Letters in Applied Microbiology 1998, 26, 35–37

Conjugal transfer of transposon Tn1545 into the cellulolyticbacterium Eubacterium cellulosolvens

K.L. Anderson 1, J.A. Megehee 1 and V.H. Varel 2

1Department of Biological Sciences, Mississippi State University, Mississippi and 2Agricultural Research Service,US Department of Agriculture, Nebraska, USA

1488/97: received 24 March 1997 and accepted 17 June 1997

K.L. ANDERSON, J.A. MEGEHEE AND V.H. VAREL. 1998. Tn1545, a self-mobilizingtransposon, was introduced into the chromosome of the ruminal cellulolytic bacteriumEubacterium cellulosolvens. This was achieved by conjugal transfer of the transposonfrom Clostridium beijerinckii at a frequency of 1 per 106 recipient cells.Transconjugants of Eu. cellulosolvens were resistant to both tetracycline anderythromycin, and were able to mobilize Tn1545 back into Cl. beijerinckii. Southern blothybridization of representative transconjugants did not reveal site-specific insertion.This potential randomness of the transposon insertion site may prove useful in thedevelopment of Tn1545 as a tool for mutagenesis of Eu. cellulosolvens.

INTRODUCTION Eubacterium cellulosolvens is a Gram-positive, cellulolyticanaerobic ruminal bacterium. While rarely encountered in

Ruminants consume large volumes of plant material, a majorruminant microbial populations in the United States, this

component of which is cellulose. As ruminants are totallyorganism appears to be prominant in European cattle (van

dependent on microbial activity to degrade this poly-Gylswyk 1990). Strains of this organism are able to degrade

saccharide, there has been much study of the physiologicalcrystalline cellulose and can be readily cultivated in the lab-

features of anaerobic cellulolytic bacteria. This has led to theoratory. Thus, it provides an appropriate model for the study

realization that bacterial cellulolytic systems are far moreof ruminal bacterial cellulose degradation.

complex than simply the production of extracellular cellulases(Beguin and Lemaire 1996). However, it is unlikely that thecomplex features of ruminal cellulolytic bacteria will be fully MATERIALS AND METHODSelucidated without systems by which they can be geneticallymanipulated. Transposons, bacterial strains and growth

Genetic manipulations, such as transposon mutagenesis, conditionsenable the analysis of gene function and significance. Such

Eubacterium cellulosolvens 5494 was obtained from M. Ras-mutational analyses have proved to be useful in answeringmussen (ARS/USDA, Ames, IA), and Clostridium beijerinckiimany questions regarding the molecular basis of poly-NCIMB 8052 and C. beijerinckii AA202 were obtained fromsaccharide degradation by non-cellulolytic bacteria (SalyersM. Young (University of Wales, Aberystwyth, UK). Theseet al. 1993 ; Reeves et al. 1996). While numerous genes fromorganisms were grown under anaerobic conditions (Holde-cellulolytic bacteria have been cloned (Beguin and Aubertman et al. 1977) on a basal medium described by Varel1994), genetic manipulation of cellulolytic bacteria has provedand Pond (1984), except that the total volume of clarifiedto be an elusive target with only limited success (Beguin andincubated rumen fluid was increased to 30% andAubert 1994 ; Flint 1994). In order to address this need, theK2HPO4 .H2O (0·3%) was added. Bacteria were incubated atpresent authors have successfully introduced the transposon39 °C in the basal medium containing 0·5% glucose (RFM).Tn1545 into the chromosome of the ruminal cellulolytic bac-

Clostridium beijerinckii AA202 contains multiple copies ofterium Eubacterium cellulosolvens.transposon Tn1545. This 25·3 kbp transposon belongs to thesame family of elements as Tn916, making it one of severalCorrespondence to : Kevin Anderson, Department of Biological Sciences, POself-transmissible conjugative elements commonly found inDrawer GY, Mississippi State University, MS 39762, USA (e-mail :

kla@ra.msstate.edu). streptococci (Murphy 1989). Tn1545 contains the genes for

© 1998 The Society for Applied Microbiology

36 K.L. ANDERSON ET AL.

resistance to tetracycline (tetM), erythromycin (ermAM) and These transconjugants were verified as Eu. cellulosolvens bycomparing the agarose electrophoretic pattern of their DNAkanamycin (aphA-3) (Courvalin and Carlier 1987). Like

Tn916, Tn1545 contains imperfect inverted repeats at the with the pattern of EcoRI digested total DNA from Cl. beijer-inckii and Eu. cellulosolvens (data not shown). The number ofends, and undergoes precise or imprecise excision (Poyart-

Salmeron et al. 1990). transposon insertions in these representative transconjugantswas then determined by Southern blot hybridization.

