The σ54 system directly regulates bacterial natural product genes

Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutics and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery controlling their transcription. Recent findings suggest that the σ54 system, which includes σ54-loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explored this idea by analyzing a selected group of putative σ54 promoters identified in Myxococcus xanthus natural product gene clusters. We show that mutations in putative σ54-RNA polymerase binding regions and in putative Nla28 EBP binding sites dramatically reduce in vivo promoter activities in growing and developing cells. We also show in vivo promoter activities are reduced in a nla28 mutant, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the Nla28 binding sites are mutated. Together, our results indicate that M. xanthus uses σ54 promoters for transcription of at least some of its natural product genes. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product loci are located within genes and not in intergenic sequences.


M. xanthus growth and development. M. xanthus strains were grown by inoculating cells into flasks
containing CTTYE broth and incubating the cultures at 32 °C with vigorous swirling. Development was induced as previously described 25 . Briefly, M. xanthus cells were grown in CTTYE broth until the cultures reached a density of approximately 5 × 10 8 cells/ml, the cells were pelleted, the supernatant was removed, and the cells were resuspended in MC7 buffer to a density of 5 × 10 9 cells/ml. Forty μl aliquots of the cell suspensions were placed into polystyrene plate wells containing 100 μl of MC7 buffer and the polystyrene plates were transferred to a 32 °C incubator for 24 h.
Standard DNA procedures. Chromosomal DNA from wild-type M. xanthus strain DK1622 46 was extracted using ZYMO Research gDNA extraction kit. Oligonucleotides used in PCR reactions were synthesized by Integrated DNA Technologies (IDT) and are listed in Table S2. Plasmid DNA was extracted using the Promega Nucleic acid purification kit. Amplified and digested DNA fragments were purified using the Gel Extraction Minipreps kit of Bio Basic. For all kits, the manufacturer's protocols were used. The compositions of all plasmids and promoter fragments were confirmed by DNA sequencing (Genewiz). www.nature.com/scientificreports/ Site-directed mutations. Site-directed mutations in putative σ 54 promoter elements were generated using the Quick Lightning Mutagenesis Kit from Agilent Technologies and the manufacturer's protocol. Briefly, promoter fragments containing the putative σ 54 -RNA polymerase binding site in the − 12 and − 24-bp regions and the upstream Nla28 tandem repeat binding site were cloned into pCR 2.1 TOPO vector (Invitrogen). Mutations in the − 12-bp region, the − 24-bp region, the spacer between the − 12 and − 24-bp regions were generated using primers carrying the appropriate nucleotide changes (Table S2), plasmids containing the promoter fragments and PfuUltra DNA polymerase. Parental plasmid DNA was removed by digesting with DpnI and transformed into E. coli for conversion into duplex form. Plasmid-borne promoter mutations were verified by DNA sequence analysis. Promoter fragments carrying Nla28 binding site mutations (P EM1286 , P EM1579 and P EM3778 ) were synthesized by IDT; the first putative Nla28 half binding site in each promoter was changed to AAA AAA AA. The mutant promoter fragments were then subcloned into the promoterless lacZ expression vector pREG1727 47 , introduced into M. xanthus strains and analyzed as described below.
In vivo analysis of wild-type and mutant promoters. Wild-type and mutant MXAN1286, MXAN1579, MXAN1603 and MXAN3778 promoter fragments were cloned into the promoterless lacZ expression vector pREG1727 to create lacZ transcriptional fusions 47 . The plasmids were introduced into strain DK1622 or a derivative of strain DK1622 carrying an insertion in the nla28 gene, and cells carrying a plasmid integrated at the Mx8 phage attachment site in the chromosome were identified via PCR. The in vivo activities of wild-type and mutant promoters were determined by measuring the specific activities of β-galactosidase in cells developing in submerged cultures for 1, 2, 6, 12 or 24 h, or growing in CTTYE broth for various amounts of time 48,49 . Mean wild-type and mutant promoter activities were compared using a two-way analysis of variance (ANOVA) and Tukey's multiple comparisons post hoc tests, considering two independent variables in each group. The significance level was set at p < 0.05 or lower. GraphPad Prism software v8.4 (GraphPad Software, La Jolla, CA, USA) was used for all analyses.
