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Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis
FEBS Letters 588 (2014) 1808–1812
journal homepage: www.FEBSLetters.org
Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis Stefani R. Mladenova, Kathryn R. Stein, Lianne Bartlett, Kelly Sheppard ⇑ Chemistry Department, Skidmore College, Saratoga Springs, NY 12831, USA
a r t i c l e
i n f o
Article history: Received 21 February 2014 Revised 17 March 2014 Accepted 18 March 2014 Available online 28 March 2014 Edited by Michael Ibba Keywords: tRNA RNA-dependent Asparagine biosynthesis Aspartyl-tRNA synthetase Staphylococcus aureus
a b s t r a c t The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNAAsn. The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNAAsn. S. aureus thus encodes both the direct and indirect routes for Asn-tRNAAsn formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The human pathogen Staphylococcus aureus in its genome does not encode either Asn synthetase (AsnA and AsnB) to amidate Asp to Asn but does encode an asparaginyl-tRNA synthetase (AsnRS) to directly ligate Asn to tRNAAsn [1]. However, the lack of an Asn synthetase does not prevent S. aureus from growing in the absence of Asn [2] suggesting it has as an alternate means of producing the amino acid. Numerous bacteria rely on an indirect two-step pathway to synthesize Asn in a tRNA-dependent manner enabled by an aspartyl-tRNA synthetase (AspRS) with relaxed tRNA specificity, recognizing both tRNAAsn as well as tRNAAsp [3]. As the first step in Asn-tRNAAsn synthesis, this non-discriminating AspRS (NDAspRS) forms Asp-tRNAAsn [4,5]. The Asp-tRNAAsn is then amidated to Asn-tRNAAsn by the amidotransferase GatCAB [4,6,7]. GatCAB in bacteria can also be used to synthesize Gln on tRNAGln in species lacking a glutaminyl-tRNA synthetase (GlnRS) [1,8–12]. GatCAB is present in S. aureus [13]. Given the presence of AsnRS but lack of Abbreviations: AsnRS, asparaginyl-tRNA synthetase; AspRS, aspartyl-tRNA synthetase; ND-AspRS, non-discriminating AspRS; D-AspRS, discriminating AspRS; GlnRS, glutaminyl-tRNA synthetase; ND-GluRS, non-discriminating glutamyl-tRNA synthetase; AsnA, asparagine synthetase A; AsnB, asparagine synthetase B; TrpA, tryptophan synthetase alpha subunit ⇑ Corresponding author. Address: Chemistry Department, Skidmore College, 815 North Broadway, Saratoga Springs, NY 12866, USA. Fax: +1 518 580 5139. E-mail address: [email protected] (K. Sheppard).
a GlnRS in S. aureus [1], the S. aureus GatCAB is predicted for only Gln-tRNAGln formation [14]. However, encoding AsnRS does not preclude an organism from also using GatCAB for tRNA-dependent Asn biosynthesis [3]. Three organisms are known to use both routes for Asn-tRNAAsn formation: Clostridium acetobutylicum, Deinococcus radiodurans, and Thermus thermophilus [4,6,12,15–18]. They encode in their genomes AsnRS, GatCAB, and at least two AspRSs. In these bacteria, one AspRS is a discriminating enzyme (D-AspRS) recognizing only tRNAAsp while the additional AspRS(s) is non-discriminating, acquired through horizontal gene transfer from archaea [4,6,12, 15–17]. Unlike those three bacteria, S. aureus uses only one AspRS. Given that S. aureus is an Asn prototroph [2] despite lacking an Asn synthetase [1], we predicted the lone S. aureus AspRS is nondiscriminating and works in concert with GatCAB to synthesize Asn-tRNAAsn. We demonstrate that S. aureus AspRS can aspartylate tRNAAsn and with GatCAB provides the organism a tRNAdependent route for Asn biosynthesis. 2. Materials and methods 2.1. General Oligonucleotides for PCR were from Integrated DNA Technologies (San Diego, California). S. aureus FPR3757 genomic DNA was
http://dx.doi.org/10.1016/j.febslet.2014.03.042 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
S.R. Mladenova et al. / FEBS Letters 588 (2014) 1808–1812
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obtained from American Type Culture Collection (Manassas, VA). Sequencing was carried out by the Yale DNA Analysis Facility on Science Hill (New Haven, CT). Nuclease P1, L-Asp, and L-Asn were from Sigma–Aldrich (St. Louis, MO). Tris and citrate buffered phenol, ATP, and chloroform were from Fischer Scientific. [a-32P]ATP (10 mmol/lCi) was from Perkin Elmer (Shelton, CT). Polyethylenimine (PEI)-cellulose thin layer chromatography (TLC) plates were from EMD Millipore (Billerica, MA). Restriction enzymes, Escherichia coli BL21(DE3) and NEB10b strains, OneTaq DNA Polymerase, and T4 DNA ligase were from New England Biolabs (Ipswich, MA). E. coli JF448 was from the Yale Coli Genetic Stock Center (New Haven, CT). E. coli trpA34 was a gift from the Söll Laboratory at Yale University (New Haven, CT).
studies with 5 nM AspRS were carried out at 37 °C with 111 nM 32Plabeled tRNA, 0–20.0 lM tRNA, and 4 mM L-Asp over 6 min. Steadystate kinetic studies with 5 nM AsnRS were carried out at 37 °C with 55 nM 32P-labeled tRNAAsn, 0–10.0 lM tRNAAsn, and 5 mM L-Asn over 6 min. Reactions were pre-incubated for 30 s at 37 °C before addition of enzyme and repeated 3 times. Time points were quenched, digested, separated by TLC, and processed as described previously [1,22,23] and repeated 3 times. For Michaelis–Menten kinetic constants, initial velocities vs. [tRNA] were plotted in KalediaGraph 3.1 (Synergy Software) and fitted to the Michaelis–Menten equation.
2.2. Over-production and purification S. aureus aaRSs
The S. aureus aspS was cloned into pCBS2 between the NdeI and BglII restriction sites (pCBS2-Sa-aspS) [24]. The pCBS2-Sa-aspS and two controls (D. radiodurans D-aspS and ND-aspS in pCBS2 [24], gifts from the Yale Söll Laboratory) were separately transformed into E. coli trpA34 cells. Cultures were grown overnight at 37 °C in LB with ampicillin (100 lg/ml). The overnight culture was used to inoculate 5.0 ml of M9 minimal media (1x M9 salts, 1 mM MgSO4, 0.4% D-glucose, 1 lg/ml thiamine, and 0.1 mM CaCl2) supplemented with all twenty amino acids (20 lg/ml each) and ampicillin (100 lg/ml). The cultures were grown shaking for 7 h at 37 °C. The cultures were centrifuged at 1500g for 3 min. To remove excess Trp, the cell pellet was resuspended in M9 minimal media (1.0 ml) with ampicillin (100 lg/ml) before centrifugation at 1500g for 3 min. The process was repeated twice. After the final spin, the cell pellets were resuspended in M9 minimal media (200 lL) with ampicillin (100 lg/ml) before being spotted (2.0 lL) onto M9 minimal media agar plates supplemented with ampicillin (100 lg/ml) and 19 amino acids (20 lg/ml) with or without L-Trp (20 lg/ml). The plates were incubated at 37 °C and scored daily for growth.
