APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1991, p. 2703-2709

0099-2240/91/092703-07$02.00/0

Vol. 57, No. 9

Conjugal Transfer of Tn916, Tn916AE, and pAM31 from Enterococcus faecalis to Butyrivibrio fibrisolvens Strains TERENCE R. WHITEHEAD Fermentation Biochemistry Research, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 N. University Street, Peoria, Illinois 61604 ROBERT B. HESPELL*

AND

Received 25 February 1991/Accepted 25 June 1991

Anaerobic filter matings of Butyrivibriofibrisolvens H17c, CF3, Dl, or GS113, representing different DNA relatedness groups, were done with Enterococcus faecalis CG110, which contains chromosomally inserted Tn916. Tetracycline-resistant transconjugants were obtained with each mating pair at average frequencies of 4.4 x 10-6 (per recipient) and 5.2 x 10-6 (per donor). The transfer frequencies of Tn916 into B. fibrisolvens varied 5- to 10-fold with mating time, strain, and growth stage. By using Southern hybridization with pAM120 as the probe, Tn916 was shown to insert at one or more separate chromosomal sites for each strain of B. fibrisolvens. Retransfer of Tn916 from B. fibrisolvens H17c or CF3 to E. faecalis OG1-X or JH 2-2 or to B. fibrisolvens Dl or GS113 could not be shown. Matings of E. faecalis RH110, which contains chromosomally inserted Tn916AE, with B. fibrisolvens 49, H17c, Dl, CF3, GS113, or VV-1 resulted in erythromycin-resistant transconjugants at average frequencies of 5.3 x 10-7 (per recipient) and 2.5 x 10-7 (per donor). Tn916AE was shown by Southern hybridization with pAM120 to insert at one or more sites in the chromosome of each strain. B. fibrisolvens H17c was anaerobically filter mated with E. faecalis JH 2-SS, which contains pAMIl. Erythromycin-resistant transconjugants were obtained at frequencies of 2 x 10-5 (per recipient) and 6 x 10'5 (per donor). The presence of pAM,ll in these transconjugants could not be shown by, agarose gel electrophoresis of plasmid minilysates but could be shown by Southern hybridization analysis. Retransfer of pAMfrl from B. fibtrisolvens H17c transconjugants in matings with B. fibrisolvens DI or GS113 or with E. faecalis JH 2-2 cotild not be shown. These data are the first to show stable transfer of a plasmid (pAMfrl), transposons (Tn916, Th9J6AE), or genes (erythromycin and tetracycline resistances) from nonruminal species to ruminal species of bacteria.

Butyrivibrio species are common inhabitants of the rumen and cecum of ruminants and are present in the gastrointestinal tract of a variety of mammals (4, 5). In addition, these bacteria are present in environments outside of animals, such as anaerobic digestors (42), and strains from human fecal material have been designated Butyrivibrio crossatus (33). All isolates obtained from domestic animals have been called strains of Butyrivibrio fibrisolvens, but recent studies have indicated that these strains may actually represent several species, based on chromosomal DNA-DNA hybridizations (31) and extracellular polysaccharide compositions (47). All characterized strains of B. fibrisolvens are strictly anaerobic and highly xylanolytic, but many strains are also pectinolytic, amylolytic, proteolytic, or lipolytic. When stained by conventional procedures, all B. fibrisolvens strains appear as gram-negative cells. However, electron microscopic studies have shown that this organism has a very thin, gram-positive cell wall structure (6), and some strains may also have an outer envelope layering (10, 44). Lipoteichoic acids are present in some B.fibrisolvens strains (22). Partial 16s rRNA sequences of some B. fibrisolvens strains indicate that these bacteria are of gram-positive phylogenetic origin (11). Thus, it would appear that B. fibrisolvens strains are in fact gram-positive bacteria but have a cell wall structure atypical of the classical grampositive or gram-negative types. Utilization of ingested feed materials, such as cellulose and xylans, by ruminants depends upon microbial digestion. The degradation of these materials in the rumen is incom*

Corresponding author.

