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Use of Corynebacterium glutamicum in industrial production of amino acids incited genetic studies on amino acid biosynthesis pathways and on central metabolism in this gram-positive species (14). However, efficient utilization of gene cloning techniques and construction of overproducing strains with precisely tuned levels of expression of individual biosynthetic genes are still impeded by the lack of data on transcription initiation signals in C. glutamicum and on elements regulating their activity.

Computer-aided analysis of C. glutamicum promoters. Recently, we isolated a number of C. glutamicum promoters and mapped the respective transcription start (TS) sites. Using the programs PROBAB and PROMSCAN, we analyzed 33 C. glutamicum promoters and defined the consensus DNA sequence of promoters of vegetative genes in C. glutamicum (10). Now we have used the same programs to analyze a set of 44 C. glutamicum promoter sequences. The result was essentially the same as that of the previous analysis, with some minor deviations. In the 35 region, the bases in hexamer TTGGCA were rather evenly conserved (48 to 55%). In the 10 region, the bases of the hexamer T₁A₂(T/G)₃A₄A₅T₆ were conserved to the following levels: T₆, 91%; T₁, 80%; A₂, 73%; A₄, 52%; and A₅, 48%. At the nonconserved third position, T and G occurred with the same frequency (32%). Outside the 10 hexamer, the G at the second base upstream and the G at the second base downstream were less highly conserved (55 and 43%, respectively). The main motifs, TTGGCA and GNTANAATNG, were substantially less conserved than the canonical hexamers TTGACA and TATAAT of Escherichia coli (1, 3) and Bacillus subtilis (4). A low level of conservation of bases was conspicuous particularly in the 35 region, which was hardly recognizable in many of the analyzed promoters. This finding suggested that the 35 region plays a less important role in the function of C. glutamicum promoters.

Cloning of P-dapA. In P-dapA, the putative 10 hexamer TAACCT shares 3 bases with the consensus sequence of the C. glutamicum 10 region, TANAAT, while the 35 hexamer is not apparent in the common 35 region (10). The sequence TTAACC (39 to 34), with 3 bases matching those found in the C. glutamicum consensus sequence might function as the 35 hexamer; however, the spacing difference of 19 bases makes it less probable. We constructed a transcriptional fusion of P-dapA, carried on the 400-bp BamHI-KpnI fragment, to the cat reporter gene in the multicopy promoter-probe vector pET2 (17) replicating in C. glutamicum. To avoid possible effects of varying the plasmid copy number, the promoter fragment was also cloned in the integrative promoter-probe vector pRIM2 (17) for assay in a single-copy system. The vector pRIM2 was transferred into C. glutamicum by the conjugation technique described by Schäfer et al. (15).

Activity of P-dapA. Activity of the wild-type (WT) promoter and of its deletion and mutation derivatives was evaluated by chloramphenicol acetyltransferase (CAT) assay done by the method of Shaw (16). One unit of enzyme activity was defined as 1 µmol of chloramphenicol acetylated per minute.

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Deletion analysis of P-dapA. Deletion analysis of the 400-bp fragment (Fig. 1) carrying P-dapA was performed to define the extent of sequence necessary for promoter function. PCR was used to create fragments of various lengths, with appropriate restriction sites attached to the ends for cloning. The truncated fragments were cloned in pET2 and tested for their promoter activity (Fig. 1). Deletions at the 5' end as far as position 86 did not affect P-dapA strength. Deletions downstream of the TS, including a deletion up to position 7 that encompassed the TS (clone DD3), also resulted in little or no effect on promoter activity. A remarkable reduction of activity (to 26%) was caused by the deletion of nucleotides between positions 59 and 35 (UD6). Deletion of 3 more bases covering the possible 35 region resulted in a negligible reduction of activity. A further deletion that removed the 10 region (UD8) abolished promoter activity completely. We conclude from these results that (i) the region necessary for full P-dapA activity is not longer than 80 bp (86 to 7), (ii) the 10 element is essential for P-dapA activity, and (iii) the 22-bp sequence upstream of position 35 is necessary for full activity of the promoter P-dapA.

View larger version (40K): [in this window] [in a new window]   FIG. 1.   Deletion mapping of the promoter P-dapA. The horizontal shaded boxes represent the relative lengths of DNA fragments carrying the promoter studied. The 5' and 3' ends of the fragments relative to the TS are shown. The thick vertical bars mark the positions of potential 35 and 10 regions of the promoter. The vertical bar with an arrow on the top indicates the position of TS. PCR-generated fragments were cloned into pET2 to construct cat transcriptional fusions for determination of promoter activity. CAT activity is expressed relative to that measured with full-length fragment (UD1). Standard deviations (SD) from three independent measurements are shown.

