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ELSEVIER FEMS Microbiology Letters 126 (1995) I-6 Isolation of insertion elements from Gram-positive Brevibacterium, Corynebacterium and Rhodococcus strains using the Bacillus subtilis sacB gene as a positive selection marker Wolfgang JZger, Andreas Schgfer, Jijrn Kalinowski *, Alfred Piihler Departmentof Genetics,Universityof Bielefeld,Postfach 100131, 33501 Bielefeld,Germany Received 21 October 1994; accepted 17 November 1994 Abstract The sacB gene of Bacillus subtilis was successfully applied in various Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains for the isolation of transposable elements. Three different insertion sequence (IS) elements entrapped in sacB were isolated. The IS elements IS-B1 and IS-Cg isolated from Brevibacterium lactofermentum and Corynebacterium glutamicum, respectively, were found to be similar in size (1.45 kb) and generated target duplications of 8 bp. Their inverted repeats showed homology. In contrast, the IS element IS-Rf isolated from Rhodococcus fascians was only 1.3 kb long and generated a 3-bp target duplication. IS-Cg and IS-Rf were not restricted to their original host strains, and we also found strains harbouring more than one element. Keywords: Arthrobacter; Brevibacterium; Corynebacterium; Rhodococcus; sacB; Insertion sequence element 1. Introduction The Bacillus subtilis sacB gene encodes levan sucrase, a secreted exoenzyme, which synthesizes levan by hydrolysation of sucrose and polymerization of the resulting fructose residues [l]. Expression of the sacB gene in Escherichia coli and other Gram-negative bacteria is known to be lethal in the presence of sucrose [2]. Therefore, the gene enables a direct selection for marker inactivation, and it has been widely used for the isolation of IS elements Corresponding author. ( + 49-521) 106 5626 l Tel.: (+49-521) 0378-1097/95/$09.50 0 1995 Federation SSDI 0378-1097(94)00513-3 106 5606; of European Fax: Microbiological entrapped in sacB by selecting for sucrose-resistant colonies [3]. In a previous paper, we published the expression of the Bacillus subtilis sacB gene in Corynebacterium glutamicum ATCC 13032, which leads, as in Gram-negative bacteria, to sucrose sensitivity [4]. Moreover, insertions in sacB within the size of typical IS elements were found. With regard to this observation, we stated that the sacB gene could act as the first conditionally lethal marker for the isolation of IS elements from C. glutamicum. In the meantime, we continued to work on IS elements from C. glutamicum and other corynebacterial species. The Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains used in Societies. All rights reserved 2 W. Jiiger et al. /FEMS Microbiology Letters 126 (1995) 1-6 this study are Gram-positive soil bacteria with a GC content of more than 51 mol% [5,6]. Several corynebacteria and brevibacteria are of great industrial importance. They are mainly used for the fermentative production of amino acids like L-glutamic acid, L-lysine, or L-aspartate [7]. Mobile genetic elements are powerful tools to achieve strain improvement. However, so far only one IS element, IS31831 from C. glutamicum ATCC 31831, has been published [8], and the DNA sequence of an IS3 related element from C. glutamicum B115 was entered into the GenBank database [9]. The aim of this study was to survey the occurrence and the dissemination of IS elements in industrially important Arthrobucter, Breuibacterium, Corynebacterium and Rhodococcus strains. 2. Materials and methods 2.1. Bacterial strains, plasmids and growth media Bacterial strains used in this study are listed in Table 1. Plasmid pECM2 as well as pWJ5 carrying sacB were described previously [4]. The mobilizing donor strain for conjugal transfer was E. coli S17-1 [lo]. Cloning experiments were done in E. coli DH5a [ll] using the vector pKl8mob [12]. Arthrobacter, Breuibacterium, Corynebacterium, Rhodococcus strains and E. coli were grown on LBG (1.0% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.2% glucose) at 30°C and 37°C respectively. Plasmid carrying strains were selected on LBG supplemented with kanamycin to a final concentration of 25 pg ml-’ for Arthrobacter, Brevibacterium, Corynebacterium, Rhodococcus, and 50 pug ml-’ for E. coli. Transconjugants were selected on LBG containing 50 pg ml-’ nalidixic acid. Sensitivity to sucrose was tested on 10% sucrose added to selective medium. 