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Keywords:

  • Bacterial promoter;
  • Bacteriophage promoter;
  • Strong promoter;
  • Promoter isolation;
  • Vector

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

DNA dependent RNA polymerase (RNAP) initiates the transcription process in bacteria after binding to a specific DNA sequence named the promoter. The efficiency of this process strongly depends on the structure of the promoter. In Escherichia coli several studies indicate that canonical hexanucleotides at the −35 (TTGACA) and −10 (TATAAT) regions are important for σ70-RNAP recognition [1]. Recently, a very A+T rich region between positions −40 and −60 (named the UP element) was also recognized in some promoters as an important determinant of promoter efficiency [2]. On the other hand, a relationship between promoter efficiency and the presence of intrinsically curved DNA upstream of the promoter has also been demonstrated [3]. Thus, promoter efficiency seems to be a complex matter related not only to the kind of nucleotides directly involved in the contact with the RNAP but also to the three-dimensional structure of the whole region.

In order to further investigate the bases for promoter efficiency we thought that it would be useful to isolate a large collection of very efficient (strong) promoters from different sources.

A possible strategy for promoter isolation is to construct a vector having a gene marker devoid of its own promoter (a silent gene). The insertion of a DNA fragment containing a promoter in place of the original promoter would activate the silent gene. Using this strategy several promoter-probe plasmids have been constructed using as reporters antibiotic resistance genes [4–7], the β-galactosidase gene [8–10], the galactokinase gene [11], the gene for a fluorescent protein [12], the β-1,4-endogluconase gene [13], the xylE gene [14], the chloramphenicol acetyltransferase gene [15–18], and the β-glucuronidase gene [19]. The main problem with this strategy is that if a very strong promoter is present in the inserted DNA fragment, the reporter gene product will accumulate in very large amounts inside the cell, resulting in reduced cell viability.

To avoid this problem, we have constructed a vector based on the ampicillin resistance gene of the pBR322 plasmid whose product is secreted into the periplasmic space of E. coli. Cell viability is probably not affected as seen in high copy number vectors such as pUC [20,21]. Three strong promoters and one very strong promoter were isolated from bacteriophages, which confirmed the correctness of this approach. To our knowledge, this is the first vector which allows the trapping of very strong bacterial or bacteriophage promoters.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

2.1Strains and growth conditions

E. coli DH5αF′Iq φ80dlacM15, Δ(lacYZA-argF)U169, recA1, endA1, hsdR17 (rk−, mk+), supE44 λ−, thi-1, gyrA, relA1/F′proAB+, lacIqZΔM15zzf::Tn5(knr) was used in cloning experiments. Bacteria were usually grown in Luria-Bertani broth (LB) liquid or solid media at 37°C. Staphylococcus aureus CDC phages ϕ 83, ϕ 85, ϕ 95 and ϕ 3E/C were used as a source of DNA to isolate promoters.

2.2Cloning and sequencing

Plasmid DNA was purified according to the method described by Birnboim and Doly [22] and was digested and ligated using standard protocols [23]. Transformations were performed according to the method described by Hanahan [21]. The screening of the inserts was performed by digestion of plasmid DNA with restriction enzymes followed by analysis of the products by agarose gel electrophoresis [23]. DNA was sequenced on both strands using the fmol DNA Sequencing kit (Promega) [24] after purification with the Wizard Clean Up system (Promega).

2.3Penicillinase activity

Penicillinase assays were performed using a colorimetric method [25]. Briefly, 50 μl of ampicillin resistant E. coli cells (OD600 0.7) were incubated for 5 min at 30°C in 0.1 M phosphate buffer pH 7,0. 6 μmol of penicillin G was added at different times (t= 0–170 min). The reactions were ended by adding 1.8 ml of a 10% iodine solution (0.32 M I2, 1.2 M potassium iodine, in 0.5 M buffer sodium acetate pH 4.0) and the resulting color was spectrophotometrically measured at 540 nm. Activity was defined as the difference between the initial OD540 and the OD540 at a given time (ΔOD540(t)), divided by the time in minutes, as a percentage of the initial OD540 (OD540(to)): Act.=[(ΔOD540(t)/t(min))/OD540(to)]×100.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

3.1Vector construction

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 [26] containing a chloramphenicol resistance gene and ORI for Gram-positive bacteria of the S. aureus plasmid pC194 [27] 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.

image

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.

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image

Figure 2. pProm vector map. Endonucleases of the MCS shown in bold letters have unique restriction sites.

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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).

image

Figure 3. Sequence of DNA fragments inserted into pProm plasmid, which activate the promoterless Ap. The EMBL accession number for X8 is Y12633.

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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
ApTransforming plasmid
 pProm-X1pProm-X2pProm-X3pProm-X8pProm-TacpProm-PLpPromE. 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. [28] as follows:

  • 1
    A −10 region between nucleotides 310 and 315, TTTAAT, which shows only one nucleotide difference to the canonical TATAAT −10 region of E. coliσ70-RNAP promoters. It also bears the TG dinucleotide upstream which increases the contact with the σ70-RNAP [30,31].
  • 2
    A −35 region between nucleotides 283 and 288, GTGTCA, shows only two mismatches with the canonical TTGACA −35 region of the E. coliσ70-RNAP promoters.
  • 3
    A homopyrimidine box TCCTCC downstream of the −35 region, which has been previously reported for promoters with longer than usual spacer length [32].
  • 4
    A −44 region between nucleotides 273 and 278, TTATTC, which shows only one nucleotide difference with TTTTTC of the −44 region previously reported as typical of the E. coliσ70-RNAP promoters [29,33].
  • 5
    A −54 region between nucleotides 262 and 267, TTTTTT, which is identical to one of the −54 regions previously reported as typical of the E. coliσ70-RNAP promoters [29,33].

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.

3.3Promoter strength evaluation

The penicillinase activity of cell suspensions was measured in order to assess the relative efficiencies of the isolated promoter sequences (Fig. 4). According to this, the promoter present in the X8 DNA fragment is stronger than the well-known PTac and Lambda PL promoters. This result confirms our initial expectation that very strong promoters could be isolated using the pProm vector and opens the possibility of collecting many such promoters.

image

Figure 4. Penicillinase assay of E. coli clones transformed with pProm derivatives containing DNA inserts which activated the promoterless ampicillin gene.

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Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

We thank Gabriela Paván for technical support and Maria Elisa Paván and Martin Vazquez for critical reading of the manuscript.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References
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