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An integron of class 1 is present on the plasmid pCG4 from Gram-positive bacterium Corynebacterium glutamicum
FEMS Microbiology Letters 169 (1998) 391^395
An integron of class 1 is present on the plasmid pCG4 from Gram-positive bacterium Corynebacterium glutamicum Jan Nesívera *, Jitka Hochmannovaè, Miroslav Paètek Institute of Microbiology, Academy of Sciences of the Czech Republic, V|èdeoéskaè 1083, CZ-14220 Praha 4, Czech Republic Received 31 August 1998 ; received in revised form 14 October 1998; accepted 27 October 1998
Abstract The streptomycin/spectinomycin resistance determinant of the 29-kb plasmid pCG4 from Corynebacterium glutamicum was found to be a part of a typical class 1 integron. The sequence analysis revealed that the integron (designated InCg) identified in this Gram-positive bacterium is almost identical to the integron InC present on the plasmid pSA1700 from the Gram-negative bacterium Pseudomonas aeruginosa. Differences in only two base pairs were found in the 3.8-kb sequence. One base substitution (GCC) is present in the streptomycin/spectinomycin resistance determinant which is thus identical to the aadA2a gene from the integron In6 of the broad-host-range plasmid pSa. The other one (CCG) is present in the extended 310 region of the integron promoter involved in expression of the antibiotic resistance gene. It was shown that this novel version of the integron promoter displays five times higher activity in both C. glutamicum and Escherichia coli than the original one. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Corynebacterium glutamicum ; Integron; Antibiotic resistance; Promoter
1. Introduction Integrons are genetic units that include site-speci¢c recombination system capable of capturing genes (most frequently determinants of resistance to antibiotics) located on mobile elements called gene cassettes. Integrons have been classi¢ed into three classes according to the similarities existing between the integron-encoded integrases; the class 1 being most numerous. The class 1 integrons are composed of two conserved segments (5P- and 3P-conserved segments) and a recombination site between them, where gene cassettes are integrated. Most of genes * Corresponding author. Tel.: +42 (2) 475-2398; Fax: +42 (2) 472-2257; E-mail: [email protected]
on integrated cassettes lack their own promoters and are expressed from a common promoter region located in the 5P-conserved segment [1]. Four versions of these integron promoters, di¡ering in the sequences of the 335 and/or 310 hexamers and in their strength, have been described so far [2]. Integrons have been found almost exclusively in Gram-negative bacteria. Only two cases of conserved integron sequences present in Gram-positive bacteria have been reported. However, in both cases, on the transposon Tn610 from Mycobacterium smegmatis [3] and in the chromosome of Rhodococcus erythropolis NI86/21 [4], only incomplete and therefore nonfunctional parts of the integron sequence are present. In this report, we describe, for the ¢rst time, presence of the class 1 integron possessing the whole
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 1 6 - 3
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Fig. 1. Physical and genetic map of 3.8-kb BamHI fragment of plasmid pCG4 having a typical structure of the class 1 integrons and being designated InCg. White box represents the integrated gene cassette and black boxes the 5P- and 3P-conserved segments (CS). Numbers below the vertical arrows represent positions of the base pairs di¡ering from those of integron InC [17]. Pant indicates the integron promoter involved in expression of the streptomycin/spectinomycin resistance gene associated with the cassette. The following genes are indicated : int, site-speci¢c integrase; aadA2a, streptomycin/spectinomycin resistance; qacEv1, antiseptics resistance; sulI, sulfonamide resistance ; ORF5, unknown function, not completely present on the BamHI fragment. We found that sulI gene is expressed in C. glutamicum (pCG4) (MIC of sulfafurazol being 6000 Wg ml31 vs. 100 Wg ml31 in the plasmidless C. glutamicum). B, BamHI; E, EcoRI; H, HindIII; P, PstI ; S, SphI.
structure necessary for its functions on the plasmid harbored in a Gram-positive bacterium. A novel version of the integron promoter involved in expression of the antibiotic resistance gene associated with the cassette is presented. Our ¢nding that the identical integron is present on replicons from the phylogenetically distant bacterial genera is documented.
