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 (G→C) 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 (C→G) is present in the extended −10 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.
Integrons are genetic units that include site-specific recombination system capable of capturing genes (most frequently determinants of resistance to antibiotics) located on mobile elements called gene cassettes. Integrons have been classified 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 (5′- and 3′-conserved segments) and a recombination site between them, where gene cassettes are integrated. Most of genes on integrated cassettes lack their own promoters and are expressed from a common promoter region located in the 5′-conserved segment . Four versions of these integron promoters, differing in the sequences of the −35 and/or −10 hexamers and in their strength, have been described so far .
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 and in the chromosome of Rhodococcus erythropolis NI86/21 , only incomplete and therefore non-functional parts of the integron sequence are present.
In this report, we describe, for the first time, presence of the class 1 integron possessing the whole 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 finding that the identical integron is present on replicons from the phylogenetically distant bacterial genera is documented.
2Materials 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 . Escherichia coli DH5α and C. glutamicum R127  were used as hosts for the constructed recombinant plasmids. The mini-derivative of the plasmid pSa, pGV1106 , served as a source of promoter of integron In6. Plasmid pUC18  served as a vector for cloning and sequencing the defined fragments of pCG4. C. glutamicum–E. coli shuttle promoter–probe vector pET2 carrying the promoterless cat gene (derivative of the plasmid pEKplCm ) was used for analysis of promoter activity.
2.2 Growth and transformation conditions
E. coli strains were grown at 37°C in LB medium and C. glutamicum strains were grown at 30°C in CY medium . Transformation of E. coli was carried out by the method of Hanahan  and electrotransformation of C. glutamicum was done according to the method of Liebl et al. . The following concentrations of antibiotics (μg ml−1) 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 Purification System (Promega). Plasmid DNA from C. glutamicum was isolated by a modified alkaline extraction procedure with lysozyme . Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturers. DNA fragments carrying the integron promoter regions were amplified by the PCR technique. The PCR-mediated site-directed mutagenesis was carried out by the method of Ito et al. . The nucleotide sequence was determined by using automatic sequencer Vistra (Amersham).
2.4 Enzyme assay and determination of minimal inhibitory concentrations
The specific activity of chloramphenicol acetyltransferase (CAT) in bacterial cell-free extracts was determined by the method of Shaw . A unit of activity was defined as 1 μmol 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. .
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.
3.1 Nucleotide sequence of Sm/Sp resistance determinant of plasmid pCG4 and its flanking 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-host-range plasmid pSa originally isolated from the Gram-negative bacterium Shigella flexneri. The nucleotide sequence of the regions flanking the Sm/Sp resistance gene was shown to be identical to that of 5′- and 3′-conserved regions of class 1 integrons. Analysis of the nucleotide sequence of the whole 3.8-kb BamHI fragment of pCG4, containing the Sm/Sp resistance gene, revealed its structure typical of the class 1 integrons described until now only in Gram-negative 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. As shown in Fig. 1, differences in only two base pairs are present on the 3.8-kb fragment. One base substitution (G→C) was found in the aadA2 gene associated with the gene cassette, the other one (C→G) is present in the extended −10 region of the integron promoter involved in expression of this gene (Table 1, first column). Both of these substitutions are also present in the integron In6 from the plasmid pSa.
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 5′- and 3′-conserved segments (CS). Numbers below the vertical arrows represent positions of the base pairs differing from those of integron InC . 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-specific integrase; aadA2a, streptomycin/spectinomycin resistance; qacE Δ1, 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 μg ml−1 vs. 100 μg ml−1 in the plasmidless C. glutamicum). B, BamHI; E, EcoRI; H, HindIII; P, PstI; S, SphI.
Activity of the integron promoters assayed by determining the MIC of CM and by measuring the specific activity of CAT in cell-free extracts of C. glutamicum and E. coli
Integron promoter in pET2
MIC of CM (μg ml−1)
CAT activitya (μmol min−1 mg protein−1)
MIC of CM (μg ml−1)
CAT activitya (μmol min−1 mg protein−1)
aValues represent the means±DS.D. from at least three experiments.
bNucleotide sequence of the extended −10 region of integron promoters; the −10 hexamer is underlined and the base substitutions are indicated by bold letters.
3.2 Positive effect of C→G substitution in the extended −10 region on activity of the integron promoter
G at a position two bases upstream of −10 hexamer is moderately conserved in C. glutamicum promoters . 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 in some promoters. We have therefore tested the effect of C→G 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 amplified 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 5′-ends). To obtain the sequence of the integron promoter of InC , which is a typical ‘weak’ integron promoter , the G→C transversion in the position two bases upstream of the −10 hexamer was prepared by site-directed mutagenesis of the 159-bp fragment containing InCg promoter and checked by sequencing. All three BamHI-fragments containing the respective integron promoters were cloned in E. coli DH5α 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 five 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 C→G two bases upstream of −10 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 C→G substitution two bases upstream of −10 hexamer of this promoter is undoubtedly responsible for the observed increase of the promoter activity.
The integron InCg present on the plasmid pCG4 from C. glutamicum is the first 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 identified in Gram-positive bacteria. In the chromosome of Rhodococcus erythropolis NI86/21, only 47 bp of the 5′-conserved segment of class 1 integron was detected . Transposon Tn610 from Mycobacterium fortuitum contains substantial parts of both 5′- and 3′-conserved integron regions; however, the key integron sequence, namely the recombination site necessary for integrating a gene cassette into the integron, is missing . Presence of the almost identical integron (InCg or InC) on different 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.
Differences in the strength of integron promoters have as yet been ascribed only to base substitutions within the −35 and/or −10 hexamers . We have found that the base substitution (C→G) two bases upstream of the −10 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 effect of G at this position on the activity of some promoters from C. glutamicum (our unpublished results) and E. coli. 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  and in the integrons of the plasmid pLST1000  and of a Citrobacter freundii Cf155 plasmid . However, none of the respective authors recognized the importance of the sequence in extended −10 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 .
We thank A. Šroglová for excellent technical assistance and P. Šebo for critical reading of the manuscript. This work was supported by Grant 204/97/0528 from the Grant Agency of the Czech Republic.