Mutations in the 59 to 35 region. To identify which bases upstream of position 35 were essential for promoter activity, 1- to 4-base changes were introduced by oligonucleotide-directed mutagenesis (5) into the sequence between positions 59 and 35. These substitutions were done in the sequences potentially important in promoter function (Fig. 2), as follows: in the AT-rich region at the 5' end of the 35 region (clone ME77), in the stretch of four G's (clones ME77 and MH1) forming part of a 6-base inverted repeat with the sequence AACCCC within the 35 region, and in the stretch of six T's (clones P4 and R2) which might induce an intrinsic bend in a DNA strand. The TAAGGG change at positions 38 to 36 in P-dapA mutant ME77 resulted in a level of activity about 70% of that of the WT promoter. This relatively small reduction of activity caused by the replacement of bases forming part of a potential 35 region (TTAACC or AACCCC with a spacing difference of 19 or 17 bp, respectively) reflected the probable minor role of the 35 region in promoter function. Disruption of the inverted repeat (Fig. 2) by a substitution of AAA for GGG at positions 46 to 44 (clone MH1) had also a small negative effect (reducing the level of activity to 80% of the WT level of activity). In contrast, both single-base (53TA) and double-base (53,52TTCC) substitutions in the six-T tract reduced the promoter activity to about 20% (promoters P4 and R2). Removing this run of six consecutive T's was therefore probably mainly responsible for the marked decrease of activity of the clone with a deletion at the 5' end at position 35. We hypothesize that the T tract induces a DNA bend (12) or serves as an UP element (13) in P-dapA.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Effects of mutations in the 59 to 35 region of P-dapA on promoter activity. The arrows mark the inverted repeat. Replacement bases are indicated in bold face. Transcriptional activity of the mutated fragments was evaluated in the vector pET2. TS marks the transcription start (underlined A). CAT activity is expressed relative to that measured with the WT promoter. Standard deviations (SD) from three independent measurements are shown.

Mutations in the 10 region. All transcriptional fusions of the promoter mutations to the cat reporter were borne by the plasmid pET2. Most of them were also integrated into the chromosome with pRIM2, and promoter strength was evaluated in both systems. To prove that the sequence TAACCT from positions 14 to 9 functions as the 10 hexamer, 14T and/or 9T, occupying the most conserved positions, were replaced with other bases. In all these cases (clones B27, J3, J2, B12, B31, B8, and B6), activity of the promoter was nearly eliminated (Fig. 3A). This result confirmed that these T's are essential for promoter function and that the sequence TAACCT is really the 10 hexamer of P-dapA. All substitutions of one, two, or three bases increasing the homology with the consensus sequence TANAAT gave rise to promoters stronger than the original one (Fig. 3A). We made all possible changes at the third position of the hexamer (which is not conserved according to the statistical analysis of C. glutamicum promoters) combined with substitutions of AA for CC at positions 11 and 10. Among them, the sequence TAAAAT (mutant A45) yielded the lowest levels of activity (1.5- and 1.7-fold times the WT promoter activity level in the single-copy and multicopy systems, respectively), while the hexamer TATAAT (A16) represented the strongest sequence variation (with 5.2- and 3.9-fold times the activity of the WT promoter). The hexamers TAGAAT (L1) and TACAAT (M3) formed promoters of intermediate strength (with 1.8- to 2.3-fold times the WT promoter activity).

View larger version (31K): [in this window] [in a new window]   FIG. 3.   Effects of mutations in the 10 region of P-dapA on promoter activity. Replacement bases are indicated in boldface. CAT activity measured by using the transcriptional fusion to the cat reporter gene in the multicopy vector pET2 is shown by open bars; activity measured after integration of the promoter fragment with the vector pRIM2 into the C. glutamicum chromosome is shown by solid bars. CAT activity is expressed relative to that measured with the WT promoter. Standard deviations (SD) from three independent measurements are indicated with error bars. (A) Mutations within the 10 hexamer; (B) mutations outside of the 10 hexamer.

Conclusion. Mutational analysis of the dapA promoter supports the results of computer analysis of the set of C. glutamicum promoters aimed at defining the consensus sequence of C. glutamicum promoters of vegetative genes. We suggest that the extended 10 region is more important for transcription initiation than the 35 region in many C. glutamicum promoters. Mutagenesis study of other promoters, in particular those dependent on the 35 region, is necessary to establish the final C. glutamicum promoter consensus sequence.