2. Materials and methods 2.1. Bacterial strains and plasmids Corynebacterium glutamicum T250 (ATCC 31830) is a natural host of the 29-kb streptomycin/spectinomycin (Sm/Sp) resistant plasmid pCG4 [5]. Escherichia coli DH5K [6] and C. glutamicum R127 [7] were used as hosts for the constructed recombinant plasmids. The mini-derivative of the plasmid pSa, pGV1106 [8], served as a source of promoter of integron In6. Plasmid pUC18 [9] served as a vector for cloning and sequencing the de¢ned fragments of pCG4. C. glutamicum^E. coli shuttle promoter^ probe vector pET2 carrying the promoterless cat gene (derivative of the plasmid pEKplCm [10]) was used for analysis of promoter activity. 2.2. Growth and transformation conditions E. coli strains were grown at 37³C in LB medium and C. glutamicum strains were grown at 30³C in CY medium [11]. Transformation of E. coli was carried
out by the method of Hanahan [6] and electrotransformation of C. glutamicum was done according to the method of Liebl et al. [7]. The following concentrations of antibiotics (Wg ml31 ) were used for selection of transformants: ampicillin, 100; kanamycin and streptomycin, 20; chloramphenicol, 10. 2.3. DNA isolation and manipulation Plasmid DNA from E. coli was isolated using the Wizard Plus Midipreps DNA Puri¢cation System (Promega). Plasmid DNA from C. glutamicum was isolated by a modi¢ed alkaline extraction procedure with lysozyme [12]. Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturers. DNA fragments carrying the integron promoter regions were ampli¢ed by the PCR technique. The PCR-mediated site-directed mutagenesis was carried out by the method of Ito et al. [13]. The nucleotide sequence was determined by using automatic sequencer Vistra (Amersham). 2.4. Enzyme assay and determination of minimal inhibitory concentrations The speci¢c activity of chloramphenicol acetyltransferase (CAT) in bacterial cell-free extracts was determined by the method of Shaw [14]. A unit of activity was de¢ned as 1 Wmol of chloramphenicol (CM) acetylated per min. The minimal inhibitory concentration (MIC) of antibiotics was determined on LB or CY plates by the method of Ozaki et al. [15].
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2.5. Nucleotide sequences accession numbers The nucleotide sequence of the reported integron InCg has been deposited in Genbank/EMBL under accession number Y14748, that of the promoter region of integron In6 under accession number Y17696.
3. Results 3.1. Nucleotide sequence of Sm/Sp resistance determinant of plasmid pCG4 and its £anking regions The nucleotide sequence of the Sm/Sp resistance determinant of 29-kb plasmid pCG4 from C. glutamicum T250 was determined and shown to be identical to that of the aadA2a gene from the broad-hostrange plasmid pSa originally isolated from the Gram-negative bacterium Shigella £exneri [16]. The nucleotide sequence of the regions £anking the Sm/ Sp resistance gene was shown to be identical to that of 5P- and 3P-conserved regions of class 1 integrons. Analysis of the nucleotide sequence of the whole 3.8kb BamHI fragment of pCG4, containing the Sm/Sp resistance gene, revealed its structure typical of the class 1 integrons described until now only in Gramnegative bacteria. The physical and genetic map of this integron of plasmid pCG4, designated InCg, is shown in Fig. 1. Its nucleotide sequence was found to be almost identical to that of the integron InC of the 33-kb plasmid pSA1700 from Pseudomonas aeruginosa [17]. As shown in Fig. 1, di¡erences in only
393
two base pairs are present on the 3.8-kb fragment. One base substitution (GCC) was found in the aadA2 gene associated with the gene cassette, the other one (CCG) is present in the extended 310 region of the integron promoter involved in expression of this gene (Table 1, ¢rst column). Both of these substitutions are also present in the integron In6 from the plasmid pSa. 3.2. Positive e¡ect of CCG substitution in the extended 310 region on activity of the integron promoter G at a position two bases upstream of 310 hexamer is moderately conserved in C. glutamicum promoters [18]. Substitution of any base to G at this position results in increase of promoter activity in both C. glutamicum (our unpublished results) and E. coli [19] in some promoters. We have therefore tested the e¡ect of CCG transversion on the activity of the integron promoter involved in expression of the cassette associated genes. DNA fragments (each 159 bp in size) containing promoters of the integrons InCg and In6, respectively, were ampli¢ed by PCR technique (the upper primer covered positions 992 to 1006, while the lower primer positions 1150^1133 on the map in Fig. 1; both primers had BamHI sites attached to their 5P-ends). To obtain the sequence of the integron promoter of InC [17], which is a typical `weak' integron promoter [2], the GCC transversion in the position two bases upstream of the 310 hexamer was prepared by site-directed mutagenesis of the 159-bp fragment containing InCg promoter and checked by sequencing. All three Bam-
