Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNAHis have distinctive identity elements, we constructed E. coli tRNAHis CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNAHis CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNAHis CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNAHis CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.


Introduction
Nonsense suppression (read-through of the stop codons UAG, UAA, or UGA) has been observed in many biological systems. Since nonsense mutations cause premature termination of translation, suppression of nonsense codons has been extensively applied in prokaryotes and lower eukaryotes for identifying gene functions by conditional expression of such genes [1,2]. Nonsense mutations in essential genes could be lethal. Moreover, eukaryotic messenger RNAs that contain premature termination codons are degraded through nonsense-mediated decay [3], and may be associated with many genetic disorders [4]. Therefore, nonsense suppression would rescue cells from deleterious effect of nonsense mutations. Genetic studies showed that nonsense suppression is often mediated by suppressor tRNAs with altered anticodons that now recognize nonsense codons and insert an amino acid in response. Such suppressors have been found in many organisms including E. coli [5], yeast [2], C. elegans [6,7], and human [8].
C. crescentus is an a-proteobacterium common in fresh water ecosystems. It has been widely used as a model organism for cell cycle and cellular differentiation studies because of its unique characteristics during cell division. Recently it was found that C. crescentus tRNA His lacks a universal feature, G-1, the major identity element recognized by HisRS in acylating tRNA His . Because of differences in the nature of the discriminator base and of position 72 of tRNA, and the presence or absence of G-1, the HisRS/ tRNA His pairs of C. crescentus and E. coli are orthogonal [9][10][11].
Therefore we reasoned that an E. coli tRNA His with a CUA anticodon (tRNA His CUA ) should act as a suppressor tRNA for UAG codons in C. crescentus in the presence of the E. coli HisRS (Fig. 1). To test our hypothesis, we decided to express three C. crescentus UAG-containing reporter genes encoding a b-lactamase, the fluorescent mCherry protein, and the C. crescentus xylonate dehydratase.

Strains and Plasmids
The strains, plasmids and primers in the study can be found in Tables S1 and S2. CB15DxylD was a gift of Craig M. Stephens, Santa Clara University. A lac promoter was prepared from pUC19 by PCR and cloned into pRVCHYN-5 [12], leading to pRV-lac2-mCherry. Ala184 of an ampicillin resistance gene, His22 of mCherry gene, His283 or His290 of xylD, and the codon encoding Phe3 of groES were mutated to amber codon by QuikChange (Stratagene).
The DNA sequence of E. coli HisRS was codon-optimized for C. crescentus by using JCat [13]. It was synthesized (GenScript) and inserted into pBXMCS-2 [12] or pBX-lac2. A precursor of E. coli tRNA His contains the sequence from -40 to +123 of tRNA His (G-1 is the first nucleotide of tRNA His ). Since E. coli tRNA His should be produced by correct processing of the precursor transcripts in C. crescentus, we constructed another transcription unit, tRNA His2 CUA , by replacing C. crescentus mature tRNA His from the precursor transcript of tRNA His with E. coli mature tRNA His . The 59 and 39 flanking regions of tRNA His2 remained those of C. crescentus sequence. The sequences of the precursor tRNA His are shown in Figure S1. The template for mature tRNA His was prepared by PCR.
The promoter sequence of C. crescentus ffs was amplified from the genomic DNA and a transcriptional terminator, rrnC, was obtained from pTECH [14]. For the expression of HisRS, tRNA His or tRNA His CUA , the appropriate fragments were combined as described in Fig. 2A. C. crescentus groES was inserted into SacII site of pBX-lac2-HisRS-tRNA His CUA by In-Fusion (Clontech).

