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Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites

Li Lia, Michal T. Bonieckib, Jacob D. Jaffec, Brian S. Imaid, Peter M. Yaud, Zaida A. Luthey-Schultena,e, and Susan A. Martinisa,b,e,1

aCenter for Biophysics and Computational Biology, bDepartment of Biochemistry, eDepartment of Chemistry, dRoy J. Carver Biotechnology Center, University of Illinois, Urbana, IL 61801; and cProteomics Platform, The Broad Institute, 7 Cambridge Center, Cambridge, MA 02142

Edited by Paul Schimmel, The Skaggs Institute for Chemical Biology, La Jolla, CA, and approved April 20, 2011 (received for review November 8, 2010)

Mycoplasma parasites escape host immune responses via me- degenerate based on substitutions at key sites in the hydrolytic chanisms that depend on remarkable phenotypic plasticity. Identi- active site. This was surprising because functional defects in fication of these mechanisms is of great current interest. The AARS editing that decrease the fidelity of tRNA aminoacylation aminoacyl-tRNA synthetases (AARSs) attach amino acids to their have been clearly shown to result in amino acid toxicities, cell cognate tRNAs, but occasionally make errors that substitute closely death, as well as neurological disease in mammals (7–9). As such, similar amino acids. AARS editing pathways clear errors to avoid in order to achieve the threshold levels of translational fidelity mistranslation during protein synthesis. We show here that AARSs that are required for cell viability, these amino acid editing func- Mycoplasma in parasites have point mutations and deletions in tions have been broadly conserved across all three domains their respective editing domains. The deleterious effect on editing of life. was confirmed with a specific example studied in vitro. In vivo In contrast, we determined that Mycoplasma mobile exhibits mistranslation was determined by mass spectrometric analysis of AARS-dependent translational infidelities. Editing-defective proteins produced in the parasite. These mistranslations are uni- AARSs mischarge tRNA, which subsequently results in mistran- form cases where the predicted closely similar amino acid replaced slation in vivo. It is possible that this AARS-dependent mechan- the correct one. Thus, natural AARS editing-domain mutations in ism could provide a unique pathway to introduce heterogeneity Mycoplasma parasites cause mistranslation. We raise the possibi- into the cell’s proteome that could confer phenotypic plasticity in lity that these mutations evolved as a mechanism for antigen diversity to escape host defense systems. Mycoplasma pathogens. Results amino acid editing ∣ fidelity ∣ quality control ∣ statistical proteins ∣ Mycoplasma Have Evolved AARSs with Inactivated Editing Domains. host-pathogen interactions Using bioinformatic approaches to broadly scrutinize genomes across the three domains of life, we identified AARSs with unu- ycoplasma are characterized by their lack of a cell wall sual amino acid editing domains. In Mycoplasma and closely Mand dependence on a vertebrate host (1). Their relationship with the host can be parasitic or they can coexist as an obligate related species, we discovered AARSs with deletions and substi- commensal. The persistent survival of Mycoplasma within their tutions that we hypothesized would abolish their editing activities host has been attributed to a phenotypic plasticity that allows these (Fig. 1). These Mycoplasma AARSs have substitutions at key pathogens to facilely alter their antigenic properties (2). Paradoxi- residues in the hydrolytic active sites of the editing domains. In cally, this phenotypic plasticity is generated in spite of the Myco- one extreme case, the editing domain was completely deleted plasma’s extremely small genomes, which have lost many of the from the M. mobile leucyl-tRNA synthetase (LeuRS). components that typically comprise signaling pathways to adapt In six different Mycoplasma and also two closely related to changing environments (2). Remarkably, these wall-less bacteria species, threonyl-tRNA synthetase (ThrRS) contained editing have a highly dynamic surface architecture comprised of mem- domains in which the editing site had amino acid substitutions at brane proteins that confer antigenic and functional versatility (2). critical residues (Fig. 1A). In each of these cases, critical histidine, As with other pathogenic and nonpathogenic organisms, aspartic acid, and cysteine residues (Fig. S1) within two distal Mycoplasma contain a complete set of AARSs (aminoacyl-tRNA peptides that folded to form the editing active site were replaced synthetases), which are essential to translate the genetic code into (10–12). The editing domains of the editing-defective ThrRSs functional proteins (3). Each AARS has evolved for specificity to have a lower average sequence identity (24.4%) compared to the a single standard amino acid to maintain the fidelity of the genetic canonical cases (44.6%), while retaining similar levels of conser- code. The AARS enzyme family activates and transfers amino vation for their aminoacylation domains (54.7% versus 56.1%). acids to their cognate tRNA isoacceptor. Once the tRNA is This suggested that the editing domain in these Mycoplasma spe- “ ” charged with an amino acid, it is shuttled to the ribosome for cies was preferentially prone to retaining substitutions (Table S1). incorporation into nascent polypeptides. About half of the AARSs are prone to mistakes by activating structurally similar amino acids and mischarging them to tRNA. Author contributions: L.L., M.T.B., and S.A.M. designed research; L.L., J.D.J., B.S.I., and To minimize the potential for creating statistical proteins that P.M.Y. performed research; L.L. contributed new reagents/analytic tools; L.L., M.T.B., J.D.J., B.S.I., P.M.Y., Z.A.L.-S., and S.A.M. analyzed data; and L.L., Z.A.L.-S., and S.A.M. wrote contain mistranslated amino acids, these AARSs have developed the paper. “ ” a second sieve (4); they have adapted to include hydrolytic The authors declare no conflict of interest. editing domains that are distinct from their canonical aminoacy- This article is a PNAS Direct Submission. lation core domains. In some cases, aminoacylation accuracy is 1To whom correspondence should be addressed at: Department of Biochemistry, 419 Roger also enhanced by independent editing domains that function as Adams Laboratory, Box B4, 600 South Mathews Avenue, University of Illinois, Urbana, tRNA-specific deacylases (5, 6). IL 61801. E-mail: [email protected]. We have identified multiple AARSs with editing domains This article contains supporting information online at www.pnas.org/lookup/suppl/ in Mycoplasma and closely related species that appeared to be doi:10.1073/pnas.1016460108/-/DCSupplemental.

