Expression of Shigella flexneri gluQ-rs gene is linked to dksAand controlled by a transcriptional terminator
© Caballero et al.; licensee BioMed Central Ltd. 2012
Received: 11 June 2012
Accepted: 11 September 2012
Published: 5 October 2012
Glutamyl queuosine-tRNAAsp synthetase (GluQ-RS) is a paralog of the catalytic domain of glutamyl-tRNA synthetase and catalyzes the formation of glutamyl-queuosine on the wobble position of tRNAAsp. Here we analyze the transcription of its gene in Shigella flexneri, where it is found downstream of dksA, which encodes a transcriptional regulator involved in stress responses.
The genomic organization, dksA-gluQ-rs, is conserved in more than 40 bacterial species. RT-PCR assays show co-transcription of both genes without a significant change in transcript levels during growth of S. flexneri. However, mRNA levels of the intergenic region changed during growth, increasing at stationary phase, indicating an additional level of control over the expression of gluQ-rs gene. Transcriptional fusions with lacZ as a reporter gene only produced β-galactosidase activity when the constructs included the dksA promoter, indicating that gluQ-rs do not have a separate promoter. Using bioinformatics, we identified a putative transcriptional terminator between dksA and gluQ-rs. Deletion or alteration of the predicted terminator resulted in increased expression of the lacZ reporter compared with cells containing the wild type terminator sequence. Analysis of the phenotype of a gluQ-rs mutant suggested that it may play a role in some stress responses, since growth of the mutant was impaired in the presence of osmolytes.
The results presented here, show that the expression of gluQ-rs depends on the dksA promoter, and strongly suggest the presence and the functionality of a transcriptional terminator regulating its expression. Also, the results indicate a link between glutamyl-queuosine synthesis and stress response in Shigella flexneri.
KeywordstRNA modification Gene expression Stringent response Osmotic stress
The fidelity of the translation process depends on the aminoacyl–tRNA synthetase enzymes (aaRS). These essential enzymes are responsible for the correct attachment of the corresponding amino acid onto the cognate tRNA, therefore organisms have at least 20 synthetases . The enzymes are divided in two classes, each class having a conserved structure. The genes encoding the aaRS are easily detected within sequenced genomes [2, 3], and some species contain synthetase gene duplications, such as the glutamyl-tRNA synthetases (GluRS) in Acidithiobacillus ferrooxidans and Helicobacter pylori (genes gltX1 and gltX2) [4, 5]. aaRS paralogs, predicted sequences with homology to fragments of synthetases, have also been identified, which is not surprising given the modular nature of the aaRS . Some of the paralogs may be pseudogenes while others have known functions. For instance HisZ from Lactococcus lactis, which has similarity with the catalytic domain of histidyl-tRNA synthetase, is involved in histidine biosynthesis . A recent study in Salmonella enterica has shown that PoxA, encoded by poxA/genX, has similarity to the carboxy-terminal catalytic domain of lysine-tRNA synthetase and is required for posttranslational aminoacylation of bacterial elongation factor P. A poxA mutant has reduced colonization and virulence, possibly due to misregulated expression of proteins encoded by the SPI-1 pathogenicity island [8, 9].
An Escherichia coli glutamyl-tRNA synthetase paralog, glutamyl queuosine-tRNAAsp synthetase (GluQ-RS) has approximately 35% amino acid similarity with the catalytic domain of GluRS. This includes the amino acids involved in recognition and activation of glutamate. Although GluQ-RS is missing the carboxyl-terminus domain responsible for the tRNA recognition, in E. coli this enzyme is able to activate the amino acid in the absence of the tRNA. Further, once the aminoacyl-adenylate has been formed, the enzyme attaches the glutamate to the nucleoside queuosine present onto the tRNAAsp. Therefore, this enzyme is involved in the synthesis of a new modified nucleoside glutamyl-queuosine (GluQ) present in tRNAAsp[10, 11]. This modification is present in tRNA isolated under acidic conditions from bacterial cells grown in rich media. However, the enzyme is not essential for growth of E. coli in rich or minimal media . Queuosine is widely distributed in bacteria, and it is present in the first base of the anticodon of tRNAAsp, tRNAAsn, tRNAHis and tRNATyr; however in E. coli only tRNAAsp is a substrate for the GluQ-RS enzyme.