S. aureus aspS was cloned between the NdeI and BamHI restriction sites in pET28a to be N-terminally His6-tagged. The AspRS was overproduced using the previously described autoinduction method [19]. The cells were pelleted by centrifugation at 2000g for 25 min and then resuspended in wash buffer (25 mM HEPES– KOH, pH 7.2, 250 mM NaCl, 10 mM imidazole, and 5 mM 2-mercaptoethanol) with 0.1% Triton X-100 and one protease inhibitor tablet (Pierce, #8861). The cell suspension was sonicated for 5 min total, alternating 2 s on and 2 s off, at 40% amplitude (Fisher Sonic Dismembrator). The homogenate was centrifuged at 21 000g for 45 min. The supernatant was added to nickel-nitrilotriacetic acid-agarose slurry and AspRS was purified according to manufacturer’s protocols (Qiagen). Following elution in wash buffer with 250 mM imidazole, AspRS was dialyzed in storage buffer (25 mM HEPES–KOH, pH 7.2, 30 mM KCl, 0.2 mM EDTA, 10% glycerol, and 5 mM 2-mercaptoethanol) overnight at 4 °C, concentrated using Amicon Ultra centrifugal filter units, and then dialyzed in storage buffer with 50% glycerol at 4 °C overnight and stored at 20 °C. The S. aureus asnS was chemically synthesized with optimized codons for over-production in E. coli (Life Technologies GeneArt) and subcloned into pET28a between the NdeI and BamHI sites. AsnRS was over-produced and purified as described for AspRS. The enzyme preparations were determined >95% pure by Coomassie-stained polyacrylamide gel. 2.3. In vitro transcription The S. aureus tRNA genes were chemically synthesized with a T7 promoter (Integrated DNA Technologies), in vitro transcribed and purified as described for the D. radiodurans tRNAs [17]. 2.4. tRNA folding and
32
P labeling
tRNAs were unfolded at 95 °C for 5 min and slowly cooled to room temperature. At 65 °C, MgCl2 was added to a 5 mM final concentration. After folding, tRNA was stored at 20 °C. Aliquots of tRNA were 32P-labeled using the E. coli CCA-adding enzyme as described previously [1]. 2.5.
32
P-based tRNA aminoacylation assay
Aminoacylation of tRNAAsn and tRNAAsp was monitored using the established 32P-based assay [1,20–23]. The aminoacylation reactions contained 50 mM HEPES–KOH, pH 7.2, 30 mM KCl, 15 mM MgCl2, 5 mM DTT and 4 mM ATP. For plateau aminoacylation of tRNA with S. aureus AspRS, reactions were carried out at 37 °C with 1.0 lM 32P-labeled tRNA, 19.0 lM tRNA, 4 mM L-Asp, and 3.0 lM enzyme. For plateau aminoacylation of tRNA with S. aureus AsnRS, reactions were carried out at 37 °C with 1.0 lM 32P-labeled tRNA, 19.0 lM tRNA, 5 mM L-Asn, and 3.0 lM AsnRS. Steady-state kinetic
2.6. E. coli trpA34 in vivo assay
2.7. E. coli JF448 in vivo assay The S. aureus aspS and gatCAB were fused into an artificial operon as described previously [11,17]. The artificial operon was subcloned into the pCBS2 plasmid between the NdeI and BglII restriction sites (pCBS2-Sa-aspS-gatCAB). pCBS2-Sa-aspS and pCBS2-Sa-aspS-gatCAB were separately transformed into E. coli JF448 cells. S. aureus pCBS2-aspS was also transformed into NEB10b cells to serve as an additional positive control. Cultures were grown shaking overnight at 37 °C in LB with ampicillin (100 lg/ ml). The starter culture was used to inoculate M9 minimal media supplemented with all twenty amino acids (20 lg/ml each) and ampicillin (100 lg/ml). The cultures were grown shaking for 4 h at 37 °C and centrifuged for 5 min at 1500g. The cell pellets were washed three times to remove excess L-Asn as described above for the trpA34 system. Afterwards, the samples were resuspended with M9 minimal media (1.0 ml) supplemented with ampicillin (100 lg/ml) and then diluted with M9 minimal media to 0.45 O.D.600. The samples were further diluted 100-fold with M9 minimal media. Aliquots of culture (2.0 lL) were spotted onto M9 minimal media agar plates supplemented with ampicillin (100 lg/ml) and 19 amino acids (20 lg/ml) in the presence or absence of L-Asn (20 lg/ml). The plates were incubated at 37 °C and scored daily. 3. Results 3.1. Sequence prediction unclear as to S. aureus AspRS tRNA specificity Bacterial GatCABs distinguish their tRNA substrates from other tRNA species in a cell by recognizing the U1-A72 base pair found in
S.R. Mladenova et al. / FEBS Letters 588 (2014) 1808–1812
(A)
(B)
S. aureus s AspRS
50 40 30
Asp
tRNA A
Asn
A tRNA
20 10 0 0
10
20
30
40
Percent aminoacylated y (%) ( )
many bacterial tRNAAsn and tRNAGln isoacceptors [13,14]. S. aureus tRNAAsn retains the U1-A72 base pair [13,14], consistent with GatCAB being used for Asn biosynthesis. However, the presence of an U1-A72 pair in tRNAAsn is not an automatic indicator that the organism synthesizes Asn on tRNAAsn. In many bacterial species without a ND-AspRS, tRNAAsn retains the U1-A72 base pair including a number of bacteria that do not encode GatCAB as well as bacteria that use GatCAB for only Gln-tRNAGln formation [14]. Given ancestral bacteria likely used the indirect two-step pathway for Asn-tRNAAsn formation [25], the U1-A72 in tRNAAsn in these modern bacteria is likely vestigial. In a similar fashion, the elements GatCAB discriminates against in tRNAAsp are retained in many bacteria that lack a ND-AspRS [14]. For tRNA-dependent Asn biosynthesis, bacteria must encode GatCAB, a tRNAAsn with a U1A72 base-pair, and a ND-AspRS. Studies of the Helicobacter pylori and the Pseudomonas aeruginosa ND-AspRSs implicated four residues (equivalent to Leu31, Glu84, Thr85 and Ile90 in the S. aureus sequence) in the anticodon recognition domain that enable bacterial ND-AspRSs to recognize tRNAAsn [26,27]. We therefore aligned the S. aureus, H. pylori, and P. aeruginosa AspRS sequences along with a representative sampling of bacterial AspRSs with predictable tRNA specificities to determine if the discriminating nature of the S. aureus enzyme could be predicted from its sequence (Fig. 1). The AspRS of a species was predicted to be discriminating if the organism’s genome coded for AsnRS but not GatCAB or if tRNAAsn did not have a U1A72 base pair when both AsnRS and GatCAB were present. NDAspRS was predicted when AsnRS was not encoded in the genomes but GatCAB was. As in a previous study [27], AspRS sequences were excluded when a species encoded both AsnRS and GatCAB in its genome when tRNAAsn had a U1-A72 base pair. Unfortunately, it was unclear from the alignment if the S. aureus AspRS was non-discriminating or discriminating. Leu31 in the S. aureus AspRS is found in many bacterial D-AspRS enzymes while in most ND-AspRS enzymes it is replaced with a His. Mutation of the His in the P. aeruginosa ND-AspRS to Leu increased specificity towards tRNAAsp by 3.5-fold using E. coli tRNA though no difference was observed when P. aeruginosa tRNA was used as a substrate [26]. However, Leu at that position is also found in the ND-AspRSs from Thermatoga maritima and Streptomyces avermitilis (Fig. 1). Glu84 and Ile90 are found in both discriminating and non-discriminating AspRS enzymes. Thr85 is found in some other ND-AspRS sequences. However, the role of Thr at that position has not been
Percent aminoacylated (%)
1810
S. aureuss AsnRS
40 30
tRNA AAsp
20
Asn
A tRNA 10 0 0
20
10
time (min.)
30
40
time ((min.)