9378–9383 ∣ PNAS ∣ June 7, 2011 ∣ vol. 108 ∣ no. 23 www.pnas.org/cgi/doi/10.1073/pnas.1016460108 Downloaded by guest on October 1, 2021 BIOCHEMISTRY

Fig. 1. Degenerated editing domains of Mycoplasma AARS. (A) Phylogenetic tree of Mycoplasma based on 16S rRNA. Bootstrap values are shown for each node and scale bar denotes substitutions per site. Predicted editing-defective AARS are indicated in boxes (Right) (see also Figs. S1–S3). (B) Alignment of LeuRS CP1 domain with key editing site residues indicated (see also Table S3). Shaded and black boxes represent conserved and homologous residues. (C) Tandem MS analysis of precursor peak m∕z ¼ 796.4 at z ¼ 3 identified a mistranslated peptide in M. mobile. A fragment peak (m∕z ¼ 411.2; y3 ion of YPIILEDGFSEHDWDA (Y)TK; phosphopyruvate hydratase) was confirmed by the balance of the fragmentation spectrum. (Inset) Predicted y2 and y3 ion positions for the genome- encoded peptide (dotted bars) and observed mistranslated product (solid bars; F → Y). (D) Identification of faithfully translated peptide YPIILEDGFSEHDWDA (F)TK for phosphopyruvate hydratase (peak of m∕z ¼ 395.2 at the y3 ion position).

In some Mycoplasma species, conserved motifs in the editing these mutational events in the three AARS genes have taken site of phenylalanyl-tRNA synthetase (PheRS) (13, 14) have also place independently multiple times (Fig. 1A). acquired substitutions at key sites (Fig. S2), and the overall sequence identity for their editing domains (19.2%) is lower than M. mobile Has a Statistical Proteome. We hypothesized that Myco- the canonical counterparts (31.3%), although the two groups plasma with editing-defective AARSs would be prone to gener- share similar identities in the aminoacylation domains (46.8% ating statistical substitutions during protein synthesis. The genome of M. mobile (15), a fish pathogen, encodes a PheRS with versus 44.1%; Table S1). In at least four organisms, PheRS and substitutions at key sites in the editing pocket. In addition, LeuRS ThrRS proteins are simultaneously encoded to express editing is completely missing its editing domain that is called CP1 domains that appear to be functionally defective (Fig. 1A). In (16, 17). In order to hunt for statistical proteins, we bioinforma- another set of Mycoplasma species, the fidelity domains of either tically rescreened data obtained for the complete proteome of PheRS (Fig. S2) or LeuRS (Fig. 1B and Fig. S3) or both have M. mobile (15) by instructing the database search program to been mutated suggesting that they are editing-deficient. Phyloge- allow for potential mistranslations corresponding to suspected netic analysis indicates that in different Mycoplasma lineages, editing-defective AARSs. These Mycoplasma were grown in rich

