BA-85 (0.45-pum pore size) was ob- Tn916AE, in case the donor strain used influences conjugal tained from Schleicher & Schuell (Keene, N.H.). All other transfer of Tn916AE. Alternatively, the erythromycin resis- chemicals were of reagent grade or better. tance gene in Tn916AE is from Enterococcus sanguis and may not be expressed in S. bovis. RESULTS AND DISCUSSION Conjugal transfer of pAM31. Erythromycin-resistant transconjugants of S. bovis were isolated after filter mating Conjugal transfer of Tn916. Filter matings of E. faecalis with E. faecalis JH2-2 containing pAM31, a plasmid orig- CG110 (chromosome-inserted Tn916) or CG180 (plasmid- inally isolated from Enterococcus pyogenes. The transfer inserted Tn916) with S. bovis JB1 produced tetracycline- frequencies for pAM31 are shown in Table 1. The results of resistant S. bovis transconjugants at transfer frequencies of Southern hybridization analyses of DNA from S. bovis JB1 10-6 or greater (see Table 1). To determine if Tn916 was and transconjugant JB1-E2 are shown in Fig. 2. The DNA inserted at one or more sites in the S. bovis chromosome, was digested with HpaII to linearize pAMB1 (26.5 kb) (lanes total DNA was isolated from S. bovis JB1 and three trans- 2 and 3) or run on the gel undigested (lanes 4 and 5). The conjugants (JB1-T1, JB1-T2, JB1-T3). The DNA was di- results demonstrated the presence of a linear and undigested gested with HindIlI and analyzed by Southern hybridization (near top of gel) form of pAM31. Of interest is the presence with the Tn916-containing plasmid pAM120. The results of at least one plasmid in JB1 (Fig. 2, lanes 4 and 5; between shown in Fig. 1 demonstrate that Tn916 was inserted in the 2.0- and 4.4-kb markers), the mobility of which is altered different locations in the chromosome (lanes 3 to 5) and that after digestion with HpaII. JB1 has no cross-hybridizing DNA (lane 2). The high- Conjugal transfer of Tn916 and pAM,(l from S. bovis. molecular-weight band in lane 3 appears to be the result of When S. bovis JB1-E2 (pAMP1) was filter mated with either incomplete digestion with Hindlll. However, no erythromy- E. faecalis OG1-X or JH2-2, erythromycin-resistant trans- cin-resistant S. bovis transconjugants were obtained after conjugants were readily obtained (Table 1). These results matings with E. faecalis RH110 containing the chromoso- indicate that both the antibiotic resistance and conjugative mally inserted Tn916AE, a derivative of Tn916 having the properties of pAM,B1 are expressed in S. bovis. Despite this, 2712 HESPELL AND WHITEHEAD APPL. ENVIRON. MICROBIOL.

TABLE 1. Transfer frequencies of Tn916, Tn9J6AE, A. B. and pAMPi after matings between E. faecalis, 1 2 3 4 5 1 2 3 4 S. bovis, B. subtilis, and B. fibrisolvens 5 Transfer frequency Cross (donor x recipient)a Element Per donor Per Kb recipient E. faecalis x S. bovis CG110 x JB1 (3) Tn916 1.4 x 10-6 6.9 x 10-6 23.1- CG180 x JB1 (1) Tn916 7.0 x 10-7 2.1 x 10-6 RH110 x JB1 (2) Tn9J6AE NDb ND 94.... JH2-2 x JB1 (3) pAMPi 8 x 10-6 1.0 x 10-6 9.4- t 0 . S. bovis x E. faecalis 6.6- JB1-E2 x OG1-X (2) pAM,1 7.3 x 10-5 9.6 x 10-5 JB1-E2 x JH2-2 (2) pAM,Bl 3.1 x 10-4 4.1 x 10 4.4- JB1-T2 x OG1-X (3) Tn916 ND ND JB1-T2 x JH2-2 (3) Tn916 ND ND S. bovis x B. subtilis 2.3- JB1-T2 x BrlSl (2) Tn9O6 2 x 10-9 1.0 x 10-7 2.0- JB1-E2 x Brl5l (2) pAMP1 5.5 x 10-10 5.0 x 10-8 S. bovis x B. fibrisolvens JB1-E2 x H17c (2) pAM,1 6 x 10-6 1.2 x 10- JB1-E2 x CF3 (2) pAMpi ND ND JB1-E2 x Dl (2) pAMpi 2.1 x 10-6 5.0 x 10-6 JB1-T2 x H17c (2) Tn9O6 3.5 x 10-7 2.5 x 10-7 JB1 x CF3 (2) Tn916 2.9 x 10-8 1.3 x 10-8 JB1 x Dl (2) Tn9O6 7.7 x 10-8 6.1 x 10-8 a Number of replicate experiments indicated in parentheses. b FIG. 2. Southern hybridization analysis of total DNA from S. ND, not detected. bovis JB1 and pAMP1 transconjugant JB1-E2. DNA was probed with biotinylated DNA synthesized from pDL216. (A) Photograph of 0.8% agarose gel stained with ethidium bromide. (B) Detection of hybridizing DNA with alkaline peroxidase-streptavidin conjugate erythromycin-resistant transconjugants of B. subtilis BrlSl after DNA transfer to nitrocellulose. Lanes: 1, biotinylated lambda- were isolated at only a very low frequency after matings with Hindlll molecular size markers; 2, Hpall-digested JB1 DNA; 3, JB1-E2 (Table 1). Similar, low transfer frequencies were HpaII-digested JB1-E2 DNA; 4, undigested JB1 DNA; 5, undi- observed with matings of S. bovis JB1-T2 (Tn916) and B. gested JB1-E2 DNA. subtilis Brl51. No tetracycline-resistant transconjugants of E. faecalis OG1-X or JH2-2 were obtained from matings with S. bovis JB1-T2. Since chromosomal location can influence ACKNOWLEDGMENTS Tn916 transfer (14), additional matings with E. faecalis and We thank Patricia O'Bryan and David Lee for excellent technical other JB1-Tn916 transconjugants were carried out. How- assistance. ever, no tetracycline-resistant transconjugants were obtained. 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Washington, D.C. VOL. 57, 1991 INTRODUCTION OF Tn916 AND pAM,1 INTO S. BOVIS JB1 2713

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