In order to isolate very strong promoters, a plasmid named pProm was constructed (Fig. 1). First, a small fragment containing the promoter of the ampicillin gene of the pBR322 was eliminated using the endonucleases SspI and EcoRI. The plasmid was reconstructed by inserting a HincII-EcoRI fragment recovered from the M13mp19 multiple cloning site (MCS). A 1700-bp ClaI DNA fragment from pRIT5  containing a chloramphenicol resistance gene and ORI for Gram-positive bacteria of the S. aureus plasmid pC194  was inserted into the AccI site of M13mp7. It was recovered from this vector by digestion with EcoRI and inserted into the EcoRI site of the modified pBR322. The resulting vector named pProm (Fig. 2) possesses the following features: an ORI(−) to replicate in Gram-negative bacteria, an ORI(+) to replicate in Gram-positive-bacteria, a promoterless ampicillin gene with an MCS including a unique SmaI site which replaces the promoter of the ampicillin resistance gene, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria.
Figure 1. Construction of the pProm plasmid for strong promoter isolation. Ap: ampicillin resistance gene; Tc: tetracycline resistance gene; Cm: chloramphenicol resistance gene; ORI+: replication origin for Gram-positive bacteria; ORI−: replication origin for Gram-negative bacteria; MCS: multiple cloning site.
Download figure to PowerPoint
Insertion into the MCS of DNA fragments containing promoters should activate the ampicillin resistance gene allowing the selection of bacteria transformed with the plasmid in ampicillin-containing agar plates.
The ORI for replication in Gram-positive bacteria was introduced into pProm in order to allow transformation into Bacillus subtilis cells where the product of the ampicillin resistance gene would be secreted. However, preliminary experiments showed that upon transformation in these bacteria pProm suffers a number of structural alterations, which limits its usefulness (data not shown).
3.2Isolation of promoters
Using a mix of restricted DNA of the S. aureus bacteriophages CDC ϕ 83, ϕ 85, ϕ 95 and ϕ 3E/C, the capability of pProm to isolate strong promoters was assayed. These phages were chosen in order to have a reasonable chance that strong, foreign promoters were isolated. Phage DNAs were purified and digested with the restriction enzymes AluI and HaeIII and inserted into the SmaI site of pProm. Recombinant plasmids were introduced into E. coli cells by transformation and bacterial clones selected on LB agar plates with a high concentration of ampicillin (1 mg ml−1) in order to: (a) isolate only strong promoters and (b) avoid a background of colonies of low resistance to ampicillin because of the chloramphenicol promoter or because a divergent promoter expressing tetracycline resistance (Fig. 3).
Figure 3. Sequence of DNA fragments inserted into pProm plasmid, which activate the promoterless Ap. The EMBL accession number for X8 is Y12633.
Download figure to PowerPoint
Four of the selected bacterial colonies (X1, X2, X3 and X8) were chosen for characterization. An assay performed using agar plates containing different amounts of ampicillin (Table 1) demonstrated that the selected clones were able to grow at very high concentrations of this antibiotic. Bacteria transformed with pProm activated by insertion of well-known strong promoters (PTac and lambda PL) were used as controls. The selected clones were able to grow at concentrations over 1 mg ml−1 of ampicillin (Table 1). The most resistant clone, X8, was able to grow on media containing up to 7 mg ml−1 of ampicillin. This level of resistance was not reached by the positive controls.
Table 1. Ap resistance of E. coli DH5αF′Iq transformed with derivatives of pProm as estimated by agar plate assay
| ||pProm-X1||pProm-X2||pProm-X3||pProm-X8||pProm-Tac||pProm-PL||pProm||E. coli|
|0.5 mg ml−1||+++||+++||+++||+++||+++||+++||+||−|
|1 mg ml−1||++||++||++||+++||+++||++||−||−|
|2 mg ml−1||+||+||++||+++||+++||+||−||−|
|3 mg ml−1||+||+||++||+++||++||−||−||−|
|4 mg ml−1||+||−||+||+++||+||−||−||−|
|5 mg ml−1||−||−||−||+++||−||−||−||−|
|7 mg ml−1||−||−||−||++||−||−||−||−|
|The number of + indicates if colonies produced after incubation of plates seeded with equal amounts of cells were abundant (+++), regular (++), scarce (+), or absent (−).|
|1.5% agar-LB medium plates with different amount of Ap were inoculated with 10 μl of a cell suspension containing about 300 viable bacteria and were evenly distributed on the agar surface using small glass spheres. Incubation was carried out for 15 h at 37°C.|
Subsequently, the DNA fragments inserted into pProm X1, X2, X3 and X8 plasmids were sequenced and putative promoter areas localized (Fig. 3). DNAs fragments X1, X2 and X3 possess hexanucleotides with different degrees of homology to the canonical −10 and −35 elements of the σ70-RNAP of E. coli promoters. In addition X1 has, in the −44 region and downstream of the −10 element, sequences coincident with motifs pointed out by Ozoline et al. [28,29] as frequently present in these E. coli promoters. The X8 fragment is particularly interesting because within it lies a putative promoter, which possesses most of the motifs described by Ozoline et al.  as follows:
These homologies, together with the fact that major RNA polymerases of Gram-positive bacteria have consensus nucleotide sequences very similar to those of E. coli promoters [34–39], strongly suggest that fragment X8 possesses a real promoter.
However, this may not be the case for X1, X2 and X3 which might be random sequences that act as good promoters. Further studies are necessary to evaluate this possibility.
Using hybridization techniques, fragment X8 was demonstrated to belong to the S. aureus bacteriophage ϕ 85 genome (not shown). According to its exceptional strength, the promoter of this DNA fragment may direct the synthesis of a highly expressed phage component. However, sequence data to corroborate this are not yet available in databases.
Although putative promoters were recognized in the X1, X2, X3 and X8 DNA fragments, it is possible that the inserted DNA sequences may be acting as enhancer elements of the basal transcriptional activity of the chloramphenicol promoter or the tetracycline divergent promoter in pProm (see above). In order to rule out this possibility, we removed these promoters in pProm-X1, X2, X3 and X8 by deletion. For this, plasmids were first digested with the SalI restriction enzyme that releases two fragments, one of them containing both promoters, the chloramphenicol and tetracycline resistance genes, and the ORI for Gram-positive bacteria and the other containing the activated ampicillin gene and the ORI for Gram-negative bacteria (Fig. 2). Recircularization of this last fragment resulted in plasmids with only one resistance marker (ampicillin). The ampicillin resistance levels were about the same in cells transformed with deleted or undeleted plasmids (not shown), a fact that supports the idea that the inserted DNA fragments are acting as promoters.