Northern Blot Analysis
Ten microgram of total RNAs from C. crescentus were separated on a 1% agarose gel and transferred into Hybond-N+ membrane (GE Healthcare Life Sciences) by the semi-dry blotter (Bio-Rad). After 2 h hybridization in Rapid-hyb (GE Healthcare Life Sciences) with 0.2 pmol of 59 end-labeled HisR1 at 42uC, the membrane was washed twice with Solution I (2xSSC and 0.1% SDS) and then twice with Solution II (0.1xSSC and 0.1% SDS) at room temperature. The membrane was analyzed by autoradiography. The control E. coli tRNA His was prepared by in vitro transcription of pUCT7/tRNA His .

Viability Assays
All incubations were performed at 30uC. For ampicillin resistance, CB15NDbla6 was transformed with pRV-lac2-AmpR-TAG, which was constructed by Ala184TAG mutation on the ampicillin resistance gene in pRV-lac2-mCherry, by electroporation. Subsequently, pBX-derived plasmids in E. coli S17-1 were introduced into the strain by mating. The cells were cultured in PYE medium (peptone-yeast extract) containing 5 mg/ml of kanamycin (Kan) and 1 mg/ml of oxytetracycline (OxyTet) overnight. Then 3 ml of cultures were spotted onto the PYE plates containing 0.2% xylose, 20 mg/ml of Kan, 2 mg/ml of OxyTet and 50 mg/ml of ampicillin. For the growth curves, 200 ml of the diluted cultures (A 660 nm = 0.01) were further incubated in the same medium in presence or absence of 50 mg/ml of ampicillin. The absorbances at 660 nm were measured at every 30 min by using Synergy HT plate reader (BioTek). The assays were repeated at least three times.
For the xylose assay, CB15DxylD was transformed with either pRV-lac2-xylDTAG847 or pRV-lac2-xylDTAG868, and obtained the pBX-lac2-derived plasmids by mating as described above. The cells were cultured in PYE media with Kan and OxyTet overnight and then diluted to A 660 = 0.2 with the same media. The diluted cultures were further incubated for another 6 h and collected by centrifugation. The cells were washed twice and then resuspended with M2 minimal media. The cells were spotted onto M2 plates containing either 10 mM glucose (M2G) or 10 mM xylose (M2X). The plates were incubated at 30uC until cell growth was able to be observed. The assays were repeated twice.

Microscopy and Data Analysis
C. crescentus cells were grown at 30uC in M2G+ media (M2G supplemented with 1% PYE) to exponential phase (A 660 = 0.3-0.4) supplemented with 5 mg/ml of Kan and 1 mg/ml of OxyTet, and then induced by adding 0.03% xylose for 2-3 h before visualization. The PYE-supplemented M2G medium was used in order to increase growth rate without interfering the microscopic analysis.
Cells were immobilized on 1% agarose pads with M2G+ supplemented with Kan, OxyTet and 0.03% xylose. Cell imaging was performed at room temperature (,22uC) using a Nikon 80i microscope equipped with a 100X NA 1.4 phase contrast objective and a Hamamatsu Orca II-ER camera controlled by MetaMorph  software or a Nikon Ti-U inverted microscope with a 100X NA1.4 phase contrast objective and a Hamamatsu Orca ER camera. Data analysis was performed using the MATLAB-based software MicrobeTracker [15]. The experiments were repeated at least twice.

Purification of GroES and Mass Spectrometric Analysis
For the expression of C. crescentus groES, CB15N with pBX-lac2-HisRS-tRNA His CUA -groESTAG was grown and then the culture was diluted 1:200 with 1 L of PYE containing 5 mg/ml of Kan and then incubated at 30uC overnight. Then the temperature was raised to 40uC for 2 h. The protein was purified by Ni-NTA

Results
Expression of the E. coli HisRS/tRNA His Pair in C. crescentus The E. coli HisRS and tRNA His genes were introduced into C. crescentus with the constructed plasmids. For expression, the HisRS and tRNA His genes were inserted consecutively into the pBXMCS-2 plasmid with a xylose-inducible promoter (P xyl ) or the pBX-lac2 plasmid with a lac promoter (P lac ) ( Fig. 2A). The ffs promoter, which is derived from the constitutive promoter of the 4.5S RNA gene in C. crescentus [16], was inserted before the tRNA His genes. To make an amber suppressor, the anticodon of tRNA His was mutated to CUA. Because G-1 is a major identity element for E. coli HisRS [17], this nucleotide was included in all tRNA His constructs. The production of mature E. coli tRNA His in C. crescentus was confirmed by Northern blot analysis (Fig. 2B).