plete, and there is substantial interest in altering ruminal fermentation to optimize digestion. A suggested approach to accomplish this alteration has been to use genetically manipulated species of ruminal bacteria (13, 18, 19, 45). Because of its biochemical diversity and numerical importance in the rumen, B. fibrisolvens is a logical choice for genetic manipulation, although few genetic studies have been done with this microorganism. Several genes encoding for enzymes involved in xylan (32, 43) or cellulose (1, 29) degradation have been cloned from B. fibrisolvens. Some strains appear to have cryptic, indigenous plasmids (30, 48). However, there have been no reports of DNA exchange mechanisms or expression of foreign genes by any strain of B. fibrisolvens. The streptococcal transposon Tn9J6 can mediate its own conjugal transfer from Enterococcus faecalis to other Enterococcus species (8, 14, 15) as well as to other grampositive species such as Bacillus anthracis (23), Bacillus thuringiensis (33), or Staphylococcus aureus (24) and anaerobic bacteria such as Clostridum acetobutylicum (2, 3, 51) or Clostridium tetani (49). Furthermore, Tn916 can serve as the mobilizing agent for conjugal transfer of nonconjugative plasmids between Bacillus species (35). The self-mobilizing streptococcal plasmid pAMP1 can also be conjugatively transferred among a number of Enterococcus species (26) and other gram-positive species (28, 36, 37). On the basis of the apparent gram-positive cell wall structure and phylogenetic origin of B. fibrisolvens, we reasoned that Tn9J6 and pAM,B1 might be used to develop genetic transfer systems with this organism. We now report that these genetic elements can be transferred to several B. fibrisolvens strains which may represent different Butyrivibrio species. 2703

2704

APPL. ENVIRON. MICROBIOL.

HESPELL AND WHITEHEAD TABLE 1. Bacterial strains and phenotypes

Phenotype'

Strain

B. fibrisolvens 49 H17c CF3 GS113 Dl VV-1 E. coli CG120 E. faecalis

Reference

or

source

5

pAM120; Ampr Tcr

B. Dehority B. Dehority 41 5 V. Varel 14

Tn9O6; Tcr Rif' Fusr pAM,1; Emr Strr Spcr Tn916AE; Emr Rif' Strr Rif Fusr Strr Spcr pAM180; Emr Tcr pDL216; Emr

14 27 39 B. White B. White B. White 15 27

Novr Novr

CG110 JH2-SS RH110 OG1-X JH2-2 JH2-SF CG180 S. sanguis DL216

aAbbreviations: Nov, novobiocin; Amp, ampicillin; Tc, tetracycline; Rif, rifampin; Fus, fusidic acid; Spc, spectinomycin; Em, erythromycin; Str, streptomycin.

(A preliminary report of this work has been presented

[20].) MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains used in this study are listed in Table 1. Escherichia coli strains were grown on LB medium (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 1 liter of distilled water) or LB containing 50 ,g of ampicillin to maintain plasmids. B. fibrisolvens strains were grown anaerobically on RGM medium (21) or DM medium (9) containing 0.4% glucose or arabinose as the energy source. E. faecalis strains were also grown anaerobically on glucose-containing RGM. DM and RGM media were supplemented with 10 p.g of erythromycin, tetracycline, chloramphenicol, rifampin, streptomycin, fusidic acid, or spectinomycin per ml as appropriate for individual strains. All cultures were incubated at 37°C. Conjugation experiments. Unless indicated otherwise, all procedures were done anaerobically at room temperature by using a glovebox having a 75% nitrogen-20% carbon dioxide-5% hydrogen atmosphere. Test tube cultures (3.5 to 10.0 ml) grown at 37°C in the glovebox to mid- or late-logarithmic-growth stage were transferred to airtight, 10-ml polycarbonate centrifuge tubes (DuPont) that were removed from the glovebox and centrifuged (6,000 x g, 15 min, 15°C). The tubes were returned to the glovebox, and the supernatant fluid was decanted. The cell pellet was suspended in carbohydrate-free RGM or DM medium (50% original culture volume), washed twice by centrifugation, and suspended to 10% original culture volume in the same wash medium. Aliquots (0.3 to 0.5 ml) of the donor and recipient strain cell suspensions were added to a 1.5-ml microfuge tube and centrifuged (6,500 rpm, 5 min, 22°C). After decanting the bulk fluid, the pellet was resuspended by vortexing by using the residual fluid (ca. 50 [ld). The concentrated cell suspension was transferred to the center of a sterile filter (type HAWP, 25-mm diameter, 0.45-pum pore size; Millipore Corp.) on an RGM agar (2.0%) plate. After adsorption of fluid onto the filter, the plates were incubated at 37°C. The optimal incubation time varied with the B. fibrisolvens strain