Fig. 2. Aminoacylation of in vitro transcribed S. aureus tRNAAsp (s) and tRNAAsn (d) by either (A) S. aureus AspRS or (B) S. aureus AsnRS. Reactions were carried out at 37 °C with 1.0 lM 32P-labeled tRNA, 19 lM tRNA, 4 mM ATP, and 3.0 lM enzyme. For the AspRS reactions 4 mM L-Asp was included, while for the AsnRS reactions 5 mM L-Asn was included.
studied, only Leu and Asn [27]. In summary, Leu31 suggests the S. aureus AspRS may be discriminating while Thr85 suggests it may be non-discriminating, and Glu84 and Ile90 are ambiguous as to the tRNA specificity of the S. aureus enzyme. 3.2. In vitro aminoacylation of tRNAAsp and tRNAAsn Given the unclear sequence alignment prediction as to the S. aureus AspRS tRNA specificity, we purified recombinant protein from E. coli (Supplemental Fig. 1) to test whether the AspRS could aminoacylate in vitro transcribed S. aureus tRNAAsn and tRNAAsp. To monitor Asp-tRNA formation, we used the established 32P-based assay [1,20–23]. The S. aureus AspRS was able to aspartylate both tRNAAsp and tRNAAsn (Fig. 2A), indicative of the enzyme being non-discriminating. Like other ND-AspRS enzymes [17,27,28], the S. aureus ND-AspRS has a slight preference (1.5-fold) for tRNAAsp over tRNAAsn (Table 1). In contrast, D-AspRS enzymes prefer tRNAAsp to tRNAAsn by 500–2250-fold [17,28]. As expected, tRNAAsn is also a substrate for the S. aureus AsnRS (Fig. 2B). S. aureus tRNAAsn is a slightly better substrate for AsnRS than ND-AspRS (1.7-fold) (Table 1). The AspRS has a higher kcat (4.7-fold) but also a higher KM (7.8-fold) than AsnRS with tRNAAsn as a substrate. The lower AsnRS kcat may explain the lower plateau tRNAAsn aminoacylation level than that of AspRS (Fig. 2).
D-AspRS C. perfringens E. coli P. gingivalis S. boydii T. thermophilus V. cholera
29 15 29 11 29 28
S. aureus
29 RDLGGLIFVDLRDREGIVQVVFNP-AFSEEALKIAETVRSEYVVEVQGTVTKRDPETVNPKIK
H. pylori N. meningitis P. aeruginosa R. belli S. avermitilis S. meliloti T. maritima T. tengcongensis ND-AspRS
28 28 90 31 29 29 29 29
RNLGGLQFIDLRDREGILQVVFND-DLGEEILEKAKSIRPEYCIAVTGEIVKR--ESVNPNMP RDLGSLIFIDMRDREGIVQVFFDP--DRADALKLASELRNEFCIQVTGTVRARDEKNINRDMA RRMGGMTFVDLRDRYGTTQLVFNRDSAPAELCDRAEDLGREWVIRATGTVMER--SSKNPNIP RDLGSLIFIDMRDREGIVQVFFDP--DRADALKLASELRNEFCIQVTGTVRARDEKNINRDMA RDLGGLIFLDLRDREGLVQLVAHP---ASPAYATAERVRPEWVVRAKGLVRLR--PEPNPRLA RDLGGLIFIDMRDREGIVQVVVDP--DMADVFAVANQLRSEFCIKLTGEVRARPESQVNKEMA
RDHGGVVFIDLRDKSGLVQLVCDP---SSKAYEKALEVRSEFVLVAKGKVRLRGAGLENPKLK RDHGGVIFIDLRDREGIVQVVIDP--DTPEAFAAADSSRNEYVLSITGRVRNRPEGTTNDKMI RDHGGVIFLDVRDREGLAQVVFDP--DRAETFAKADRVRSEFVVKITGKVRLRPEGARNPNMA RDHGNLVFVDLRDHYGITQIVFTD--QNPQLMDDASRLRYESVVTVIGKVVARSEETINNTLP RDLGGILFIDLRDHYGITQLVARP---GTPAYEALDKVSKESTVRVDGKVVSRGTENVNPDLP RDHGGVLFIDLRDHYGMTQVVADP---DSPAFKTAETVRGEWVIRVDGAVKARTDDTVNKNMP RDLGGVRFIDLRDRYGETQIVCDV---NSEAYSVVDELTRESVVLVEGTVRKRPEGTENPNIE RDHGGLVFIDLRDRTGIVQIVFSE-QVSKEVFEKVQSVRSEYVLAVEGEVVKRLPENVNPKIP
Fig. 1. Sequence alignment of representative bacterial AspRS anticodon binding domains. A phylogentically diverse sampling of 152 bacterial AspRS sequences were aligned in Geneious v. 4.7.4. For clarity only a select sampling of sequences is shown. D-AspRS enzymes (orange) were grouped together (38 total sequences). A D-AspRS was predicted based on either previous biochemical evidence or if the bacteria encoded AsnRS but not GatCAB in its genome. A D-AspRS was also predicted when AsnRS was present and tRNAAsn did not have a U1-A72 base pair. ND-AspRS enzymes (green) were similarly grouped together (82 sequences). A ND-AspRS prediction was based on either biochemical evidence or if the bacteria encoded GatCAB but not AsnRS. AspRS sequences (32 total) were excluded from the analysis due to their tRNA specificity being unclear based on genomic content as these organisms encoded both AsnRS and GatCAB as well as a tRNAAsn with a U1-A72 base pair. The four residues implicated previously [26,27] in AspRS recognition of tRNAAsn are in red. Shading is based on sequence similarity for the overall alignment in Geneious.
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S.R. Mladenova et al. / FEBS Letters 588 (2014) 1808–1812 Table 1 Aminoacylation kinetics of S. aureus AspRS and AsnRS at 37 °C. kcat (s
1
)
KM (lM)
kcat/KM (s
+ Asn 1
lM 1)
L*
- Asn
1 2
AspRS tRNAAsp tRNAAsn
0.24 ± 0.07 0.20 ± 0.03
2.4 ± 1.3 3.1 ± 1.3
AsnRS tRNAAsn
0.043 ± 0.004
0.40 ± 0.19
(10 ± 6) 10 2 (7 ± 3) 10–2
1.5 1.0
2
1.7
(11 ± 5) 10
L = Specificity relative to the catalytic efficiency of AspRS with tRNAAsn as a substrate, (kcat/KM)/(kcat/KM) of AspRS for tRNAAsn. Experiments were repeated three times and standard deviations are reported.
*
+ Trp
- Trp
1
3 4 5 Fig. 4. Co-production of S. aureus AspRS and GatCAB rescues the Asn auxotrophy of E. coli JF448. E. coli JF448 was transformed with pCBS2 coding for either (1) the D. radiodurans ND-aspS and gatCAB or (2) the D. radiodurans D-aspS and gatCAB as positive and negative controls, respectively. (3) E. coli NEB10b, an Asn prototroph, was transformed with pCBS2-S. aureus aspS as an additional positive control. E. coli JF448 was also transformed with pCBS2 encoding either (4) the S. aureus aspS alone or (5) the S. aureus aspS and gatCAB. The resultant strains were grown in triplicate on M9 minimal media agar plates in the presence (+Asn) or absence of Asn ( Asn) at 37 °C for three days.
2 3 Fig. 3. The E. coli trpA34 strain transformed with S. aureus aspS is able to grow in the absence of Trp. E. coli trpA34 was transformed with pCBS2 coding for either (1) the ND-aspS from D. radiodurans, (2) the D-aspS from D. radiodurans, or (3) the aspS from S. aureus. The cells were grown in triplicate on M9 minimal media agar plates in the presence (+Trp) or absence ( Trp) of Trp at 37 °C for three days.