UAG Suppression by the E. coli HisRS/tRNA His CUA Pair
The orthogonality of the E. coli HisRS/tRNA His CUA pair and its efficiency for UAG (amber) suppression in C. crescentus were investigated using three reporter genes with an in-frame UAG codon. Since natural suppressor tRNAs are absent in C. crescentus   [18], the productions of full-length proteins indicate that successful suppression resulted from the E. coli HisRS/tRNA His CUA pair. First, a b-lactamase gene (bla) with a permissive amber mutation (Ala184TAG) [19,20] was introduced into the ampicillin-sensitive C. crescentus strain CB15NDbla6, which does not survive ampicillin concentrations at or greater than 50 mg/ml [21]. When E. coli HisRS and tRNA His CUA were expressed together, the cells survived 50 mg/ml of ampicillin in both solid (Fig. 3A) and liquid media (Fig. 3C), although the presence of ampicillin in the medium delayed the growth of the strain at lag phase (Figs. 3B and 3C). The other precursor construct of the suppressor, tRNA His2 -CUA , showed similar resistance, while the growth of the strain was slower than in the HisRS-tRNA His CUA -containing strain (Figs. 3A and 3C). All the strains grew at similar rates in the medium lacking ampicillin (Fig. 3B). Those results indicate that both constructs were able to produce active E. coli tRNA His in C. crescentus. The increase of ampicillin resistance suggests production of the active, full length b-lactamase resulted from amber suppression. On the other hand, neither E. coli HisRS nor tRNA His CUA alone increased ampicillin resistance (Figs. 3A and 3C). This indicates that the E. coli HisRS and tRNA His CUA did not cross-react with the host tRNAs and aminoacyl-tRNA synthetases, respectively.
Second, the efficiency of amber suppression was determined with the production of the fluorescent protein mCherry [22]. When wild-type mCherry was expressed from the plasmid pRV-lac2-mCherry, fluorescence was clearly detected (Fig. 4A) as expected. Introduction of an amber mutation (His22TAG) into the mCherry gene resulted in no fluorescence detected in cells (Fig. 4B). The co-expression with the E. coli HisRS/tRNA His CUA pair restored 462% (in two independent experiments) of the fluorescence intensities compared to the wild-type mCherry ( Fig. 4C and 4D). Although the E. coli HisRS/tRNA His CUA pair only partially restored the fluorescence, this result indicated effective amber suppression under no growth selection pressure. The growth rates of the strains that contained either pBXMCS-2 (the empty vector) or pBX-HisRS-tRNA His CUA were similar (Fig.  S2), which implies that the production of the HisRS/tRNA His CUA pair does not affect the protein synthesis significantly in the strain.
Third, the effect of amber suppression was evaluated by rescuing a C. crescentus xylD nonsense mutation. The C. crescentus xylD encodes a xylonate dehydratase that is essential for xylose metabolism. The C. crescentus xylD deletion mutant strain (CB15DxylD) cannot utilize xylose as a sole carbon source [23]. For complementation, two constructs of xylD with amber mutation (His283TAG and His290TAG) were individually introduced into the CB15DxylD strain pre-transformed with pBX-lac2-HisRS-tRNA His CUA for expression of the E. coli HisRS/tRNA His CUA pair. As we expected, all strains grew on M2G plates supplemented with glucose, but only the complementation strains expressing both the E. coli HisRS and tRNA His CUA were able to grow on M2X plates with xylose as the sole carbon source (Fig. 5). The strain containing xylDTAG847 (His283TAG) grew faster than that containing xylDTAG868 (His290TAG); this suggests that the suppression efficiency is different depending on the position of the amber mutation. The growth of the strain that contained HisRS alone seemed slower than those of the other strains (lane 2 in the M2G plate), however, in another sets of the experiment, the growth of the strain was similar to those of the other strains in both an M2G plate and PYE liquid medium (data not shown). GroES is a chaperonin that forms a complex with GroEL [24]. In C. crescentus, GroES is expressed from groESL operon and the expression of groES is elevated at 40uC [25]. We cloned C. crescentus groES including the intrinsic promoter and mutated the phenylalanine codon at the third position to amber codon. The mutated gene was expressed with E. coli HisRS/tRNA His CUA pair in C. crescentus. Histidine incorporation at amber codon was confirmed by LC-MS/MS analysis. Only a few ions were detected but they confirmed the fragment peptide, HRPLGDR, which contained histidine at the UAG codon of GroES (Fig. 6). We detected a histidine immonium ion from the N-terminus of the peptide, contributing to the identification of the fragment. We did not observe other amino acids at position 3 from the LC-MS/MS analysis.