Li et al. PNAS ∣ June 7, 2011 ∣ vol. 108 ∣ no. 23 ∣ 9379 Downloaded by guest on October 1, 2021 media under conditions that would not be expected to bias a A typical rate for translational fidelity has been estimated to bacteria to incorporate mutations (15). However, analysis of be 1∕3;000 (18), and AARSs that cannot meet this threshold the proteome identified examples of statistical mutations includ- have evolved editing domains (19). In our peptide pool for the ing an F322Y substitution in phosphopyruvate hydratase (Fig. 1C) M. mobile proteome, we screened 138 phenylalanines and 518 that would be indicative of an editing-defective PheRS, which leucines to determine an error of 1 and 2 substitutions, respec- generates mischarged Tyr-tRNAPhe (13). In parallel, a faithfully tively. The probability that these statistical substitutions result translated peptide from phosphopyruvate hydratase was also from an editing-defective PheRS and LeuRS (Model E, Eqs. 1 2 detected that did not contain the F322Y mutation via mass spec- and below) is significant with values of 0.35 and 0.25, respec- trometry analysis of the peptide pool (Fig. 1D), which supported tively, if we assume that the error rate of these aaRSs are approxi- 1∕200 the statistical nature of these mistakes. mately (vide infra): LeuRS fidelity is challenged by a broader scope of amino acids 1 137 1 1 199 (9). Notably, the most predominant expected replacement for pðPhejEÞ¼C138 × × ≈ 0.35; [1] leucine would be isoleucine (9), but this change would not be 200 200 detected by mass spectrometry because these isomers have iden- tical molecular weights. However, examples where valine, which 1 2 199 516 pðLeujEÞ¼C2 × × ≈ 0.25: [2] is only weakly misactivated by LeuRS (9) (Table S2), was substi- 518 200 200 tuted for leucine in phosphopentomutase (L318V; Fig. 2A) and phosphotransacetylase (L14V; Fig. 2B) provided additional This would compare to very low probabilities of less than 0.05 evidence for statistical substitutions in the M. mobile proteome. if these substitutions were based on normal mistranslation rates

Fig. 2. Tandem MS analysis of single peptides from M. mobile and E. coli that express M. mobile LeuRS demonstrate mistranslation of leucine codons. An L → V substitution of M. mobile peptides LTKLINS(L/V)K from phosphopentomutase (A) and IKTSVKN(L/V)AK from phosphotransacetylase (B) respectively generated peaks of m∕z ¼ 869.2 [confirmatory b8 ion of LTKLINS(V)K; precursor peak m∕z ¼ 508.5 at z ¼ 2] and m∕z ¼ 870.2 [confirmatory b8 ion of IKTSVKN(V)AK; precursor peak m∕z ¼ 544.3 at z ¼ 2]. (C)AnL→ V substitution of E. coli peptide LINQGMI(L)GK generated a peak of m∕z ¼ 303.2 corresponding to the confirmatory y3 ion of LINQGMI(V)GK. An L → M substitution of E. coli peptides HLENKIIKKEIYIAKKI(L)NFII (D) and SKDKFYA(L)D MFPYPSGSGLHVGHPEGY- þþ ox TATDIISR (E) respectively generated peaks of m∕z ¼ 1105.7 [confirmatory b18 ion of HLENKIIKKEIY IAKKI(M )NFII] and m∕z ¼ 953.5 [confirmatory b8 ion of SKDKFYA(Mox)DMFPYPSGSGLHVGHPEGYTATDIISR]. Methionine was typically oxidized to methionine sulfoxide (see also Fig. S4).