Table 1 Activity of the integron promoters assayed by determining the MIC of CM and by measuring the speci¢c activity of CAT in cell-free extracts of C. glutamicum and E. coli Integron promoter in pET2
C. glutamicum MIC of CM (Wg ml31 )
None InCg TGGTAAGCTb In6 TGGTAAGCTb InC TCGTAAGCTb
5 50 50 30
E. coli CAT activitya (Wmol min31 mg protein31 ) 6 0.001 1.29 þ 0.13 1.19 þ 0.02 0.26 þ 0.01
a
MIC of CM (Wg ml31 ) 10 1300 1300 800
CAT activitya (Wmol min31 mg protein31 ) 6 0.001 1.21 þ 0.17 1.35 þ 0.17 0.27 þ 0.02
Values represent the means þ DS.D. from at least three experiments. Nucleotide sequence of the extended 310 region of integron promoters ; the 310 hexamer is underlined and the base substitutions are indicated by bold letters. b
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HI-fragments containing the respective integron promoters were cloned in E. coli DH5K into the promoter probe vector pET2 (having a promoterless cat gene) linearized with BamHI. The constructed recombinant plasmids were then transferred into C. glutamicum R127. The activity of the respective promoters was measured as MIC of CM and as CAT activity in both hosts. As shown in Table 1, the promoter of integron InCg displays ¢ve times higher activity in both C. glutamicum and E. coli than the InC integron promoter. The same CAT activity was found when the promoter of integron In6 (having the same base substitution CCG two bases upstream of 310 hexamer) was used as a control. The promoters of the integrons InCg and In6 thus represent a new version of the integron promoters involved in expression of antibiotic resistance genes present on the cassettes. The CCG substitution two bases upstream of 310 hexamer of this promoter is undoubtedly responsible for the observed increase of the promoter activity.
4. Discussion The integron InCg present on the plasmid pCG4 from C. glutamicum is the ¢rst class 1 integron found in the Gram-positive bacteria which possesses all parts necessary for its function. Until now, only in two cases, incomplete parts of an integron were identi¢ed in Gram-positive bacteria. In the chromosome of Rhodococcus erythropolis NI86/21, only 47 bp of the 5P-conserved segment of class 1 integron was detected [4]. Transposon Tn610 from Mycobacterium fortuitum contains substantial parts of both 5P- and 3P-conserved integron regions; however, the key integron sequence, namely the recombination site necessary for integrating a gene cassette into the integron, is missing [3]. Presence of the almost identical integron (InCg or InC) on di¡erent replicons (pCG4 or pSA1700) suggests that this integron may be a part of a transposon. Our observation that the identical integron was found on replicons isolated from phylogenetically distant bacterial genera (Corynebacterium vs. Pseudomonas) is an additional indirect evidence for the intergeneric horizontal transfer of genes in natural environment. Di¡erences in the strength of integron promoters
have as yet been ascribed only to base substitutions within the 335 and/or 310 hexamers [2]. We have found that the base substitution (CCG) two bases upstream of the 310 hexamer of the integron promoter present in both integrons InCg and In6 dramatically increased the activity of the so called `weak' integron promoter present in 27 known integrons of class 1, including the proposed ancestor integron In0 [2,20]. This result is in agreement with the positive e¡ect of G at this position on the activity of some promoters from C. glutamicum (our unpublished results) and E. coli [19]. Analysis of the Genbank/EMBL data revealed that, besides the integrons InCg and In6, this novel version of the integron promoter involved in expression of the antibiotic resistance genes is also present in the integron In17 of the enterobacterial plasmid pLMO150 [21] and in the integrons of the plasmid pLST1000 [22] and of a Citrobacter freundii Cf155 plasmid [23]. However, none of the respective authors recognized the importance of the sequence in extended 310 region for the activity of the integron promoter. The supposed evolutionary advantage of the described mutation is supported by the fact that the plasmid pLST1000 carrying this version of the integron promoter is one of the most widely distributed multi-resistant plasmids [22].
Acknowledgments We thank A. Síroglovaè for excellent technical assistance and P. Síebo for critical reading of the manuscript. This work was supported by Grant 204/97/ 0528 from the Grant Agency of the Czech Republic.
