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Nucleic Acids Res. Mar 2011; 39(4): 1294–1309.
Published online Oct 21, 2010. doi:  10.1093/nar/gkq986
PMCID: PMC3045617

Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae

Abstract

Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ54-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ70-promoters overlap the σ54-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ70-promoter. Thus, transcription of glmY and glmZ is controlled by σ54 and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ70-dependent transcription. In addition, we show that activity of the σ54-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

INTRODUCTION

Post-transcriptional gene regulation involving regulatory RNAs has emerged as a widespread principle occurring in all three domains of life. In bacteria, one important mode of riboregulation involves trans-encoded small RNAs (sRNAs), which appear to be involved in regulation of almost every important physiological function (1–5). The majority of sRNAs acts by base-pairing with target mRNAs usually in the vicinity of the ribosome binding site (4,6). Most often, this interaction represses translation and/or stimulates mRNA degradation, although a few cases are known where sRNA–mRNA interaction increases gene expression (7). One example is provided by the sRNA GlmZ in Escherichia coli. Binding of GlmZ to its target mRNA glmS destroys an inhibitory stem loop that sequesters the Shine–Dalgarno sequence of glmS. GlmZ is also an unusual case, because it works in concert with a second homologous sRNA, GlmY (1,8,9). However, while other homologous sRNAs regulate their targets redundantly or additively (6), GlmY/GlmZ act hierarchically to activate expression of the glmS gene, which encodes glucosamine-6-phosphate (GlcN6P) synthase GlmS. GlmS catalyzes formation of GlcN6P, which initiates the pathway that generates precursors of cell wall synthesis. Of both sRNAs, only GlmZ is able to base-pair with glmS mRNA. However, ongoing processing removes most of the base-pairing residues and thereby inactivates GlmZ. Upon depletion of GlcN6P, the second sRNA GlmY accumulates and counteracts processing of GlmZ. This activates synthesis of GlmS, which re-synthesizes GlcN6P. Hence, both sRNAs work in a cascade to mediate feedback control of GlmS (8–11).

To understand the impact of sRNAs on bacterial physiology, it is important to identify the signals and mechanisms that control expression of a particular sRNA. sRNA transcription is often controlled by transcriptional regulatory proteins similar to that of protein-coding genes [for an overview, see (4)]. Some sRNA genes are controlled by two-component systems (TCS) and/or alternative sigma factors, which are the key devices for perception of environmental signals and their conversion into gene expression changes (3,12,13). Evidence is accumulating that sRNAs are also members of the modulon controlled by σ54 involving genes important for nitrogen and carbon-utilization, uptake of metal ions, stress responses and other apparently unrelated functions. Transcription of sRNA genes from σ54-dependent promoters has been demonstrated in Pseudomonas aeruginosa and Vibrio harveyi (14,15). It is estimated that there are ~70 σ54-dependent promoters in E. coli (16,17). σ54 is unique among σ factors since it is not related to other σ factors and recognizes a different sequence composed of −24/−12 motifs (18). The σ54-RNAP holo-enzyme is unable to catalyze formation of the open promoter complex. This reaction requires interaction with an activator protein that usually binds to activating binding sites (ABS) located far upstream of the promoter.

Despite the parallels in the transcriptional control of protein-coding and sRNA genes, there appears to be at least one difference: many protein-coding genes are transcribed from multiple promoters that can be activated by different σ factors and use different transcriptional start sites (19,20). While differing 5′ sequences of mRNAs are without consequences for the nature of the encoded protein, they have functional consequences for sRNAs as shown for the IstR-1 and IstR-2 sRNAs, which are transcribed from consecutive promoters (21). To allow transcription of identical sRNA species from alternative promoters, these promoters must overlap to allow transcription initiation at the same nucleotide. Such an unorthodox arrangement has recently been identified for the E. coli glmY gene, where overlapping σ70- and σ54-promoters start transcription at the same site (22). The σ54-promoter requires activation by the TCS GlrR/GlrK (alternative names: QseF/QseE or YfhA/YfhK), which is encoded downstream of glmY and transcribed independently (22). The activator protein GlrR consists of an N-terminal response regulatory domain, a central σ54-interaction module and a C-terminal helix-turn-helix DNA-binding motif. GlrR binds three TGTCN10GACA motifs located more than 100 bp upstream of glmY and thereby activates the σ54-promoter, while activity of the σ70-promoter is unaffected. Both promoters are moderately active during the exponential growth phase. Their activities interfere since binding of σ54 represses activity of the overlapping σ70-promoter to some extent (22).

In this work, we analyzed the transcriptional regulation of glmY and glmZ. The TCS GlrR/GlrK as well as glmY and glmZ are conserved in Enterobacteriaceae. In silico analyses of the glmY and glmZ promoter sequences identified three groups within the enterobacterial species. Yersinia pseudotuberculosis, Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 and E. coli K12 are representatives of each group and were analyzed. We show that both, glmY and glmZ, are controlled by GlrR and σ54 in Y. pseudotuberculosis, S. typhimurium and presumably in many other species. In these species, both sRNAs are expressed from σ54-dependent promoters that require activation by GlrR. However, overlapping σ70-promoters additionally contribute to expression in S. typhimurium. In E. coli, glmY is transcribed from overlapping σ54- and σ70-promoters, while glmZ is expressed from a single σ70-promoter that is constitutively active. In conclusion, glmY and glmZ appear to be strictly σ54-dependent genes in one subgroup of Enterobacteriaceae, while σ54-dependency is lost in favor of unregulated σ70-promoters in a second subgroup. Furthermore, we show for E. coli glmY that activity of the σ54-promoter requires the integration host factor IHF, which presumably binds to two conserved sites flanking the proximal GlrR binding site. Finally, our data indicate that phosphorylation of GlrR increases its affinity for its target sites on the DNA.

