To obtain a system producing polycistronic mRNA transcripts where expression from cotranscribed ORFs is easily quantified we inserted a stop codon between the gfp and DsRed parts in the pGRFP double reporter plasmid 8. Also, to improve prospects for translational coordination between the genes, the intergenic distance was kept short and a strong SD sequence was included 9. The resulting pGRFPsep construct (Figure 1, Figure 2A) was introduced into E. coli AS19. After treatment with varying concentrations of anti-gfp, anti-DsRed or two unrelated control PNAs (anti-lacZ and anti-lacY, see Table 1 for details) protein expression was determined. As seen in Figure 2B, treatment with the anti-gfp PNAs resulted in a reduction of GFP levels, whereas RFP levels remained largely unaffected (upper panel). Similarly, the anti-DsRed PNAs only reduced the expression of RFP (Figure 2B, middle panel). Moreover, the control experiments with PNAs directed to unrelated mRNA targets did not inhibit the expression of GFP or DsRed (Figure 2B, lower panel). Therefore, the results show a specific downregulation of expression even though the coding sequences belong to the same transcriptional unit.

The above result describes the effect of antisense PNA targeted to cotranscribed genes within a system where translation is discoordinated. However, as this system is an artificial construct lacking naturally evolved regulatory components and sequences we decided to continue the study with an endogenous operon. We targeted the genes in the chromosomally expressed lac-operon and determined the effect on the expression of its three structural genes; lacZ, lacY and lacA (see Figure 3A). Treatment with anti-lacZ PNA resulted in a strong inhibition of the lacZ encoded protein (Figure 3B). Also, expression from the two downstream genes lacY and lacA was markedly reduced, however not to the same extent as lacZ expression. When bacteria were incubated with anti-lacY PNA, lacY and lacA gene expression was reduced, whereas expression from the upstream lacZ gene was unaffected (Figure 3C). Unfortunately, we did not obtain any data with anti-lacA PNA as this displayed strong growth inhibitory effects. Nevertheless, the results show that antisense targeted to an upstream gene in the lac-operon has a large impact on the expression from the downstream genes. In contrast, no upstream coordination was observed, suggesting a directional inhibitory effect in the lac-operon.

To ensure that the anti-lacZ and anti-lacY PNAs did not interfere with bacterial growth and examine whether expression of an unrelated protein was affected by the presence of antisense, we determined growth rate and Lux activity in cells carrying the plasmid pLux1 following treatment with PNAs that target the lac-operon. Neither growth nor Lux expression was altered by the PNAs when added in concentrations up to 1000 nM (see Additional file 1).

To better understand the expression pattern observed at the protein level, we used quantitative real time PCR (qPCR) to study the impact of antisense PNA inhibition at the mRNA level. Samples from bacteria treated with varying concentrations of either anti-lacZ (0, 200 and 400 nM) or anti-lacY PNA (0, 100 and 200 nM) were analysed for their lacZ and lacY mRNA content (Figure 4). Treatment with anti-lacZ PNA significantly reduced lacZ mRNA at both 200 nM and 400 nM (p < 0.01 and p < 0.001, respectively) (Figure 4A). LacY mRNA was less affected and a significant reduction was only obtained with the higher anti-lacZ PNA concentration (p < 0.05). Moreover, there was a significantly higher reduction of lacZ mRNA than lacY mRNA after treatment with the lower antisense dose (p < 0.05), but not at the higher dose (p = 0.066). Interestingly, a comparison between protein and mRNA levels after anti-lacZ PNA treatment reveals a coupled reduction (cf. Figure 3B with 4A). Anti-lacY PNA significantly reduced the level of lacY mRNA at both 100 and 200 nM concentrations (p < 0.05 and p < 0.005, respectively), whereas lacZ mRNA levels were comparable to untreated cells (Figure 4B). Again, a coupled reduction of transcript and protein levels is demonstrated as LacY but not LacZ protein concentrations were reduced by anti-lacY PNA (cf. Figure 3C with 4B).

The above results demonstrate a dose dependent gene expression inhibition at both the mRNA and protein levels, with a proportionally stronger reduction of protein. To further examine this relationship we performed a more detailed analysis of lacZ gene expression with anti-lacZ PNA doses ranging from 0 to 400 nM concentrations at 50 nM intervals. Again, a coupled reduction of both protein and mRNA levels was observed (Figure 5A). Supporting our previous observations (Figure 3B and Figure 4A), increasing doses of anti-lacZ PNA resulted in a negative exponential decrease in LacZ protein activity whereas the decrease in lacZ mRNA was linear (Figure 5B).

E. coli strain AS19 was used throughout this study. Due to its hyper permeable nature lower antisense concentrations are required for gene expression inhibition in this strain. PNAs were designed to target the start codon regions of gfp, luc, lacZ and lacY (and lacA), with the peptide (KFF)₃K attached for improved cellular uptake 28 and were obtained from Panagene (Daejeon Metropolitan City, Korea). Peptide-PNA sequences are listed in Table 1. Importantly, none of the peptide-PNAs (except anti-lacA PNA) displayed any alteration in bacterial growth under the conditions and with the concentrations used in this study.

Cloning vector pGRFP 8 expressing GFP and DsRed as a fusion protein was kindly provided by Stephanie Mohr at Dana-Farber/Harvard Cancer Center DNA Resource Core, US. A GFP stop codon, a BglII cleavage site, a Shine-Dalgarno sequence and a unique PNA binding site were introduced between gfp and DsRed using upstream primer 5'-gcgagatctcatttgtatagttcatccatgccatgtgtaatcccagcagc-3' and downstream primer 5'-tatagatctaaggaggaaacagaatggcctcctccgaggacgtcatca-3'. Cleavage with BglII and ligation yielded pGRFPsep, expressing gfp and DsRed as a single mRNA encoding two different proteins (Figure 1). pGRFPsep was transformed into E. coli AS19 where the double reporter system was constitutively expressed.