Expression and purification of Nla28-DBD. A fragment of the nla28 gene corresponding to the Nla28 DNA binding domain (Nla28-DBD) 30 was PCR amplified using gene-specific primers (Table S2), and then cloned into the pMAL-c5x vector. The resulting plasmid, which creates an N-terminal Maltose Binding Protein (MBP) fusion to Nla28-DBD, was introduced into E. coli strain BL21 (DE3) using chemical transformation. Cells containing the Nla28-DBD expression plasmids were grown in rich LB broth to a density of 2 × 10 8 cells/ ml. Protein expression was induced by the addition of 0.3 mM IPTG to the culture and the subsequent incubation of the culture for 12 h at 15 °C. Cells were pelleted via centrifugation and resuspended in 25 ml column buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 5 U/ml DNase I) per liter of culture. The resuspended cells were lysed by a combination of freeze-thawing and sonication, and pelleted by centrifugation. The crude extract (supernatant) containing Nla28-DBD was diluted by adding 125 ml of cold column buffer to every 25 ml aliquot of crude extract. For purification of Nla28-DBD from the diluted crude extract, 15 ml of amylose resin was placed in a 2.5 × 10 cm column and the amylose resin was washed with 250 ml cold column buffer. Diluted crude extract containing Nla28-DBD was loaded onto amylose columns at a flow rate of 5 ml/min and washed with 600 ml cold column buffer at a flow rate of 10 ml/min. Nla28-DBD was eluted using 100 ml cold column buffer containing 10 mM maltose; the flow rate was 5 ml/min and 20 fractions containing 5 ml were collected. The presence of eluted Nla28-DBD was detected by UV absorbance at 280 nm. Nla28-DBD-containing fractions were pooled and incubated with 1 mg of Factor Xa at 4 °C overnight to cleave the MBP tag. Nla28-DBD was separated from MBP and concentrated to about 1 mg/ml using Amicon Ultra centrifugal filter units (EMD Millipore). SDS-PAGE and Bradford assays were used to determinate the purity and concentration of Nla28-DBD.

Results
Identifying putative σ 54 promoter elements in M. xanthus natural product gene clusters. In a previous study 38 , the algorithm developed by Studholme et al. 50 was used to examine whether the σ 54 system might be a common regulator of bacterial natural product genes. Namely, 180 annotated PK and NRP gene clusters from 58 bacterial species were analyzed for sequences that closely match the σ 54 promoter consensus in the − 12-bp region and in the − 24-bp region (i.e., the regions of σ 54 -RNA polymerase binding). The results, which uncovered 124 clusters with at least one σ 54 promoter based on consensus matching, supported the idea that the σ 54 system might be a general regulator of bacterial natural product genes. The goal of the work presented here was to examine whether a major producer of bacterial natural products (M. xanthus) uses the σ 54 system for transcription of PK and NRP gene clusters, as predicted in the bioinformatics www.nature.com/scientificreports/ analysis of Stevens et al. 38 . We focused on the putative σ 54 promoters upstream of MXAN1286, MXAN1579, MXAN1603 and MXAN3778, as these genes were linked to natural product biosynthetic genes/clusters. Namely, the MXAN1286 gene is embedded in a putative NRP gene cluster and flanked by genes that are likely to be involved in NRP biosynthesis. MXAN1579 and MXAN1603 are predicted to be part of another M. xanthus NRP gene cluster. MXAN1603 is a putative non-ribosomal peptide synthetase gene. MXAN1579 is embedded in the cluster and flanked by genes such as MXAN1603 that are likely to be involved in NRP biosynthesis. MXAN3778 is adjacent to a putative hybrid non-ribosomal peptide synthetase/type I polyketide synthase gene and possibly part of the same operon 38 . The MXAN1286, MXAN1579, MXAN1603 and MXAN3778 genes were also identified as potential targets of the EBP Nla28 30,39 ; 8-bp repeat sequences, which are close matches to the consensus Nla28 half binding site, were identified upstream of the putative − 12 and − 24-bp regions (Fig. 1). It is notable that six of the eight putative Nla28 half binding sites and three of the four putative σ 54 -RNA polymerase binding sites are located within protein coding sequences. Indeed, residence in an intragenic region is common among the putative PK and NRP σ 54 promoters identified in the M. xanthus genome (Fig. 2, Table S3 and Figure S1), and among the σ 54 promoters known to be regulated by the EBP Nla28 30,39 . It is also noteworthy that many of the putative σ 54 promoter elements are located within operons; they might serve as internal promoters (Fig. 2, Table S3 and Figure S1).