used, but generally a time of 12 to 16 h was used. After incubation, the filter was transferred to a sterile 30-ml Corex centrifuge tube, 1 ml of carbohydrate-free RGM or DM medium was added, and the cells were washed off the filter surface by vortexing. After appropriate dilution (usually 10to 100-fold) in the same medium, 50-pd aliquots were spread on DM medium containing L-arabinose and 10 ,ug of the appropriate antibiotic(s) per ml. For E. faecalis and B. fibrisolvens matings, remazol brillant blue xylan was often included (2.5 mg/ml) in the selective media to aid in the detection of minute (0.1-mm diameter or less) colonies of B. fibrisolvens transconjugants by the occurrence of clearing zones around the colonies. Counterselection of E. faecalis donor strains was obtainable because these organisms do not grow on arabinose-containing DM medium and are nonxylanolytic. For matings with only B. fibrisolvens strains, RGM medium containing glucose or L-arabinose plus 10 pug of the appropriate antibiotic(s) per ml was used as the selective medium. Selected transconjugant strains were obtained by picking colonies and sequentially streaking colonies twice on the selective medium to insure that the appropriate antibiotic resistances were present and that the strain was free of any contaminating donor cells. Transfer frequencies were calculated on the basis of the number of viable cells placed on the filter at the beginning of the mating. Viable cell counts were determined by plating aliquots (20 or 50 pI per plate) of dilutions made in carbohydrate-free RGM medium of samples taken from cell cultures before harvesting. The plating medium was RGM containing glucose and 2.0% agar. Purification of DNA. Total cellular DNA was isolated from various strains by using the method of Saito and Miura (40). Plasmid DNA was purified from E. coli by a modified alkaline lysis procedure with the Circle Prep kit (Bio 101, La Jolla, Calif.). DNAs were analyzed by digestion with restriction endonucleases according to the instructions of the manufacturer and electrophoresed through agarose gels prepared in buffer (89 mM Tris, 68 mM phosphoric acid, 2 mM EDTA [pH 8.5]). DNA fragments in gels were detected by staining with ethidium bromide (2.0 pug/ml) and visualized by UV transillumination. Restriction analysis and hybridization. For hybridization with biotinylated probes, DNA was transferred from agarose gels to nitrocellulose by use of a vacuum blotter (model 785; Bio-Rad Laboratories, Richmond, Calif.) with 10x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate [pH 7.0]). Plasmid DNA was labeled with [14-biotin]dATP by using a random primed DNA labeling kit according to the instructions of the manufacturer (Boehringer-Mannheim, Indianapolis, Ind.). Hybridizations were carried out at 67°C for 16 h with standard hybridization buffer (46). After hybridization, the nitrocellulose sheets were sequentially washed as follows: 2x SSC-0.1% sodium dodecyl sulfate (SDS), room temperature, 5 min; 0.2x SSC-0.1% SDS, room temperature, 5 min; 0.16x SSC-0.1% SDS, 500C, 30 min; and 2x SSC-0.1% SDS, room temperature, 2 min. The resultant washed sheets were stained with the BluGene DNA detection kit (Bethesda Research Laboratories, Gaithersburg, Md.) by using streptavidin-alkaline phosphatase for detection of the probe according to the instructions of the manufacturer. Materials. Restriction endonucleases, [14-biotin]dATP, and biotinylated lambda-HindIII molecular size markers were purchased from Bethesda Research Laboratories. Remazol brilliant blue xylan, oatspelt xylan, and other routine biochemicals were obtained from Sigma Chemical Co. (St. Louis, Mo.). Nitrocellulose sheets (BA-85; 0.45-,um pore

CONJUGAL TRANSFER FROM E. FAECALIS TO B. FIBRISOLVENS

VOL. 57, 1991

TABLE 2. Frequencies of Tn9O6 transfer to B. fibrisolvens strains from E. faecalis CG110 B. fibrisolvens

Transfer frequencya (10-6)

A. 1

2

R 4

R6

7 8 9

B. 1

2 3 4

2705

5 6 7 8 9

No. of

strain

Per recipient

Per donor

replicates

H17c CF3 Dl GS113

6.6 3.7 1.9 5.2

3.2 6.7 6.2 5.0

6 4 5 3

a Data are average values calculated from the indicated number of replicate experiments with an average mating time of 16 ± 2 h.

Kb

12.2 10.28.1 6.1-

size) were purchased from Schleicher & Schuell (Keene, N.H.).

