Identification of transcriptional units within the sxt biosynthetic gene cluster
Reverse-transcriptase PCR revealed that the sxt BGC in D. circinale AWQC131C is transcribed as five transcriptional units from two bidirectional promoter regions (Figure 1). All five transcripts appear to be constitutively expressed under standard laboratory conditions, since sxt mRNA was detected across all time points. Operon 1, sxtDV*EABC (* indicates disrupted ORF of sxtV [34]), spans 7.3 kb, is transcribed in the reverse direction and encodes several proteins predicted to be involved early in PST biosynthesis. Operon 2, sxtPQR, spans 3.5 kb and is transcribed in the forward direction. The catalytic functions of SxtP, SxtQ and SxtR are unknown, but they are likely to be essential for PST biosynthesis as their presence and organisation is conserved amongst all reported sxt clusters. The third transcriptional unit is monocistronic and encodes SxtPER, a putative permease of the drug/metabolite transporter family of proteins and is transcribed from two promoters, as discussed further below. Operon 4, is transcribed in the forward direction and spans 12.8 kb. Operon 4 encodes a protein of unknown function, Orf24 that is conserved in most sxt clusters, followed by genes encoding twelve enzymes involved in PST biosynthesis, resulting in the polycistron orf24sxtSTUNGHMIJKLO.
The 3′ ends of operons 1-4 were bioinformatically screened for putative Rho-dependent and Rho-independent transcriptional termination sites using the programs TransTerm and TranstermHP, respectively [46, 47]. Rho-independent transcription termination sites were identified in the non-coding regions of three out of four sxt mRNA transcripts (Table S1). Rho-dependent or Rho-independent termination sites were not identified in the sequence of mRNA encoding operon 1.
Transcription start sites and promoter regions of the sxt operons
The transcriptional start sites (TSSs) of each operon were experimentally identified via 5′ rapid amplification of cDNA ends (5′ RACE) (Table 1; Supporting Information). The upstream region of each TSS was screened for a promoter sequence consistent with conserved binding sequences of group 1, 2 and 3 sigma factors [31]. All promoters identified in this study displayed sequence similarity to the consensus -10 hexamer (Pribnow box) of the prokaryotic RNA polymerase binding site, while there was sporadic presence of the -35 hexamer binding site (Table 3.1). These results suggest that the sxt promoters of D. circinale AWQC131C are activated by an RNA polymerase core enzyme in conjunction with a group 1 or group 2 sigma factor [31, 48]. For the -10 promoter sequences identified, a search was conducted for an extended -10 binding site and upstream (UP) element. The 5′ untranslated region (UTR) of each operon was also bioinformatically screened for the presence of consensus ribosomal binding site (RBS) sequences, although previously reported bioinformatics surveys of cyanobacterial genomes were not able to identify the consensus RBS sequence in all genes [49, 50]. Based on the 5′ RACE and bioinformatics data, the D. circinale AWQC131C sxt BGC includes of a total of five TSSs under standard culture conditions (Figure 2).
Table 1: Characteristics of promoter regions in the sxt biosynthetic gene cluster of D. circinale AWQC131C.
|
Promoter
|
Promoter sequence 5′ à 3′
|
PositionΔ
|
PsxtD
|
tgtcTTGTGG....(14bp).....GAgTATACTtgactagtA
|
-32
|
PsxtP
|
gtatCTATCA....(12bp).....GTgTATACTagtcaagtA
|
-34
|
PsxtPER1
|
ttccTTGCAA....(15bp).....AGtTACAATtacatgA
|
-91
|
PsxtPER2
|
tgagATGACA....(21bp).....CGaTATATTttgggtG
|
+94
|
Porf24
|
aaaaTTTCCT....(15bp).....TGcTATAATgaaatcT
|
-160
|
E. coli σ70 consensus
|
....TTGACA....(14bp).....TGnTATAAT......N
|
|
|
|
|
|
The -10 and -35 hexamers are capitalised and conserved nucleotides are in bold print. N indicates transcriptional start site (TSS). Δ Position of promoter relative to TSS.
Operon 1 (sxtDV*EABC) contains a short 5′ UTR of -32 bp upstream of the translation start site and a promoter (PsxtD) with high sequence similarity to the E. coli σ70 -10 and -35 hexamers. PsxtP initiates the transcription of operon 2, possesses a short 5′ UTR spanning 34 bp and contains both -10 and -35 regions. The transcript initiated by PsxtP also displayed a likely RBS (AAGA) 6 nucleotides upstream of the sxtP translation start site. A conserved -35 sequence was also identified 21 bp upstream of the extended -10 sequence, resulting in an unusually long distance between the two hexamers. Porf24 has a perfectly conserved -10 consensus sequence, including the extended -10 TGn motif (Table 1). The 5′ UTR for orf24 is 160 bp in length.
