Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator
© Kaw and Blumenthal; licensee BioMed Central Ltd. 2010
Received: 2 July 2010
Accepted: 19 November 2010
Published: 19 November 2010
Most type II restriction-modification (RM) systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease) initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene.
Expression levels of lacZ translational fusions to pvuIIR or pvuIIC were determined, with the native pvuII promoter having been replaced by one not controlled by C.PvuII. In-frame pvuIIC insertions did not substantially decrease either pvuIIC-lacZ or pvuIIR-lacZ expression (with or without C.PvuII provided in trans). In contrast, a frameshift mutation in pvuIIC decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of pvuIIR-lacZ was unaffected when the pvuIIC stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the pvuIIR initiation codon. Disrupting the putative hairpins had no significant effects.
The initiation of translation of pvuIIR appears to be independent of that for pvuIIC. Direct tests failed to detect regulatory rules for either gene overlap or the putative hairpins. Thus, at least during balanced growth, transcriptional control appears to be sufficiently robust for proper regulation of this RM system.
Bacterial type II restriction-modification (RM) systems are abundant in both the bacterial and the archaeal worlds . Many play important roles in defense against phage , but they also appear to modulate horizontal gene transfer , and to act as "selfish" toxin-antitoxin addiction modules [4, 5]. Type II RM systems generally specify separate DNA methyltransferase (MTase) and restriction endonuclease (REase) proteins . Many type II RM systems are on mobile genetic elements, but even chromosomal RM systems can move into new host cells via transduction, transformation or conjugation [7–10].
PvuII is a plasmid-borne type II RM system from the Gram-negative bacterium Proteus vulgaris . The MTase (M.PvuII) modifies the cognate DNA sequence CAGCTG by methylating the internal cytosine , generating N4-methylcytosine ; while the REase (R.PvuII) cleaves the central GpC if this sequence is unmethylated [13–15]. The REase and MTase act independently of each other in type II RM systems. As a result, strict regulation is needed after the genes enter a new cell in order to delay REase accumulation until the MTase has had time to protect the new host's DNA. The basis for this regulation is unknown for most RM systems.
A subset of type II RM systems are controlled by a third gene, that was designated as "C" (controller) protein when first discovered in the BamHI and PvuII systems [16, 17]. Sequence comparisons quickly identified orthologs in the SmaI and EcoRV systems , and since then C proteins have been identified (and in some cases confirmed) in a wide variety of other RM systems .
The transcriptional regulation of C-controlled RM systems is understood in outline, from the structure of the C proteins [19–22], through their action at conserved "C boxes" upstream of the C genes [7, 9, 23–27], to their dual function as activators and repressors  and possible interaction with RpoD (σ70) [7, 20]. The temporal behavior of one of these systems has also been studied, following its introduction into new host cells .
The RBS for pvuIIC is very poor (Figure 1B), based on the very limited similarity to an AGGAGG motif . The expected poor translation initiation would synergize with the lack of transcriptional autogenous activation by C.PvuII , by slowing the elongation of any transcripts that did get produced . In contrast, when C.PvuII eventually begins to accumulate, it activates transcription from the second promoter, rapidly boosting transcription of pvuIIC and pvuIIR via a positive feedback loop [7, 28]. Furthermore, the resulting C.PvuII-dependent transcript is leaderless , beginning at the pvuIIC initiator codon, and is thus independent of the RBS [34–36].
A key area of uncertainty in this model is whether transcription-level regulation is sufficient to protect the cell. Early transcription from the weak C-independent promoter appears to proceed into pvuIIR . If pvuIIR mRNA can be translated well, independent of the rate at which the upstream pvuIIC gene is translated, this might lead to unacceptably rapid early accumulation of restriction activity. Unlike pvuIIC, pvuIIR has an obvious RBS motif (Figure 1B). We accordingly investigated whether there is some form of translational control of pvuIIR.