4.1-

RESULTS

2.0-

Antibiotic resistances and growth of B. fibrisolvens strains. When inoculated into broths or streaked onto agar plates of RGM or DM medium containing 5 ,ug of either tetracycline or erythromycin per ml, no growth resulted for B. fibrisolvens H17c, 49, CF3, Dl, GS113, or VV-1. Plating of about 5 x 10 cells onto similar media resulted in no formation of colonies after incubation for 3 to 4 days. With further incubation, a few pinpoint colonies were occasionally detected, but the majority of these colonies, when restreaked on the same medium, did not show growth. These results indicated that the frequency of occurrence of spontaneous tetracycline or erythromycin mutants was very low (i.e., 10-9). Testing of a number of other B. fibrisolvens strains indicated that they all were sensitive to erythromycin, but strains E21c, R28, IL631, and NOR37 were resistant to 10 to 15 ,u.g of tetracycline per ml. All B. fibrisolvens strains tested were also sensitive to (10 ,ug/ml or less) ampicillin, gentamycin, kanamycin, streptomycin, and rifampin. However, all B. fibrisolvens strains were highly resistant to nalidixic acid (40 to 100 p,g/ml), as were E. faecalis strains. Growth studies indicated that RGM or DM medium with glucose or L-arabinose as the carbon source supported growth of all B. fibrisolvens strains. E. faecalis strains grew well on RGM medium with glucose added, formed pinpoint colonies on RGM with or without added L-arabinose, but showed no colony formation on DM medium with L-arabinose. On the basis of the patterns of antibiotic sensitivity and substrate utilization of B. fibrisolvens strains, the use of DM medium with added L-arabinose was chosen as a means to counterselect against donor E. faecalis strains in cell suspensions obtained after mating. Remazol brilliant blue xylan was also included in this medium since minute (0.1-mm diameter) B.fibrisolvens colonies could be easily detected by the clearing zones (1- to 3-mm diameter) surrounding these colonies, whereas colonies of E. faecalis strains showed no clearing of remazol brilliant blue xylan when included in RGM medium containing glucose. Conjugal transfer of Tn916 to B. fibrisolvens strains. When E. faecalis CG110, which contains a chromosomally located copy of Tn9J6 (16), was used as the donor in matings with B. fibrisolvens H17c, tetracycline-resistant transconjugant colonies of the recipient were readily detected within 48 to 72 h after plating. The transfer frequencies were always greater than 10-6 (Table 2). A survey of 10 randomly picked transconjugants were examined for tetracycline resistance levels by using RGM broth cultures. All of these were resistant to high levels of tetracycline (40 to 80 ,ug/ml) and, in

FIG. 1. Southern hybridization analysis of genomic DNA from B. fibrisolvens H17c and Tn9O6 transconjugants with random-

primed DNA synthesized from pAM120. (A) Photograph of 0.8% agarose gel stained with ethidium bromide. (B) Detection of hybridizing DNA with alkaline phosphatase-strepavidin conjugate after DNA transfer to nitrocellulose. Lanes: 1, 1-kb DNA molecular size ladder; 2, 0.2 ,ug of EcoRI-digested pAM120; 3 to 9, 15 ,ug of Hindlll-digested genomic DNA (3, H17c; 4, H17c-Tl; 5, H17c-T2; 6, H17c-T3; 7, H17c-T4; 8, H17c-T5; 9, H17c-BX3).

addition, were resistant to 20 vig of minocycline per ml, consistent with the acquisition of tetM from Tn916. In comparison with the parental strain, all transconjugants appeared to have normal cell morphologies or other apparent characteristics when grown in tetracycline-containing RGM broth medium. Total DNA preparations were made from randomly selected strains and were digested with HindlIl. When the digested DNAs were subjected to agarose gel electrophoresis, ethidium bromide staining revealed the presence of large DNA fragments in transconjugant strains that were absent in the parental H17c strain (Fig. 1A). Further analysis of these gels by Southern hybridization with biotin-labeled pAM120 (16) as the probe showed the presence of two or more hybridizing DNA bands. Since Tn916 contains a single HindlIl site, two junction fragments can be generated for each chromosomal copy of Tn916. Many of the transconjugants showed only the presence of two hybridizing bands, but the DNA fragment sizes varied between strains. Thus, it appears that Tn916 inserts at different sites in the chromosome of B. fibrisolvens H17c. For some transconjugants; multiple Tn916 chromosomal insertions occurred as demonstrated by four hybridizing bands. To determine whether conjugal transfer of Tn9O6 to B. fibrisolvens was of general occurrence, further matings were done with E. faecalis CG110 as the donor and strain CF3, Dl, or GS113 as the recipient. Each of these strains and strain H17c are representatives of various groups of B. fibrisolvens strains that can be considered separate species (31). Tetracycline-resistant B. fibrisolvens transconjugants were obtained from matings with each of these pairs. Although the frequency of tetracycline resistance transfer varied between strains, the frequency was always greater than 10-6 (Table 2). As found with strain H17c transconjugants, the transconjugants from these other strains were resistant to high levels of tetracycline and displayed normal

APPL. ENVIRON. MICROBIOL.

HESPELL AND WHITEHEAD

2706

B.

A.

1

Kb

2

3

5

4

6

7

1

3

2

4

5

6

7

23.1-

TABLE 4. Frequencies of Tn916AE transfer to B. fibrisolvens strains from E. faecalis RHllO B. fibrisolvens strain

9.46.6-

49 H17c CF3 Dl GS113 VV-1

4.4-

2.32.0-

Transfer frequencya (10-) Per recipient Per donor

10.2 2.6 10.4 5.7 1.9 1.2

10.8 1.1 2.1 0.6 0.3 0.2

No. of replicates

3 2 2 2 1 2

a Data are average values calculated from the indicated number of replicate experiments with an average mating time of 12 h.