3.3. Asp-tRNAAsn formation in vivo To test whether the S. aureus AspRS recognizes modified tRNAAsn in vivo, we used the established trpA34 E. coli complementation assay [24,29]. The trpA gene encodes the a-subunit of Trp synthetase (TrpA) and Asp60 is essential for activity. The E. coli trpA34 mutant strain is a Trp auxotroph due to mutation of codon 60 from an Asp codon to an Asn codon [30]. ND-AspRS production in the strain rescues the Trp auxotrophy as the enzyme forms AsptRNAAsn, a missense suppressor, enabling decoding of Asn codons with Asp and synthesis of active TrpA (Fig. 3) [24,29]. The assay thus provides an in vivo test as to whether an AspRS can recognize tRNAAsn [24,29]. We therefore subcloned the S. aureus aspS into pCBS2 and transformed the vector into the trpA34 strain. The S. aureus aspS supported growth in the absence of Trp (Fig. 3), consistent with the S. aureus AspRS being non-discriminating and able to form the missense suppressor Asp-tRNAAsn. 3.4. Rescue of E. coli Asn auxotroph We hypothesized S. aureus encodes a ND-AspRS to form the Asp-tRNAAsn required for tRNA-dependent Asn biosynthesis. Like other bacterial GatCABs [1,6,9–11,16], the S. aureus GatCAB can in vitro amidate Asp to Asn on tRNAAsn [31]. To determine in vivo whether S. aureus AspRS and GatCAB can work in concert to synthesize Asn, we co-produced both enzymes in E. coli JF448 [17,32], an Asn auxotrophic strain due to mutations to both the AsnA and AsnB genes [32]. Co-production of a ND-AspRS and GatCAB rescues the phenotype as Asn is synthesized on tRNAAsn (Fig. 4) [17]. Consistent with S. aureus synthesizing Asn in a tRNA-dependent manner, co-production of the S. aureus AspRS and GatCAB rescued the Asn auxotrophy of the JF448 strain (Fig. 4). 4. Discussion The S. aureus AspRS has relaxed tRNA specificity, recognizing tRNAAsn almost as well as tRNAAsp, and can work with GatCAB to
synthesize Asn-tRNAAsn. The presence of a ND-AspRS and GatCAB to synthesize Asn in a tRNA-dependent manner explains why S. aureus is not an Asn auxotroph [2] despite lacking AsnA and AsnB [1]. As Asn is made on tRNAAsn, the pathway allows S. aureus to directly couple Asn biosynthesis with its use during translation. Given the presence of AsnRS, S. aureus encodes the indirect and direct pathways for Asn-tRNAAsn formation like C. acetobutylicum, D. radiodurans, and T. thermophilus [4,6,12,15–17]. The role of AsnRS in S. aureus is unclear since the bacterium does not synthesize Asn in a tRNA-independent manner and the indirect pathway could presumably meet the cellular demands for Asn-tRNAAsn. It is tempting to speculate that when S. aureus is growing on humans it uses AsnRS to take advantage of the available Asn in human sweat and blood plasma (on average 164 and 62 lM, respectively) [33]. Under this scenario, the indirect pathway would meet the basal needs for Asn-tRNAAsn when S. aureus is growing away from a host, while the AsnRS would allow S. aureus to take advantage of the environmentally available Asn when growing on a host. AsnRS may also be used to recycle Asn from degraded proteins as predicted for T. thermophilus and D. radiodurans [4,6,17]. It is unclear how production of ND-AspRS, AsnRS, ND-GluRS and GatCAB in S. aureus is regulated to meet the cellular demands for Asn-tRNAAsn and Gln-tRNAGln. Upon infecting a mammalian host, S. aureus gatC expression doubles, while the expression of the genes coding for GatA, GatB, AsnRS, ND-AspRS, and ND-GluRS remains unchanged [34]. In C. acetobutylicum, one set of ND-AspRS and GatCAB genes are in an operon together regulated by a Tbox riboswitch that may indirectly monitor Asn levels in the cell that may be tied to the organism switching from acidogenesis to solventogenesis [12]. The regulatory control may also explain why C. acetobutylicum acquired additional aspS genes that are not needed in S. aureus. In S. aureus, the lone aspS is in an operon with hisS coding for histidyl-tRNA synthetase, not gatCAB. The lack of a signature sequence in bacterial AspRSs to differentiate a D-AspRS from a ND-AspRS makes it difficult to establish how many other bacteria use both routes for Asn-tRNAAsn formation when tRNAAsn has a U1-A72 base pair [1]. For D-AspRS to evolve in an organism, it must encode an AsnRS in its genome to aminoacylate tRNAAsn. Given the distribution of AsnRS in bacteria [1,35], it is likely D-AspRS is a case of convergent evolution with tRNA specificity for tRNAAsp evolving separately in different bacterial lineages. Clarifying the discriminating nature of bacterial AspRSs will help in understanding how Asn metabolism and use of the amino acid in protein synthesis are integrated into bacterial life cycles.
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Acknowledgments We thank Ariel Alperstein, Mark Benhaim, Melanie Feen, Nilendra Nair, Brittany Ulrich, and Sanida Lukovic at Skidmore College for support. We also thank at Skidmore College Dr. Sarita Lagalwar and Dr. Sylvia McDevitt for critical discussions, and Dr. Patricia Hilleren for use of her Storm 820 phosphorimager. This work was supported by the National Science Foundation (MCB1244326 to K.S.) and the Skidmore Schupf Scholars Program (to K.R.S.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2014.03. 042. References [1] Sheppard, K., Akochy, P.M., Salazar, J.C. and Söll, D. (2007) The Helicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNAGln. J. Biol. Chem. 282, 11866– 11873. [2] Charles, B.G. and Rawal, B.D. (1976) Cultivation of Staphylococcus aureus in a synthetic medium of low ultraviolet absorptivity. Antimicrob. Agents Chemother. 10, 915–917. [3] Yuan, J., Sheppard, K. and Söll, D. (2008) Amino acid modifications on tRNA. Acta Biochim. Biophys. Sin. (Shanghai) 40, 539–553. [4] Becker, H.D. and Kern, D. (1998) Thermus thermophilus: a link in evolution of the tRNA-dependent amino acid amidation pathways. Proc. Natl. Acad. Sci. USA 95, 12832–12837. [5] Cathopoulis, T., Chuawong, P. and Hendrickson, T.L. (2007) Novel tRNA aminoacylation mechanisms. Mol. BioSyst. 3, 408–418. [6] Becker, H.D., Min, B., Jacobi, C., Raczniak, G., Pelaschier, J., Roy, H., Klein, S., Kern, D. and Söll, D. (2000) The heterotrimeric Thermus thermophilus AsptRNAAsn amidotransferase can also generate Gln-tRNAGln. FEBS Lett. 476, 140– 144. [7] Curnow, A.W., Ibba, M. and Söll, D. (1996) tRNA-dependent asparagine formation. Nature 382, 589–590. [8] Curnow, A.W., Hong, K., Yuan, R., Kim, S., Martins, O., Winkler, W., Henkin, T.M. and Söll, D. (1997) Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation. Proc. Natl. Acad. Sci. USA 94, 11819–11826. [9] Raczniak, G., Becker, H.D., Min, B. and Söll, D. (2001) A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA in Chlamydia trachomatis. J. Biol. Chem. 276, 45862–45867. [10] Salazar, J.C., Zúñiga, R., Raczniak, G., Becker, H., Söll, D. and Orellana, O. (2001) A dual-specific Glu-tRNAGln and Asp-tRNAAsn amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans. FEBS Lett. 500, 129–131. [11] Wu, J., Bu, W., Sheppard, K., Kitabatake, M., Kwon, S.T., Söll, D. and Smith, J.L. (2009) Insights into tRNA-dependent amidotransferase evolution and catalysis from the structure of the Aquifex aeolicus enzyme. J. Mol. Biol. 391, 703–716. [12] Saad, N.Y., Schiel, B., Brayé, M., Heap, J.T., Minton, N.P., Dürre, P. and Becker, H.D. (2012) Riboswitch (T-Box)-mediated control of tRNA-dependent amidation in Clostridium acetobutylicum rationalizes gene and pathway redundancy for asparagine and asparaginyl-tRNAAsn synthesis. J. Biol. Chem. 287, 20382–20394. [13] Nakamura, A., Yao, M., Chimnaronk, S., Sakai, N. and Tanaka, I. (2006) Ammonia channel couples glutaminase with transamidase reactions in GatCAB. Science 312, 1954–1958.