Translational lacZ fusions to pvuIIC and pvuIIR
Assessing translational coupling of pvuIIC and pvuIIR
Nevertheless, previous findings  and the results from the WT translational fusions indicate that the translation of pvuIIR is very similar to that of pvuIIC under several conditions, consistent with their translation being coupled. Accordingly we further tested this possibility.
Frameshift mutation in pvuIIC reduces translation of pvuIIR
Esp19 is an in-frame insertion of one leucine codon into pvuIIC, within the first helix of the DNA-binding helix-turn-helix motif  (see Figure 3A, and Additional file 1, Figure S1). Like the "WT" fusions, the pvuIIC-Esp19 fusions gave similar translational activity in both reading frames. Furthermore, the levels of translation were fairly similar to those when pvuIIC was WT.
Cla35 is a frameshift mutation in pvuIIC that results in a termination codon ~70 nt upstream of the normal stop codon  (see Figure 3A, and Additional file 1, Figure S1). The new stop codon is UAG followed by A, which in one in vivo assay system gives a termination rate of about 50% . This mutation led to a roughly fivefold decrease in β-galactosidase expression, relative to the WT fusions, when lacZ was fused in either reading frame. This effect appears to be due to transcriptional polarity (see below), but the important point here is that pvuIIR- and pvuIIC- frame translation changed in parallel.
Quantitative real-time RT-PCR (QRT-PCR) reveals transcriptional polarity
The parallel fivefold drop in pvuIIC and pvuIIR translation in the pvuIIC- Cla35 frameshift mutant (Figure 6A) could reflect premature translation termination leading to premature transcript termination [43, 44]. To test this, relative lacZ mRNA levels were determined by QRT-PCR for the WT and both mutant pvuIIR-lacZ fusion strains (see lines 3-5 in Figure 3B). In the case of Cla35, the amount of lacZ mRNA dropped by the same fivefold amount as did translation (Figure 6B), consistent with transcriptional polarity (and, for that matter, with coupling between the rates of pvuIIC and pvuIIR translation).
However, surprisingly, the levels of lacZ mRNA dropped by about the same extent in the strains bearing an in-frame single-codon insertion (Esp19; Figure 6B). The junction sequences of the Cla35 and Esp19 fusions are identical (line 5, Figure 3B). Esp19 only minimally changed the level of pvuIIR-lacZ translation (Figure 6), but this implies that it substantially increased the apparent translational initiation at the downstream pvuIIR initiator, relative to WT, as there is only about an eighth as much mRNA (gray bars in Figure 6B).
Putative hairpins upstream of pvuIIR have no obvious effect on its translation
If this model is correct, altering Arm 2 (orange-shaded sequence in Figure 7A) should disrupt both hairpins and thus increase pvuIIR translation, while altering Arm 1 should disrupt the 5' hairpin, promote formation of the 3' hairpin, and thus decrease translation of pvuIIR. We made an in-frame deletion of Arm 2 from the WT construct ("Δ2" in Figure 7A, and Additional file 1, Figure S1) and tested the effects on a pvuIIR-lacZ translational fusion (Figure 7B). Rather than the predicted increase in translation, this deletion reduced expression somewhat. [Supplying WT C.PvuII in trans had no effect (as expected, since a C-independent promoter was being used).] In contrast, the ΔArm1 deletion (Figure 7A, and Additional file 1, Figure S1) was predicted to reduce pvuIIR-lacZ translation, but did so only mildly, and even then only in the presence of WT C.PvuII supplied in trans (Figure 7C).
In bacteria such as E coli, genes are often transcribed into polycistronic mRNAs . Ribosomes from an upstream gene can reinitiate translation at the next initiator in a process called translational coupling. The extent of dependence on reinitiation (as opposed to initiation by newly-bound ribosomes) varies, but tends to vary inversely with the distance between the termination and reinitiation codon . In most polycistronic operons, termination codons are close to the initiation codon of the downstream gene; in many cases they even overlap , as in the BcnI and AhdI R-M systems (Figure 2A). A high degree of coupling results in coordinated expression of the genes, as illustrated by the gal operon [43, 52].