MATERIALS AND METHODS

Growth conditions and strains

LB was used as standard medium for cultivation of bacteria. Escherichia coli and S. typhimurium LT2 were grown routinely under agitation (200 r.p.m.) at 37°C and Y. pseudotuberculosis YPIII was cultivated at 25°C. When necessary, antibiotics were added to the medium (ampicillin 100 µg/ml, kanamycin 30 µg/ml, chloramphenicol 15 µg/ml, spectinomycin 50 µg/ml). For induction of the PAra promoter on pBAD plasmids, 0.2% l-arabinose was added. The E. coli strains are listed in Table 1, including a description of their relevant genotypes. The ΔihfA::kan and ΔihfB::kan alleles were transduced to strains Z190 and Z197 using bacteriophage T4GT7 (23). Most of the lacZ reporter fusions used in this study were first established on plasmids and subsequently integrated into the λattB-site on the E. coli chromosome by site-specific recombination yielding the strains as indicated in Table 1. Recombination was achieved using helper plasmid pLDR8 as described (24). Briefly, origin-less DNA-fragments encompassing the respective lacZ fusion, the aadA spectinomycin resistance gene and the λattP-site were isolated by BamHI digestion and agarose gel-electrophoresis. The DNA-fragments were self-ligated and subsequently introduced into target strains carrying the temperature-sensitive λ-integrase expression plasmid pLDR8. Recombinants were obtained by selection on spectinomycin-plates at 42°C. Correct integration was verified by PCR using appropriate primers and loss of plasmid pLDR8 was confirmed by sensitivity to kanamycin.

Table 1.
E. coli strains used in this study

Construction and site-directed mutagenesis of plasmids

DNA cloning was carried out in E. coli strain DH5α following standard procedures. The plasmids and oligonucleotides used in this study are listed in Supplementary Tables S1 and S2, respectively (see ‘Supplementary data’). Plasmid constructions are also described under ‘Supplementary data’.

Analysis of glmY and glmZ transcription (β-galactosidase assays)

Overnight cultures of E. coli were inoculated into fresh LB medium to an OD600 of 0.1 and grown to an OD600 of 0.5–0.7. Subsequently, the cells were harvested and the β-galactosidase activities were determined as previously described (25). β-Galactosidase activities were determined from Y. pseudotuberculosis cells as described recently (26). The presented values are the average of at least three measurements using independent cultures.

Protein purification

C-terminally His-tagged E. coli and Y. pseudotuberculosis GlrR proteins were overproduced in E. coli DH5α carrying plasmid pBGG219 or pBGG397, respectively. Cells were grown in 1 l LB-ampicillin to an OD600 = 0.5–0.8. After addition of 1 mM IPTG for the induction of GlrR::His10 synthesis, growth was continued for one additional hour. Cells were harvested and washed in ZAP-buffer (50 mM Tris–HCl, 200 mM NaCl, pH 7.5). The crude lysate was prepared using a one shot cell disrupter at 2600 Ψ (Constant systems Ltd.) and subsequently cleared by low speed centrifugation followed by ultracentrifugation. The cleared lysates were loaded onto pre-equilibrated Ni-NTA Superflow columns (Qiagen) and proteins were eluted with a gradient of imidazol solved in ZAP buffer. Samples of the different purification steps and elution fractions were analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. The 250 mM imidazol fractions contained the pure GlrR-His10 proteins. These fractions were dialysed two times for 24 h against buffer (20 mM Tris–HCl pH 7.5, 100 mM KCl, 2 mM DTT). In the second dialysis step, the buffer additionally contained 25% (v/v) glycerol. The purified proteins were aliquoted and stored at −20°C until their use.

Electrophoretic mobility shift assay

Electrophoretic mobility shift assays (EMSAs) were carried out as described previously (22,27). The DNA fragments tested in the EMSAs were amplified by PCR using the same oligonucleotides that were used for construction of the corresponding glmY’- and glmZ’-lacZ gene fusions (Supplementary Table S1). The 200 and 400 bp lacZ promoter fragments, which were used as internal controls, were generated by PCR using primer pairs BG580/BG581 and BG578/BG579, respectively. DNA concentrations were determined with the NanoDrop Spectrometer ND-1000 (Peqlab). Binding assays were carried out in 10 µl volume containing binding buffer (20 mM Tris–HCl, pH 7.5, 100 mM KCl, 2 mM DTT, 10% glycerol), 30 ng of each DNA-fragment and the protein concentrations as indicated in the Figures. The reactions were incubated at 30°C for 20 min and subsequently 6 µl of the samples were separated at 4°C alongside with a DNA size marker on non-denaturing 8% acrylamide gels prepared in 0.5 × TBE. Gels were stained with ethidium bromide for visualization of the DNA. For testing the effect of acetyl phosphate, GlrR protein was incubated for 1 h at 37°C in binding buffer containing 50 mM acetyl phosphate and then used for the binding assays.

RESULTS

Conservation and gene synteny of glmY and glmZ in Enterobacteriaceae

In E. coli, transcription of glmY is controlled by overlapping σ70- and σ54-dependent promoters. Activity of the σ54-promoter is governed by the TCS GlrR/GlrK, which is encoded downstream of glmY (22). To investigate, whether this unusual promoter architecture is conserved in other bacteria and to increase our understanding of regulation of glmZ transcription, we compared the promoter sequences of glmY and glmZ from a comprehensive number of genomes. To retrieve these sequences, we used the sRNA sequences of Escherichia coli K12 (strain MG1655) as queries in NCBI Blast analyses. This search generated a list of species, all belonging to the Enterobacteriaceae family, which coincidently contained both sRNA genes. Inspection of gene synteny using the MicrobesOnline tool (28) and the KEGG database (29) revealed conserved localization of glmZ downstream of the divergently orientated hemCDXY operon encoding enzymes involved in tetrapyrrole synthesis, whereas the region upstream of glmZ is variable and may carry insertion elements (Figure 1A and Supplementary Figure S1). Gene glmY is always located upstream of the gene cluster glrK-yfhG-glrR (Figure 1A and Supplementary Figure S2). Collectively, these observations suggest that sRNA genes glmY and glmZ are elements of the core genome conserved in Enterobacteriaceae. The conserved co-localization of glmY with the genes encoding the sensor kinase GlrK and the response regulator GlrR suggests that regulation of glmY expression by this TCS might be likewise conserved.