References [1] Recchia, G.D. and Hall, R.M. (1995) Gene cassettes: a new class of mobile elements. Microbiology 141, 3015^3027. [2] Leèvesque, C., Brassard, S., Lapointe, J. and Roy, P.H. (1994) Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons. Gene 142, 49^ 54. [3] Martin, C., Timm, J., Rauzier, J., Gomez-Luz, R., Davies, J. and Gicquel, B. (1990) Transposition of an antibiotic resistance element in mycobacteria. Nature 345, 739^743. [4] Nagy, I., Schoofs, G., Vanderleyden, J. and de Mot, R. (1997)
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Transposition of the IS21-related element IS1415 in Rhodococcus erythropolis. J. Bacteriol. 179, 4635^4638. Katsumata, R., Ozaki, A., Oka, T. and Furuya, A. (1984) Protoplast transformation of glutamate-producing bacteria with plasmid DNA. J. Bacteriol. 159, 306^311. Hanahan, D. (1985) Techniques for transformation of E. coli. In : DNA Cloning, a Practical Approach (Glover, D.M., Ed.), Vol. 1, pp. 109^135. IRL, Oxford, UK. Liebl, W., Bayerl, A., Schein, B., Stillner, U. and Schleifer, K.H. (1989) High e¤ciency electroporation of intact Corynebacterium glutamicum cells. FEMS Microbiol. Lett. 65, 299^ 304. Leemans, J., Langenakens, J., De Greve, H., Deblaere, R., Van Montagu, M. and Schell, J. (1982) Broad-host-range cloning vectors derived from the W-plasmid Sa. Gene 19, 361^364. Vieira, J. and Messing, J. (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259^268. Eikmanns, B.J., Kleinertz, E., Liebl, W. and Sahm, H. (1991) A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors, for cloning, controlled gene expression, and promoter probing. Gene 102, 93^98. Paètek, M., Nesívera, J., Hochmannovaè, J. and Sítokrovaè, J. (1988) Transfection of Brevibacterium £avum with bacteriophage BFB10 DNA. Folia Microbiol. 33, 247^254. Paètek, M., Nesívera, J. and Hochmannovaè, J. (1989) Plasmid cloning vectors replicating in Escherichia coli, amino acid-producing coryneform bacteria and Methylobacillus sp. Appl. Microbiol. Biotechnol. 31, 65^69. Ito, W., Ishiguro, H. and Kurosawa, Y. (1991) A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction. Gene 102, 67^70. Shaw, W.V. (1975) Chloramphenicol acetyl transferase from chloramphenicol-resistant bacteria. Methods Enzymol. 43, 737^755. Ozaki, A., Katsumata, R., Oka, T. and Furuya A. (1984)
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Functional expression of the genes of Escherichia coli in gram-positive Corynebacterium glutamicum. Mol. Gen. Genet. 196, 175^178. Bito, A. and Susani, M. (1994) Revised analysis of aadA2 gene of plasmid pSa. Antimicrob. Agents Chemother. 38, 1172^ 1175. Kazama, H., Kizu, K., Iwasaki, M., Hamashima, H., Sasatsu, M. and Arai, T. (1995) Isolation and structure of a new integron that includes a streptomycin resistance gene from the R plasmid of Pseudomonas aeruginosa. FEMS Microbiol. Lett. 134, 137^141. Paètek, M., Eikmanns, B.J., Paètek, J. and Sahm, H. (1996) Promoters from Corynebacterium glutamicum : cloning, molecular analysis and search for a consensus motif. Microbiology 142, 1297^1309. Voskuil, M.I., Voepel, K. and Chambliss, G.H. (1995) The 316 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli, Mol. Microbiol. 17, 271^279. Bisonette, L. and Roy, P.H. (1992) Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gramnegative bacteria. J. Bacteriol. 174, 1248^1257. Sundstroëm, L. and Skoëld, O. (1990) The dhfrI trimethoprim resistance gene of Tn7 can be found at speci¢c sites in other genetic surroundings. Antimicrob. Agents Chemother. 34, 642^650. Hopkins, J.D., O'Brien, T.F. and Syvanen, M. (1988) Functional and structural map of pLST1000: a multiresistance plasmid widely distributed in Enterobacteriaceae. Plasmid 20, 163^166. Hannecart-Pokorni, E., Depuydt, F., de Wit, L., van Bossuyt, E., Content, J. and Vanhoof, R. (1997) Characterization of the 6P-N-aminoglycoside acetyltransferase gene aac(6P)-Il associated with a sulI-type integron. Antimicrob. Agents Chemother. 41, 314^318.