9380 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1016460108 Li et al. Downloaded by guest on October 1, 2021 of 1∕3;000. Thus, we propose that this high level of mistranslation an alternate pretransfer editing pathway (20). Also, M. mobile is statistical for M. mobile and is caused by editing-defective LeuRS has not evolved the higher threshold of amino acid dis- PheRS and LeuRS that cannot clear their own mischarged tRNA crimination that has been measured for human mitochondrial products. LeuRS, which lacks editing activity (21). We also tested for M. mobile LeuRS-dependent mistranslations in Escherichia coli. Previously, we showed that LeuRS Discussion misactivates valine, isoleucine, and methionine (9). Because In all three domains of life, LeuRS contains the CP1 editing isoleucine cannot be distinguished from leucine via mass spectro- domain as does isoleucyl (IleRS)- and valyl (ValRS)-tRNA metry, we focused on valine and methionine substitutions. To synthetases (22). These homologous proteins have differentiated increase sensitivity, we induced expression of M. mobile LeuRS to accommodate different amino acid specificities for both ami- in E. coli BL21 cells (17 nmol∕g cells) in the presence of increas- noacylation and editing. They also can rely selectively or com- ing valine or methionine concentrations of up to 10 mM. Tandem binatorially on pre- and posttransfer editing mechanisms to mass spectrometry of purified M. mobile LeuRS that was trypsin- respectively target the aminoacyl-adenylate intermediate or mis- digested identified statistical substitutions for multiple sets of charged tRNA product to clear misactivated amino acids (23–25). peptides, where valine or methionine was substituted for leucine To our knowledge, M. mobile LeuRS is the only known example (Fig. 2) as well as correlating examples where these specific sites for LeuRS, IleRS, or ValRS that is completely missing its CP1 had been faithfully translated (Fig. S4). In comparison, there was editing module (Fig. 1B). Previously, we had shown that the no mistranslation detected in BL21 cells that expressed E. coli 22 ∕ fidelity of E. coli and yeast mitochondrial LeuRS were protected LeuRS ( nmol g cells) under the same condition, even at high in the absence of its CP1 module (LeuRS-ΔCP1) by a latent concentrations of noncognate amino acids. In addition, E. coli pretransfer editing activity that cleared misactivated aminoa- BL21 cells that expressed M. mobile LeuRS had a lengthy lag cyl-adenylate intermediate (20). In this case, M. mobile LeuRS phase that slowed cell growth (Fig. S5). with its naturally missing CP1 editing module failed to recapture Statistical Proteome Substitutions Correlate to AARS Amino Acid an alternate editing mechanism to posttransfer editing that was sufficient to completely protect the proteome. Editing Defect. Preparation of pure ðαβÞ2 PheRS and dimeric ThrRS for in vitro analysis could be complicated by heterogeneity Statistical proteins have been proposed to provide an advan- of their respective quaternary structures. Thus, we focused on the tage in primitive cells during the evolution of the modern protein synthesis machinery (26, 27). They have also been suggested to

monomeric M. mobile LeuRS for enzymatic characterization of BIOCHEMISTRY its putative editing defect to understand the molecular mechan- increase fitness in contemporary bacteria by providing a protein ism underlying error-prone translation in M. mobile. The gene reservoir that is phenotypically diverse (28). In a transcription- was synthesized in order to convert TGA triplets (which are used dependent error-prone experimental model for Bacillus subtilis, to encode tryptophan in M. mobile) to TGG and also to optimize proteome diversity caused by frame-shift or nonsense mutations codon usage frequencies for expression in E. coli. The monomeric enabled the organism to adapt to fluctuations in the environment, LeuRS was purified by affinity chromatography via an N-terminal such as changes in temperature (28). In yeast, CUG codon ambi- six-histidine tag and the enzyme robustly aminoacylated in vitro guity originating in Candida albacans was experimentally intro- transcribed M. mobile tRNALeu (Fig. S6) as well as E. coli duced to confer an adaptive advantage under stress conditions Leu k ∕K tRNA (Fig. S7). Based on the cat M for amino acid activa- via induction of a unique set of stress proteins (29). Under spe- tion by M. mobile LeuRS (Table S2), the discrimination factors cific stress conditions in mammalian systems, codon ambiguity for noncognate amino acids versus leucine were well below the increases significantly via activation of MetRS misacylation of accepted threshold of 3,000. Indeed, the discrimination factor noncognate tRNA isoacceptors (30). An artificial mutation in the of 200 for isoleucine was consistent with our threshold calcula- ValRS editing domain also allowed E. coli to adapt for subsis- tions for misaminoacylation in Mycoplasma (vide supra). It also tence on the nonstandard amino acid α-aminobutyrate, rather suggests that M. mobile LeuRS is even more prone to isoleucine than valine (31). misactivation than a typical LeuRS (Table 1). We have identified natural examples in Mycoplasma species As would be expected, M. mobile LeuRS failed to dea- Leu Leu Leu and related species in which the gene encoding AARSs contains cylate mischarged Val-tRNA , Ile-tRNA , and Met-tRNA mutations that disrupt the amino acid editing function. The shed- (Fig. 3), because it does not have a CP1 editing domain. These ding of the editing domain of LeuRS in M. mobile was likely noncognate amino acids can be charged to M. mobile tRNA facilitated by existing mechanisms in Mycoplasma that have (Fig. 3). Consistent with the introduction of leucine to methionine or valine statistical substitutions in the E. coli proteome resulted in the dramatic evolutionary reduction of its genomes. (Fig. 2), M. mobile LeuRS also mischarged E. coli tRNALeu with Accordingly, the erosion of AARS-dependent translational fide- methionine or valine (Fig. S7). lity may be a stepwise response based on the LeuRS CP1 do- ’ The propensity of M. mobile LeuRS to produce stably mis- main s massive sequence degeneration in Mycoplasma agalactiae charged tRNALeu contrasts sharply with other examples of and Mycoplasma synoviae, and then complete elimination of the LeuRSs that were determined to rely on additional mechanisms entire module in M. mobile (Fig. 4). to ensure fidelity. E. coli or yeast mitochondrial LeuRS main- Editing-defective AARSs in M. mobile introduce errors at tained fidelity in the absence of their CP1 modules by activating the translational level of protein synthesis. Because of the strong selection pressure throughout the three domains of life to main- Table 1. Apparent kinetic parameters for amino acid activation tain translational fidelity, we hypothesize that the loss of AARS by LeuRS editing in Mycoplasma is likely due to idiosyncratic demands on the organism, which are benefited by the introduction of statis- k ∕K cat m, −1 −1 −1 tical proteomes (19). It is possible that this mechanism to intro- KM,mM k ,s mM s cat duce statistical proteins could confer antigenic diversity and Enzyme Leu Ile Leu Ile Leu Ile phenotypic plasticity (2). This unique AARS-dependent mechan- Ec LeuRS* 0.018 ± 0.008 1.0 ± 0.3 71 ± 25 4.7 ± 1.3 3.9 × 103 4.7 ism at the translational level would be akin to the replication- Ec LeuRS ΔCP1* 0.021 ± 0.006 3.4 ± 0.9 0.23 ± 0.07 0.23 ± 0.07 11 0.65 dependent high rates of retroviral mutations that confer virus 2 Mm LeuRS 0.072 ± 0.025 6.0 ± 1.7 11.7 ± 4.2 4.8 ± 1.3 1.6 × 10 0.80 population heterogeneity and resistance (32) to escape host *Values measured previously (20). defense mechanisms.