Mutations in the putative − 12-bp region, − 24-bp region or spacer region impair the in vivo
activities of natural product promoters. σ 54 promoters typically have a GC dinucleotide in the − 12-bp region and a GG dinucleotide in the − 24-bp region 1,2 . These dinucleotides and the 4-bp spacer between the − 12 and − 24-bp regions are often referred to as the hallmarks of σ 54 promoters. Indeed, the putative σ 54 promoters in the MXAN1286, MXAN1579 and MXAN1603 natural product loci appear to have these hallmarks (Fig. 1). As for the σ 54 promoter identified in the MXAN3778 locus, one hallmark variation is apparent. Namely, the − 24-bp region has GA instead of a GG dinucleotide. Despite this variation in − 24-bp region dinucleotide, MXAN3778 was classified as a potential σ 54 promoter, as a 1-bp change in either the GC or GG dinucleotide has been identified in a number of characterized σ 54 promoters, including the σ 54 promoters in the M. xanthus asgE, spi and nla6 loci 30,[51][52][53] .
To confirm that the natural product loci have bona fide σ 54 promoter elements, we analyzed the putative σ 54 promoter hallmarks via mutational analysis. In particular, a 446-bp DNA fragment of the MXAN1286 promoter www.nature.com/scientificreports/ region, a 402-bp fragment of the MXAN1579 promoter region, a 518 bp fragment of the MXAN1603 promoter region and a 600-bp fragment of the MXAN3778 promoter region were used to generate mutations in the putative − 12-bp, − 24-bp and spacer regions. We should note that the MXAN1286 promoter fragment contains both of the putative σ 54 promoters located in the 5′ end of the MXAN1286 gene and the MXAN1579 promoter fragment contains the two putative intergenic σ 54 promoters that are oriented in the appropriate direction with respect to MXAN1579 transcription (Table S3 and Figure S1). However, we focused the mutational analysis on the putative σ 54 promoter that is the farthest downstream in the MXAN1286 coding sequence (+ 111 bp; see Table S3) and the putative σ 54 promoter that is farthest upstream of the predicted transcriptional start site of MXAN1579 gene (− 104 bp; see Table S3) after considering a number of factors, including the presence of σ 54 promoter hallmarks, the overall match to the σ 54 promoter consensus sequence [TGG CAC G-4 N-TTGC(T/A)] and the distance from putative Nla28 binding sites 1,2 . The mutations that we generated are the following: the GC dinucleotide in the − 12-bp region was replaced with a TT, the GG (GA in MXAN3778) dinucleotide in the − 24-bp region was replaced with a TT, or 1 bp in the spacer between the − 12 and − 24-bp regions was deleted. Subsequently, wildtype and mutant promoter fragments were fused to the promoterless lacZ gene in plasmid pREG1727 47 and the lacZ transcriptional fusion plasmids were introduced into wild-type M. xanthus strain DK1622 (the plasmids integrated at the Mx8 phage attachment site in the chromosome). Wild-type and mutant promoter activities during growth in CTTYE broth and fruiting body development in MC7 starvation buffer were inferred from the levels of lacZ expression. As shown in Fig. 3A-C, the in vivo activities of the MXAN1286, MXAN1579 and MXAN3778 promoters increased about 4.6-fold, 1.8-fold and twofold, respectively, during growth in CTTYE broth. It is notable that peak levels of promoter activity occurred at the highest cell densities, which correspond to the transition into stationary phase and presumably nutrient depletion. This of course agrees with the data shown in Fig. 3D-F, which revealed a 1.8-to 2.5-fold increase in the vivo activities of the three promoters during development in MC7 starvation buffer. Mutations in the − 12-bp region, the − 24-bp region and spacer dramatically reduced (about 3.2-to 11.1-fold) the activities of these promoters at all cell densities during growth and time points in development. Thus, mutations in the putative sites of σ 54-RNA polymerase binding substantially impacted the activities of the MXAN1286, MXAN1579 and MXAN3778 promoters in growing and developing cells, supporting the prediction that the three natural product loci use σ 54 promoter elements for transcription.