Figure 1.
Organization of the glmY and glmZ genes in Enterobacteriaceae. (A) Diagram illustrating gene synteny of the glmY and glmZ regions in Enterobacteriaceae. The gene cluster glmY-glrK-yfhG-glrR-glnB is conserved in Enterobacteriacea, but in some species e.g. ...

Sequences for a σ54-promoter and for binding sites of the response regulator GlrR as well as of IHF are shared features of the glmY and glmZ promoter regions of many, but not all Enterobacteriaceae

We performed sequence alignments of the promoter regions of the glmY as well as glmZ genes retrieved from 39 genome sequences representing the most important genera of Enterobacteriaceae. The σ54-dependent promoter of E. coli glmY is conserved in all species (Supplementary Figure S3). The GlrR binding sites are likewise conserved although sequence deviations from the consensus TGTCN10GACA occur in a few cases, in particular in ABS 1 and 3. Two additional regions flanking ABS3 exhibit a higher degree of conservation and show similarity to binding sites of IHF, which are represented by the consensus WATCARXXXXTTR (30). The previously characterized −10 sequence (CATAAT) of the σ70-promoter, which overlaps with the −12 sequence of the σ54-promoter of glmY in E. coli, is conserved only in a subset of genera, i.e. in Escherichia (which includes Shigella strains), Klebsiella, Salmonella, Enterobacter, Citrobacter and Cronobacter. Putative −35 sequences are also detectable at the appropriate positions. In contrast, in other genera, such as Erwinia, Photorhabdus, Serratia and Yersinia, overlapping potential σ70-promoter sequences are not detectable (Supplementary Figure S3).

The analysis of the promoter of the second sRNA gene glmZ revealed two groups of sequences, which exhibit no similarity and could not be aligned with each other (Supplementary Figure S4). In the group comprising the majority of sequences, the glmZ promoter region is strongly reminiscent of the organization of the glmY promoter. Sequence motifs of a σ54-promoter, three GlrR binding sites and two IHF binding sites are detectable. The putative ABS1 and IHF-sites are less conserved in comparison to the glmY promoters (compare Supplementary Figures S3 and S4). In a subset of genera, i.e. Cronobacter, Citrobacter, Enterobacter and Salmonella, putative σ70-promoters overlapping with the σ54-promoters are also detectable upstream of glmZ (Supplementary Figure S4). Interestingly, these species also possess overlapping σ70- and σ54-promoter sequences upstream of glmY (Supplementary Figure S3). The second group comprised the genera Klebsiella and Escherichia. In these cases, sequence motifs for σ54-promoters and for GlrR- and IHF-binding sites are lacking. Instead of that, putative σ70-promoter sequences (ATGTTA-N15-tggCATAAT in Escherichia sp. and Shigella strains and ATGCAA-N15-tgcGATAAT in Klebsiella pneumoniae) are present at the appropriate positions.

From these analyses we hypothesized that enterobacterial species can be classified into three groups in respect to control of glmY and glmZ expression (Figure 1B): (i) Species of the genera Pantoea, Erwinia, Pectobacterium, Arsenophonus, Photorhabdus, Serratia, Proteus, Yersinia and Dickeya may transcribe both, glmY and glmZ, from σ54-dependent promoters, which might be controlled by GlrR/GlrK. (ii) This may also apply to species of the genera Cronobacter, Citrobacter, Enterobacter and Salmonella, but as a difference, additional overlapping σ70-promoters are present, which may start transcription at the same site. (iii) Overlapping σ54- and σ70-promoters also control expression of glmY in Klebsiella and Escherichia species. In contrast, transcription of glmZ is driven exclusively from σ70-promoters.

Finally, IHF might be important for the activities of the σ54-dependent glmY and glmZ promoters. To address these hypotheses, we selected one species per group to experimentally analyse the glmY and glmZ promoters (Figure 1B). These were Y. pseudotuberculosis YPIII (group I), S. enterica subsp. enterica serovar typhimurium str. LT2 (group II) and E. coli K12 (group III).

Response regulator GlrR binds to the glmY promoter regions of S. typhimurium and Y. pseudotuberculosis

First, we wanted to verify if the putative σ54-dependent glmY promoters of S. typhimurium and Y. pseudotuberculosis are controlled by the response regulator GlrR. Therefore, we tested whether purified GlrR protein is able to bind to these promoters. EMSAs were carried out using purified GlrR protein from E. coli and DNA fragments covering the glmY promoter regions of these species. For comparison, binding of GlrR to the corresponding DNA fragment of E. coli was tested. Different concentrations of purified His-tagged GlrR protein were incubated with the various glmY promoter fragments, respectively. In order to verify binding specificity, an additional DNA fragment, which covered the lacZ promoter and had a size of either 400 or 200 bp was simultaneously present in these assays. Protein/DNA-complexes and unbound DNA were separated by polyacrylamide gel electrophoresis (Figure 2A). The glmY promoter fragments of all three species were shifted to distinct slower migrating bands indicating DNA/GlrR complexes, while the lacZ control fragments were not bound. Comparable protein concentrations were required to achieve binding, indicating that GlrR binds with similar affinities to all these glmY fragments. GlrR of E. coli shares 95% and 87% amino acid sequence identity with its homologs from S. typhimurium and Y. pseudotuberculosis, respectively. To confirm that the results obtained with the heterologous GlrR protein are valid, we additionally performed EMSAs using purified Y. pseudotuberculosis GlrR. This protein also bound the glmY promoter DNA fragments of both, Y. pseudotuberculosis and E. coli, with comparable affinities (Supplementary Figure S5). However, in comparison to GlrR from E. coli higher protein concentrations were required to achieve binding.