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An integron of class 1 is present on the plasmid pCG4 from Gram-positive bacterium Corynebacterium glutamicum Jan Nesívera *, Jitka Hochmannovaè, Miroslav Paètek Institute of Microbiology, Academy of Sciences of the Czech Republic, V|èdeoéskaè 1083, CZ-14220 Praha 4, Czech Republic Received 31 August 1998 ; received in revised form 14 October 1998; accepted 27 October 1998
Abstract The streptomycin/spectinomycin resistance determinant of the 29-kb plasmid pCG4 from Corynebacterium glutamicum was found to be a part of a typical class 1 integron. The sequence analysis revealed that the integron (designated InCg) identified in this Gram-positive bacterium is almost identical to the integron InC present on the plasmid pSA1700 from the Gram-negative bacterium Pseudomonas aeruginosa. Differences in only two base pairs were found in the 3.8-kb sequence. One base substitution (GCC) is present in the streptomycin/spectinomycin resistance determinant which is thus identical to the aadA2a gene from the integron In6 of the broad-host-range plasmid pSa. The other one (CCG) is present in the extended 310 region of the integron promoter involved in expression of the antibiotic resistance gene. It was shown that this novel version of the integron promoter displays five times higher activity in both C. glutamicum and Escherichia coli than the original one. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Corynebacterium glutamicum ; Integron; Antibiotic resistance; Promoter
1. Introduction Integrons are genetic units that include site-speci¢c recombination system capable of capturing genes (most frequently determinants of resistance to antibiotics) located on mobile elements called gene cassettes. Integrons have been classi¢ed into three classes according to the similarities existing between the integron-encoded integrases; the class 1 being most numerous. The class 1 integrons are composed of two conserved segments (5P- and 3P-conserved segments) and a recombination site between them, where gene cassettes are integrated. Most of genes * Corresponding author. Tel.: +42 (2) 475-2398; Fax: +42 (2) 472-2257; E-mail: [email protected]
on integrated cassettes lack their own promoters and are expressed from a common promoter region located in the 5P-conserved segment [1]. Four versions of these integron promoters, di¡ering in the sequences of the 335 and/or 310 hexamers and in their strength, have been described so far [2]. Integrons have been found almost exclusively in Gram-negative bacteria. Only two cases of conserved integron sequences present in Gram-positive bacteria have been reported. However, in both cases, on the transposon Tn610 from Mycobacterium smegmatis [3] and in the chromosome of Rhodococcus erythropolis NI86/21 [4], only incomplete and therefore nonfunctional parts of the integron sequence are present. In this report, we describe, for the ¢rst time, presence of the class 1 integron possessing the whole
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 1 6 - 3
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Fig. 1. Physical and genetic map of 3.8-kb BamHI fragment of plasmid pCG4 having a typical structure of the class 1 integrons and being designated InCg. White box represents the integrated gene cassette and black boxes the 5P- and 3P-conserved segments (CS). Numbers below the vertical arrows represent positions of the base pairs di¡ering from those of integron InC [17]. Pant indicates the integron promoter involved in expression of the streptomycin/spectinomycin resistance gene associated with the cassette. The following genes are indicated : int, site-speci¢c integrase; aadA2a, streptomycin/spectinomycin resistance; qacEv1, antiseptics resistance; sulI, sulfonamide resistance ; ORF5, unknown function, not completely present on the BamHI fragment. We found that sulI gene is expressed in C. glutamicum (pCG4) (MIC of sulfafurazol being 6000 Wg ml31 vs. 100 Wg ml31 in the plasmidless C. glutamicum). B, BamHI; E, EcoRI; H, HindIII; P, PstI ; S, SphI.
structure necessary for its functions on the plasmid harbored in a Gram-positive bacterium. A novel version of the integron promoter involved in expression of the antibiotic resistance gene associated with the cassette is presented. Our ¢nding that the identical integron is present on replicons from the phylogenetically distant bacterial genera is documented.