In contrast to the other promoters, the MXAN1603 promoter only showed a slight increase (about 1.3-fold) in activity during growth in CTTYE ( Figure S2A) and during development in MC7 starvation buffer ( Figure S2B). Furthermore, mutations in the − 12-bp region, the − 24-bp region and the spacer caused a modest, but statistically significant decrease (P < 0.001) in promoter activity at all cell densities during growth and after 1 h of www.nature.com/scientificreports/ development ( Figure S2). Thus, mutations in the putative σ 54 -RNA polymerase binding site in the MXAN1603 promoter region only had a modest impact on growth-related and developmental activities. Our interpretation of this result is that the fragment of the MXAN1603 promoter region contains an unidentified promoter element that is responsible for the majority of the observed development and growth-related activities and that the putative σ 54 -type promoter element only makes a minor contribution to the observed activities.
The in vivo activities of natural product promoters are impacted by inactivation of the nla28 gene. EBPs are essential for transcription at σ 54 promoters, as EBP-mediated ATP hydrolysis opens the σ 54 -RNA polymerase promoter complex so that transcription can initiate [3][4][5] . Since the σ 54 promoters in the MXAN1286, MXAN1579, and MXAN3778 loci were identified as potential targets of Nla28, we determined whether the activities of wild-type promoter fragments are reduced as predicted in a mutant containing an inactivated nla28 gene 25 (Note that the MXAN1603 σ 54 promoter was not analyzed further because it is unlikely to be the primary promoter used during growth or development). MXAN1286, MXAN1579, and MXAN3778 promoter activities in wild-type and nla28 mutant cells grown in CTTYE broth are shown in Fig. 4A-C. As predicted, inactivation of nla28 abolished the growth phase regulation of all three promoters and caused about 3.1to fourfold reduction in peak promoter activities at the highest cell densities. Inactivation of nla28 also abolished the developmental activities of the promoters, as the promoters did not show the typical increases in activities when nla28 cells were placed in MC7 starvation buffer (Fig. 4D-F). Indeed, the peak developmental promoter activities in nla28 mutant cells were reduced about 3.4-to 5.0-fold relative to the corresponding peak activities in wild-type cells. These findings indicate that Nla28 is crucial for the observed growth-related and developmental activities of the MXAN1286, MXAN1579, and MXAN3778 natural product promoters.

Mutations in putative Nla28 half sites impact the in vivo activities of natural product promoters.
As noted above, we identified 8-bp repeat sequences, which are close matches to the consensus Nla28 half binding site [CT(C/G)CG(C/G)AG], in the σ 54 promoters under study here (see Fig. 1). To examine whether the σ 54 promoters are directly regulated by Nla28 and to further confirm that the promoters are members of the σ 54 family, mutations were generated in the putative Nla28 binding sites in the MXAN1286, MXAN1579, and MXAN3778 promoter fragments noted above. Namely, the distal (relative to the − 12 and − 24-bp regions) Nla28 half binding site in each promoter fragment was converted to all A nucleotides. Wild-type and mutant promoters were introduced into wild-type strain DK1622 and promoter activities during growth in CTTYE broth and development in in MC7 starvation buffer were determined (Fig. 4). The data revealed that Nla28 binding site mutations abolish the growth phase regulation of all three promoters (Fig. 4A-C). Indeed, the peak mutant promoter activities, which were observed at the highest cell density, were reduced about 2.6-to fivefold compared to www.nature.com/scientificreports/ that of the corresponding wild-type promoter. Similarly, wild-type MXAN1286, MXAN1579, and MXAN3778 promoters showed increased activities during development and the Nla28 binding site mutations abolished this developmental regulation (Fig. 4D-F). Furthermore, the peak developmental activities of the mutant promoters were reduced from about 2.8-to 4.8-fold. These findings are consistent with the idea that Nla28 directly regulates the MXAN1286, MXAN1579, and MXAN3778 σ 54 promoters, that the 8-bp repeats that we identified are Nla28 binding sites and that Nla28 is crucial for growth-related and developmental promoter activities.