Figure 2.
Comparison of the roles of GlrR and σ54 for expression of glmY from E. coli, S. typhimurium and Y. pseudotuberculosis. (A) EMSAs to test binding of E. coli GlrR protein to the glmY promoter regions of E. coli (−238 to +22), S. typhimurium ...

Analysis of S. typhimurium and Y. pseudotuberculosis glmY expression

The EMSAs suggested that glmY expression is regulated by GlrR in all three species. To validate this conclusion and to determine whether single or overlapping σ70- and σ54-promoters control expression of glmY, we constructed fusions of the glmY genes of all three species to the lacZ reporter gene. The fusions were integrated into the chromosome of E. coli wild-type and isogenic ΔglrR and ΔrpoN mutants (rpoN encodes σ54). The resulting strains were grown to exponential phase and the β-galactosidase activities were determined. The E. coli glmY’-lacZ fusion was readily expressed in the wild-type, while its expression was 6-fold lower in the ΔglrR mutant reflecting the lack of activity of the σ54-promoter (Figure 2B, columns 1 and 2). However, a certain level of expression was retained in the ΔglrR mutant, which is due to the activity of the overlapping σ70-promoter (22). Complementation of the ΔglrR mutant with a plasmid carrying E. coli glrR under PAra promoter control restored expression of glmY’-lacZ to wild-type levels (Figure 2B, columns 1 and 3), while a somewhat lower activity was obtained when a plasmid carrying glrR from Y. pseudotuberculosis was used (Figure 2B, column 4). This effect was also seen in all subsequent complementation experiments suggesting that GlrR from Y. pseudotuberculosis is less active than the E. coli GlrR protein. In agreement with previous data (22), the E. coli glmY’-lacZ fusion was expressed at higher levels in the ΔrpoN mutant in comparison to the ΔglrR mutant (Figure 2B, columns 2 and 5). This difference results from repression of the σ70-dependent promoter by binding of σ54-RNAP to the overlapping σ54-promoter in the ΔglrR mutant (22).

Similar results were obtained using the S. typhimurium glmY’-lacZ fusion (Figure 2B, columns 6–10). However, expression of this fusion was almost completely abolished in the ΔglrR mutant (Figure 2B, columns 2 and 7). A considerable level of expression was detectable in the ΔrpoN mutant as it was also observed for the E. coli glmY’-lacZ fusion (Figure 2B, columns 5 and 10). Hence, the data are compatible with overlapping σ70- and σ54-promoters, as predicted by the sequence alignment (Figure 1B, Supplementary Figure S3). The σ70-promoter of S. typhimurium glmY appears to be completely repressed by binding of σ54 to the overlapping σ54-promoter. The Y. pseudotuberculosis glmY’-lacZ fusion exhibited a different pattern of expression (Figure 2B, columns 11–15). This fusion was neither expressed in the ΔglrR nor in the ΔrpoN mutant. Complementation of the ΔglrR mutant with plasmids encoding glrR either from E. coli or Y. pseudotuberculosis restored expression to higher levels than in the wild-type strain (Figure 2B, columns 11, 13, 14). Collectively, the data support the conclusions drawn from the sequence alignments: The glmY genes of all three species are transcribed from σ54-dependent promoters that require activation by GlrR. An additional σ70-promoter overlapping the σ54-promoter exists in E. coli and S. typhimurium, but not in Y. pseudotuberculosis.

The response regulator GlrR binds the glmZ promoter region of S. typhimurium and Y. pseudotuberculosis, while the E. coli glmZ promoter is not bound

The sequence alignment analysis of the glmZ promoter regions had revealed putative σ54-promoters and GlrR binding sites in S. typhimurium and Y. pseudotuberculosis, while these elements are missing upstream of E. coli glmZ (Figure 1B, Supplementary Figure S4). To determine whether GlrR is able to bind to these promoter regions, EMSAs were carried out using E. coli GlrR protein and DNA fragments encompassing the respective glmZ promoter regions. These experiments showed that GlrR binds the glmZ promoters of S. typhimurium and Y. pseudotuberculosis with comparable affinities, whereas the E. coli glmZ promoter is not bound (Figure 3A). In addition, EMSAs were carried out using GlrR from Y. pseudotuberculosis (Supplementary Figure S6). Binding of the glmZ promoter fragment from Y. pseudotuberculosis was detectable, but four times higher protein concentrations were required in comparison to GlrR from E. coli, as already observed in the EMSAs using the glmY promoter fragments (Supplementary Figure S5). In contrast, the E. coli glmZ promoter fragment was not bound (Supplementary Figure S6). In conclusion, GlrR binds the glmZ promoters of Y. pseudotuberculosis and S. typhimurium, but not of E. coli.

Figure 3.
Comparison of the roles of GlrR and σ54 for expression of glmZ from E. coli, S. typhimurium and Y. pseudotuberculosis. (A) EMSAs to test binding of E. coli GlrR protein to the glmZ promoter regions of E. coli (−424 to +32), S. typhimurium ...

Analysis of E. coli, S. typhimurium and Y. pseudotuberculosis glmZ expression

To obtain further evidence that σ54 and GlrR regulate the glmZ genes of S. typhimurium and Y. pseudotuberculosis and are not involved in E. coli glmZ regulation, lacZ fusions of the glmZ genes were constructed and integrated into the chromosome of E. coli wild-type, ΔglrR and ΔrpoN strains. Expression of the E. coli glmZ’-lacZ fusion was neither affected by the ΔglrR nor by the ΔrpoN mutation and expression of glrR from a plasmid had also no stimulatory effect (Figure 3B, columns 1–5). Hence, expression of E. coli glmZ is not controlled by GlrR or σ54. Expression of the S. typhimurium glmZ’-lacZ fusion was also not decreased in the ΔglrR mutant. In contrast to the E. coli glmZ’-lacZ fusion, expression was significantly increased when glrR was expressed from a plasmid (Figure 3B, compare columns 6–9 and 1–4). Interestingly, expression of this fusion was also strongly increased in the ΔrpoN mutant (Figure 3B, columns 6 and 10). These results can be explained by the existence of overlapping σ70- and σ54-promoters. The high levels of glmZ transcription detected in the ΔglrR and ΔrpoN mutants (Figure 3B, columns 7 and 10) suggest that this σ70-promoter is stronger than the σ70-promoter preceding the glmY gene in S. typhimurium.