FIG. 2. Southern hybridization analysis of genomic DNA from B. fibrisolvens CF3 and Tn916 transconjugants with random-primed DNA synthesized from pAM120. (A) Photograph of 0.8% agarose gel stained with ethidium bromide. (B) Detection of hybridizing DNA with alkaline phosphatase-strepavidin conjugate after DNA transfer to nitrocellulose. Lanes: 1, biotinylated lambda-HindIII molecular size markers; 2 to 7, 15 ,ug of Hindlll-digested genomic DNA (2, CF3; 3, CF3-T1; 4, CF3-T2; 5, CF3-3; 6, CF3-T5; 7, 0.2 ,ug of pAM120 digested with HindlIl).

growth characteristics. Total DNAs were isolated from randomly selected transconjugants and subjected to analysis by digestion and Southern hybridization as described for strain H17c transconjugants. The results of these studies indicated that Tn916 had inserted into the chromosome of each B. fibrisolvens strain tested, and representative data are shown for strain CF3 (Fig. 2). Optimization of Tn916 transfer to B. fibrisolvens. To further characterize the conjugal transfer of Tn916, E. faecalis CG110 was mated with B. fibrisolvens strains for various times. The results indicated that the number of transconjugants varied both with the mating time and particular B. fibrisolvens strain used (Table 3). Surprisingly, transconjugants were obtained at 0 h (or as soon as the cells could be resuspended from the filters) for all three tested strains. For strain CF3, maximal numbers of transconjugants were obtained with 0- to 1-h mating times and decreased to about 50 and 20% of these values with 3- and 22-h mating times, respectively. In contrast, maximal transconjugant numbers were obtained with a 6-h mating time for strain Dl, the type species for B. fibrisolvens. For strain H17c, maximal transconjugants were obtained with about 3-h mating times, with a gradual decrease in numbers with mating times up to 34 h. Culture conditions used to prepare the cells before mating TABLE 3. Effect of mating time on frequency of Tn916 transfer to B. fibrisolvens strains from E. faecalis CG110 No. of transconjugantsa Time (h) Strain H17c

Strain CF3

487 555 188 145 164 100 90

1,500

0 3 6 12 22 28 34 a

Data are expressed

fibrisolvens recipients.

as

693 520 531 285

Strain Dl

115 136

1,804 275 69

the total number of transconjugants per 108 B.

also influenced the numbers of transconjugants obtained. No differences in transfer frequencies were detectable when E. faecalis CG110 cells were taken from either mid-log- or early-stationary-phase cultures. However, with B. fibrisolvens H17c or CF3, cells taken from early-stationary-phase cultures resulted in 4- to 10-fold lower transfer frequencies compared with cells taken from early- to mid-logarithmicphase cultures. Growth of B. fibrisolvens H17c and CF3 on RGM or DM medium with either glucose or arabinose as the energy source did not appear to have any influence on Tn916 transfer frequencies. Conjugal transfer of Tn916AE to B. fibrisolvens strains. Tn9J6AE is Tn916 with the tetracycline resistance gene replaced with an erythromycin resistance gene derived from Streptococcus sanguis and displays transposition properties similar to those of Tn9O6 (39). E. faecalis RH110, which contains two chromosomal copies of Tn916AE, transfers Tn916AE at high frequencies to other E. faecalis strains. When strain RH110 was used as the donor in matings with B. fibrisolvens strains, erythromycin-resistant transconjugants of B. fibrisolvens were readily detected (Table 4). These results demonstrate that the S. sanguis erythromycin resistance gene could be expressed in B. fibrisolvens. The transfer frequencies varied more than 10-fold, depending upon which B. fibrisolvens strain served as the recipient. The highest frequencies were observed with strain CF3, and the lowest frequencies were observed with strain VV-1. In general, the transfer frequencies of Tn916AE (Table 4) were about 10-fold less than those observed for Tn916 (Table 2). About five to six erythromycin-resistant transconjugants obtained with each B. fibrisolvens strain were randomly selected and examined for erythromycin resistance levels. All of these transconjugants were found to be resistant to at least 100 ,ug of erythromycin per ml when grown in RGM broth but tended to form shorter cells and some chains. In addition, examination of four transconjugants of B. fibrisolvens H17c indicated that these strains were also resistant to 20 ,ug of clindamycin per ml, as expected for the macrolidelincosamide-streptogramin B resistance of Tn916AE. Genomic DNAs were prepared from selected transconjugants obtained from these matings and subjected to Southern hybridization analysis. The results indicated that transconjugants from B. fibrisolvens VV-1, H17c, or CF3 possessed chromosomal inserts corresponding to Tn9J6AE. Similar to that found with Tn9O6, single and multiple inserts at various chromosomal locations were observed (data not shown). Retransfer of Tn916 or Tn916AE from B. fibrisolvens strains. Transconjugants of B. fibrisolvens H17c harboring Tn916 were selected to serve as donors for matings with B. fibrisolvens Dl and GS113. These strains were chosen as