2.2. DNA isolation, transfer and manipulation Plasmid DNA was prepared by using the QIAGEN Plasmid Mini Kit (QIAGEN Inc., Chatsworth, CA), with an additional incubation step for 2 h at 37°C for Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus after resuspension in buffer Pl containing 20 mg ml-’ lysozyme. Chromosomal DNA was isolated as described by Altenbuchner and Cullum [13]. Vectors pWJ5 and pECM2 were transferred to Arthrobacter, Brevibacterium, Corynebac- Table 1 Strain Sucrose-resistant colonies by insertions in sacB a Identification of insertions in sacB as IS elements b Hybridization of IS element probes to chromosomal DNA ’ Arthrobacter albidus DSM 20128 Brevibacterium divaricatum DSM 20297 Brevibacterium frnvum DSM 20411 Brevibacterium lactofermentum ATCC 13869 Brevibacterium stationis DSM 20302 Corynebacterium acetoacidophilum ATCC 13870 Coryebacterium ammoniagenes DSM 20305 Corynebacterium callunae DSM 20147 Corynebacterium glutamicum AS019 Corynebacterium glutamicum ATCC 13032 Cotynebocterium glutamicum ATCC 13058 Corynebacterium herculis DSM 20301 Corynebacterium lilium DSM 20137 Corynebacterium melassecola ATCC 17965 Rhodococcus fascians DSM 20131 - n.a. n.a. n.t. IS-B1 n.a. n.a. n.a. n.a. n.t. IS-cg n.a. n.t. n.a. n.a. IS-Rf n.t. n.t. IS-Q IS-BI, IS-Q, IS-Rf nt. n.t. n.t. wt. IS-cg IS-cg n.t. IS-Q, IS-Rf n.t. n.t. IS-Q, IS-Rf + + + + + + a Insertions were identified by enlargement of sacB of plasmid pWJ5. ’ Insertions were analysed by sequencing, results are presented in Fig. 1. IS-Bl, IS-Cg and IS-R?, IS elements first found in B. lactofermentum, C. glutumicum and R. fascians, respectively. n.a., not applicable; n.t., not tested. ’ DNA probes of IS-Bl, IS-Cg and IS-Rf were used in Southern hybridization experiments, results are shown in Fig. 2. W. Jiiger et al. / FEMS Microbiology Letters 126 (I 995) l-6 3 Boehringer (Mannheim, Germany), following the manufacturers’ instructions. Stringency washes were done at 68°C with washing buffer containing 0.1% ssc. terium and Rhodococcus strains by conjugation [14] using 1 X 10’ cells of donor and recipient strains. E. coli was transformed by the CaCl, method [ll]. In vitro analysis of plasmid and chromosomal DNA was carried out by standard procedures [l-51. Hybridization experiments were performed according to Southern [16] using Hybond-N nylon membranes purchased from Amersham (Braunschweig, Germany), the vacuum blotter VacuGene LKB2016 from Pharmacia (Freiburg, Germany) and the DNA Labeling and Detection Kit Nonradioactive from 2.3. DNA sequencing DNA sequences were determined by the chain termination method [17] using the T7 Sequencing Kit (Pharmacia, Freiburg, Germany). Sequencing gels were prepared as described by Garoff and Ansorge IS-Bl IS-cg IS-Rf (I.45 kb) (1.45 kb) (1.3kb) a) ClaI Clal .... pWJ5 b) sacB tgattgagctaaac)gatgatta$iGCCCm IS-Q ATGCACTCTAAAACAGGAAGAGC~atgatt+acactgaaaaaag tatgtttctaatltctttaa+GCTCTTCCGTGTTTAGAGTGCATTG IS-B1 CAATGCACTCTAAAAACGGAAGAGCClm$ggcccatacaa tggatcttgatcctaacgatgta*GACC-GACCCCGATATGG IS-Rf CCAAATGGGGGTCAGGTCCmttacttactcacacttcgctgtac Fig. 1. Localization and terminal sequences of three IS elements entrapped in the sacB gene. (a) The insertions are shown of IS elements isolated from B. lactofemaentum ATCC 13869, C. glutamicum ATCC 13032 and R. faxinns DSM 20131, named IS-B& IS-Cg, and IS-Rf, respectively, into the sacB coding region of plasmid pWJ5. The sizes of the IS elements are given in brackets. (b) The terminal sequences of the IS elements (capital letters) and the adjacent sacB sequences (lower case letters) are presented. Arrows indicate inverse repetitive ends (bold face was used for matches). Boxed italic letters show duplicated sequences at the target sites. 4 W. Jiiger et al. / FEMS Microbiology Letters 126 (1995) l-6 [18]. We searched for homologies in EMBL and GenBank databases by means of the FASTA [19] E-mail Server (EMBL, Heidelberg, Germany) or the BLAST [20] Server (NCBI, Bethesda, MD). 