Patent Application
20110217282
Sequence listings in written and computer readable form are submitted herewith. The information recorded in computer readable form is identical to the written sequence listings.
It has been reported that quorum sensing is involved in biofilm formation (1-4). MqsR expression was found to be induced eightfold in biofilms (5) and also by the quorum sensing signal autoinducer-2 (AI-2), which is a species-nonspecific signaling molecule produced by both gram-negative and gram-positive bacteria, including E. coli (6). It was reported that induction of MqsR activates a two-component system, the qseBqseC operon, which is known to play an important role in biofilm formation (6). Thus, it has been proposed that MqsR (98 amino acid residues) is a regulator of biofilm formation since it activates qseB, which controls the flhDC expression required for motility and biofilm formation in E. coli (6). However, the cellular function of MqsR has remained unknown.
Interestingly, all free-living bacteria examined to date contain a number of suicide or toxin genes in their genomes (7,8). Many of these toxins are co-transcribed with their cognate antitoxins in an operon (termed as toxin-antitoxin or TA operon), and form a stable complex in the cell so that their toxicity is subdued under normal growth conditions (9-11). However, the stability of antitoxins is substantially lower than that of their cognate toxins so that any stress causing cellular damage or growth inhibition that induces proteases alters the balance between toxin and antitoxin, leading to toxin release in the cell.
To date, sixteen (24) TA systems have been reported on the E. coli genome, including relB-relE (12,13), chpBI-chpBK (14), mazE-mazF (15-17), yefM-yoeB (18,19), dinJ-yafQ (20,21), hipB-hipA, hicA-hicB (25,26), prlF-yhaV (27) and ybaJ-hha (28). Interestingly, all of these TA operons appear to use similar modes of regulation; the formation of complexes between antitoxins and their cognate toxins to neutralize toxin activity and the ability of TA complexes to autoregulate their expression. The cellular targets of some toxins have been identified: CcdB directly interacts with gyrase A and blocks DNA replication (29,30); RelE, which by itself has no endoribonuclease activity, appears to act as a ribosome-associating factor that promotes mRNA cleavage at the ribosome A-site (12,31,32). PemK (33), ChpBK (14) and MazF (34) are unique among toxins, since they target cellular mRNAs for degradation by functioning as sequence-specific endoribonucleases to effectively inhibit protein synthesis and thereby cell growth.
MazF, ChpBK and PemK have been characterized as sequence-specific endoribonucleases, which cleave mRNA at the ACA, ACY (Y is U, A, or G) and UAH (H is C, A, or U) sequences, respectively. They are completely different from other known endoribonucleases such as RNases E, A, and T1, as these toxins function as protein synthesis inhibitors by interfering with the function of cellular mRNAs. It is well known that small RNAs, such as micRNA (mRNA-interfering-complementary RNA) (37), miRNA (38), and siRNA (39), interfere with the function of specific RNAs. These small RNAs bind to specific mRNAs to inhibit their expression. Ribozymes also act on their target RNAs specifically and interfere with their function (40). Therefore, MazF, ChpBK and PemK homologues form a novel endoribonuclease family which exhibits a new mRNA-interfering mechanism by cleaving mRNAs at specific sequences. Thus, they have been termed “mRNA interferases” (2).
All references described herein are incorporated by reference in their entireties for al purposes.
It has been discovered on the E. coli genome that the MqsR gene is co-transcribed with a downstream gene, YgiT. These two genes appear to function as a TA system, as their size is small (98 residues for MqsR and 131 residues for YgiT) and their respective open reading frames are separated by one base-pair. As disclosed herein, MqsR/YgiT is a new E. coli TA system consisting of a toxin, MqsR and an antitoxin, YgiT. Moreover, as disclosed herein, MqsR is a novel mRNA interferase, which does not exhibit homology to MazF. This toxin cleaves RNA at GCU sequences in vivo and in vitro and therefore has implications in cell physiology and biofilm formation as disclosed herein.