Li et al. PNAS ∣ June 7, 2011 ∣ vol. 108 ∣ no. 23 ∣ 9381 Downloaded by guest on October 1, 2021 Fig. 3. Wild-type M. mobile LeuRS mischarges tRNALeu. Deacylation reactions contained approximately 20 μM[3H]-Val-tRNALeu (A), approximately 6.5 μM[3H]- Ile-tRNALeu (B) or 100 μM[35S]-Met-tRNALeu (C) and 100 nM M. mobile, E. coli wild-type or mutant LeuRS. Mischarging assays incorporated 160 μM[14C]-valine (50 μCi∕mL; D), 21 μM[3H]-isoleucine (166 Ci∕mmol; E)or20μM[35S]-methionine (20 μCi∕mL; F) and 1 μM M. mobile or E. coli LeuRS. Editing-defective E. coli LeuRS (Ec-Ed) mutant is a positive control. (G) Acid gel of tRNALeu charged with [35S]-methionine. Abbreviations: (square), Ec-WT; (inverted triangle), Mm; (triangle), Ec-Ed; and (diamond), no enzyme control (No E). Error bars represent standard deviations from triplicated reactions.

Materials and Methods homology mode substitutions F → Y and L → V were defined. The data were Bioinformatic Analysis and M. mobile Proteome Mass Spectral Data. Sequences searched against all annotated trypsin-digested ORFs as described (15). Indi- were retrieved from the Swiss-Prot database (http://ca.expasy.org/), aligned vidual peptides with SpectrumMill scores >13 and scored percent identity > and edited in MultiSeq/VMD (33) using CLUSTALW (34), and shaded by 80% were further considered. These peptide spectra were searched against “ ” Biology Workbench (http://workbench.sdsc.edu). The phylogenetic tree of the M. mobile database in no enzyme mode as a test of specificity. Con- Mycoplasma and related species was constructed by a maximum-likelihood cordant results were verified by manual inspection. method based on 16S rRNA sequences using RAxML (35). The GTR model Leu was used for nucleotide substitution and the Γ model for rate heterogeneity. Cloning and in Vitro Transcription of the M. mobile tRNA Gene. The gene for M. mobile tRNALeu , the most abundant M. mobile tRNALeu isoacceptor, was The support values for the bipartitions were estimated from 1,000 nonpara- UAA cloned into pUC18 using BamHI and HindIII restriction sites to yield metric bootstrap runs. pUC18LiMmtRNALeu. The plasmid (450 μg) was digested overnight with Previous mass spectral data (15) were analyzed via SpectrumMill (Agilent) 25 U of SacII and then used as a template for in vitro runoff transcription using the default parameters for ThermoFinnigan LCQ ion trap data. The (36, 37). The SacII digestion cleaves the tRNA template after the penultimate nucleotide at the 3′ end to generate tRNALeu without the terminal A76 Leu Leu (tRNA ΔA76). The tRNA ΔA76 transcript was purified by PAGE, and the terminal A76 was added as described previously (20).