E. coli AS19 and AS19/pGRFPsep were grown for around 12 h in 2 ml Mueller Hinton (MH) broth at 37°C with constant shaking at 225 rpm. Optical density (OD) was measured at 550 nm and the cultures were diluted to 1.25 × 10⁶ bacteria per ml in MH. Aliquots (80 μl) were added to the wells of a 96 well plate (COSTAR^® 3474, Corning Inc., NY). Plates were incubated in a VERSAmax spectrophotometer (Molecular Devices Corporation, CA, USA) at 37°C with shaking and OD measurements taken each five minutes. When OD₅₅₀ had increased by ~0.025, dilutions of anti-gfp or anti-DsRed PNAs were added to make total culture volumes of 100 μl. PNAs directed to lacZ and lacY mRNA were also included to provide controls. Cultures were harvested in late logarithmic phase after an increase in OD₅₅₀ of ~0.1.

For GFP/DsRed level determinations, AS19 and AS19/pGRFP-sep were lysed by mixing 50 μl cell samples with 50 μl lysis buffer (Pierce Biotechnology, prod. no. 78990) in a 96-well assay plate ("Costar 3632", Corning Inc.). Fluorescence measurements were carried out in a NOVOstar fluorescence/luminescence reader (BMG Labtechnologies, NC, USA). Lysates were excited at 485/560 nm and fluorescence emission was detected at 520/590 nm. Total fluorescence was determined by 100 flashes with maximum capacity of 65000 relative fluorescence units (RFU). Background fluorescence was determined with plasmid free AS19 cell lysates.

E. coli AS19 cells were pre-grown overnight in 2 ml MH broth at 37°C with constant shaking at 225 rpm. Cultures were diluted to 1.25 × 10⁶ bacteria per ml in MH and aliquots (200 μl) corresponding to approximately 2.5 × 10⁵ cells were added to the wells of a 96 well plate. Dilutions of PNAs were added together with IPTG to make a total culture volume of 250 μl with 100 μM IPTG. IPTG- and PNA-free cultures were used as controls. Plates were incubated in a VERSAmax spectrophotometer 37°C with shaking and OD measurements each five minutes until an increase in OD₅₅₀ of ~0.1 was reached. Samples of the cultures were treated as follows:

E. coli AS19 treated with different concentrations of anti-lacZ or anti-lacY PNA in the presence or absence of 100 μM IPTG were incubated until the OD₅₅₀ increased by ~0.1. Cells with equal treatment were pooled to get two separate 1 ml samples for each treatment. Total RNA was extracted with TRIZOL^®Reagent following the manufacturer's instructions (Invitrogen). Samples were dissolved in 34 μl RNase-free water (Invitrogen) and treated with RNase-free DNase (Sigma Aldrich). RNA concentrations were determined by OD-readings on an Ultrospec 3000 (Pharmacia Biotech, Uppsala, Sweden). Reverse transcription was performed using random hexamers with 1 μg RNA in 50 μl reactions following the manufacturers protocol (Applied Biosystems). Aliquots derived from 50 ng RNA were analyzed by qPCR on an ABI PRISM 7000 (Applied Biosystems) according to the manufacturers protocol. Samples were run in triplicates and the data obtained were analyzed with ABI Prism SDS Software (Applied Biosystems). The ΔΔCt method was used to calculate lacZ and lacY gene expression using rRNA as an internal standard 30. Sequences for the lacZ and lacY primers were selected to amplify regions near the 5' end of the coding regions to increase the chances of detecting functional transcripts as lac mRNAs are degraded in a 5' to 3' direction 2033, and were: lacZ forward 5'-ccctggcgttacccaacttaa-3'; lacZ reverse 5'-gcgggcctcttcgctattac-3'; lacY forward 5'-cgcaaatacctgctgtggatta-3' and lacY reverse 5'-tggcccgaagataaaaataaagaa-3'. The primer binding sites did not interfere with antisense PNA target sites (see Figure 3A). Primer sequences for rRNA were taken from 34 and were: rRNA forward 5'-catgccgcgtgtatgaagaa-3' and rRNA reverse 5'-cgggtaacgtcaatgagcaaa-3'. RNA isolation and quantification was repeated at four different occasions giving eight samples and 24 data points per PNA concentration. Data comparisons were made to a normalised control value obtained by calculating the mean at each experimental occasion. Statistical calculations were performed in Excel using unpaired t-tests assuming equal variance among groups and data are presented as the mean with error bars reflecting the standard deviation. Significance was set at p < 0.05.

E. coli AS19 treated with anti-lacZ PNA concentrations ranging from 0 to 400 nM at 50 nM intervals were grown in six double replicas in the presence or absence of 100 μM IPTG. Cultures were incubated under the same conditions as described above and harvested when OD₅₅₀ had increased by ~0.1. Samples of 20 μl were removed from each well to be used for separate LacZ activity measurements as described above, and the remainder of the cultures were pooled into groups with equal treatment. Total RNA was extracted and treated with DNase I using RiboPure Bacteria kit (Ambion), according to the manufacturer's protocol. Quantification of lacZ mRNA was performed as described above. Mean values from the six replicates of each treatment were used to determine relative LacZ activity and the averages of three replicates derived from each sample pool were used to estimate mRNA abundance. The experimental procedure was repeated at three different occasions, and mean values with error bars were plotted as a histogram. Regression analysis was conducted using VassarStats 35.