As disclosed herein, the MqsR induction is highly toxic, and its toxicity is blocked by co-expression of YgiT and cellular mRNAs are degraded when MqsR is induced. This in-vivo result was substantiated in vitro using purified MqsR. E. coli total RNA was incubated with MqsR for 30 min at 37° C., clearly indicating that purified MqsR cleaves RNA. Importantly this endoribonuclease activity was completely inhibited when its presumed antitoxin, YgiT was added in the reaction mixture. With use of 3.5-kbase phage MS2 RNA, we have identified the major cleavage sites by this toxin. Thus, it appears to be a highly sequence-specific mRNA interferase, that recognizes a triplet sequence, GCU.
This sequence may be either underrepresented or overrepresented in some genes, and the genes may be associated with quarum sensing and/or biofilm formation.
Accordingly, this invention relates to a new TA system, MqsR YgiT in E. coli. The induction of MqsR was highly toxic in E. coli and caused a degradation of mRNA in vivo. Purified MqsR showed endoribonuclease activity and YgiT neutralized the activity in vitro. MqsR cleaves MS2 phage RNA at GCU.
The invention can be used in single-protein production in prokaryotic and eukaryotic cells, such as E. coli and mammalian cells. It also has applications in gene therapy by using the MqsR/YgiT system to treat various human diseases such as cancer, bacterial infection and viral infection including AIDS. The invention can be used as an RNA restriction enzyme for RNA structural study.
In certain embodiments, the invention is directed to a method of inhibiting cell function comprising inducing the expression of a mRNA interferase that cleaves mRNA at GCx, wherein x is A, C, G, or U. The mRNA interferase can be ribosome-independent and is preferably MqsR or a homolog thereof. In alternative embodiments, the induction is capable of being inhibited by an antitoxin, e.g., YgiT. The cell can be, e.g., E. coli or Homo sapiens.
In embodiments disclosed herein, the inhibition of mRNA interferase can be either in-vitro or in-vivo.
In certain embodiments, the invention is directed to a method of inhibiting cell function comprising inducing the expression of MqsR or a homolog thereof.
In certain embodiments, the invention is directed to a plasmid comprising a gene encoding MqsR or a homolog thereof. The expression of MqsR can be induced, e.g., with IPTG and can be, e.g., a pET28a plasmid. In alternative embodiments, the gene has a sequence according to SEQ ID NO: 1.
In certain embodiments, the invention is directed to a plasmid comprising a gene encoding YgiT or a homolog thereof. The expression of YgiT can be induced, e.g., with arabinose and can be, e.g., a pBAD24 plasmid. In alternative embodiments, the gene has a sequence according to SEQ ID NO: 3.
In certain embodiments, the invention is directed to a plasmid comprising: a) a gene encoding MqsR or a homolog thereof; and b) a gene encoding YgiT or a homolog thereof. In alternative embodiments, the gene encoding MqsR has a sequence according to SEQ ID NO: 1 and the gene encoding YgiT has a sequence according to SEQ ID NO: 3.
In certain embodiments, the invention is directed to a cell (e.g., E. coli or Homo sapiens) transformed with one or more plasmids disclosed herein.
In certain embodiments, the invention is directed to a method of inhibiting MqsR endoribonuclease activity comprising contacting MqsR with YgiT. In alternative embodiments, the method comprises pre-incubating MqsR with YgiT.
In certain embodiments, the invention is directed to the use of YgiT as an antitoxin for MqsR.
In certain embodiments, the invention is directed to a method of inhibiting cell lysis of E. coli comprising inactivating MqsR. In alternative embodiments, the MqsR is inactivated by YgiT.
In certain embodiments, the invention is directed to an isolated YgiT polypeptide having an amino acid sequence according to SEQ ID NO: 4. In alternative embodiments, the polypeptide has an amino acid sequence which has 90% homology with this amino acid sequence and has antitoxin activity.