The Y. pseudotuberculosis glmZ’-lacZ fusion showed an expression pattern that was reminiscent of the results obtained with the cognate glmY’-lacZ fusion. Expression of both fusions was abolished in ΔglrR as well as ΔrpoN mutants (columns 12 and 15 in Figures 2B and and3B,3B, respectively). Complementation of the ΔglrR mutant with plasmids carrying glrR either from E. coli or Y. pseudotuberculosis restored expression to levels that were even higher than in the wild-type strain (columns 11, 13 and 14 in Figures 2B and and3B).3B). In conclusion, Y. pseudotuberculosis glmY as well as glmZ appear to be expressed exclusively from σ54-dependent promoters that require activation by GlrR. Apparently, overlapping σ70-promoters do not exist in these cases.

Expression of glmY and glmZ in Y. pseudotuberculosis

Among Enterobacteriaceae, E. coli and Y. pseudotuberculosis are distantly related (31). Although the transcriptional machinery and all elements involved in regulation of glmY and glmZ expression are conserved in both species, one might argue that the patterns of Y. pseudotuberculosis glmY and glmZ expression, as observed here in E. coli, do not appropriately reflect expression of these sRNAs in the authentic host. To address this possibility, we transformed Y. pseudotuberculosis with plasmids carrying either the Y. pseudotuberculosis glmY’-lacZ or the glmZ’-lacZ fusion or with the empty fusion vector. The cells carrying the glmY’-lacZ or the glmZ’-lacZ fusion displayed significantly higher β-galactosidase activities than the transformant carrying the empty lacZ fusion plasmid (Supplementary Figure S7A). Thus, both fusions are expressed in Y. pseudotuberculosis. The glmY’-lacZ fusion was approximately two-fold higher expressed than the glmZ’-lacZ fusion. The same difference was observed in E. coli (compare columns 11 in Figures 2B and and3B).3B). Next, a second compatible plasmid carrying either glrR from E. coli or Y. pseudotuberculosis or no gene (empty vector) under control of the PAra promoter was introduced. Presence of the glrR expression plasmids strongly increased expression of the lacZ fusions (Supplementary Figure S7B). Expression of E. coli glrR resulted in higher expression levels of the lacZ fusions in comparison to Y. pseudotuberculosis glrR. These differences were also detected in E. coli (Figures 2B and and3B).3B). Taken together, it appears justified to conclude that the data obtained with these lacZ fusions in E. coli reflect their expression in Y. pseudotuberculosis.

E. coli glmZ is exclusively transcribed from a σ70-promoter, while Y. pseudotuberculosis glmZ transcription depends on σ54 and GlrR

Our data suggested that glmZ of Y. pseudotuberculosis is transcribed from a single promoter that requires activation by σ54 and GlrR, whereas expression of E. coli glmZ is not affected by these factors. To confirm this conclusion, we mutated the left half-site of each of the three putative ABS of GlrR individually or in combination (Figure 4A, left). Fusions of Y. pseudotuberculosis glmZ’ to lacZ carrying these mutations were integrated into the chromosome of the E. coli ΔglrR mutant. These strains were subsequently complemented with the plasmid carrying Y. pseudotuberculosis glrR under PAra promoter control and the β-galactosidase activities were determined (Figure 4A, right). Mutation of ABS 1 had no negative impact on Y. pseudotuberculosis glmZ’-lacZ transcription, whereas mutation of ABS 2 or ABS 3 reduced expression more than two-fold. Expression was completely abolished, when all three ABS were simultaneously mutated. To corroborate these data, we performed EMSA experiments using Y. pseudotuberculosis glmZ promoter fragments carrying a mutation in ABS3 or simultaneously in all three ABS. These EMSAs were carried out using purified GlrR from E. coli (Figure 4B) or from Y. pseudotuberculosis (Supplementary Figure S8). In addition, a truncated glmZ promoter fragment lacking all three ABSs was tested in EMSA with Y. pseudotuberculosis GlrR (Supplementary Figure S8). The data show that mutation of ABS3 decreased binding efficiency significantly. Finally, binding of GlrR was completely prevented, when all three ABS were truncated or simultaneously mutated (Figure 4B, Supplementary Figure S8). These results show that Y. pseudotuberculosis glmZ is transcribed from a single σ54-promoter, which requires activation by binding of GlrR to its upstream located ABS.

Figure 4.
Transcription of Y. pseudotuberculosis glmZ depends on binding of GlrR to its three target sites upstream of the promoter. (A) β-Galactosidase activities of E. coli strains carrying mutated GlrR binding sites in the chromosomal Y. pseudotuberculosis ...

To further confirm that E. coli glmZ expression is independent of upstream activating sequences, a promoter deletion analysis was performed. For this purpose, DNA fragments carrying gradually 5′-truncated versions of the aslA-glmZ intergenic region were fused to lacZ (Figure 5A). Plasmids carrying these various fusions were subsequently introduced into E. coli wild-type and the β-galactosidase activities were determined. The data show that the region upstream of position −40 relative to glmZ is dispensable for promoter activity (Figure 5B). Deletion of the sequences upstream position −20, which removes the −35 motif of the putative σ70-promoter (Figure 5A and Supplementary Figure S4), abrogates expression. To verify if the assumed −35 and −10 sequences are indeed elements of a functional σ70-promoter, these sequence elements were mutated. Mutation of the three bases matching the consensus sequence TTGACA within the putative −35 sequence (Figure 5A) reduced expression of the fusion drastically (Figure 5C). Mutation of the right half site of the putative −10 motif completely abolished expression (Figure 5C). These data confirm that E. coli glmZ is transcribed from a single σ70-promoter, which is constitutively active and apparently unregulated, at least under the tested conditions.