Discussion
In this study, we demonstrated that the orthogonal E. coli HisRS/tRNA His CUA pair suppressed amber codons in C. crescentus. In three reporter systems, the amber suppression led to increased ampicillin resistance, production of the fluorescent protein mCherry, and complementation of xylD mutations. Based upon the fluorescence intensities of cells expressing mCherry, the suppression efficiency was about 4% (Fig. 4D). To account for the suppression efficiency, two factors are to be considered. First, alteration of the tRNA His anticodon to CUA decreases about 20fold decrease the in vitro aminoacylation efficiency (k cat /K m values) of E. coli HisRS [26]; thus tRNA His CUA is a poorer substrate than wild-type tRNA His . Second, the bases around termination codons have been suggested to affect the efficiencies of both termination [27,28] and suppression [29,30]. Indeed, the suppression efficiencies were different depending on the positions of amber mutations in xylD (Fig. 5). Therefore, the suppression efficiencies of mutant mCherry and other systems may also be influenced by context effects.
Orthogonal tRNA synthetase/tRNA pairs have been applied in bacterial and eukaryotic systems for translational incorporation of non-canonical amino acids (reviewed in [31][32][33]). Since the E. coli HisRS/tRNA His CUA pair is orthogonal in C. crescentus, this pair can serve as a new tool for protein engineering in the important model organism C. crescentus. In this organism protein constructs can be secreted to the cell surface by fusion of the recombinant protein with RsaA, an abundant surface layer protein in C. crescentus. This process facilitates high yield and convenient protein purification by filtration [34,35] and makes C. crescentus an ideal organism for production of some engineered proteins. Second, C. crescentus has been widely used for studying cell cycle, cell division, and cellular differentiations. These studies could be aided by the application of engineered E. coli HisRS/tRNA His CUA pairs that would efficiently acylate histidine analogs and lead to incorporation of noncanonical amino acids with desirable properties. Figure S1 The precursor tRNA His sequences. A. The sequence of tRNA His CUA that corresponds to E. coli precursor tRNA His from 240 to +123. The sequence of mature tRNA His is shown in uppercase. The anticodon for amber codon is labeled in red. B. The sequence of the tRNA His2 CUA . It contains the identical E. coli mature tRNA His sequence. In contrast to tRNA His CUA , the flanking sequences originated from the C. crescentus sequences. (PDF) Figure S2 rowth curves of CB15N. Both strains carried pRV-lac2-mCherryTAG as well as either empty vector (pBXMCS-2) or the vector containing HisRS and tRNA His CUA (pBX-HisRS-tRNA His CUA ). The growth curve in blue came from the strain with pBXMCS-2 and the growth curve of the strain that has pBX-HisRS-tRNA His CUA is shown in red. The cultures grew in PYE medium with 1 mg/ml oxytetracycline, 5 mg/ml kanamycin and 0.2% xylose. The error bars indicate standard deviations. (PDF)