In certain embodiments, the invention is directed to an isolated YgiT polynucleotide having a DNA sequence according to SEQ ID NO: 3. In alternative embodiments, the polynucleotide has a DNA sequence which has 90% homology with this DNA sequence and encodes a polypeptide having antitoxin activity.
In certain embodiments, the invention is directed to a complex comprising MqsR and YgiT, or homologs thereof. In alternative embodiments, the complex comprises a polypeptide according to SEQ ID NO: 2 and a polypeptide according to SEQ ID NO: 4.
In certain embodiments, the invention is directed to a method of producing a polypeptide having endoribonuclease activity comprising: a) transforming a cell by introducing a polynucleotide encoding MqsR into the cell, and b) culturing the transformed cell.
In certain embodiments, the invention is directed to a method of producing a polypeptide having antitoxin activity comprising: a) transforming a cell by introducing a polynucleotide encoding YgiT into the cell, and b) culturing the transformed cell.
In certain embodiments, the invention is directed to a method of cleaving mRNA comprising contacting an mRNA interferase with mRNA wherein the mRNA interferase is not homologous to MazF. In alternative embodiments, the mRNA is cleaved at GCx, wherein x is A, C, G, or U.
In certain embodiments, the invention is directed to a method of altering cell function comprising manipulating the expression of one or both of MqsR and YgiT.
In certain embodiments, the invention is directed to a method of treating a patient with a disease comprising administering to the patient a mRNA interferase that cleaves mRNA at GCx, wherein x is A, C, G, or U. The disease can be, e.g., cancer, bacterial infection or viral infection. The viral infection can be, e.g., caused by HIV or a retrovirus.
In certain embodiments, the invention is directed to a method of treating a patient with a disease comprising administering to the patient a gene encoding a mRNA interferase that cleaves mRNA at GCx, wherein x is A, C, G, or U. The disease can be, e.g., cancer, bacterial infection or viral infection. The viral infection can be an infection caused by a virus having a single-stranded RNA genome, e.g., HIV or a retrovirus.
In certain embodiments, the invention is directed to a primer according to any one of SEQ ID NOs 5-36
The invention is further described by the following non-limiting experimental procedures.
Toxicity of MqsR in E. coli.
E. coli BL21 cells were transformed with pET-MqsR and pBAD-YgiT or pBAD and pET plasmids. The cells were spread on glycerol-M9-casamino acids agar plates with and without inducers [arabinose (0.2%) and IPTG (0.1 mM)] and these plates were incubated at 37° C. for 24 h. as shown in
Effect of MqsR on mRNA Stability and Protein and DNA Synthesis
Total cellular RNA was extracted from E. coli BL21 cells containing pBAD-MqsR at various time points as indicated after the addition of arabinose and subjected to Northern blot analysis using radiolabeled lpp, ompF, and ompA ORF DNA as probes.
MazG protein synthesis was performed in the E. coli T7 S30 extract system (Promega) with pET-11a-MazG as template. The results are shown in
E. coli total RNA was incubated with purified His-tagged MqsR for 30 min at 37° C. In the last lane, purified YgiT was added. RNA was analyzed in 1.2% TBE agarose gel and the gel was stained with ethidium bromide (EtBr), as shown in
Cleavage of MS2 ssRNA and its Inhibition by YgiT.
MS2 ssRNA (0.8 μg; 3569 bases; Roche) was digested by His-MqsR in 20 at 37° C. His-MqsR was preincubated with purified YgiT for 10 min on ice and then further incubated with MS2 RNA for 30 min. Denatured products in urea were separated on 1.2% TBE native agarose gel. The gel was stained with EtBr. The results are shown in
In vitro cleavage of the MS2 RNA with His-MqsR. Lane 1, MS2 RNA with His-MqsR; lane 2, represents a control reaction in which no proteins were added; Cleavage sites are indicated by red arrows on the RNA sequence and were determined using the RNA ladder shown on the left. The results are shown in
Bacterial strains and plasmids—E. coli BL21(DE3) and C43 were used. Both MqsR and YgiT genes in the MqsRYgiT operon, were separately amplified by PCR using the E. coli genomic DNA as template and first cloned into pET28a (Novagen). The MqsRYgiT operon was also amplified by PCR with MqsR-Fw and YgiT-Rv primers using the E. coli genomic DNA as template and cloned into pET28a to express the MqsR-YgiT complex. Subsequently, the MqsR and YgiT genes were separately cloned into pBAD24 creating pBAD-MqsR and pBAD-YgiT, respectively. The promoter region of MqsRYgiT was amplified by PCR with RT-proF and RT-proR primers and cloned into pCR®2.1-Topo® vector (invitrogen).