The presence of modifications within the anticodon loop of the tRNA, could enhance the accuracy of the codon binding . Then the tRNAAspQ34 might improve recognition of both GAC and GAU codons  and stimulate the binding of the GAU codon to the ribosome . In Shigella flexneri it has been shown that mutations in genes required for tRNA modifications, miaA and tgt decreased virulence. miaA is required for 2-methylthio-N6-isopentenyladenosine modification at position 37 of the anticodon loop and tgt is involved in queuosine modification at position 34 within the anticodon loop [16–18]. In this study, we determined the role of the genome organization and its effect on the expression of the gluQ-rs gene in the major human pathogen, S. flexneri.
Genomic organization of the S. flexneri gluQ-rsgene
S. flexneri gluQ-rs gene is co-transcribed with dksAgene
Bacterial strains and plasmids used in this work
Bacterial strains or plasmid
Source or reference
S. flexneri 2457T
Wild type strain
S. flexneri 2457T ΔgluQ-rs::kan
Deletion mutant of gluQ-rs gene
E. coli W3110 ΔgluQ-rs::kan
Deletion mutant of gluQ-rs gene
F - ϕ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK-, mK+) phoA supE44 λ-thi-1 gyrA relA1
F - ompT gal dcm lon hsdS B (r B - m B - ) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])
bla, pMB1 ori, lacZ peptide, f1 phage ori
bla, pMB1 ori, lacZ gene without promoter
Empty vector, a modified version of pET15b
S. flexneri fragment from nucleotide +58a of dksA gene to beginning of gluQ-rs gene (+590) cloned into pQF50. Pair of primers used were PgluQF/PdksARCT.
S. flexneri fragment from nucleotide −506 of dksA gene to beginning of gluQ-rs gene (+590) cloned into pQF50. Pair of primers used were PdksAF/PdksARCT.
S. flexneri fragment from nucleotide −506 of dksA gene to beginning of gluQ-rs gene (+590) cloned into pQF50, with the terminator mutated by the nucleotides indicated in Figure 4a.
S. flexneri fragment from nucleotide −506 of dksA gene to nucleotide +527 (end of dksA gene) cloned into pQF50. Pair of primers used were PdksAF/PdksARST.
S. flexneri gene from nucleotide +509 (stop codon of dksA) to nucleotide +1469 (last codon of gluQ-rs without stop codon). Pair of primers used were ATGGQRSF/ATGGQRSR.
The S. flexneri gluQ-rsgene has an upstream transcription terminator
Identification of the first methionine
Phenotype of the S. flexneri gluQ-rsmutant
Because expression of dksA is required for S. flexneri virulence , and growth of Shigella in the intracellular environment may induce a stress response, we also measured invasion and plaque formation by the gluQ-rs mutant. However, no significant differences were noted (data not shown), suggesting that GluQ-RS is not essential for invasion or intracellular growth of S. flexneri.
Conserved dksA-gluQ-rsgenomic organization in gammaproteobacteria
GluQ-RS, a paralog of GluRS synthetase, is involved in the formation of GluQ, the nucleoside located at the wobble position of tRNAAsp in bacteria. The protein is present in Firmicutes, Actinobacteria, Cyanobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria (Figure 1). From the phylogenetic analysis we distinguished the three subgroups described previously  based on the HIGH motif that is present in the class I aminoacyl-tRNA synthetases . As was described previously , all GluQ-RS enzymes are characterized by the replacement of a threonine in GluRS enzymes, which is involved in the recognition of the amino acid and the terminal adenosine of the tRNAGlu (Thr133 of Methanocaldococcus jannaschii GluRS enzyme) by isoleucine, leucine or valine at that position (Ile47 of S. flexneri GluQ-RS). This substitution is also conserved in all enzymes analyzed here, including those from the Firmicutes group.
The gluQ-rs gene is widely distributed in the bacterial domain; however, its genome organization is variable. We observed that only in members of the gammaproteobacteria, namely Aeromonadales, Alteromonadales, Pseudomonadaceae, Enterobacteriaceae and Vibrionaceae, the gluQ-rs gene is located immediately downstream of the dksA gene (Figure 1). A more detailed analysis shows that even within this genomic organization there are differences. In some species of Pseudomonadaceae, such as P. aeruginosa, P. entomophila, and P. fluorescens, we observed the same genomic structure as in E. coli or S. flexneri, with a distinctive terminator between the genes. In contrast, while the dksA gene is also upstream of gluQ-rs in some P. syringae, there are insertions of an encoded transposase or more than a 400 base pairs separating both genes without a detectable terminator. However, using bioinformatics tools we detected a possible promoter within this region in P. syringae (data not shown), indicating that the expression of the gluQ-rs gene may be under control of its own promoter, a question that remains to be addressed.