In vitro analyses indicate that purified Nla28-DBD binds to natural product promoter fragments carrying a wild-type Nla28 binding site, but not to fragments carrying a mutated Nla28 binding site. Electrophoretic mobility shift assays (EMSAs) were used to confirm that the MXAN1286, MXAN1579, and MXAN3778 natural product promoters are targets of the Nla28 EBP. In particular, we used EMSAs to determine whether the purified DNA binding domain of Nla28 (Nla28-DBD) is capable of binding a fragment of the MXAN1286 promoter, MXAN1579 promoter, and MXAN3778 promoter. Each promoter fragment, which corresponded to DNA upstream of − 12 and − 24-bp regions, contained a putative binding site for a Nla28 dimer. As shown in Fig. 5, Nla28-DBD is capable of binding to a MXAN1286, MXAN1579 and MXAN3778 promoter fragment that has a wild-type Nla28 binding site. However, when the distal Nla28 half binding site in each promoter fragment was converted to all A nucleotides, no Nla28-DBD binding was detected (Fig. 5). These findings provide further support that the Nla28 EBP directly regulates the σ 54 promoter elements of the MXAN1286, MXAN1579 and MXAN3778 natural product loci and that the tandem repeats that we identified in the σ 54 promoter elements are Nla28 binding sites.

Discussion
For decades, bacterial-derived PK and NRP natural products have been a crucial source of therapeutic agents such as antibiotics and yet little information about the regulation of these genes has been uncovered. In a notable study in 2012, Volz et al. showed that two M. xanthus EBPs (HsfA and MXAN4899) are capable of binding to fragments of natural product gene promoters 40 . With this information and the preliminary data from Nla28 studies in mind, Stevens et al. asked if the σ 54 system might be a common regulator natural product genes 38 . Namely, a bioinformatics analysis was used to search for putative σ 54 promoters in the sequences of 180 PK or NRP gene clusters from 58 bacterial species. The results, which revealed that about 70% of natural product www.nature.com/scientificreports/ gene clusters have at least one putative σ 54 promoter, suggested that the σ 54 system might indeed be a common regulator of natural product genes.
One of the goals of this study was to analyze the bioinformatics data experimentally and the putative natural product promoter targets of Nla28 seemed particularly well suited for such a study, given our knowledge of Nla28-mediated regulation. Furthermore, M. xanthus is an excellent system to study natural product gene regulation, as this bacterium is a major producer of PKs and NRPs and over 80 putative σ 54 promoters were identified in the PK and NRP gene clusters of strain DK1622 38 (Table S3 and Figure S1).
Our in vivo and in vitro data indicate that three of the M. xanthus natural product promoter regions characterized here (MXAN1579, MXAN3778 and MXAN1286) contain bona fide σ 54 promoter elements and that the σ 54 promoters are targets of the EBP Nla28. Our results also indicate that the σ 54 promoters are crucial for both growth-related and developmental activities. We propose that the fourth natural product promoter region that we characterized, MXAN1603, also contains a σ 54 promoter. However, it seems likely that the MXAN1603 promoter region contains an unidentified promoter element that is responsible for the majority of the observed developmental and growth-related activities and the σ 54 -type promoter only makes a minor contribution to these activities.
Previous work indicated that the Nla28 EBP is a response regulator that forms a two component signal transduction system with the membrane-bound sensor histidine kinase Nla28S 54,55 . Nla28 begins modulating gene expression in the early stages of starvation-induced fruiting body development 25,30,39 , which led to the suggestion that the Nla28/Nla28S signal transduction system might be a general regulator of starvation-induced or stress-responsive genes 55 . The findings presented here are consistent with this idea. In particular, the MXAN1286, MXAN1579, and MXAN3778 σ 54 promoters are induced in the early-middle stages of fruiting body development and the developmental activities of these promoters are dependent on Nla28 (Fig. 4D-F). The activities of the three promoters also increase during growth, with peaks corresponding to the transition into stationary phase and presumably nutrient depletion. As shown in Fig. 4A-C, this growth-phase regulation is abolished when Nla28 is absent, further linking the Nla28/Nla28S signal transduction system to the starvation response.