VOL. 57, 1991

CONJUGAL TRANSFER FROM E. FAECALIS TO B. FIBRISOLVENS

recipients because of their naturally occurring resistance to novobiocin (20 ,ug/ml) and ability to degrade rutin, both traits that were absent in strain H17c. The results of four separate matings employing six H17c strains harboring either single or multiple copies of Tn916 showed no transfer of Tn916 as indicated by the inability to obtain tetracycline- and novobiocin-resistant transconjugants. Similar matings were conducted with five different B. fibrisolvens CF3 strains harboring Tn916 as the donor and either strain Dl or 113 as the recipient. Again, no Tn916 transfer was detected (less than 10-8), even though these strains served as recipients in matings with E. faecalis CG110 (Table 2). As an alternative approach, matings were set up with Tn916-bearing strains of B. fibrisolvens H17c or CF3 as the donor and E. faecalis JH 2-2 (rifampin resistant) or OGX-1 (streptomycin resistant) as the recipient. Although a few tetracycline- and rifampinresistant or tetracycline- and streptomycin-resistant colonies were occasionally obtained with platings from these matings, further restreaking and microscopic examination of these putative transconjugants indicated that these were spontaneous tetracycline- or streptomycin-resistant mutants of the B. fibrisolvens donor strain cells. Tn916AE has been reported to have similar mobilization and transfer properties as Tn916 (39). Because of these observations and the lack of transfer of Tn916 from various B. fibrisolvens strains, attempts to transfer Tn916AE from B. fibrisolvens strains were not undertaken. Stability of Tn.916 and Tn916AE in B. fibrisolvens strains. To assess the stability of Tn916 in B. fibrisolvens, selected transconjugant strains were grown in RGM medium in the absence of tetracycline for several consecutive transfers, and the viable cell counts were determined by plating on RGM medium with (10 ,ug/ml) or without added tetracycline. After approximately 30 doublings, only 34 and 10% of the total cell numbers were tetracycline resistant for strains T2 (single copy of Tn9O6) and BX3 (two copies of Tn9O6) of B. fibrisolvens H17c, respectively. For B. fibrisolvens Dl transconjugant strains Ti and T2, less than 1% of the total cell numbers were tetracycline resistant after approximately 25 doublings. In contrast, Tn916 transconjugants of B. fibrisolvens CF3 and GS113 were quite stable and displayed no loss of tetracycline resistance even after about 25 doublings in the absence of tetracycline. A survey of representative B. fibrisolvens transconjugants harboring Tn916AE also showed variation in stability of erythromycin resistance. After 25 doublings in erythromycin-free RGM medium, 72, 80, 89, and 95% of the total cell numbers were erythromycin resistant for B. fibrisolvens transconjugants of strains Dl, H17c, VV-1, and CF3, respectively. Conjugal transfer of pAM131 to B. fibrisolvens strains. The enterococcal plasmid pAMP1 is a large (26.5-kb), self-mobilizing plasmid transmissible to all Enterococcus species (7) and strains of Bacillus subtilis (25), Lactobacillus casei (17), and Staphylococcus aureus (12). When E. faecalis JH 2-SS containing pAMP1 was used as the donor in matings with B. fibrisolvens H17c, erythromycin-resistant transconjugants were readily obtained. The average transfer frequencies from two replicate matings were 2 x 10-5 per recipient and 6 x 10-5 per donor. With RGM broth cultures, six randomly picked transconjugants were resistant to at least 100 ,ug of erythromycin per ml, and no changes in cell morphology or other characteristics were apparent with these transconjugants when compared with the parental strain. Attempts to demonstrate the presence of pAM,B1 in a number of transconjugants by using agarose gel electrophoretic analysis of plasmid preparations by many minilysate techniques were