3. Results and discussion The positive selection system used in this study is based on the Bacillus subtilis sacB gene which causes sucrose sensitivity in many Gram-negative bacteria and in the Gram-positive Corynebacterium glutamicum ATCC 13032 [3,4]. To enlarge the list of Gram-positive bacterial species we analysed the sacB selection system in several Arthrobacter, Breuibacterium, Corynebacterium and Rhodococcus strains (Table 1). For this purpose, the sacB vector pWJ5 as well as plasmid pECM2 [4] lacking the sacB gene were introduced into these bacteria via conjugation. The effect of sacB expression in the resulting clones was analysed by investigating their growth in the presence of sucrose. Transconjugants were plated in parallel on selective LBG medium with and without 10% sucrose, and were incubated for 24 h at 30°C. When plasmid pWJ5 was present, all bacterial strains kb 123456 123456 probe: tested were unable to grow on sucrose medium, whereas no growth inhibition occurred in the absence of sucrose, nor in cells harbouring vector pECM2. The sucrose sensitive phenotype is consistent with the previously described lethal effect for C. glutamicum ATCC 13032 on sucrose medium caused by expression of sacB [4]. Nevertheless, our results demonstrate that the sacB system is functional in the other Gram-positive organisms used in this study, and is not restricted to C. glutamicum. The observed lethal effect caused by sacB in the presence of sucrose provided the basis for the isolation of IS elements from Brevibacterium, Corynebacterium and Rhodococcus strains by selecting for sucrose-resistant colonies and analysing the sacB gene for insertional inactivation. The strains harbouring plasmid pWJ5 were spread onto selective LBG agar plates containing 10% sucrose, in a density of lo8 cells per plate. After an incubation for 48-72 h at 30°C sucrose-resistant colonies arose from every strain. Restriction analysis of vector pWJ5 isolated from these clones, revealed in some cases the presence of DNA insertions in sacB (Table 1). In some strains, however, no insertions in sacB were found. For some of these sucrose-resistant mutants it IS-B/ 123456 IS-cg Fig. 2. Distribution of the IS elements IS-B& IS-Cg and IS-Rf in six different Breuibacterium, Coryebucterium and Rhodococcus strains. Southern hybridizations to &I-restricted chromosomal DNAs from B. flauum DSM 20411 (lanes 11, B. luctofermentum ATCC 13869 (lanes 21, C. glutumicum ATCC 13032 (lanes 31, C. glutamicum AS019 (lanes 4), C. herculis DSM 20301 (lanes 5) and R. fascians DM200-2 (lanes 6) were performed using the subcloned IS-H, IS-Cg and IS-Rf elements as probes. The size of the molecular mass marker is given in kb. W. JZiger et al. / FEMS Microbiology was shown that they carry the unaffected vector pWJ5. This was shown by introducing the plasmids in question into E. coli and by testing for sucrose sensitivity (data not shown). Therefore, besides mutation of the sacB gene, at least one other, so far unknown resistance mechanism should exist. Since in E. coli the lethal effect parallels with an active levan sucrase in the periplasm [2], chromosomal mutations preventing enzyme or substrate transport may be responsible for resistance while the sacB gene remains unaffected. Three different insertions in the sacB gene originating from B. lactofermentum ATCC 13869, C. glutumicum ATCC 13032 and R. fuscians DSM 20131, which were named IS-B& IS-Cg and IS-Rf according to their host strains, were analysed in more detail. The sizes of the insertions ranged from 1.3 to 1.45 kb (Fig. la) as calculated by restriction pattern analysis (data not shown). The ClaI/HindIII fragments of the sacB gene carrying the inserted IS-B1 or IS-Rf elements as well as the ClaI/SspI fragment harbouring IS-Cg were isolated, blunt-ended and subcloned into the SmaI site of vector pK18mob [12]. By sequencing the ends of the cloned fragments, the target sites within the sacB gene and the terminal sequences of the insertions were determined (Fig. lb). The sequence data displayed typical features of IS elements, including target site duplications of 8 bp for IS-B1 and IS-Cg, and 3 bp for IS-Rf, as well as terminal inverted repeats (Fig. lb), verifying that IS elements were isolated. Homology searches revealed that the ends of IS-B1 are similar to the recently published element IS32831 isolated from C. glutumicum ATCC 31831 [S], while IS-Cg ends are identical to this element. The terminal sequences of IS-Rf showed 78% homology to an insertion sequence isolated from C. glutumicum B115 [91. The strains in which insertional