The above results point to nutrient depletion/starvation as the potential trigger for Nla28/Nla28S-mediated activation of the MXAN1286, MXAN1579 and MXAN3778 promoters. We suggest that the stringent response and accumulation of (p)ppGpp might also be a key inducing factor, as accumulation of this starvation signal is known to initiate M. xanthus fruiting body development 56,57 . Furthermore, it is reasonable to speculate that (p) ppGpp accumulates when M. xanthus transitions from exponential growth to stationary phase, since this seems to be the case in other bacterial species 58 . In addition, bacterial natural product gene expression was previously linked to (p)ppGpp accumulation 59 and stringent response-associated genes such as relA and spoT have been linked to synthesis of bacterial natural products 41,42,60 .
It is interesting that three of the four natural product σ 54 promoters that we characterized are located within the coding sequences of genes (intragenic) and not in intergenic regions (Fig. 1). In the MXAN1286 and MXAN1603 loci, the σ 54 promoters are located in the coding sequence of the first gene of an operon and in the 5′ end of a single gene, respectively. In the other case (MXAN3778), the σ 54 promoter is located in the coding sequence of an upstream gene (Fig. 1). With the exception of MXAN1603, the Nla28 binding sites of the natural product promoters are also intragenic, located in the coding sequence of an upstream gene (Fig. 1). These findings are counter to the commonly held view that bacterial promoter elements are typically located in intergenic regions [61][62][63] , but are supported by additional pieces of bioinformatic and experimental data. First, the vast majority of the putative natural product σ 54 promoters shown in Figure S1 and listed in Table S3 are intragenic. Secondly, the majority of the characterized σ 54 promoter targets of the M. xanthus EBP Nla6 are intragenic, located in the coding sequence of an upstream gene 53 . Thirdly, the vast majority of the developmental σ 54 promoter targets of Nla28, which we recently characterized, are located in the coding sequence of an upstream gene or appear to be internal promoters in operons 30,39 . Lastly, genomic-wide studies in Escherichia coli and Salmonella enterica www.nature.com/scientificreports/ serovar Typhimurium uncovered an abundance of potential intragenic σ 54 promoters and a number of these promoters were subsequently confirmed experimentally 64,65 . Together, these results suggest that σ 54 promoter elements might indeed be commonly located in intragenic regions. Many of the natural product σ 54 promoters shown in Figure S1 and listed in Table S3 appear to be internal promoters; promoters located within a gene in an operon instead of upstream of the first gene in the operon. There are examples of internal promoters oriented in the antisense direction relative to the genes in an operon [65][66][67][68] ; however, this does not seem to be the case for the above internal σ 54 promoters. Instead, each promoter is predicted to yield mRNA corresponding to a subset of an operon's genes. As noted in a recent review, one potential advantage of using internal promoters would be the ability to express only the genes needed in a particular environment 69 .
A second and relatively large group of putative natural product σ 54 promoters is apparent from scanning Figure S1 and Table S3. Namely, many of the putative σ 54 promoters are located in the coding sequence of a gene that is upstream of the single gene or operon they are predicted to regulate, even though the single gene or operon has an upstream and adjacent intergenic region that is long enough to accommodate a σ 54 promoter (σ 54 -RNA polymerase binding site). The question that arises is, why would a σ 54 promoter be located so far upstream of the single gene or operon whose transcription it aims to regulate? One possibility is that the natural product σ 54 promoter is positioned at a distant location to produce a relatively long 5′ untranslated region (UTR) in the mRNA and that the 5′ UTR provides an additional layer of regulation for natural product gene expression. We should note that a substantial number of the internal σ 54 promoters are predicted to yield relatively long 5′ UTRs as well. Why would many natural product genes need multiple levels of regulation? Natural product synthesis is often energy expensive and many natural products are toxic at high levels; hence, tight and signal-responsive regulation of natural product gene expression might be important in many cases.
Of course, addressing questions about the role and mechanism of σ 54 -mediated regulation of natural product genes and other types of bacterial genes will require researchers to go beyond predicting where σ 54 promoters are located. Indeed, we argue that the work of confirming putative σ 54 promoters and analyzing the mRNAs under σ 54 control is crucial, as the results will help address a number of long-held assumptions about bacterial promoters and bacterial gene regulation in general. www.nature.com/scientificreports/