2707

unsuccessful. Therefore, total cellular DNA preparations were made, digested with HpaII, and subjected to agarose gel electrophoresis. Southern hybridization analysis of these gels with pDL216 (27) as the probe DNA revealed the presence of linearized pAM,B1 DNA (data not shown). Several matings were conducted with E. faecalis JH 2-SS-pAM31 as the donor and B. fibrisolvens 49, which shows more than 95% DNA-DNA homology with strain H17c (31). However, erythromycin-resistant transconjugants were not obtained. Similar results were obtained in other matings with B. fibrisolvens CF3, Dl, and VV-1, each of which is representative of a separate B. fibrisolvens DNA relatedness group (31). To determine whether pAM,1 could be retransferred from B. fibrisolvens, the novobiocin-sensitive, pAM31-containing strain H17c-E1 was used as a donor in matings with the novobiocin-resistant B. fibrisolvens strains Dl and GS113. No erythromycin-resistant transconjugants were obtained from these matings nor from matings of strain H17c-E1 with E. faecalis JH 2-2. DISCUSSION The results presented here show that pAM,31 can be conjugatively transferred to B. fibrisolvens H17c from E. faecalis at a frequency of about 10-5. This transfer rate is approximately the same as that reported for transfer of this plasmid from E. faecalis to various species of oral streptococci (26). The conjugal transfer of pAMP1 to another anaerobe, C. acetobutylicum, has also been reported. However, the transfer frequency appears to be dependent upon the donor species used. With E. faecalis as the donor, a high transfer frequency (6.8 x 10-5) was observed (52), whereas with B. subtilis as the donor in matings with C. acetobutylicum (39) the frequencies were low (ca. 6 x 10-6 or less). In contrast, our results indicate that the particular strain of B. fibrisolvens used as the recipient in matings with E. faecalis has a dramatic influence on the transfer of pAMP1. No erythromycin-resistant transconjugants were found when B. fibrisolvens 49, CF3, Dl, or VV-1 was used in matings in which a transfer frequency of about 10-8 would have been detectable. Although pAM,B1 can apparently replicate in B. fibrisolvens H17c, significant amounts of this plasmid could not easily be recovered from this organism, suggesting that the copy number is relatively low. This was not unexpected since, in E. faecalis, the copy number is approximately 7 for the intact pAMP1 but can be increased to more than 40 for pAM,B1 derivatives that retain both replication and erythromycin resistance functions (27). Both of these functions are located in regions of pAM,31 that are separate from the segment encoding for the conjugative functions. By using various techniques, nonconjugative plasmids containing the pAM,1 replicon have been transferred from E. coli to C. acetobutylicum (50) and plasmids integrated into pAM,B1 have transferred from B. subtilis to C. acetobutylicum (36). We are currently using similar strategies to develop shuttle vectors based on pAM,1 to introduce genes into B. fibrisolvens. The transposon Tn916 and its derivative, Tn916AE, were conjugatively transferred from E. faecalis to a number of B. fibrisolvens strains by use of filter mating techniques. In general, the transfer frequency of Tn916 into B. fibrisolvens strains was about 5 x 10-6 (Table 2), which is about the same as noted for Tn916 transfer between E. faecalis strains

(14-16). This frequency is about 10-fold higher than the transfer frequency of Tn916 from E. faecalis to B. thuringi-

2708

HESPELL AND WHITEHEAD

ensis

(34) or to B. anthracis (23) when the

APPL. ENVIRON. MICROBIOL. same

chromoso-

mally located Tn916 donor strain (E. faecalis CG110) was used and about 100-fold higher than that observed for Tn916 transfer into C. acetobutylicum (2) when E. faecalis CG180, which carries Tn916 on a plasmid (16), was used. However, the Tn916 transfer frequency from E. faecalis CG180 to C. tetani was about 10-4 (49). The transfer frequencies of Tn916AE from E. faecalis to B. fibrisolvens strains were more than 10-fold less than those observed for Tn916 transfer and ranged from about 1 x 10' to 10 x 1i-' (Table 4), depending on the recipient strain used. Transfer frequencies of Tn916.AE between E. faecalis strains have been reported to be about 5 x 10-6 (39). The variation in transfer frequencies of chromosomally borne Tn916 or Tn916AE can be partially explained by the number of transposon copies present in the chromosome and possibly by the nature of the flanking DNA sequences. As shown by our data (Table 3), the length of mating time can alter the number of transconjugants obtained by five- to eightfold. In fact, the recovery of transconjugants immediately after mating suggests that some transfer might be occurring on the selective plates. Decreases in transfer were also noted when stationary-phase cells of B. fibrisolvens were used as recipients. Since all B. fibrisolvens strains used in these studies produce an extracellular polysaccharide (47), part of which is associated with the cell (6), changes in this material and/or cell wall structure might result in a decreased ability of stationary-phase cells to take up DNA. When examined in previous studies, chromosomally inserted Tn9J6 appeared to be relatively stable as shown when the harboring bacterial species is grown in the absence of tetracycline. The reversion frequency of Tn916-induced amino acid auxotrophs of Streptococcus mutans grown in the absence of tetracycline is about 5 x 10-8, which is about the same rate of loss observed with growth in tetracyclinecontaining media (38). With B. thuringiensis transconjugants, Tn916 also shows a high stability with growth in the absence of tetracycline (35). Our results based on loss of tetracycline resistance with B. fibrisolvens transconjugants indicate that the stability of Tn916 is quite variable, depending on which strain harbors the transposon. A high stability occurs with strains CF3 and GS113, but rapid loss of Tn916 was observed for strains H17c and Dl. It is unclear whether strain, insertion site, or both account for the Tn916 instabilities. However, Tn916AE was relatively stable in all four of these strains. While Tn916 could be introduced into several B. fibrisolvens strains, retransfer of this transposon from B. fibrisolvens strains could not be demonstrated. No tetracyclineresistant transconjugants were detected in matings with either E. faecalis or other B. fibrisolvens strains used as recipients. At present, the basis for this nontransfer is not clear. Since Tn916 was lost at a high rate from B. fibrisolvens H17c transconjugants with growth in the absence of tetracycline, it would appear that at least the initial processes