Figure 5.
Analysis of the E. coli glmZ promoter (A) Schematic representation of the aslA-hemY intergenic region comprising the E. coli glmZ gene. DNA fragments extending until position +32 relative to the glmZ start site and with the 5′ ends indicated by ...

Activity of the σ54-dependent glmY promoter requires binding of IHF

The sequence alignment analyses detected two additional sequence motifs with similarity to the binding site of the global transcriptional regulator IHF. These sequence elements were detectable in all species, except for the glmZ promoters of Escherichia, Shigella and Klebsiella (Supplementary Figures S3 and S4), which according to all evidence are transcribed from single σ70-promoters. This suggested a role of these sites for activities of the σ54-promoters upstream of glmY and glmZ (Figure 6A). Therefore, we tested whether IHF is able to bind to the promoter fragments of E. coli glmY and Y. pseudotuberculosis glmZ. Both DNA fragments were bound by IHF protein (Figure 6B). The lacZ promoter fragments, which served as internal controls, were also bound, but at higher protein concentrations. The lacZ promoter is not known to contain any IHF site indicating unspecific binding. To confirm this conclusion, we repeated the experiments using a DNA fragment covering the ptsG promoter from Bacillus subtilis as internal control. B. subtilis does not possess IHF. Once more, efficient binding of the glmY and glmZ promoters could be observed, while the ptsG promoter was only bound at higher protein concentrations (Supplementary Figure S9). Hence, binding of IHF to the lacZ and ptsG promoters is unspecific, which is in line with previous data reporting that IHF binds DNA with lower affinity also in sequence-independent manner (30).

Figure 6.
Role of IHF for expression of glmY. (A) Schematic representation of the E. coli glmY promoter region and location of GlrR and putative IHF binding sites. The sequences of the putative IHF binding sites upstream of E. coli glmY and Y. pseudotuberculosis ...

Next, we determined whether IHF is important for the activities of the σ54-promoters. Therefore, we examined the role of IHF for expression of E. coli glmY. Expression of the chromosomally encoded E. coli glmY’-lacZ fusion was determined in mutants lacking ihfA or ihfB, which encode the subunits of IHF (30). In both mutants, expression of the fusion was reduced ~four-fold (Figure 6C, columns 1–3). The remaining activities were comparable with the expression level of this fusion in the ΔglrR mutant (Figure 2B, column 2), suggesting that it is caused by activity of the overlapping σ70-promoter (22). Therefore, we repeated the experiment using a glmY’-lacZ fusion in which the −10 sequence of the σ70-promoter is mutated, while the σ54-promoter is unaffected (22). Expression of this fusion was abolished in the ΔihfA and ΔihfB mutants (Figure 6C, columns 4–6). This demonstrates that IHF is essential for activity of the σ54-promoter of glmY.

To assess whether the two sequence elements resembling IHF binding sites are important for σ54-promoter activity, we mutated the putative IHF-site 1 in the E. coli glmY’-lacZ fusion. Four highly conserved nucleotides (Supplementary Figure S3) were exchanged within the putative IHF site 1 (Figure 6A). This mutation yielded the same effects as the ΔihfA and ΔihfB mutations. Expression of the glmY’-lacZ fusion dropped five-fold and the remaining expression was comparable with the expression obtained in the ΔglrR mutant, in which solely the σ70-promoter is active (Figure 6D, columns 1–3). Mutation of the putative IHF-1 site had no further negative impact on the residual expression of the fusion in the ΔglrR mutant (Figure 6D, columns 2 and 4) suggesting that activity of the σ70-promoter is unaffected by this mutation. To verify the role of site 1 for activity of the σ54-promoter, the experiments were repeated using the glmY’-lacZ fusion in which the σ70-promoter had been mutated. Mutation of IHF site 1 abolished expression of this fusion and therefore had the same effect as a ΔglrR or the Δihf mutations (Figure 6D, columns 5–8; Figure 6C, columns 4–6). Hence, site 1 is essential for activity of the σ54-promoter. Collectively, these data show that activity of the σ54-promotor of E. coli glmY requires binding of IHF to the promoter region. The two sites identified by sequence alignment are likely candidates for these IHF binding sites. In contrast, activity of the overlapping σ70-promoter appears to be unaffected by IHF.

Phosphorylated GlrR is active and stimulates sRNA expression

GlrR contains a response regulatory domain including the conserved putative phosphorylation site aspartate 56 at its N-terminus. Phosphorylation of GlrR by its cognate kinase GlrK has been previously demonstrated in vitro (32). Furthermore, a ΔglrK mutation was shown to abolish activity of the σ54-promoter of glmY in E. coli, suggesting that GlrK controls activity of this promoter through modulation of the phosphorylation state of GlrR (22). In many TCS, the histidine kinase is capable of phosphorylating as well as dephosphorylating the response regulator. Phosphorylation of the response regulator results in structural changes, which in most cases activate the protein and stimulate interaction with the target DNA (33). In a few cases the dephosphorylated protein was shown to be active (34). We wanted to discriminate, whether phosphorylated or dephosphorylated GlrR is active. Therefore, we exploited the fact that many response regulators can autophosphorylate in vitro using small molecules such as acetyl phosphate as phosphoryl group donors (35). Therefore, EMSAs were carried out using the E. coli glmY promoter fragment and the E. coli GlrR protein that was pre-incubated with 50 mM acetyl phosphate for 1 h at 37°C prior to EMSA. Since ongoing incubation of GlrR at 37°C resulted in increasing inactivation of the protein (compare left panels in Figures 2A and and7A),7A), a control experiment was performed in which GlrR was treated the same way but acetyl phosphate was omitted. These experiments revealed that binding affinity of GlrR was somewhat increased by the acetyl phosphate treatment relative to the control (compare panels in Figure 7A).