Results of Southern blot analysis (Fig. 1) revealed that theConjugation conditionstransconjugants contained a single copy of Tn1545. No cross-

Clostridium beijerinckii AA202 was grown to an optical density hybridization of the tetM probe and total DNA from Eu.of 0·5 at 650 nm in RFM containing tetracycline (Tc ; 10 mg cellulosolvens 5494 was detected (Fig. 1, lane B). All trans-ml−1), and Eu. cellulosolvens was grown in RFM containing conjugants and the donor strain contained the same 9·5 kbpnalidixic acid (Nal ; 600 mg ml−1), to an optical density of ClaI fragment. The tetM of Tn1545 contains an internal ClaI0·4–0·5 at 650 nm. site (Martin et al. 1986) ; therefore, the 9·5 kbp ClaI fragment

Conjugal transfer of Tn1545 from Cl. beijerinckii to Eu. is an internal piece of the transposon. The second band is acellulosolvens was achieved using the anaerobic procedure ClaI fragment with one end at a ClaI site in the chromosome.described previously (Shoemaker et al. 1991). Following incu- Thus, of those transconjugants shown in Fig. 1, no sitebation of the mating pairs for 16 h, selection for trans- specificity for Tn1545 insertion was detected.conjugants of Eu. cellulosolvens was accomplished using Tn1545 is reported to have an integration-excision systemanaerobic roll tubes (Holdeman et al. 1977) containing RFM analogous to the lambdoid phages (Poyart-Salmeron et al.plus Nal (400 mg ml−1), and Tc (3 mg ml−1) or erythromycin 1990 ; Trieu-Cuot et al. 1993). Thus, the transposon insertion(Em ; 3 mg ml−1). sites represent putative chromosomal ‘att’ sites (Poyart-Sal-

Conjugal transfer of Tn1545 from transconjugants of Eu. meron et al. 1990). Assuming transposon insertion into Eu.cellulosolvens to Cl. beijerinckii NCIMB 8052 was achieved in cellulosolvens DNA also involves these putative ‘att’ sites,the same manner. Selection of Cl. beijerinckii transconjugants the randomness of insertion (Fig. 1) suggests that thesewas done using roll tubes containing Tc (3 mg ml−1) and sites may be broadly distributed throughout this organism’sxylose (0·5%) as the sole energy source. chromosome.

Since the core region of the putative ‘att’ site changes asDNA hybridization the element transposes to different locations (Trieu-Cuot et

al. 1993), a donor with multiple copies of the transposonPutative Eu. cellulosolvens transconjugant colonies from dif-(such as the donor strain used here) should increase theferent conjugal matings were subsequently inoculated intonumber of potential insertion sites. However, screening ofRFM containing Tc (5 mg ml−1) and Nal (400 mg ml−1).numerous transconjugants will be necessary before the trueThese cells were then grown in 200 ml of RFM containingrandomness of the insertion sites can be fully assessed.Tc (10 mg ml−1). Clostridium beijerinckii AA202 and Eu. cel-

In addition, Eu. cellulosolvens readily expressed both tetMlulosolvens 5494 were also grown in 200 ml of RFM withoutantibiotics. Total DNA was obtained from each of theseorganisms by the lysis method of Saito and Miura (1963),except that each cell suspension was frozen rapidly andthawed three times to facilitate cell lysis. This DNA wasdigested with either EcoRI or ClaI according to the manu-facturer’s specifications (Promega, Madison, WI).

ClaI digests of total DNA were analysed further for thepresence of Tn1545 by Southern blot hybridization, whichwas performed as described previously (Shoemaker et al.1985). Blots were probed with about 107 c.p.m. of 32P-labelledtetM isolated from a HincII restriction digest of pAM120.The molecular weight standard was HindIII digested l DNA,which was visualized by adding 104 c.p.m. of 32P-labelled l

DNA to the hybridization step. Fig. 1 Southern hybridization analysis of total DNA fromClostridium beijerinckii AA202 (a), Eubacterium cellulosolvens5494 (b), and transconjugants of Eu. cellulosolvens (c–g). DNARESULTS AND DISCUSSIONwas digested with ClaI and probed with the tetM gene of

The frequency of transfer was found to vary, but was typically pAM120. Arrows denote location of molecular weight markersof 23·1, 9·4 and 6·7 kbpone transconjugant for every 106 cells of Eu. cellulosolvens.

© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 35–37

TRANSFER OF Tn1545 TO E. CELLULOSOLVENS 37

Flint, H.J. (1994) Molecular genetics of obligate anaerobes from theand ermAM. Following adaptation, a resistance to antibioticrumen. FEMS Microbiology Letters 121, 259–268.levels of 35 mg ml−1 of tetracycline or erythromycin was

Holdeman, L.V., Cato, E.P. and Moore, W.E.C. (1977) Anaerobeobtained with representative transconjugants. Tn1545 wasLaboratory Manual, 4th ed. Blacksburg, VA : Anaerobe Labora-also found to be relatively stable in Eu. cellulosolvens 5494,tory, Virginia Polytechnic Institute and State University.

because at least 90% of the transconjugants retained the Martin, P., Trieu-Cuot, P. and Courvalin, P. (1986) Nucleotidetransposon after 20 generations of growth in antibiotic-free sequence of the tetM tetracycline resistance determinant of theRFM. streptococcal conjugative shuttle transposon Tn1545. Nucleic

Using a transconjugant of Eu. cellulosolvens, it was possible Acids Research 14, 7047–7058.to mobilize Tn1545 back into Cl. beijerinckii NCIMB 8052 Murphy, E. (1989) Transposable elements in Gram-positive

bacteria. In Mobile DNA. ed. Berg, D. and Howe, M. pp. 269–(parent strain of Cl. beijerinckii AA202). The transfer288. Washington DC : American Society for Microbiology.occurred at a frequency of 1 per 107 Cl. beijerinckii, although

Poyart-Salmeron, C., Trieu-Cuot, P., Carlier, C. and Courvalin, P.no attempt was made to optimize the transfer frequency. This(1990) The integration-excision system of the conjugative trans-raises the possibility that Eu. cellulosolvens may serve as aposon Tn1545 is structurally and functionally related to those of

conjugal donor to other Eubacterium spp. as well as those lambdoid phages. Molecular Microbiology 4, 1513–1521.polysaccharolytic species of clostridia that are phylo- Rainey, F.A. and Stackebrandt, E. (1993) 16S rDNA analysis revealsgenetically related to Eu. cellulosolvens (Rainey and Stracke- phylogenetic diversity among the polysaccharolytic clostridia.brandt 1993). FEMS Microbiology Letters 113, 125–128.

The conjugal transfer of Tn1545 into Eu. cellulosolvens Reeves, A.R., D’elia, J.N., Frias, J. and Salyers, A.A. (1996) ABacteroides thetaiotaomicron outer membrane protein that is essen-provides a key first step towards genetic manipulation of atial for utilization of maltooligosaccharides and starch. Journal ofruminal cellulolytic bacterium. Both tetM and ermAM areBacteriology 178, 823–830.readily expressed by Eu. cellulosolvens, providing genetic

Saito, H. and Miura, K.I. (1963) Preparation of transforming deoxy-marker genes for a variety of future genetic constructs. Inribonucleic acid by phenol treatment. Biochimica et Biophysica

addition, the potential randomness of the insertion site and Acta 72, 619–629.subsequent stability of the transposon insertion may prove Salyers, A.A., Valentine, P. and Hwa, V. (1993) Genetics of poly-useful in the development of Tn1545 as a tool for mutagenesis saccharide utilization pathways of colonic Bacteroides species. Inof Eu. cellulosolvens. The ability of Eu. cellulosolvens to serve Genetic and Molecular Biology of Anaerobic Bacteria ed. Sebald,as a conjugal donor may also prove useful in future studies. M. pp. 505–516. New York : Springer-Verlag.

Shoemaker, N.B., Anderson, K.L., Smithson, S.L., Wang, G. andSalyers, A.A. (1991) Conjugal transfer of a shuttle vector fromthe human colonic anaerobe Bacteroides uniformis to the ruminal

ACKNOWLEDGEMENTS anaerobe Prevotella (Bacteroides) ruminicola B14. Applied andEnvironmental Microbiology 57, 2114–2120.This research was supported by grant 94-37208-1225 from

Shoemaker, N.B., Guthrie, E.P., Salyers, A.A. and Gardner, J.F.the United States Department of Agriculture. The authors(1985) Evidence that the clindamycin-erythromycin resistancethank Matthew Fields for his laboratory assistance. They alsogene of Bacteroides plasmid pBF4 is on a transposable element.

thank M. Young for graciously providing the Clostridium Journal of Bacteriology 162, 626–632.strains. Trieu-Cuot, P., Poyart-Salmeron, C., Carlier, C. and Courvalin, P.

(1993) Sequence requirements for target activity in site-specificrecombination mediated by the Int protein of transposon Tn1545.Molecular Microbiology 8, 179–185.REFERENCES

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© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 35–37