associated with excision of Tn916 from the chromosome to allow transfer would not be a limiting factor with this strain. However, the lack of transfer was also observed with Tn916 transconjugants of B. fibrisolvens CF3 or Dl, which show low DNA relatedness to strain H17c (31), and could be viewed as other species. Currently, it is known that a closed covalent circular form is an intermediate in Tn916 transposition and can be used to introduce Tn916 into cells by transformation (41). Further experiments are needed to determine whether a similar form is detectable with B. fibrisolvens strains. If so, then perhaps one of the factors

limiting Tn916 transfer to other cells from B. fibrisolvens is transfer of this form across the B. fibrisolvens cell wall. The observed changes in frequency of transfer of Tn916 into B. fibrisolvens strains which are dependent on both growth stage and mating times would be consistent with the possible occurrence of cell wall alterations for optimal DNA transfer across this structure. In summary, the data presented in this paper provide not only the first evidence for introduction of a plasmid from nonruminal species of bacteria into ruminal species of bacteria but also a demonstration of introduction of transposons from nonruminal species. Tn9O6, Tn9J6Ae, and pAM31 can be introduced by filter mating into B. fibrisolvens strains representing different DNA relatedness groups. The transfer frequencies found (Tables 2 and 3) reflect minimal ones since selection of transconjugants had to be done by using defined media. Thus, Tn916-induced auxotrophs for amino acids or other nutritional factors would not have been detected. However, these rates along with multiple chromosomal insertion sites make it appear possible to carry out transposon mutagenesis with B. fibrisolvens. Experiments are currently in progress to screen for Tn916 transconjugants with mutations in genes encoding for proteins associated with starch or xylan degradation in order to clone these genes by previously outlined strategies (16) and to study their regulation. In addition, attempts to introduce Tn916 or other DNA elements into B. fibrisolvens by transformation or electroporation approaches are being undertaken to allow for selection of transposon mutants by using rich media. ACKNOWLEDGMENTS We thank Patricia O'Bryan and David Lee for their excellent technical assistance. REFERENCES 1. Berger, E., W. A. Jones, D. T. Jones, and D. R. Woods. 1989. Cloning and sequencing of an endoglucanase (end 1) gene from Butyrivibriofibrisolvens H17c. Mol. Gen. Genet. 219:193-198. 2. Bertram, J., and P. Durre. 1989. Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum. Arch. Microbiol. 151:551-557. 3. Bertram, J., A. Kuhn, and P. Durre. 1990. Tn916-induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation. Arch. Microbiol. 153:373-377. 4. Brown, D. W., and W. E. C. Moore. 1960. Distribution of Butyrivibriofibrisolvens in nature. J. Dairy Sci. 43:1570-1574. 5. Bryant, M. P., and N. Small. 1956. The anaerobic monotrichous butyric acid-producing curved rod-shaped bacteria of the rumen. J. Bacteriol. 72:16-21. 6. Cheng, K.-J., and J. W. Costerton. 1977. Ultrastructure of Butyrivibriofibrisolvens: a gram-positive bacterium?. J. Bacteriol. 129:1506-1512. 7. Clewell, D. B. 1981. Plasmids, drug resistance, and gene transfer in the genus Streptococcus. Microbiol. Rev. 45:409-436. 8. Clewell, D. B., and C. Gawron-Burke. 1986. Conjugative transposons and the dissemination of antibiotic resistance in streptococci. Annu. Rev. Microbiol. 40:635-659. 9. Cotta, M. A., and R. B. Hespell. 1986. Proteolytic activity of the ruminal bacterium Butyrivibrio fibrisolvens. Appl. Environ. Microbiol. 52:51-58. 10. Dibbayawan, T., G. Cox, K. Y. Cho, and D. M. Dwarte. 1985. Cell wall and plasma membrane architechure of Butyrivibrio spp. J. Ultrastruct. Res. 90:286-293. 11. Dore, J., D. A. Stahl, and M. P. Bryant. 1988. Phylogenetic study of Syntrophococcus sucromutans, a representative of a new genus of the Veillonellaceae family, abstr. R-17, p. 240. Abstr. 88th Annu. Meet. Am. Soc. Microbiol. 1988. American Society for Microbiology, Washington, D.C. 12. Engle, H. W. B., N. Soedirman, A. Rost, W. J. van Leeuwen, and

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