In RM systems that rely on upstream C genes to mediate the delay in REase expression, allowing the MTase time to protect a new host's chromosome ; translational coupling could play an important role in the regulatory design. The C and R genes of another C-activated RM system, Esp1396I, are in fact translationally coupled . Furthermore, as shown in Figure 1, translation of pvuIIC from the early pvuIICR transcripts relies on a poor RBS (a feature not shared by most of the C genes). Translational coupling of pvuIIR to pvuIIC would ensure that very little R.PvuII is made from the early transcripts (though in rare cases transcripts lacking RBSs in the leader can still be translated efficiently ). Once C.PvuII begins to accumulate, it not only generates a positive feedback loop by activating a second promoter upstream of pvuIIC, but translation of these later transcripts should be much more efficient as they are leaderless  and do not rely on the weak RBS.
However our evidence provides no consistent evidence that the translation of pvuIIR is coupled to that of pvuIIC. Most significantly, moving the pvuIIC termination codon in either direction relative to the pvuIIR initiator (Figures 3B, 5) had no apparent effect. In contrast, coupled genes are quite sensitive to the stop-start codon spacing (e.g., see Additional file 1, Figure S2, replotted from data in ).
This lack of coupling is consistent with previous work from our laboratory. We had demonstrated a sharp decrease in the expression of pvuIIR in each of four pvuIIC null mutants , including two in-frame (EspI) and two frameshift (ClaI) mutants (two of which were used in the current study). These mutants showed greatly-impaired restriction of infection by unmethylated bacteriophage λ, or of transformation by plasmid DNA, and the in vitro R.PvuII activity was ~104-fold less than in strains that carried the intact parental plasmid. Providing pvuIIC in trans had no effect on the WT strain, but providing it to any of the pvuIIC mutants resulted in full restoration of in vivo or in vitro R.PvuII activity. However boosting transcription of frameshift mutants, that terminate translation ~70 nt upstream of the pvuIIR initiation codon (Figure 3A, and Additional file 1, Figure S1), would not have restored a flow of translating ribosomes to the pvuIIR initiator, so these results conflict with coupling.
C proteins themselves can have regulatory effects at the translational level. In the Eco72I RM system , providing C.Eco72I in trans to eco72IR-lacZ translational fusions restored REase gene expression even when the upstream eco72IC gene included a frameshift mutation. However, in the case of PvuII, neither pvuIIC-lacZ nor pvuIIR-lacZ translational fusions showed substantial effects of supplying C.PvuII in trans.
Hairpins in the RNA that occlude RBSs can reduce translation initiation [45, 47–49], though initiating ribosomes have the capacity to unfold mRNA  and may also be prepositioned to slide into place when an RNA hairpin spontaneously unfolds . In-frame deletions in pvuIIC, that would affect predicted alternative hairpins upstream of pvuIIR and thus alter the availability of the pvuIIR RBS (Figure 7A), had no major effect (Figure 7B, C). However, the hairpins might nonetheless reduce the entry of new ribosomes at the pvuIIR RBS, while not affecting progress of translating ribosomes. Searching for potential RBS-occluding hairpins in other C-dependent R-M systems showed that 9/11 had such hairpins with ΔG values ≤ -4.0 kcal/mol (Additional file 1, Figure S3). For comparison, the equivalent PvuII hairpin has a predicted ΔG of -4.6 kcal/mol.
The genes for RM systems are often associated with mobile genetic elements, and temporal control is critical following the entry of these genes into a new cell, to avoid restriction of the host's chromosome. The role of translation-level regulation in this process has not been well studied. In examining the PvuII RM system, we found that translation of the downstream REase gene (pvuIIR) is independent of that of the upstream gene for the autogenous activator/repressor (pvuIIC), at least under the conditions used. This independence was despite the overlapping genes and the presence of putative RNA hairpins involving the pvuIIR RBS. This suggests that the temporal control of PvuII transcription , together with the repair capabilities of the bacteria [55–57], is sufficiently robust to protect the new host cell following PvuII gene transfer.