[14] Bailly, M., Giannouli, S., Blaise, M., Stathopoulos, C., Kern, D. and Becker, H.D. (2006) A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine. Nucleic Acids Res. 34, 6083–6094. [15] Becker, H.D., Reinbolt, J., Kreutzer, R., Giegé, R. and Kern, D. (1997) Existence of two distinct aspartyl-tRNA synthetases in Thermus thermophilus. Structural and biochemical properties of the two enzymes. Biochemistry 36, 8785–8797. [16] Curnow, A.W., Tumbula, D.L., Pelaschier, J.T., Min, B. and Söll, D. (1998) Glutamyl-tRNAGln amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis. Proc. Natl. Acad. Sci. USA 95, 12838– 12843. [17] Min, B., Pelaschier, J.T., Graham, D.E., Tumbula-Hansen, D. and Söll, D. (2002) Transfer RNA-dependent amino acid biosynthesis: an essential route to asparagine formation. Proc. Natl. Acad. Sci. USA 99, 2678–2683. [18] Saad, N.Y., Stamatopoulou, V., Brayé, M., Drainas, D., Stathopoulos, C. and Becker, H.D. (2013) Two-codon T-box riboswitch binding two tRNAs. Proc. Natl. Acad. Sci. USA 110, 12756–12761. [19] Studier, F.W. (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234. [20] Wolfson, A.D. and Uhlenbeck, O.C. (2002) Modulation of tRNAAla identity by inorganic pyrophosphatase. Proc. Natl. Acad. Sci. USA 99, 5965–5970. [21] Bullock, T.L., Uter, N., Nissan, T.A. and Perona, J.J. (2003) Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants. J. Mol. Biol. 328, 395–408. [22] Sheppard, K., Akochy, P.M. and Söll, D. (2008) Assays for transfer RNAdependent amino acid biosynthesis. Methods 44, 139–145. [23] Silva, G.N., Fatma, S., Floyd, A.M., Fischer, F., Chuawong, P., Cruz, A.N., Simari, R.M., Joshi, N., Kern, D. and Hendrickson, T.L. (2013) A tRNA-independent mechanism for transamidosome assembly promotes aminoacyl-tRNA transamidation. J. Biol. Chem. 288, 3816–3822. [24] Min, B., Kitabatake, M., Polycarpo, C., Pelaschier, J., Raczniak, G., Ruan, B., Kobayashi, H., Namgoong, S. and Söll, D. (2003) Protein synthesis in Escherichia coli with mischarged tRNA. J. Bacteriol. 185, 3524–3526. [25] Sheppard, K. and Söll, D. (2008) On the evolution of the tRNA-dependent amidotransferases, GatCAB and GatDE. J. Mol. Biol. 377, 831–844. [26] Bernard, D., Akochy, P.M., Beaulieu, D., Lapointe, J. and Roy, P.H. (2006) Two residues in the anticodon recognition domain of the aspartyl-tRNA synthetase from Pseudomonas aeruginosa are individually implicated in the recognition of tRNAAsn. J. Bacteriol. 188, 269–274. [27] Chuawong, P. and Hendrickson, T.L. (2006) The nondiscriminating aspartyltRNA synthetase from Helicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity. Biochemistry 45, 8079–8087. [28] Becker, H.D., Roy, H., Moulinier, L., Mazauric, M.H., Keith, G. and Kern, D. (2000) Thermus thermophilus contains an eubacterial and an archaebacterial aspartyl-tRNA synthetase. Biochemistry 39, 3216–3230. [29] Cathopoulis, T.J., Chuawong, P. and Hendrickson, T.L. (2008) Conserved discrimination against misacylated tRNAs by two mesophilic elongation factor Tu orthologs. Biochemistry 47, 7610–7616. [30] Shirvanee, L., Horn, V. and Yanofsky, C. (1990) Escherichia coli mutant trpA34 has an Asp–Asn change at active site residue 60 of the tryptophan synthetase alpha chain. J. Biol. Chem. 265, 6624–6625. [31] Nakamura, A., Sheppard, K., Yamane, J., Yao, M., Söll, D. and Tanaka, I. (2010) Two distinct regions in Staphylococcus aureus GatCAB guarantee accurate tRNA recognition. Nucleic Acids Res. 38, 672–682. [32] Felton, J., Michaelis, S. and Wright, A. (1980) Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli. J. Bacteriol. 142, 221–228. [33] Gitlitz, P.H., Sunderman Jr., F.W. and Hohnadel, D.C. (1974) Ion-exchange chromatography of amino acids in sweat collected from healthy subjects during sauna bathing. Clin. Chem. 20, 1305–1312. [34] Date, S.V., Modrusan, Z., Lawrence, M., Morisaki, J.H., Toy, K., Shah, I.M., Kim, J., Park, S., Xu, M., Basuino, L., et al. (2013) Global gene expression of methicillinresistant Staphylococcus aureus USA 300 during human and mouse infection. J. Infect. Dis., http://dx.doi.org/10.1093/infdis/jit668. [35] Charrière, F., O’Donoghue, P., Helgadóttir, S., Marechal-Drouard, L., Cristodero, M., Horn, E.K., Söll, D. and Schneider, A. (2009) Dual targeting of a tRNAAsp requires two different aspartyl-tRNA synthetases in Trypanosoma brucei. J. Biol. Chem. 284, 16210–16217.
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Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis Stefani R. Mladenova, Kathryn R. Stein, Lianne Bartlett, Kelly Sheppard ⇑ Chemistry Department, Skidmore College, Saratoga Springs, NY 12831, USA
a r t i c l e
i n f o
Article history: Received 21 February 2014 Revised 17 March 2014 Accepted 18 March 2014 Available online 28 March 2014 Edited by Michael Ibba Keywords: tRNA RNA-dependent Asparagine biosynthesis Aspartyl-tRNA synthetase Staphylococcus aureus
a b s t r a c t The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNAAsn. The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNAAsn. S. aureus thus encodes both the direct and indirect routes for Asn-tRNAAsn formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The human pathogen Staphylococcus aureus in its genome does not encode either Asn synthetase (AsnA and AsnB) to amidate Asp to Asn but does encode an asparaginyl-tRNA synthetase (AsnRS) to directly ligate Asn to tRNAAsn [1]. However, the lack of an Asn synthetase does not prevent S. aureus from growing in the absence of Asn [2] suggesting it has as an alternate means of producing the amino acid. Numerous bacteria rely on an indirect two-step pathway to synthesize Asn in a tRNA-dependent manner enabled by an aspartyl-tRNA synthetase (AspRS) with relaxed tRNA specificity, recognizing both tRNAAsn as well as tRNAAsp [3]. As the first step in Asn-tRNAAsn synthesis, this non-discriminating AspRS (NDAspRS) forms Asp-tRNAAsn [4,5]. The Asp-tRNAAsn is then amidated to Asn-tRNAAsn by the amidotransferase GatCAB [4,6,7]. GatCAB in bacteria can also be used to synthesize Gln on tRNAGln in species lacking a glutaminyl-tRNA synthetase (GlnRS) [1,8–12]. GatCAB is present in S. aureus [13]. Given the presence of AsnRS but lack of Abbreviations: AsnRS, asparaginyl-tRNA synthetase; AspRS, aspartyl-tRNA synthetase; ND-AspRS, non-discriminating AspRS; D-AspRS, discriminating AspRS; GlnRS, glutaminyl-tRNA synthetase; ND-GluRS, non-discriminating glutamyl-tRNA synthetase; AsnA, asparagine synthetase A; AsnB, asparagine synthetase B; TrpA, tryptophan synthetase alpha subunit ⇑ Corresponding author. Address: Chemistry Department, Skidmore College, 815 North Broadway, Saratoga Springs, NY 12866, USA. Fax: +1 518 580 5139. E-mail address: [email protected] (K. Sheppard).