Stringent response and tRNA modification
Our bioinformatic analysis shows the presence of a transcriptional terminator and lack of a gluQ-rs-specific promoter. This is consistent with our results, in which we did not detect any activity from promoters other than those upstream of the dksA gene (Figure 3). This unusual arrangement suggests that gluQ-rs expression is dependent on dksA-regulated conditions. Because DksA is a key member of the stringent response in bacteria and regulates a number of processes in the cell, including its own expression [25, 28], the data suggest that there is coordinate regulation of tRNA modification and other DksA targets. Although we could not detect any promoter activity specific for gluQ-rs in the growth conditions tested (i.e. altering the pH, presence of glutamate), we cannot discount the possibility that the gene is specifically regulated under some other conditions. The regulon database (http://regulondb.ccg.unam.mx/index.jsp) indicates that the E. coli gluQ-rs gene has a recognition site for the σ24 subunit of RNA polymerase. From our analysis, this sequence is identical to S. flexneri, but there is no experimental evidence of this recognition. Interestingly, when the gluQ-rs gene was deleted in S. flexneri, the mutant showed impaired growth in the presence of osmolytes (Figure 6). A recent publication demonstrated that σ24 and σS proteins from Salmonella enterica serovar Typhi are important for the expression of several genes induced by osmotic stress in this bacterium . Moreover, the expression of the gene encoding σ24 in E. coli is regulated by the stringent response . The possible role of σ24 on the expression of gluQ-rs under osmotic stress might be interesting to study.
GluQ-RS is an enzyme responsible for the formation of the GluQ tRNA modification, and two independent groups [10, 11] have shown that this enzyme required a high concentration of glutamate to be activated and transferred to the queuosine base present on the tRNAAsp. Interestingly, one of the first events to occur when bacteria are subject to high osmolyte stress is an increase in glutamate levels within the cytoplasm . Our observation indicates an important role of the tRNA modification for the growth of S. flexneri in the presence of osmolytes (Figure 6). Other tRNA modifications might play a similar role in this stress condition. In E. coli, inactivation of the yfiC gene, responsible for the modification at the adenosine 37 present on the tRNAVal, leads to a high sensitivity to osmotic stress .
Transcription of gluQ-rsis regulated by a terminator
The results obtained in the present work show the presence of a terminator and suggested the functionality of this structure (Figure 3 and Figure 4). To our knowledge, there are few examples of bacterial genes that have similar structures. There is a terminator structure upstream of the DNA primase gene, dnaG, which also has an unusual Shine Dalgarno sequence . Another example is the recX gene in E. coli, where readthrough accounts for approximately 10% of its transcription .
The two characteristics of gluQ-rs described in this work, co-transcription with the upstream gene and the presence of a terminator immediately upstream, allow us to propose that both the transcription and translation process could be regulated in the gluQ-rs gene. It has been described, that the presence of terminators upstream of the coding region might be part of a regulatory system such as a riboswitch . Riboswitches for genes involved in queuosine formation have been described, in which the precursor preQ1 is the ligand of the mRNA structure . Using the riboswitch server (ribosw.mbc.nctu.edu.tw ), we did not identify any potential riboswitch (data not shown). However we cannot discount that the terminator described here might be part of a regulatory circuit similar to a riboswitch, or that an unidentified protein might bind the terminator structure.
GluQ modification and codon bias
tRNA modifications present at the anticodon loop might be important for the accuracy of codon reading during the translation processes . Morris et al., 1999  proposed, based on molecular modeling, that the tRNAAspQ34 might improve recognition of both GAC and GAU codons, consequently the interaction of the codon GAU with the anticodon of tRNAAspG34 could be less efficient. In fact, in S. flexneri there are a few genes such as sitA, virF and proX (an inducible gene under osmotic stress) that have a bias toward those codons that favor the modified tRNA. Thus, while there is no obvious loss of plaque formation in the gluQ-rs mutant (data not shown), the absence of GluQ-RS may influence the expression of proteins such as SitA that are required for fitness of Shigella in the host .