2. Materials and methods 2.1. Bacterial strains and plasmids Corynebacterium glutamicum T250 (ATCC 31830) is a natural host of the 29-kb streptomycin/spectinomycin (Sm/Sp) resistant plasmid pCG4 [5]. Escherichia coli DH5K [6] and C. glutamicum R127 [7] were used as hosts for the constructed recombinant plasmids. The mini-derivative of the plasmid pSa, pGV1106 [8], served as a source of promoter of integron In6. Plasmid pUC18 [9] served as a vector for cloning and sequencing the de¢ned fragments of pCG4. C. glutamicum^E. coli shuttle promoter^ probe vector pET2 carrying the promoterless cat gene (derivative of the plasmid pEKplCm [10]) was used for analysis of promoter activity. 2.2. Growth and transformation conditions E. coli strains were grown at 37³C in LB medium and C. glutamicum strains were grown at 30³C in CY medium [11]. Transformation of E. coli was carried
out by the method of Hanahan [6] and electrotransformation of C. glutamicum was done according to the method of Liebl et al. [7]. The following concentrations of antibiotics (Wg ml31 ) were used for selection of transformants: ampicillin, 100; kanamycin and streptomycin, 20; chloramphenicol, 10. 2.3. DNA isolation and manipulation Plasmid DNA from E. coli was isolated using the Wizard Plus Midipreps DNA Puri¢cation System (Promega). Plasmid DNA from C. glutamicum was isolated by a modi¢ed alkaline extraction procedure with lysozyme [12]. Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturers. DNA fragments carrying the integron promoter regions were ampli¢ed by the PCR technique. The PCR-mediated site-directed mutagenesis was carried out by the method of Ito et al. [13]. The nucleotide sequence was determined by using automatic sequencer Vistra (Amersham). 2.4. Enzyme assay and determination of minimal inhibitory concentrations The speci¢c activity of chloramphenicol acetyltransferase (CAT) in bacterial cell-free extracts was determined by the method of Shaw [14]. A unit of activity was de¢ned as 1 Wmol of chloramphenicol (CM) acetylated per min. The minimal inhibitory concentration (MIC) of antibiotics was determined on LB or CY plates by the method of Ozaki et al. [15].
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2.5. Nucleotide sequences accession numbers The nucleotide sequence of the reported integron InCg has been deposited in Genbank/EMBL under accession number Y14748, that of the promoter region of integron In6 under accession number Y17696.
3. Results 3.1. Nucleotide sequence of Sm/Sp resistance determinant of plasmid pCG4 and its £anking regions The nucleotide sequence of the Sm/Sp resistance determinant of 29-kb plasmid pCG4 from C. glutamicum T250 was determined and shown to be identical to that of the aadA2a gene from the broad-hostrange plasmid pSa originally isolated from the Gram-negative bacterium Shigella £exneri [16]. The nucleotide sequence of the regions £anking the Sm/ Sp resistance gene was shown to be identical to that of 5P- and 3P-conserved regions of class 1 integrons. Analysis of the nucleotide sequence of the whole 3.8kb BamHI fragment of pCG4, containing the Sm/Sp resistance gene, revealed its structure typical of the class 1 integrons described until now only in Gramnegative bacteria. The physical and genetic map of this integron of plasmid pCG4, designated InCg, is shown in Fig. 1. Its nucleotide sequence was found to be almost identical to that of the integron InC of the 33-kb plasmid pSA1700 from Pseudomonas aeruginosa [17]. As shown in Fig. 1, di¡erences in only
393
two base pairs are present on the 3.8-kb fragment. One base substitution (GCC) was found in the aadA2 gene associated with the gene cassette, the other one (CCG) is present in the extended 310 region of the integron promoter involved in expression of this gene (Table 1, ¢rst column). Both of these substitutions are also present in the integron In6 from the plasmid pSa. 3.2. Positive e¡ect of CCG substitution in the extended 310 region on activity of the integron promoter G at a position two bases upstream of 310 hexamer is moderately conserved in C. glutamicum promoters [18]. Substitution of any base to G at this position results in increase of promoter activity in both C. glutamicum (our unpublished results) and E. coli [19] in some promoters. We have therefore tested the e¡ect of CCG transversion on the activity of the integron promoter involved in expression of the cassette associated genes. DNA fragments (each 159 bp in size) containing promoters of the integrons InCg and In6, respectively, were ampli¢ed by PCR technique (the upper primer covered positions 992 to 1006, while the lower primer positions 1150^1133 on the map in Fig. 1; both primers had BamHI sites attached to their 5P-ends). To obtain the sequence of the integron promoter of InC [17], which is a typical `weak' integron promoter [2], the GCC transversion in the position two bases upstream of the 310 hexamer was prepared by site-directed mutagenesis of the 159-bp fragment containing InCg promoter and checked by sequencing. All three Bam-