a GlnRS in S. aureus [1], the S. aureus GatCAB is predicted for only Gln-tRNAGln formation [14]. However, encoding AsnRS does not preclude an organism from also using GatCAB for tRNA-dependent Asn biosynthesis [3]. Three organisms are known to use both routes for Asn-tRNAAsn formation: Clostridium acetobutylicum, Deinococcus radiodurans, and Thermus thermophilus [4,6,12,15–18]. They encode in their genomes AsnRS, GatCAB, and at least two AspRSs. In these bacteria, one AspRS is a discriminating enzyme (D-AspRS) recognizing only tRNAAsp while the additional AspRS(s) is non-discriminating, acquired through horizontal gene transfer from archaea [4,6,12, 15–17]. Unlike those three bacteria, S. aureus uses only one AspRS. Given that S. aureus is an Asn prototroph [2] despite lacking an Asn synthetase [1], we predicted the lone S. aureus AspRS is nondiscriminating and works in concert with GatCAB to synthesize Asn-tRNAAsn. We demonstrate that S. aureus AspRS can aspartylate tRNAAsn and with GatCAB provides the organism a tRNAdependent route for Asn biosynthesis. 2. Materials and methods 2.1. General Oligonucleotides for PCR were from Integrated DNA Technologies (San Diego, California). S. aureus FPR3757 genomic DNA was
http://dx.doi.org/10.1016/j.febslet.2014.03.042 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
S.R. Mladenova et al. / FEBS Letters 588 (2014) 1808–1812
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obtained from American Type Culture Collection (Manassas, VA). Sequencing was carried out by the Yale DNA Analysis Facility on Science Hill (New Haven, CT). Nuclease P1, L-Asp, and L-Asn were from Sigma–Aldrich (St. Louis, MO). Tris and citrate buffered phenol, ATP, and chloroform were from Fischer Scientific. [a-32P]ATP (10 mmol/lCi) was from Perkin Elmer (Shelton, CT). Polyethylenimine (PEI)-cellulose thin layer chromatography (TLC) plates were from EMD Millipore (Billerica, MA). Restriction enzymes, Escherichia coli BL21(DE3) and NEB10b strains, OneTaq DNA Polymerase, and T4 DNA ligase were from New England Biolabs (Ipswich, MA). E. coli JF448 was from the Yale Coli Genetic Stock Center (New Haven, CT). E. coli trpA34 was a gift from the Söll Laboratory at Yale University (New Haven, CT).
studies with 5 nM AspRS were carried out at 37 °C with 111 nM 32Plabeled tRNA, 0–20.0 lM tRNA, and 4 mM L-Asp over 6 min. Steadystate kinetic studies with 5 nM AsnRS were carried out at 37 °C with 55 nM 32P-labeled tRNAAsn, 0–10.0 lM tRNAAsn, and 5 mM L-Asn over 6 min. Reactions were pre-incubated for 30 s at 37 °C before addition of enzyme and repeated 3 times. Time points were quenched, digested, separated by TLC, and processed as described previously [1,22,23] and repeated 3 times. For Michaelis–Menten kinetic constants, initial velocities vs. [tRNA] were plotted in KalediaGraph 3.1 (Synergy Software) and fitted to the Michaelis–Menten equation.
2.2. Over-production and purification S. aureus aaRSs
The S. aureus aspS was cloned into pCBS2 between the NdeI and BglII restriction sites (pCBS2-Sa-aspS) [24]. The pCBS2-Sa-aspS and two controls (D. radiodurans D-aspS and ND-aspS in pCBS2 [24], gifts from the Yale Söll Laboratory) were separately transformed into E. coli trpA34 cells. Cultures were grown overnight at 37 °C in LB with ampicillin (100 lg/ml). The overnight culture was used to inoculate 5.0 ml of M9 minimal media (1x M9 salts, 1 mM MgSO4, 0.4% D-glucose, 1 lg/ml thiamine, and 0.1 mM CaCl2) supplemented with all twenty amino acids (20 lg/ml each) and ampicillin (100 lg/ml). The cultures were grown shaking for 7 h at 37 °C. The cultures were centrifuged at 1500g for 3 min. To remove excess Trp, the cell pellet was resuspended in M9 minimal media (1.0 ml) with ampicillin (100 lg/ml) before centrifugation at 1500g for 3 min. The process was repeated twice. After the final spin, the cell pellets were resuspended in M9 minimal media (200 lL) with ampicillin (100 lg/ml) before being spotted (2.0 lL) onto M9 minimal media agar plates supplemented with ampicillin (100 lg/ml) and 19 amino acids (20 lg/ml) with or without L-Trp (20 lg/ml). The plates were incubated at 37 °C and scored daily for growth.
S. aureus aspS was cloned between the NdeI and BamHI restriction sites in pET28a to be N-terminally His6-tagged. The AspRS was overproduced using the previously described autoinduction method [19]. The cells were pelleted by centrifugation at 2000g for 25 min and then resuspended in wash buffer (25 mM HEPES– KOH, pH 7.2, 250 mM NaCl, 10 mM imidazole, and 5 mM 2-mercaptoethanol) with 0.1% Triton X-100 and one protease inhibitor tablet (Pierce, #8861). The cell suspension was sonicated for 5 min total, alternating 2 s on and 2 s off, at 40% amplitude (Fisher Sonic Dismembrator). The homogenate was centrifuged at 21 000g for 45 min. The supernatant was added to nickel-nitrilotriacetic acid-agarose slurry and AspRS was purified according to manufacturer’s protocols (Qiagen). Following elution in wash buffer with 250 mM imidazole, AspRS was dialyzed in storage buffer (25 mM HEPES–KOH, pH 7.2, 30 mM KCl, 0.2 mM EDTA, 10% glycerol, and 5 mM 2-mercaptoethanol) overnight at 4 °C, concentrated using Amicon Ultra centrifugal filter units, and then dialyzed in storage buffer with 50% glycerol at 4 °C overnight and stored at 20 °C. The S. aureus asnS was chemically synthesized with optimized codons for over-production in E. coli (Life Technologies GeneArt) and subcloned into pET28a between the NdeI and BamHI sites. AsnRS was over-produced and purified as described for AspRS. The enzyme preparations were determined >95% pure by Coomassie-stained polyacrylamide gel. 2.3. In vitro transcription The S. aureus tRNA genes were chemically synthesized with a T7 promoter (Integrated DNA Technologies), in vitro transcribed and purified as described for the D. radiodurans tRNAs [17]. 2.4. tRNA folding and
32
P labeling
tRNAs were unfolded at 95 °C for 5 min and slowly cooled to room temperature. At 65 °C, MgCl2 was added to a 5 mM final concentration. After folding, tRNA was stored at 20 °C. Aliquots of tRNA were 32P-labeled using the E. coli CCA-adding enzyme as described previously [1]. 2.5.