Cloning and generation of mutants
Plasmids used in this study
WT pvuIIC gene with its own promoter, ΔpvuIIM,
ΔpvuIIR; TetR, pACYC177
pDK200 with pvuIIC- Esp19 in-frame insertion
pDK200 with pvuIIC- Esp33 in-frame insertion
pDK200 with pvuIIC- Cla35 frameshift mutation
WT PvuII R-M system; AmpR, pBR322
pPvuIIRM3.4 with pvuIIC- Esp19 in-frame insertion
Plasmid from ATCC (#87200) used to create translational fusions, to lacZ; AmpR, pMB1 origin
For this study, DNAs from WT pvuIIC, mutant Esp19 (with insertion of a leucine codon) and mutant Cla35 (frameshift insertion), were purified using the QIAprep plasmid kit (Qiagen, Germantown, MD). Oligonucleotide primers were purchased from Integrated DNA technology company (IDT, Coralville, IA). The amplified region began upstream of the pvuIIC ATG initiator and ended a few codons into pvuIIR (including the entire overlap between pvuIIC and pvuIIR, ~285 bp). High fidelity Pfx polymerase (New England Biolabs, Ipswich, MA) was used to amplify the fragment, which was then purified using a Qiagen gel extraction kit.
The amplified fragments were cloned into TOPO 2.1 vector (Invitrogen, Carlsbad, CA), and ligation products were used to transform TOP-10 chemically competent E. coli (Invitrogen). Transformants were plated onto LB agar with 100 μg/ml ampicillin. Colonies were inoculated into LB broth with appropriate antibiotics, and clones demonstrating the expected restriction pattern were confirmed by sequencing.
LacZ translational fusions
Inserts from sequence confirmed clones in TOPO 2.1 were again amplified using a second set of primers (black arrows in Figure 3A). The secondary amplification began with 'TG' of the ATG initiator of pvuIIC at the 5' end, and extended into pvuIIR (downstream of the pvuIIC termination codon). An EcoRI site was added to the 3' end of the insert. Plasmid pPvuIIRM3.4 carries the WT PvuII RM system in vector pBR322 , and pvuIIC contains unique restriction sites (ClaI and EspI/BlpI) that were used to generate mutants  (Figure 3A). Wild type and mutant versions of pPvuIIRM3.4 were used as templates. The amplified segments were cloned in-frame with lacZ in vector pLEX3B . PCR amplification used high fidelity Pfx polymerase (New England Biolabs). The EcoRI digested fragment was purified by electrophoresis and the Qiagen gel extraction kit. A similar protocol was followed for all three mutants and the wild type.
We prepared fusions in which the pvuIIC initiation codon was the optimal distance from the RBS of pLEX3B (ATCC # 87200, ATCC, Manassas, VA). Insert DNA was cloned between the XmnI and EcoRI site of the pLEX3B vector, such that on the 5' end 'A' of the XmnI site was blunt ligated to the 'TG' of the insert; the 3' end ligation used the EcoRI site. To facilitate converting these clones from pvuIIC to pvuIIR being in-frame with lacZ, a 30 bp oligonucleotide was acquired with a BglII site at the 5' end (sense strand) and EcoRI at the 3' end (see Figure 3B). In the middle was a unique XbaI site to facilitate identification of the desired clones. This 30 mer duplex was cloned between the BglII and EcoRI sites of the pvuIIR-lacZ fusions (see Figure 3B).
The WT pvuIIC translational fusion contains the full ORF and few codons from pvuIIR in-frame with lacZ. We removed the stop codon from the pvuIIC ORF. In the Cla35 frameshift mutant of pvuIIC, lacZ is in-frame with the shifted pvuIIC reading frame (though there are intervening nonsense codons). Two nucleotides (AG) were deleted to shift the frame, such that pvuIIR was in-frame with lacZ. This shifted the stop codon for pvuIIC by few codons relative to the pvuIIR initiator, so we used site directed mutagenesis to restore the stop codon to its original position by substitution. Primers (IDT) were designed for this purpose, to function with the Stratagene QuickII site-directed mutagenesis protocol and kit (Agilent, Santa Clara, CA).