inactivation of sacB was detected, were further analysed for the presence of IS-Bl, IS-Cg and IS-Rf. Southern hybridizations to chromosomal DNAs were performed using the IS elements as probes (Fig. 2). IS-Cg and IS-Rf were not restricted to their original host strains alone. The most disseminated one was IS-Cg, found to be present in the six strains analysed, whereas sequences homologous to IS-Rf existed in B. lactofermentum, C. herculis and R. fascians. IS-B1 Letters 126 (1995) I-6 5 only occurred in B. lactofermentum. Prolonged staining of the filters indicated weak homology between IS-Cg and IS-B& whereas no cross-reaction of the IS-Rf probe to IS-Cg nor IS-B1 DNAs occurred. The occurrence of identical IS elements in different strains refers to a close relationship of these bacteria, or indicates that gene transfer events must have happened. IS-Cg and IS-B1 probably have a common ancestor, since both are similar in size, share homologous sequences and generate target site duplications of 8 bp (Fig. lb). In this study we extended the application range of the sacB system that was shown so far to be functional in only one Gram-positive organism, C. glutamicum ATCC 13032 [4], to several Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains. Additionally, the application of the system was demonstrated in a positive selection procedure, which resulted in the isolation of three different IS elements. Acknowledgements We thank Michaela Behrendt for technical assistance. W.J. and A.S. acknowledge the receipt of scholarships of the Deutsche Forschungs Gemeinschaft (Graduiertenkolleg) and of the Studienstiftung des deutschen Volkes, respectively. This work was supported by the Bundesministerium fur Forschung und Technologie Grant 031925A and by the BRIDGE BIOT CT91-0264 (RZJE) project of the Commission of the European Communities. References [l] Dedonder, R. (1966) Levan sucrase from Bacillus subtilis. Methods Enzymol. 8, 500-505. [2] Steinmetz, M., Le Coq, D., Djemia, H.B. and Gay, P. (1983) Analyse genetique de sacB, gene de structure d’une enzyme secrttte, la ltvane-saccharase de Bacillus subtilis Marburg. Mol. Gen. Genet. 191, 138-144. [3] Gay, P., Le Coq, D., Steinmetz, M., Berkelmann, T. and Kado, C.I. (1985) Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J. Bacterial. 164, 918-921. [4] Jager, W., Scfifer, A., Pihler, A., Labes, G. and Wohlleben, W. (1992) Expression of the Bucilhs subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces liuiduns. .I. Bacterial. 174, 5462-5465. 6 W. Jiiger et al. / FEMS Microbiology [5] Jones, D. and Collins, M.D. (1986) Irregular, nonsporing Gram-positive rods. In: Bergey’s Manual of Systematic Bacteriology (Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G., Eds.), Vol. 2, pp. 1261-1434, Williams and Wilkins, Baltimore, MD. [6] Goodfellow, M. (1986) Genus Rhodococcus Zopf 1891. In: Bergey’s Manual of Systematic Bacteriology (Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G., Eds.), Vol. 2, pp. 1472-1481, Williams and Wilkins, Baltimore, MD. [7] Kleemann, A., Leuchtenberger, W., Hoppe, B. and Tanner, H. (1985) Amino acids. In: Ullmans Encyclopedia of lndustrial Chemistry, 5th edn., Vol. A2, pp. 57-97, VCH publishers, Weinheim. [8] VertZs, A.A., lnui, M., Kobayashi, M., Kurusu, Y. and Yukawa, H. (1994) Isolation and characterization of lS31831, a transposable element from Corynebacterium glutumicum. Mol. Micobiol. 11, 739-746. [9] Bonamy, C.A.M. (1992) C. ghtamicum IS3 related insertion element. Accession number X69104, GenBank database. [lo] Simon, R., Priefer, U. and F’iihler, A. (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio/Technology 1, 784-794. [ll] Hanahan, D. (1985) Techniques for transformation of E. coli. In: DNA Cloning (Glover, D.M., Ed.), Vol. 1, pp. 109-136. IRL press, Oxford. [12] Schlfer, A., Tauch, A., Jgger, W., Kalinowski, J., Thierbach, G. and Piihler, A. (1994) Small mobilizable multi-purpose Letters 126 (1995) l-6 [13] [14] [15] [16] [17] [18] [19] [20] cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 14.5, 69-73. Altenbuchner, J. and Cullum, J. (1984) DNA amplification and an unstable arginine gene in Streptomyces liuidans 66. Mol. Gen. Genet. 195, 134-138. Schlfer, A., Kalinowski, J., Simon, R., Seep-Feldhaus, A.H. and F’iihler, A. 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