Figure 7.
Phosphorylation increases activity of response regulator GlrR. (A) Effect of acetyl phosphate on the DNA binding activity of GlrR as revealed by EMSA. EMSAs were performed using purified E. coli GlrR and the E. coli glmY promoter fragment. To test the ...

To obtain in vivo evidence that phosphorylated rather than dephosphorylated GlrR is active, we replaced the phosphorylation site Asp 56 in GlrR with an alanine and a glutamate residue, respectively. An Ala replacement is reported to mimic the dephosphorylated form of a response regulator, while a Glu replacement is able to mimic the phosphorylated Asp in some response regulators resulting in kinase-independent activation (35). Plasmids carrying the various glrR variants or no gene (empty vector control) under PAra promoter control were used to complement the ΔglrR mutant that carries the E. coli glmY’-lacZ fusion on the chromosome. Subsequently the β-galactosidase activities were determined from these transformants. Expression of the glrR-D56A allele resulted in ~two-fold lower activity when compared with wild-type glrR (Figure 7B, columns 2 and 3). In contrast, expression of glrR-D56E enhanced glmY’-lacZ expression five-fold. Taken together, the data indicate that phosphorylation of GlrR increases its DNA binding activity and thereby expression of the sRNA.

DISCUSSION

In this study we addressed the transcriptional regulation of two sRNA genes, glmY and glmZ, which are conserved in Enterobacteriaceae. Our analysis reveals three different scenarios of control of glmY and glmZ expression operative in enterobacterial species as described for Y. pseudotuberculosis, S. typhimurium and E. coli. Sequence alignment analyses (Supplementary Figures S3 and S4) suggest that these species are representatives for other species showing similar glmY and glmZ promoter architectures, respectively (Figure 8). Most importantly, our results suggest that in most species expression of both sRNAs is controlled by σ54 and the response regulator GlrR (Figure 8). This adds two sRNA genes to the regulon governed by σ54 in Enterobacteriaceae. The glmY and glmZ genes of Y. pseudotuberculosis exhibit all features of canonical σ54-dependent genes. Their expression depends on σ54 (Figures 2 and and3)3) and on binding of the activator protein GlrR to ABS present upstream of the σ54-promoter, as demonstrated for Y. pseudotuberculosis glmZ (Figure 4 and Supplementary Figure S8). In conclusion, transcription is initiated from single σ54-promoters that require activation by GlrR and the same may also hold true for species of the genera Arsenophonus, Dickeya, Erwinia, Pectobacterium, Photorhabdus, Proteus and Serratia (Figure 8). A somewhat different scenario is operative in the case of S. typhimurium glmY and glmZ. The corresponding promoter regions also contain three ABS and a σ54-promoter. Accordingly, GlrR specifically binds to these regions and stimulates transcription (Figures 2 and and3).3). However, both genes are still expressed in mutants lacking σ54, which is at first glance incompatible with the properties of genuine σ54-dependent genes. The expression in the absence of σ54 is explained by additional σ70-promoters that overlap the σ54-promoters and can potentially start transcription at the same site. According to the sequence alignment, such overlapping σ70- and σ54-promoters may also exist in Citrobacter, Cronobacter and Enterobacter species (Figure 8). We have recently shown that in E. coli transcription of glmY is controlled by a similar mechanism (22). In contrast, E. coli glmZ is not controlled by GlrR or σ54 and accordingly GlrR does not bind the E. coli glmZ promoter (Figure 3). A single constitutively active σ70-promoter directs expression of glmZ in E. coli (Figure 5) and presumably also in Klebsiella and other Escherichia species (including Shigella) (Figure 8). In sum, our work suggests that glmY and glmZ transcription is controlled by σ54 and the TCS GlrR/GlrK in most Enterobacteria, but in a subset of species this relation is gradually lost in favor of unregulated σ70-dependent transcription.

Figure 8.
Model illustrating the roles of the TCS GlrR/GlrK, σ54 and IHF for transcription of sRNA genes glmY and glmZ in Enterobacteriaceae. Histidine kinase GlrK phosphorylates response regulator GlrR, which stimulates binding of GlrR to its target sites ...

How did these different scenarios evolve? GlmY and GlmZ are homologous sRNAs (8,9). A sequence alignment of the glmY/glmZ genes of several species reveals sequence elements that are conserved in both sRNAs, while the glmS binding site is exclusively present in GlmZ species (Supplementary Figure S10). A phylogenetic tree built from this sequence alignment clusters glmZ genes together, while the glmY genes form a distinct group (Supplementary Figure S11). A similar clustering can be observed when the sequences of the corresponding promoter regions are used for tree construction (Supplementary Figure S12). Accordingly, glmY and glmZ most likely originated from duplication of a single sRNA locus in an ancestor of Enterobacteriaceae and transcription of this ancient sRNA was presumably already controlled by σ54 and GlrR. Following duplication, divergence of the promoter regions by mutation might have generated the different promoter architectures detectable in recent bacteria.