Assay of in vivo DNA and protein synthesis—E. coli BL21(DE3) cells harboring pBAD-MqsR were grown in M9 medium with 0.5% glycerol (no glucose) and 1 mM of each amino acids except for methionine. When the O.D.600 value of the culture reached 0.3, arabinose was added to a final concentration of 0.2% to induce MqsR. Aliquots of the cell cultures (0.4 ml) were taken at time intervals as indicated in
RNA isolation and Northern blotting analysis—E. coli BL21(DE3) cells containing pBAD-MqsR were grown at 37° C. in M9 medium with 0.2% glycerol (no glucose). When the O.D.600 value reached 0.4, arabinose was added to a final concentration of 0.2%. The samples were taken at different intervals as indicated in
Primer extension analysis in vivo—For primer extension analysis of mRNA cleavage sites in vivo, total RNAs were extracted from the E. coli BL21(DE3) cells containing pBAD-MqsR at different time points after MqsR induction as indicated in
Protein purification—To purify N-terminal histidine-tagged MqsR (H-MqsR) and C-terminal histidine-tagged YgiT (YgiT-H), pET-MqsRYgiT and pET-YgiT were introduced into E. coli BL21(DE3). The expression of H-MqsRYgiT complex and YgiT-H was induced with 1 mM isopropyl-b-D-1-thiogalactoside (IPTG) for 3 h, respectively. The H-MqsRYgiT complex and YgiT-H were purified with Ni-NTA agarose (Qiagen) following the manufacture's protocol. Subsequently, the H-MqsRYgiT complex was denatured with 6M guanidine HCl. Denatured H-MqsR was then purified with Ni-NTA agarose and refolding of H-MqsR was carried out by stepwise dialysis as previously described for MazF (16).
Assay of protein synthesis in vitro—Cell-free protein synthesis was performed with an E. coli T7 S30 Extract System for Circular DNA (Promega). The reaction mixture was prepared as described in the manufacture's protocol. Then, different amounts of H-MqsR and YgiT-H were added in a final volume of 29 The reaction was started by the addition of pET11a-mazG plasmid DNA (18,37) and the mixture was incubated for 1 h at 37° C. Proteins were precipitated with acetone and analyzed by 15% SDS-PAGE. The dried gel was analyzed by autoradiography.
mRNA interferase activity of MqsR—MS2 phage RNA (Roche) was incubated with H-MqsR in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM dithiothreitol (DTT) at 37° C. for 10 min. In order to examine the antitoxin function of YgiT, H-MqsR was preincubated with YgiT-H for 10 min on ice and then further incubated with MS2 RNA for 10 min. After denaturation in urea, the products were separated on 1.2% agarose gel in 0.5×TBE buffer (44.5 mM Tris borate and 1 mM EDTA)(38).
Primer extension analysis in vitro—MS2 RNA was incubated with or without purified H-MqsR in 10 mM Tris-HCl (pH 8.0) containing 1 mM DTT at 37° C. for 15 min and the digested MS2 RNA (0.8 μg) was used for primer extension as described above.