Ligation products were used for transformation as described above, except that IPTG (1 mM), and X-gal (bromo-chloro-indolyl-galactopyranoside, 40 μg/ml) were also added to the agar. Transformants that turned blue were patched and also inoculated into LB broth with the appropriate antibiotics. Putative lacZ translational fusions were isolated and confirmed by restriction-digestion as well as colony PCR. Clones that demonstrated the desired restriction pattern were confirmed by sequencing.
We prepared in-frame arm1 and arm 2 deletions in the predicted hairpins (Figure 7A) using site-directed mutagenesis kit and protocols as described above. Again this set was confirmed by sequencing (MWG, High Point, NC).
We measured hydrolysis of the substrate ο-nitrophenyl-β-D-galactoside (ONPG). Overnight cultures were grown at 37°C in MOPS rich defined medium (TEKNOVA, Hollister, CA), in the presence of tetracycline (10 μg/ml) and the inducer IPTG (1 mM). This culture was diluted 1:100 into the same medium, without tetracycline, and grown to exponential phase. Samples were collected at regular intervals and the optical density (OD) at 600 nm was measured for every 1 ml sample. Two drops of chloroform and one drop of 0.1% SDS were added, and the sample vortexed for 10 s to open the cells. Samples were placed in a 28°C water bath for 5 min, and 200 μl ONPG (4 mg/ml) were added. Reactions were stopped with 500 μl 1 M Na2CO3, and OD420 nm was determined. The formula of Miller  was used to calculate units of β-galactosidase. Miller units were plotted against the culture OD600 nm. The slopes from linear regression indicate relative specific activity; linearity indicates that the culture was in balanced growth.
RNA isolation and quantitation
Cultures were grown as described above, to an OD 600 nm of ~0.3, in the presence of IPTG (1 mM). Samples were taken from the flasks and directly added to two volumes of RNA stabilization buffer (RNA Protect Bacteria Reagent, Qiagen, Valencia, CA). Samples were mixed and left at room temperature for 10 min. The RNeasy miniprep kit (Qiagen) was used to isolate total RNA. Cells in stabilization buffer were harvested by centrifugation at 4°C for 15 min at 5,000 rpm. Pellets were resuspended in 1× TE buffer containing lysozyme (400 μg/ml) after removing the supernatant. Ethyl alcohol was added to precipitate the RNA, which was then dissolved and loaded onto the columns. Following column washing and elution, RNA was treated with RQ1 RNAse-free DNAse (Promega, Madison, WI) to remove DNA. cDNA was synthesized using total RNA as template, random hexamers (Invitrogen), and ImPromII reverse transcriptase (Promega). The random primers were annealed at 25°C for 5 min, and the first strand was then extended at 42°C for 1 h. The reverse transcriptase was then inactivated by heating at 70°C for 1 h.
Red arrows in Figure 3A indicate the primer pair used to quantitate pvuIIC cDNA. Dilutions of cDNA were tested to determine the maximally-efficient concentration for amplification, and also the efficiency of each primer pair. recA expression was also measured, to provide an internal baseline. Cycle threshold (Ct) values were determined using a Roche Light Cycler with SYBR Green. Melting curve analysis was used to confirm the formation of specific products. The standard curve method was used to determine the relative amounts of mRNA, which were normalized to recA .
We thank Drs. Robert Lintner and Iwona Mruk for help with the real-time QRT-PCR, and Dr. Mruk for critically reviewing the manuscript. MKK was supported in part by graduate stipends from the University of Toledo and the Department of Medical Microbiology & Immunology. This material is based upon work supported by the US National Science Foundation under Grant No. MCB-0964728.
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