32
P-based tRNA aminoacylation assay
Aminoacylation of tRNAAsn and tRNAAsp was monitored using the established 32P-based assay [1,20–23]. The aminoacylation reactions contained 50 mM HEPES–KOH, pH 7.2, 30 mM KCl, 15 mM MgCl2, 5 mM DTT and 4 mM ATP. For plateau aminoacylation of tRNA with S. aureus AspRS, reactions were carried out at 37 °C with 1.0 lM 32P-labeled tRNA, 19.0 lM tRNA, 4 mM L-Asp, and 3.0 lM enzyme. For plateau aminoacylation of tRNA with S. aureus AsnRS, reactions were carried out at 37 °C with 1.0 lM 32P-labeled tRNA, 19.0 lM tRNA, 5 mM L-Asn, and 3.0 lM AsnRS. Steady-state kinetic
2.6. E. coli trpA34 in vivo assay
2.7. E. coli JF448 in vivo assay The S. aureus aspS and gatCAB were fused into an artificial operon as described previously [11,17]. The artificial operon was subcloned into the pCBS2 plasmid between the NdeI and BglII restriction sites (pCBS2-Sa-aspS-gatCAB). pCBS2-Sa-aspS and pCBS2-Sa-aspS-gatCAB were separately transformed into E. coli JF448 cells. S. aureus pCBS2-aspS was also transformed into NEB10b cells to serve as an additional positive control. Cultures were grown shaking overnight at 37 °C in LB with ampicillin (100 lg/ ml). The starter culture was used to inoculate M9 minimal media supplemented with all twenty amino acids (20 lg/ml each) and ampicillin (100 lg/ml). The cultures were grown shaking for 4 h at 37 °C and centrifuged for 5 min at 1500g. The cell pellets were washed three times to remove excess L-Asn as described above for the trpA34 system. Afterwards, the samples were resuspended with M9 minimal media (1.0 ml) supplemented with ampicillin (100 lg/ml) and then diluted with M9 minimal media to 0.45 O.D.600. The samples were further diluted 100-fold with M9 minimal media. Aliquots of culture (2.0 lL) were spotted onto M9 minimal media agar plates supplemented with ampicillin (100 lg/ml) and 19 amino acids (20 lg/ml) in the presence or absence of L-Asn (20 lg/ml). The plates were incubated at 37 °C and scored daily. 3. Results 3.1. Sequence prediction unclear as to S. aureus AspRS tRNA specificity Bacterial GatCABs distinguish their tRNA substrates from other tRNA species in a cell by recognizing the U1-A72 base pair found in
S.R. Mladenova et al. / FEBS Letters 588 (2014) 1808–1812
(A)
(B)
S. aureus s AspRS
50 40 30
Asp
tRNA A
Asn
A tRNA
20 10 0 0
10
20
30
40
Percent aminoacylated y (%) ( )
many bacterial tRNAAsn and tRNAGln isoacceptors [13,14]. S. aureus tRNAAsn retains the U1-A72 base pair [13,14], consistent with GatCAB being used for Asn biosynthesis. However, the presence of an U1-A72 pair in tRNAAsn is not an automatic indicator that the organism synthesizes Asn on tRNAAsn. In many bacterial species without a ND-AspRS, tRNAAsn retains the U1-A72 base pair including a number of bacteria that do not encode GatCAB as well as bacteria that use GatCAB for only Gln-tRNAGln formation [14]. Given ancestral bacteria likely used the indirect two-step pathway for Asn-tRNAAsn formation [25], the U1-A72 in tRNAAsn in these modern bacteria is likely vestigial. In a similar fashion, the elements GatCAB discriminates against in tRNAAsp are retained in many bacteria that lack a ND-AspRS [14]. For tRNA-dependent Asn biosynthesis, bacteria must encode GatCAB, a tRNAAsn with a U1A72 base-pair, and a ND-AspRS. Studies of the Helicobacter pylori and the Pseudomonas aeruginosa ND-AspRSs implicated four residues (equivalent to Leu31, Glu84, Thr85 and Ile90 in the S. aureus sequence) in the anticodon recognition domain that enable bacterial ND-AspRSs to recognize tRNAAsn [26,27]. We therefore aligned the S. aureus, H. pylori, and P. aeruginosa AspRS sequences along with a representative sampling of bacterial AspRSs with predictable tRNA specificities to determine if the discriminating nature of the S. aureus enzyme could be predicted from its sequence (Fig. 1). The AspRS of a species was predicted to be discriminating if the organism’s genome coded for AsnRS but not GatCAB or if tRNAAsn did not have a U1A72 base pair when both AsnRS and GatCAB were present. NDAspRS was predicted when AsnRS was not encoded in the genomes but GatCAB was. As in a previous study [27], AspRS sequences were excluded when a species encoded both AsnRS and GatCAB in its genome when tRNAAsn had a U1-A72 base pair. Unfortunately, it was unclear from the alignment if the S. aureus AspRS was non-discriminating or discriminating. Leu31 in the S. aureus AspRS is found in many bacterial D-AspRS enzymes while in most ND-AspRS enzymes it is replaced with a His. Mutation of the His in the P. aeruginosa ND-AspRS to Leu increased specificity towards tRNAAsp by 3.5-fold using E. coli tRNA though no difference was observed when P. aeruginosa tRNA was used as a substrate [26]. However, Leu at that position is also found in the ND-AspRSs from Thermatoga maritima and Streptomyces avermitilis (Fig. 1). Glu84 and Ile90 are found in both discriminating and non-discriminating AspRS enzymes. Thr85 is found in some other ND-AspRS sequences. However, the role of Thr at that position has not been
Percent aminoacylated (%)
1810
S. aureuss AsnRS
40 30
tRNA AAsp
20
Asn
A tRNA 10 0 0
20
10
time (min.)
30
40
time ((min.)
Fig. 2. Aminoacylation of in vitro transcribed S. aureus tRNAAsp (s) and tRNAAsn (d) by either (A) S. aureus AspRS or (B) S. aureus AsnRS. Reactions were carried out at 37 °C with 1.0 lM 32P-labeled tRNA, 19 lM tRNA, 4 mM ATP, and 3.0 lM enzyme. For the AspRS reactions 4 mM L-Asp was included, while for the AsnRS reactions 5 mM L-Asn was included.
studied, only Leu and Asn [27]. In summary, Leu31 suggests the S. aureus AspRS may be discriminating while Thr85 suggests it may be non-discriminating, and Glu84 and Ile90 are ambiguous as to the tRNA specificity of the S. aureus enzyme. 3.2. In vitro aminoacylation of tRNAAsp and tRNAAsn Given the unclear sequence alignment prediction as to the S. aureus AspRS tRNA specificity, we purified recombinant protein from E. coli (Supplemental Fig. 1) to test whether the AspRS could aminoacylate in vitro transcribed S. aureus tRNAAsn and tRNAAsp. To monitor Asp-tRNA formation, we used the established 32P-based assay [1,20–23]. The S. aureus AspRS was able to aspartylate both tRNAAsp and tRNAAsn (Fig. 2A), indicative of the enzyme being non-discriminating. Like other ND-AspRS enzymes [17,27,28], the S. aureus ND-AspRS has a slight preference (1.5-fold) for tRNAAsp over tRNAAsn (Table 1). In contrast, D-AspRS enzymes prefer tRNAAsp to tRNAAsn by 500–2250-fold [17,28]. As expected, tRNAAsn is also a substrate for the S. aureus AsnRS (Fig. 2B). S. aureus tRNAAsn is a slightly better substrate for AsnRS than ND-AspRS (1.7-fold) (Table 1). The AspRS has a higher kcat (4.7-fold) but also a higher KM (7.8-fold) than AsnRS with tRNAAsn as a substrate. The lower AsnRS kcat may explain the lower plateau tRNAAsn aminoacylation level than that of AspRS (Fig. 2).
D-AspRS C. perfringens E. coli P. gingivalis S. boydii T. thermophilus V. cholera
29 15 29 11 29 28
S. aureus
29 RDLGGLIFVDLRDREGIVQVVFNP-AFSEEALKIAETVRSEYVVEVQGTVTKRDPETVNPKIK
H. pylori N. meningitis P. aeruginosa R. belli S. avermitilis S. meliloti T. maritima T. tengcongensis ND-AspRS
28 28 90 31 29 29 29 29
RNLGGLQFIDLRDREGILQVVFND-DLGEEILEKAKSIRPEYCIAVTGEIVKR--ESVNPNMP RDLGSLIFIDMRDREGIVQVFFDP--DRADALKLASELRNEFCIQVTGTVRARDEKNINRDMA RRMGGMTFVDLRDRYGTTQLVFNRDSAPAELCDRAEDLGREWVIRATGTVMER--SSKNPNIP RDLGSLIFIDMRDREGIVQVFFDP--DRADALKLASELRNEFCIQVTGTVRARDEKNINRDMA RDLGGLIFLDLRDREGLVQLVAHP---ASPAYATAERVRPEWVVRAKGLVRLR--PEPNPRLA RDLGGLIFIDMRDREGIVQVVVDP--DMADVFAVANQLRSEFCIKLTGEVRARPESQVNKEMA
RDHGGVVFIDLRDKSGLVQLVCDP---SSKAYEKALEVRSEFVLVAKGKVRLRGAGLENPKLK RDHGGVIFIDLRDREGIVQVVIDP--DTPEAFAAADSSRNEYVLSITGRVRNRPEGTTNDKMI RDHGGVIFLDVRDREGLAQVVFDP--DRAETFAKADRVRSEFVVKITGKVRLRPEGARNPNMA RDHGNLVFVDLRDHYGITQIVFTD--QNPQLMDDASRLRYESVVTVIGKVVARSEETINNTLP RDLGGILFIDLRDHYGITQLVARP---GTPAYEALDKVSKESTVRVDGKVVSRGTENVNPDLP RDHGGVLFIDLRDHYGMTQVVADP---DSPAFKTAETVRGEWVIRVDGAVKARTDDTVNKNMP RDLGGVRFIDLRDRYGETQIVCDV---NSEAYSVVDELTRESVVLVEGTVRKRPEGTENPNIE RDHGGLVFIDLRDRTGIVQIVFSE-QVSKEVFEKVQSVRSEYVLAVEGEVVKRLPENVNPKIP
Fig. 1. Sequence alignment of representative bacterial AspRS anticodon binding domains. A phylogentically diverse sampling of 152 bacterial AspRS sequences were aligned in Geneious v. 4.7.4. For clarity only a select sampling of sequences is shown. D-AspRS enzymes (orange) were grouped together (38 total sequences). A D-AspRS was predicted based on either previous biochemical evidence or if the bacteria encoded AsnRS but not GatCAB in its genome. A D-AspRS was also predicted when AsnRS was present and tRNAAsn did not have a U1-A72 base pair. ND-AspRS enzymes (green) were similarly grouped together (82 sequences). A ND-AspRS prediction was based on either biochemical evidence or if the bacteria encoded GatCAB but not AsnRS. AspRS sequences (32 total) were excluded from the analysis due to their tRNA specificity being unclear based on genomic content as these organisms encoded both AsnRS and GatCAB as well as a tRNAAsn with a U1-A72 base pair. The four residues implicated previously [26,27] in AspRS recognition of tRNAAsn are in red. Shading is based on sequence similarity for the overall alignment in Geneious.