Electrophoretic mobility shift assays (EMSA)—Complementary strands (Table 1) were annealed, and purified to get palindrome 1 and 2 double-stranded DNA, respectively. The double stranded DNA fragments were end-labeled with [g-32P]ATP by T4 kinase (Takara Bio). The binding reactions were carried out at 4° C. for 30 min in 50 mM Tris-HCl (pH 7.2) buffer containing 50 mM KCl, 5% glycerol, 100 ng poly(dI-dC), labeled DNA fragment and purified proteins. Electrophoresis was performed at 4° C. in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.2) at 110 V in 5% acrylamide/bisacrylamide (40:1.2) gel. After electrophoresis, the gel was dried and analyzed by autoradiography.(39).
Reverse transcription (RT)-PCR—Total RNA from E. coli was extracted at exponential phase (O.D.600 of 0.8) as described above and treated with 100 units of RNase-free DNase I (Promega) in the presence of 0.5 μl (20 units) RNase inhibitor (Roche). The RT reaction was carried out at 47° C. for 1 h using total RNA (20 μg) and the primer YT-Rv (20 μmol) with 10 units AMV-RT (Roche). PCR was carried out using the synthesized cDNA as template with RT-Fw and RT-Rv primers (Table 1).
The MqsR and YgiT genes are in an operon—The location of the MqsRYgiT operon at 68 min on the E. coli K-12 chromosome is shown in
The effect of MqsR on cell growth—The MqsR and YgiT genes were cloned into an IPTG inducible pET28a plasmid (Novagen) and an arabinose inducible pBAD24 plasmid (40), respectively. E. coli C43 cells harboring pET-MqsR and pBAD-YgiT could not form colonies on M9-glycerol-casamino acids agar plates in the presence of arabinose (0.2%)(
Next, we examined the effect of MqsR induction on protein synthesis as measured by [35S]methionine incorporation. Within 5 min of MqsR induction, the protein synthesis was almost completely inhibited (
In vivo cleavage of the ompA, ompF and lpp mRNAs by MqsR—Next, we examined the MqsR-mediated cleavage of the ompA, ompF and lpp mRNAs by primer extension experiments. Primer extension analysis of ompA, ompF and lpp using different primers identified distinct bands that appeared 2 min after induction of MqsR corresponding to the specific cleavage sites in each mRNA (Table 2 and
The mRNA interferase activity of MqsR in vitro—In order to obtain purified MqsR, N-terminal histidine-tagged MqsR(H-MqsR) was first expressed as the H-MqsRYgiT complex from the E. coli BL21(DE3) cells harboring pET-MqsRYgiT and the complex was purified with Ni-NTA agarose. Then, the purified H-MqsRYgiT complex was denatured using 6 M guanidine HCl. Denatured H-MqsR was re-trapped on Ni-NTA agarose, eluted and refolded by stepwise dialysis (16). C-terminal histidine-tagged YgiT (YgiT-H) was expressed in E. coli and purified as described in Experimental Procedures. The molecular mass of the purified H-MqsR, YgiT-H and H-MqsRYgiT complex were determined to be 26, 32 and 90 kDa by gel filtration, respectively (data not shown). The results suggests that both MqsR and YgiT exist as dimer and that the MqsRYgiT complex likely consists of two MqsR dimers and one YgiT dimer, which is also the case of the MazEMazF complex (16).
We next examined the effect of H-MqsR and H-MqsRYgiT on cell-free protein synthesis using an E. coli T7 S30 extract system (Promega). The synthesis of MazG protein was almost completely inhibited by 40 nM or higher concentration of MqsR (
To further prove that the in vivo cleavage of ompA, ompF and lpp mRNAs observed above was due to the mRNA interferase activity of MqsR, MS2 phage RNA (3569 bases) was cleaved with purified MqsR-H in vitro. The purified MqsR preparation clearly showed endoribonuclease activity (
The ability of MqsR to cleave RNA in the absence of ribosomes is distinctly different from RelE or YoeB whose mRNA interferase activities are dependent on ribosomes (12,18,41). The activity of MqsR activity was inhibited by MgCl2 (data not shown) as described previously for MazF (34).