Table 1 Activity of the integron promoters assayed by determining the MIC of CM and by measuring the speci¢c activity of CAT in cell-free extracts of C. glutamicum and E. coli Integron promoter in pET2
C. glutamicum MIC of CM (Wg ml31 )
None InCg TGGTAAGCTb In6 TGGTAAGCTb InC TCGTAAGCTb
5 50 50 30
E. coli CAT activitya (Wmol min31 mg protein31 ) 6 0.001 1.29 þ 0.13 1.19 þ 0.02 0.26 þ 0.01
a
MIC of CM (Wg ml31 ) 10 1300 1300 800
CAT activitya (Wmol min31 mg protein31 ) 6 0.001 1.21 þ 0.17 1.35 þ 0.17 0.27 þ 0.02
Values represent the means þ DS.D. from at least three experiments. Nucleotide sequence of the extended 310 region of integron promoters ; the 310 hexamer is underlined and the base substitutions are indicated by bold letters. b
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HI-fragments containing the respective integron promoters were cloned in E. coli DH5K into the promoter probe vector pET2 (having a promoterless cat gene) linearized with BamHI. The constructed recombinant plasmids were then transferred into C. glutamicum R127. The activity of the respective promoters was measured as MIC of CM and as CAT activity in both hosts. As shown in Table 1, the promoter of integron InCg displays ¢ve times higher activity in both C. glutamicum and E. coli than the InC integron promoter. The same CAT activity was found when the promoter of integron In6 (having the same base substitution CCG two bases upstream of 310 hexamer) was used as a control. The promoters of the integrons InCg and In6 thus represent a new version of the integron promoters involved in expression of antibiotic resistance genes present on the cassettes. The CCG substitution two bases upstream of 310 hexamer of this promoter is undoubtedly responsible for the observed increase of the promoter activity.
4. Discussion The integron InCg present on the plasmid pCG4 from C. glutamicum is the ¢rst class 1 integron found in the Gram-positive bacteria which possesses all parts necessary for its function. Until now, only in two cases, incomplete parts of an integron were identi¢ed in Gram-positive bacteria. In the chromosome of Rhodococcus erythropolis NI86/21, only 47 bp of the 5P-conserved segment of class 1 integron was detected [4]. Transposon Tn610 from Mycobacterium fortuitum contains substantial parts of both 5P- and 3P-conserved integron regions; however, the key integron sequence, namely the recombination site necessary for integrating a gene cassette into the integron, is missing [3]. Presence of the almost identical integron (InCg or InC) on di¡erent replicons (pCG4 or pSA1700) suggests that this integron may be a part of a transposon. Our observation that the identical integron was found on replicons isolated from phylogenetically distant bacterial genera (Corynebacterium vs. Pseudomonas) is an additional indirect evidence for the intergeneric horizontal transfer of genes in natural environment. Di¡erences in the strength of integron promoters
have as yet been ascribed only to base substitutions within the 335 and/or 310 hexamers [2]. We have found that the base substitution (CCG) two bases upstream of the 310 hexamer of the integron promoter present in both integrons InCg and In6 dramatically increased the activity of the so called `weak' integron promoter present in 27 known integrons of class 1, including the proposed ancestor integron In0 [2,20]. This result is in agreement with the positive e¡ect of G at this position on the activity of some promoters from C. glutamicum (our unpublished results) and E. coli [19]. Analysis of the Genbank/EMBL data revealed that, besides the integrons InCg and In6, this novel version of the integron promoter involved in expression of the antibiotic resistance genes is also present in the integron In17 of the enterobacterial plasmid pLMO150 [21] and in the integrons of the plasmid pLST1000 [22] and of a Citrobacter freundii Cf155 plasmid [23]. However, none of the respective authors recognized the importance of the sequence in extended 310 region for the activity of the integron promoter. The supposed evolutionary advantage of the described mutation is supported by the fact that the plasmid pLST1000 carrying this version of the integron promoter is one of the most widely distributed multi-resistant plasmids [22].
Acknowledgments We thank A. Síroglovaè for excellent technical assistance and P. Síebo for critical reading of the manuscript. This work was supported by Grant 204/97/ 0528 from the Grant Agency of the Czech Republic.
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