In this work we have shown that the expression of gluQ-rs, a gene codifying an enzyme involved in the formation of GluQ present on the tRNAAsp, is under the control of the dksA promoter. Also, we show the presence of a functional terminator that controls the expression of gluQ-rs. Finally, we present data that suggest that the presence of modification of the tRNAAsp is important for survival of the human pathogen Shigella flexneri under osmotic stress conditions.
Bacterial growth conditions
The bacterial strains and plasmids used in this study are described in Table 1. E. coli strains were maintained on LB-agar (10 g of tryptone per liter, 5 g of yeast extract per liter, 10 g of NaCl per liter and 15 g of agar per liter), Shigella strains were maintained on Trypticase Soy Agar plus 0.01% congo red. All strains were stored at −80°C in LB broth plus 20% glycerol. The bacteria were grown in LB broth adjusted to pH 7.4 with 40 mM MOPS (Merck) or M9 minimal media . When necessary, ampicillin was added to a final concentration of 100 μg/ml. Bacterial growth was monitored by optical density at 600 nm (OD600).
Bioinformatics tools to construct the phylogenetic tree
The protein sequences were obtained from the Uniprot database (http://www.uniprot.org/) and then were searched in the GenomeNet (http://www.genome.jp/) to confirm the genomic organization. A selected number of GluQ-RS enzymes were aligned using the MUSCLE algorithm  and analyzed using the maximum-likelihood method based on the JTT matrix-based model. The percentage of trees in which the associated proteins clustered together is shown next to the branches. The analysis involved 54 amino acid sequences, including the GluRS proteins from Methanocaldococcus jannaschii and Archaeoglobus fulgidus as an outgroup. All positions containing gaps and missing data were eliminated. There were a total of 199 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 .
RNA isolation and synthesis of cDNA
Sequence 5′- 3′a
Reference and characteristics
This work. RT-PCR of dksA operon from nucleotide +40 to +1477b
This work. RT-PCR of dksA gene from nucleotide +54 to +488
This work, RT-PCR of intergenic region from nucleotide +368 to +863
This work, RT-PCR of gluQ-rs gene from nucleotide +567 to +1074
 RT-PCR of ribosomal RNA 16S
Reverse of pcnB gene from nucleotide +1993
BamHI site, from nucleotide −506
HindIII site, to nucleotide +527
HindIII site, to nucleotide +590
BamHI site, from nucleotide +58
Recognition from nucleotide +555
Recognition from nucleotide +560, underline sequence are nucleotides changed
Recognition site in pTZ57R/T
BamHI site, from nucleotide +507. Underline nucleotides correspond to the stop codon of dksA
XhoI site, to nucleotide +1469
Construction of transcriptional fusions
Transcriptional fusions were constructed to study the expression control of gluQ-rs. Fragments of the dksA gluQ-rs region were fused to lacZ in the vector pQF50 by using the BamHI and HindIII restriction sites . Each fragment was amplified from S. flexneri genomic DNA using the indicated primers (Tables 1 and 2) with the High Fidelity PCR Enzyme Mix polymerase (Fermentas) and cloned into pQF50 (Table 1). Once the sequence of each clone was confirmed, the recombinant plasmid was introduced into S. flexneri 2457T by electroporation. The nomenclature of the recombinants plasmids is: P for promoter of the dksA gene, D for the dksA gene and T for a terminator structure.
S. flexneri transformed with the corresponding constructs were cultured overnight in LB, a 1:50 dilution was inoculated into 10 ml culture of LB pH 7.4 and grown to an OD600 of 0.5. Aliquots of 0.5 ml of each strain containing the clone or the empty vector were assayed for β-galactosidase activity according to Miller . The data were analyzed using the software GraphPad Prism V5.01.
Site directed mutagenesis
A possible transcription terminator between dksA and gluQ-rs was identified using the program Mfold . Site directed mutagenesis by overlap PCR was performed to disrupt the predicted terminator . Using the fragment VCPDT cloned in the vector pTZ57R/T as template, was amplified a 1,072 bp fragment, which include the mutation, using the primers PdksAF and TERMGQ3, while a second fragment of 162 bp overlapping the mutated region, was obtained with primers TERGQ2 and M13R (Table 2). Both fragments (1,072 bp and 162 bp) were digested with DpnI, purified and mixed at equimolar quantities to carry out a PCR reaction using the 5′ and 3′ ends primers (PdksAF and PdksARCT). The 1,110 bp amplified fragment was cloned in the vector pTZ57R/T and sequenced to verify the mutation. This plasmid was digested with BamHI and HindIII and the fragment subcloned in to the vector pQF50.