Preparation and Characterization of M. mobile LeuRS. The M. mobile LeuRS gene was amplified via PCR (30 cycles) from 50 ng M. mobile genomic DNA. The DNA sequence confirmed the originally reported DNA sequence (15). The gene sequence was then optimized (Geneart) for expression in E. coli, particularly with TGA codons changed to TGG, and synthesized with flanking NdeI and BamHI sites to subclone the DNA fragment into pET14b plasmid. The resulting p14LiMmoLeuRS plasmid was used to transform E. coli BL21 (DE3) strain, and LeuRS was expressed and purified (38). Enzy- matic assays were carried out in vitro as described previously in detail (38). All biochemical results were plotted using Python and Matplotlib plotting library (39).

Fig. 4. Proposed evolutionary scheme for LeuRS CP1 editing module degen- Mass Spectrometry Analysis of Mistranslation in E. coli Expressing M. mobile eration in Mycoplasma. M. synoviae and M. agalactiae LeuRS were acquired LeuRS. E. coli BL21 (DE3) transformed with p14LiMmoLeuRS was grown in from Proteobacteria via horizontal gene transfer (Fig. S8). Primary structure LB containing excess methionine or valine (0 mM, 1 mM, 5 mM, and degeneration of the CP1 domain resulted in M. agalactiae and M. synoviae 10 mM). The expressed protein was purified via its six-histidine tag (38) LeuRS CP1a and CP1s (Left). Genome reduction in M. mobile completely and then with Perfect-Focus (G-Biosciences). The LeuRS was digested with eliminated the CP1 editing module in LeuRS (ΔCP1; Right). mass spectrometry grade trypsin (G-Biosciences) at 1∶50 wt∕wt ratio in

9382 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1016460108 Li et al. Downloaded by guest on October 1, 2021 25 mM ammonium bicarbonate. Digestion was performed using a CEM filtering, smoothing, and deisotoping. The refined peak lists were analyzed Discover Microwave Digestor (CEM Corporation) for 15 min at 75 W and with Mascot 2.2 (Matrix Science) using a tolerance of 0.4 Da for both the 55 °C. The digested peptides were lyophilized to dryness and reconstituted precursor ions and fragment ions. The searches were conducted against in 5% acetonitrile with 0.1% formic acid at 200 μg∕mL. A 10-μL aliquot the National Center for Biotechnology Information nonredundant protein was used for liquid chromatography/mass spectrometry (LC/MS) analysis. database. Specific modifications such as L → VorL→ M (or its oxidized state) LC/MS spectrometry was performed using a Waters Q-ToF connected substitutions were analyzed as variable modifications in conjunction with to a nanoAcquity UPLC (Waters Corporation) and an Atlantis dC18 nanoAc- the error-tolerant mode in Mascot. quity UPLC column (75 μm × 150 mm; 3-μm particle size) with a flow rate of 250 nL∕ min. The 60-min gradient was from 100% A to 60% B A ¼ þ 0 1% B ¼ þ 0 1% ACKNOWLEDGMENTS. We thank Ke Chen (University of Illinois, Urbana, IL) ( water . formic acid; acetonitrile . formic acid). Data for providing aligned 16S rRNA sequences and Dr. Makoto Miyata (Osaka collection was performed with MassLynx 4.1 using Data Directed Analysis. City University, Osaka, Japan) for his gift of M. mobile genomic DNA. This The top four intensive precursor ions from each survey scan were subjected work was supported by grants from the National Science Foundation to MS/MS by collision-induced dissociation. The raw mass spectrometric (MCB-0843611 and MCB-0844670) and the National Institutes of Health data were processed using ProteinLynx Global Server 2.2.5 (Waters) for data (GM063789 and P41RR05964).

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