What is the physiological meaning of regulation of glmY/glmZ transcription by GlrR/GlrK? In E. coli, GlmYZ feedback-regulate synthesis of the enzyme GlmS and are therefore crucial for maintaining the intracellular GlcN6P concentration required for undisturbed synthesis of the cell wall and the outer membrane (8,10). This important role of GlmYZ may also apply to other Enterobacteriacea, since the GlmZ/glmS base-pairing appears to be conserved (7). In E. coli, a decrease in the intracellular GlcN6P concentration induces accumulation of GlmY, which in turn increases concentration of the full-length form of GlmZ that is competent in glmS base-pairing (8,10). Most likely, GlmY acts on GlmZ through sequestration of a protein that targets GlmZ to processing (8,9), but it is unknown whether this mechanism is also operative in other species. In conclusion, up-regulation of the GlmYZ cascade in response to GlcN6P depletion occurs at the post-transcriptional level and involves stabilization of the sRNAs rather than activation of their transcription in E. coli (22). Accordingly, the basal level of transcription of the sRNAs, as observed in the exponential growth phase, is sufficient for this function. However, GlrR/GlrK strongly up-regulate glmY expression through activation of the σ54-promoter, when cells enter the stationary growth phase (22). In contrast, GlmZ levels decrease, i.e. stabilization of GlmZ as a consequence of accumulation of GlmY does not occur in this growth phase (8). Hence, GlmY accumulates in E. coli when growth ceases and ongoing cell wall synthesis and up-regulation of glmS are not required. This indicates a second function of GlmY, which requires a higher concentration of the sRNA and becomes relevant during transition to the stationary growth phase. We speculate that GlmY may have multiple functions and this may also hold for GlmZ in those species, which control expression of both sRNA through GlrR/GlrK: GlmYZ regulate glmS and thereby GlcN6P synthesis during the exponential growth phase and basal expression levels are sufficient for this purpose. In addition, they might have another function that requires further up-regulation of the sRNAs through the TCS GlrR/GlrK. What is this additional function? Interestingly, GlrR/GlrK have been implicated to play a role for virulence: Mutants of Y. pseudotuberculosis lacking GlrR exhibited reduced pathogenicity in mice (36). In enterohemorrhagic E. coli (EHEC) GlrR/GlrK (QseF/QseE) are required for transcription of espFU, which is an EHEC-specific gene and encodes an effector protein translocated to the host cell. Consequently, loss of GlrR/GlrK results in the inability to form attaching and effacing lesions that are required for destruction of microvilli, pedestal formation and rearrangement of the cytoskeleton of host cells (37,38). In conclusion, GlrR/GlrK controls functions important for interaction with eukaryotic cells in at least two different bacteria. Whether this also holds for other Enterobacteriaceae and involves GlmY(Z) remains to be determined.

What is the reason for the existence of additional σ70-promoters overlapping with the σ54-dependent glmY/glmZ promoters in a subgroup of Enterobacteriaceae? They may allow better fine-tuning of the expression to meet the requirements of the multiple functions of these sRNAs, e.g. the σ70-promoters ensure sRNA expression when the activating signal for GlrR/GlrK is absent and the σ54-promoter is inactive. Alternatively, the σ70-promoters could also be regulated and may allow regulation of the sRNAs in response to another yet unknown process. It is also possible, that the functional overlap of σ54- and σ70-dependent promoters is a more global phenomenon in certain species such as E. coli. Extensive functional overlap with σ70-promoters has been observed for σ24- and σ32-dependent genes in E. coli (20). Both, σ24 and σ32 recognize distinct promoter sequences. However, many of these promoters also contain matches to overlapping σ70-promoters. Thus, the majority of the σ32-promoters and about half of the σ24-promoters are also recognized by σ70-RNAP and transcription initiation at the same start site was demonstrated for some of these promoters (20). This was interpreted to means that the primary function of alternative σ factors is to increase transcription of σ70-dependent genes. A recent study reported that 14% of the σ54-dependent genes in E. coli can also be transcribed by σ70-RNAP in vitro (17). Whether this occurs from overlapping or consecutive promoters is not known. However, our studies prove that arrangements of overlapping σ70- and σ54-promoters exist [(22); the present study]. It remains to be elucidated whether functional overlap between σ70 and σ54 is a peculiarity of E. coli and its closest relatives or may apply to a wider range of bacterial species.

Activation of the σ54-dependent glmY and glmZ promoters requires binding of GlrR to ABS located upstream of the promoter. However, the impact of each of the three ABS on the promoter activity appears to vary from case to case, e.g. ABS2 and ABS3 were shown to be essential for activity of the σ54-promoter of E. coli glmY (22), while mutation of one of these sites upstream of Y. pseudotuberculosis glmZ reduced promoter activity only two-fold (Figure 4A). ABS1 appears to be dispensable for promoter activity in both cases, as reflected by its lower degree of conservation. Interaction of activator proteins with σ54-RNAP requires bending of the DNA, which is usually induced by IHF (18,30). IHF might also be required for the activities of the σ54-dependent glmY and glmZ promoters as demonstrated for the σ54-promoter of E. coli glmY (Figure 6). Two putative IHF binding sites were detected and we demonstrated an essential role for σ54-promoter activity for the distal site (Figure 6D). We also provided evidence that phosphorylation of GlrR enhances glmY expression (Figure 7). Substitution of the phosphorylation site Asp 56 with Ala reduced glmY expression two-fold, whereas a Glu exchange mimicking phosphorylation led to much stronger expression (Figure 7B). In addition, pre-incubation of GlrR with acetyl phosphate increased its binding affinity for the glmY promoter (Figure 7A). Taken together this indicates that the DNA-binding activity of GlrR is activated by its phosphorylation although it cannot be excluded yet that the mutations in GlrR affected the stability rather than activity of the protein. Our data indicate that just a minor fraction of GlrR is phosphorylated by GlrK during exponential growth, which is in line with previous data suggesting that this TCS drastically increases glmY expression at the on-set of the stationary growth phase in E. coli (22). So far, glmY and glmZ are the only known direct targets of GlrR/GlrK suggesting that this TCS acts predominantly through these sRNAs.

FUNDING

DFG Priority Program SPP1258 ‘Sensory and Regulatory RNAs in Prokaryotes’ grants (to B.G. and P.D.). Funding for open access charge: German Research Foundation (DFG).

Conflict of interest statement. None declared.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

Supplementary Data:

ACKNOWLEDGEMENTS

The authors thank Fabio Pisano, Karin Schnetz and Vanessa Sperandio for critical reading of the manuscript. They are grateful to Jacqueline Plumbridge for the gift of purified IHF protein and to Karin Schnetz for S. typhimurium LT2. They thank Sabine Lentes for excellent technical assistance, Jörg Stülke for lab space and support and Konstantin Albrecht and Sabine Zeides for help with the construction of plasmids or strains.

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