Determination of first methionine of GluQ-RS
In order to establish which is the first AUG codon of gluQ-rs, the recombinant plasmid pATGGQRS was constructed. A PCR reaction was performed using the primers ATGGQRSF and ATGGQRSR (Table 2) and genomic DNA from S. flexneri. The amplified fragment, containing the BamHI site, stop codon of dksA, the intergenic region with the terminator, the gluQ-rs reading frame without its stop codon and the XhoI site was cloned into pET15c, a modified version of pET15b, which was constructed by inserting the 290 bp XbaI and BlpI fragment of pET20b containing the polylinker into pET15b. This construct allowed the synthesis of a C-terminal histidine tagged protein under the transcription control of the T7 promoter. The construct was transformed in BL21(DE3) strain and the His-tagged protein was partially purified by affinity chromatography as described previously . The eluted protein was transferred to a PVDF membrane and stained with Coomassie blue. The predominant band of the expected size (34.6 kDa) was sequenced at the Protein Core Facility of the Institute for Cellular and Molecular Biology, University of Texas at Austin.
Construction of the plasmid for complementation of the gluQ-rsmutation
This plasmid was constructed from the pATGGQRS plasmid in which the T7 promoter was removed by digestion with BglII and NcoI enzymes and replaced by the TRC promoter obtained from pTRC99a plasmid by amplification and digestion with BamHI and NcoI to obtain the pTRCGQ plasmid. The empty plasmid (pCM) was constructed by incorporating the TRC promoter into the pET15c plasmid.
Inactivation of gluQ-rs gene in S. flexneri
Deletion of gluQ-rs was carried out using the λ red recombinase method  with the following modifications. S. flexneri 2457T carrying pKD46 and prepared as described elsewhere  was transformed with a purified PCR fragment amplified from the E. coli ΔgluQ-rs::kan mutant strain using primers dksAF and pcnBR (Table 2), increasing the homologous DNA region to more than 450 bp at each side. The mutant was isolated following overnight growth at 37°C on LB-agar containing kanamycin (50 μg/ml). The deletion was confirmed by PCR using the same pair of primers (dksAF-pcnBR) and using each primer together with an internal primer as described previously . The presence of the S. flexneri virulence plasmid was also confirmed by PCR amplification of the virF gene using primers virFF and virFR (Table 2).
Effect of the absence of gluQ-rs gene in S. flexnerimetabolism
The effect of the deletion of the gluQ-rs gene on the metabolism of S. flexneri was analyzed by Biolog phenotype MicroArrays following the manufacturer’s instructions (Biolog, Inc., Almeda, CA). Strains were grown at 30°C overnight and 5 ml of LB was inoculated with a 1:100 dilution and grown at 37°C to reach an OD650nm of 0.5. The cells were then washed and resuspended to 2.5 x 107 cfu/ml and diluted 200 fold in to a solution of IF-10a medium (Biolog). Each well was inoculated with 1.2 x 104 cfu (0.1 ml per well) into the corresponding plates and incubated for 24 hrs at 37°C. The metabolism was recorded and analyzed by the Omnilog software (V 1.20.02) (Biolog, Inc., Almeda, CA).
Glutamyl queuosine-tRNAAsp synthetase and its codifying gene
Transfer ribonucleic acid codifying aspartic acid
Pathogenicity islands of Salmonella
- miaA :
tRNA Δ(2)-isopentenylpyrophosphate transferase
- tgt :
tRNA guanine transglycosylase
Reverse transcription-polymerase chain reaction
Moloney Murine Leukemia Virus Reverse Transcriptase
We are grateful to Dr. Dieter Söll from Yale University, USA, for providing the E. coli strains BL21(DE3) and W3110 ΔgluQ-rs::kan. Also, we would like to thank to Dr. Claude Parsot from the Institute Pasteur, France, for providing the pQF50 plasmid and advice in the determination of the N-terminal sequence of GluQ-RS. We appreciate Dr. Elizabeth Wyckoff for her critical review of this manuscript. This publication was funded by Grants from the Department of Research, University of Chile DI I2 06/04-2 and Fondo Nacional de Desarrollo Científico y Tecnólogico (FONDECYT) 1080308 to J.C.S. and Grant AI 169351 from the National Institutes of Health to S.M.P.
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