In vitro cleavage site of MS2 RNA by purified MqsR—The in vitro MqsR activity on MS2 RNA was also analyzed by primer extension. The MS2 RNA was incubated at 37° C. for 10 min with MqsR. The product was used as template for primer extension. MqsR cleaved the MS2 RNA at five cleavage sites and the sequences of all of the cleaved sites were determined to be GCU (Table 2). Taken together, the results of the in vivo and in vitro primer extension experiments (
The binding of the MqsRYgiT complex to the MqsRYgiT promoter region—There are palindromic sequences in the promoter regions of many other TA systems including ccdAB (42,43), parDE (44), mazEF (45) and relBE (46). These antitoxins or toxin-antitoxin complexes bind to their cognate palindromic sequence to negatively regulate their own operons. Since there are two palindromic sequences in the 5′-UTR region of the MqsRYgiT operon (
As disclosed herein, we demonstrated that the MqsR and YgiT genes on the E. coli chromosome are co-transcribed and MqsR-YgiT is a new toxin-antitoxin system. In contrast to most of other TA systems, the first gene in the operon encodes the toxin, MqsR, and the second gene encodes the antitoxin, YgiT. Although MqsR has no homology to the well-characterized mRNA interferase MazF, which specifically cleaves at ACA sequences in mRNAs (29), MqsR was found to be an mRNA interferase that cleaved mRNAs at GCU sequences. Notably, MqsR is a ribosome-independent mRNA interferase like MazF, which is distinctly different from ribosome-dependent mRNA interferases such as RelE (12,46), YoeB (18) and HigB (47).
It has been reported that MqsR is induced during biofilm formation (1) and by the addition of quorum-sensing autoinducer-2, AI-2 (2). The activation of MqsR, in turn, activates a two-component system, qseBC, which is known to play an important role in biofilm formation (2). QseC is a sensor histidine kinase and QseB is a transcription regulator, which binds to the 5′-UTR region of the qseBC operon and activates transcription of this operon (48,49). The MqsR-YgiT complex is able to bind two palindromic sequences present in the 5′-UTR of the MqsRYgiT operon and seems to repress transcription of the MqsRYgiT. We examined the possibility that the MqsR-YgiT complex may also regulate expression of the qseBC operon. However, the H-MqsR-YgiT complex was unable to bind the qseBC promoter region including the QseB binding site (data not shown). Both palindromic sequences (palindrome 1 and 2;
We analyzed all the 4,226 ORFs on the E. coli genome (NCBI RefSeq; accession No. NC 000091) for the existence of the GCU sequences and found that there are only 14 ORFs which do not contain a single GCU sequence (Table 3). Out of these 14 genes, six genes, pheL, tnaC, trpL, yciG, ygaQ and ralR have been shown to be induced during biofilm formation in E. coli (50). Of special interest is YgaQ (330 bp), which is induced 32 fold in biofilms and has also been shown to be involved in the swarming mobility of E. coli (51). Since these genes are resistant to MqsR mRNA inteferase activity, MqsR induction during biofilm formation may inactivate all E. coli mRNAs except for these 14 genes, which in turn may play an important role in biofilm formation. Almost all cells die during biofilm formation in Pseudomonas aeruginosa (52). MqsR induction during biofilm formation may cause the cells to enter a quasi-dormant state similar to that caused by MazF (8,53), and eventually lead to cell death.
The discovery of the MqsR-YgiT system as a new TA system in E. coli in the present paper increases the total number of the E. coli TA systems to as many as 16, which includes MazF-MazE (16,34), RelE-RelB (12,13), ChpBK-ChpBI (14), YafQ-DinJ (21), YoeB-YefM (18,19), HipA-HipB (22,23), HicA-HicB (25,26), YhaV-PrIF (27) and YafO-YafN (24).
The present invention is not to be limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments that are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.
This application claims priority to U.S. Provisional Application No. 61/189,639, filed Aug. 20, 2008, the disclosure of which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/54503 | 8/20/2009 | WO | 00 | 5/13/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61189639 | Aug 2008 | US |