Unusually, transcription of the putative transporter, sxtPER, was initiated from two promoters, PsxtPER1 and PsxtPER2. PsxtPER1 is located 91 bp upstream of the annotated TSS of sxtPER (Figure 2) and contains a highly conserved -10 and -35 RNA polymerase binding site. PsxtPER2 is located 94 bp downstream of the translational start site and contains a highly conserved -10 sequence, including the single nucleotide seen in extended -10 promoters as well as a RBS (AAAGAAG). While uncommon, the use of a second TSS to produce two protein isoforms has previously been reported
Promoter activity in E. coli
The five promoters identified in the D. circinale sxt cluster using 5′ RACE, PsxtP, PsxtD, PsxtPER1, PsxtPER2 and Porf24, were amplified by PCR and cloned into the E. coli expression vector, pET28b (Novagen), directly in front of a luciferase reporter (lux) operon (Figure 3). Expression of luciferase from each of these promoters was measured and compared with negative controls; pET28-lux harbouring a non-promoter region from within the sxtO gene and the pET28-lux plasmid with no added promoter. Unpaired t-tests showed that all promoters exhibited significant levels of expression (Table S2) when compared to the pET28-lux negative control. Under the described culture conditions, the heterologous PsxtD, PsxtP, and PsxtPER1 promoters mediated the highest levels of luciferase expression in E. coli (Table S3). There was a statistically significant difference (p<0.0001) between the highest performing promoter PsxtD and all the other promoters, as well as the controls (sxtO and pET28-lux) (Table S4).
When luciferase expression from each promoter was compared to expression from the non-promoter sxtO fragment control, the strongest promoters (PsxtD, PsxtP, and PsxtPER1) resulted in a 1,000 to 10,000-fold increase in expression (Figure 3B).
The expression of PsxtP is an interesting example of the promoter elements required for heterologous expression of cyanobacterial promoters in E. coli. PsxtP does not seem to have a discernible -35 binding region yet does have a RBS and promoted high expression levels in E. coli. Previous studies have shown that while the distance between the -10 and -35 sequences can effect transcription in cyanobacteria, the -35 hexamer is not always required [59-61]. Thus, the competing preferences between the TSS sequence and position, taken together with other elements of the promoter such as -10, and -35 sequences, transcription factors, the sequence length between the -10 and -35 regions, and the RBS, highlights the complexity of transcriptional regulation and shows the importance of experimental validation of promoter activation data to further improve bioinformatics databases.
The promoter responsible for the transcription of orf24 and the second promoter of sxtPER, PsxtPER2 were weaker than the other promoters, but still significantly stronger than the controls (Figure 3A).The incorporation of both promoters into the lux expression constructs resulted in a 12-27-fold increase in luciferase expression over sxtO-lux (Figure 3B), and 810- 1770-fold increase in luciferase expression over the pET28-lux control. These results indicate that the promoters are active, albeit weaker than the other three promoters.
The activity of sxt promoters in Synechocystis sp. PCC 6803
Four sxt promoters were active in Synechocystis sp. PCC 6803 (Figure 4). Unpaired t-tests showed that expression of luciferase from the PsxtP, PsxtPER1, and Porf24 were significantly different to expression in the control strain (Table S6), while expression from PsxtD was not statistically different to the control strain (P<0.05; Table S7).
The sxtD promoter regulates the transcription of operon 1 of the D. circinale sxt cluster, which carries the core biosynthetic genes, including the polyketide synthase-like enzyme, sxtA. Strains harbouring PsxtD had very low luciferase expression levels that were only 1.3-fold higher than expression levels in the promoter-less control strain. The lack of statistically significant expression from PsxtD indicates the promoter as the only candidate for exchange for heterologous expression of PSTs in Synechocystis sp. PCC 6803.
PsxtP and Porf24 mediated consistent levels of luciferase expression per OD730 throughout the experiment (Figure 4B) PsxtPER1 mediated expression levels that were initially up to three-fold higher than PsxtP, however, the rate of expression decreased over the course of growth. This indicates the majority of toxins could be exported from the cell early in culture and retained in the cell as the culture progresses. This would allow future research to optimise PST extraction at different culture stages, either from the cell free component or the cell mass. Alternatively, since PsxtPER1 is active at the early growth stages within the heterologous host, it could be a target for repression to limit toxin export and therefore retaining toxin within the cell. This will increase the efficiency of toxin isolation from the cell biomass. [44]
It is known that gene expression levels will have significant impact on the amount of PST molecule produced. Here, the promoters that regulate PST biosynthesis mediated lowered expression of luciferase in Synechocystis sp. PCC 6803 compared to E. coli. The significant decrease in luciferase expression by the cyanobacterial promoters in Synechocystis sp. PCC 6803 has previously been observed in studies of the zinc inducible promoter, Psmt, from Synechococcus sp. PCC7002. Psmt mediated higher levels of protein synthesis and therefore higher levels of ethylene production in E. coli, compared to Synechocystis sp. PCC 6803, which only produced residual levels [62]. Conversely, high expression levels impacted lynbyatoxin biosynthesis in E. coli. Heterologous expression of lyngbyatoxin (ltxA-D) in E. coli was only successful when the strong T7 phage promoter was replaced with the weaker PtetO promoter [27]. PtetO has since been exploited for the heterologous expression of multiple cyanobacterial BGCs in E. coli [21, 22, 25]. Subsequent heterologous expression of lyngbyatoxin in Anabaena using non-native promoters was more successful than the E. coli system while expression from the native promoter did not occur in either case [23]. Together, these results suggest that native promoters are recognised differently in heterologous hosts and that while successful transcription of cyanobacterial BGCs in heterologous hosts is important, other factors play a role in the efficiency of the host production of secondary metabolites. This study has identified each of the five native sxt promoters and established through the use of the lux reporter, which of those were not recognised in both E. coli and Synechocystis.