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1
)
KM (lM)
kcat/KM (s
+ Asn 1
lM 1)
L*
- Asn
1 2
AspRS tRNAAsp tRNAAsn
0.24 ± 0.07 0.20 ± 0.03
2.4 ± 1.3 3.1 ± 1.3
AsnRS tRNAAsn
0.043 ± 0.004
0.40 ± 0.19
(10 ± 6) 10 2 (7 ± 3) 10–2
1.5 1.0
2
1.7
(11 ± 5) 10
L = Specificity relative to the catalytic efficiency of AspRS with tRNAAsn as a substrate, (kcat/KM)/(kcat/KM) of AspRS for tRNAAsn. Experiments were repeated three times and standard deviations are reported.
*
+ Trp
- Trp
1
3 4 5 Fig. 4. Co-production of S. aureus AspRS and GatCAB rescues the Asn auxotrophy of E. coli JF448. E. coli JF448 was transformed with pCBS2 coding for either (1) the D. radiodurans ND-aspS and gatCAB or (2) the D. radiodurans D-aspS and gatCAB as positive and negative controls, respectively. (3) E. coli NEB10b, an Asn prototroph, was transformed with pCBS2-S. aureus aspS as an additional positive control. E. coli JF448 was also transformed with pCBS2 encoding either (4) the S. aureus aspS alone or (5) the S. aureus aspS and gatCAB. The resultant strains were grown in triplicate on M9 minimal media agar plates in the presence (+Asn) or absence of Asn ( Asn) at 37 °C for three days.
2 3 Fig. 3. The E. coli trpA34 strain transformed with S. aureus aspS is able to grow in the absence of Trp. E. coli trpA34 was transformed with pCBS2 coding for either (1) the ND-aspS from D. radiodurans, (2) the D-aspS from D. radiodurans, or (3) the aspS from S. aureus. The cells were grown in triplicate on M9 minimal media agar plates in the presence (+Trp) or absence ( Trp) of Trp at 37 °C for three days.
3.3. Asp-tRNAAsn formation in vivo To test whether the S. aureus AspRS recognizes modified tRNAAsn in vivo, we used the established trpA34 E. coli complementation assay [24,29]. The trpA gene encodes the a-subunit of Trp synthetase (TrpA) and Asp60 is essential for activity. The E. coli trpA34 mutant strain is a Trp auxotroph due to mutation of codon 60 from an Asp codon to an Asn codon [30]. ND-AspRS production in the strain rescues the Trp auxotrophy as the enzyme forms AsptRNAAsn, a missense suppressor, enabling decoding of Asn codons with Asp and synthesis of active TrpA (Fig. 3) [24,29]. The assay thus provides an in vivo test as to whether an AspRS can recognize tRNAAsn [24,29]. We therefore subcloned the S. aureus aspS into pCBS2 and transformed the vector into the trpA34 strain. The S. aureus aspS supported growth in the absence of Trp (Fig. 3), consistent with the S. aureus AspRS being non-discriminating and able to form the missense suppressor Asp-tRNAAsn. 3.4. Rescue of E. coli Asn auxotroph We hypothesized S. aureus encodes a ND-AspRS to form the Asp-tRNAAsn required for tRNA-dependent Asn biosynthesis. Like other bacterial GatCABs [1,6,9–11,16], the S. aureus GatCAB can in vitro amidate Asp to Asn on tRNAAsn [31]. To determine in vivo whether S. aureus AspRS and GatCAB can work in concert to synthesize Asn, we co-produced both enzymes in E. coli JF448 [17,32], an Asn auxotrophic strain due to mutations to both the AsnA and AsnB genes [32]. Co-production of a ND-AspRS and GatCAB rescues the phenotype as Asn is synthesized on tRNAAsn (Fig. 4) [17]. Consistent with S. aureus synthesizing Asn in a tRNA-dependent manner, co-production of the S. aureus AspRS and GatCAB rescued the Asn auxotrophy of the JF448 strain (Fig. 4). 4. Discussion The S. aureus AspRS has relaxed tRNA specificity, recognizing tRNAAsn almost as well as tRNAAsp, and can work with GatCAB to
synthesize Asn-tRNAAsn. The presence of a ND-AspRS and GatCAB to synthesize Asn in a tRNA-dependent manner explains why S. aureus is not an Asn auxotroph [2] despite lacking AsnA and AsnB [1]. As Asn is made on tRNAAsn, the pathway allows S. aureus to directly couple Asn biosynthesis with its use during translation. Given the presence of AsnRS, S. aureus encodes the indirect and direct pathways for Asn-tRNAAsn formation like C. acetobutylicum, D. radiodurans, and T. thermophilus [4,6,12,15–17]. The role of AsnRS in S. aureus is unclear since the bacterium does not synthesize Asn in a tRNA-independent manner and the indirect pathway could presumably meet the cellular demands for Asn-tRNAAsn. It is tempting to speculate that when S. aureus is growing on humans it uses AsnRS to take advantage of the available Asn in human sweat and blood plasma (on average 164 and 62 lM, respectively) [33]. Under this scenario, the indirect pathway would meet the basal needs for Asn-tRNAAsn when S. aureus is growing away from a host, while the AsnRS would allow S. aureus to take advantage of the environmentally available Asn when growing on a host. AsnRS may also be used to recycle Asn from degraded proteins as predicted for T. thermophilus and D. radiodurans [4,6,17]. It is unclear how production of ND-AspRS, AsnRS, ND-GluRS and GatCAB in S. aureus is regulated to meet the cellular demands for Asn-tRNAAsn and Gln-tRNAGln. Upon infecting a mammalian host, S. aureus gatC expression doubles, while the expression of the genes coding for GatA, GatB, AsnRS, ND-AspRS, and ND-GluRS remains unchanged [34]. In C. acetobutylicum, one set of ND-AspRS and GatCAB genes are in an operon together regulated by a Tbox riboswitch that may indirectly monitor Asn levels in the cell that may be tied to the organism switching from acidogenesis to solventogenesis [12]. The regulatory control may also explain why C. acetobutylicum acquired additional aspS genes that are not needed in S. aureus. In S. aureus, the lone aspS is in an operon with hisS coding for histidyl-tRNA synthetase, not gatCAB. The lack of a signature sequence in bacterial AspRSs to differentiate a D-AspRS from a ND-AspRS makes it difficult to establish how many other bacteria use both routes for Asn-tRNAAsn formation when tRNAAsn has a U1-A72 base pair [1]. For D-AspRS to evolve in an organism, it must encode an AsnRS in its genome to aminoacylate tRNAAsn. Given the distribution of AsnRS in bacteria [1,35], it is likely D-AspRS is a case of convergent evolution with tRNA specificity for tRNAAsp evolving separately in different bacterial lineages. Clarifying the discriminating nature of bacterial AspRSs will help in understanding how Asn metabolism and use of the amino acid in protein synthesis are integrated into bacterial life cycles.
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