Pseudogene Recoding Revealed from Proteomic Analysis of

Article pubs.acs.org/jpr

Pseudogene Recoding Revealed from Proteomic Analysis of Salmonella Serovars Ye Feng,*,†,¶ Kun-Yi Chien,‡,¶ Hsiu-Ling Chen,§ and Cheng-Hsun Chiu*,‡,§ †

Genomics Research Center, Harbin Medical University, Harbin, P. R. China Graduate Institute of Biomedical Sciences, Chang Gung University College of Medicine, Taoyuan, Taiwan § Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan ‡

S Supporting Information *

ABSTRACT: Recoding refers to the reprogramming of mRNA translation by nonstandard read-out rules. In this study, we used stable isotope labeling with amino acids in cell culture (SILAC) technology to investigate the proteome of host-adapted Salmonella serovars, which are characteristic of accumulation of pseudogenes. Interestingly, a few annotated pseudogenes were indeed able to express peptides downstream of the inactivation site, suggesting the occurrence of recoding. Two mechanisms of recoding, namely, programmed frameshifting and codon redefinition, were both found. We believe that the phenomena of recoding are not rare in bacteria. More studies are required for a better understanding of bacterial translation and the implication of pseudogene recoding in Salmonella serovars. KEYWORDS: Salmonella, serovar, recoding, pseudogene

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INTRODUCTION Salmonella are important pathogens in humans and animals. Taxonomically, Salmonella organisms are considered as a genus which includes more than 2500 different serovars. While most serovars are host-generalist, a few serovars are host-restricted (or adapted), namely, able to cause disease in one or a small group of hosts only. Loss of function through pseudogene formation has been considered as a hallmark of host-restricted Salmonella serovars.1−6 For example, the host-generalist S. Typhimurium contains less than 30 pseudogenes, whereas the host-adapted serovars S. Typhi, S. Paratyphi A, and S. Choleraesuis each have more than 150 pseudogenes. A popular explanation of the phenomena is that the host-generalist pathogens may require a large gene inventory to maintain the ability of infecting multiple host species, whereas the hostspecialist pathogens, established in specialized niches, do not have to keep a high level of genetic and phenotypic plasticity. As a result, the redundant genes will finally undergo the decaying process. Currently, bacterial annotation is mostly based on in silico predictions. When the query gene cannot match the correct open reading frame as the reference ortholog, it is considered as a pseudogene. Frameshift and nonsense mutation are two main causes of pseudogenization in bacteria. The annotated pseudogenes cannot be translated because of the corrupted coding frame. However, a small proportion of them are able to utilize recoding to adjust the abnormality of the inactivation site © 2012 American Chemical Society

and allow the downstream translation. Programmed frameshifting and codon redefinition are the two mechanisms corresponding to frameshift and nonsense mutation, respectively. While the former enables ribosomes to slip across the aberrant site by adopting an alternative frame and then continuing the triplet decoding, the latter can assign the internal stop codon to a special amino acid, often selenocysteine or pyrrolysine, so that the whole translation process can be read through.7−10 Although discovered in many organisms, recoding is generally thought to rarely occur in nature.11 Since the nucleotide sequence of recoded genes and the true pseudogenes make little difference, the identification and annotation of recoded genes lag far behind. The aim of this study was to develop an appropriate experimental approach to detect recoding at the genomic scale and meanwhile to find the real extent of its occurrence. We identified peptides from Salmonella serovars by stable isotope labeling with amino acids in cell culture (SILAC) technology. Consequently, a few annotated pseudogenes were found to be able to translate normal peptides, suggesting that the recoding mechanism may actually be common in Salmonella or even in the whole bacteria kingdom. Received: September 7, 2011 Published: February 1, 2012 1715

dx.doi.org/10.1021/pr200904c | J. Proteome Res. 2012, 11, 1715−1719

Journal of Proteome Research

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Article

MATERIALS AND METHODS

stopped, and the six-port valve switched to allow the loading pump to wash away the residual salt solution in the flow path of the RP-trapping column. After the trapping column switched to the RP analytical column, the bound peptides were eluted with a complete acetonitrile gradient (elution, regeneration, and then re-equilibration) in the presence of 0.05% formic acid over 84 min.

Bacterial Strains

S. Typhimurium LT2 (abbr. STM), S. Typhi ATCC33458 (abbr. STY), and S. Choleraesuis SC-B67 (collected from Chang Gung Medical Fundation, abbr. SCS) were used in this study. All of the bacteria were maintained in LB plates before being cultured in SILAC medium.

Mass Spectrometry

SILAC and Treatments

The effluent of the online 2D LC was analyzed by a LTQOrbitrap hybrid mass spectrometer (Thermo Electron, Bremen, Germany). The mass spectrometer was operated in the information-dependent acquisition (IDA) mode. Survey full scan MS spectra (from m/z 300 to 2000) are acquired in the Orbitrap at a resolution of 60 000 with lock mass function enabled. Ten most intense ions in each MS spectrum are selected for isolation and fragmentation in the linear ion trap (MS/MS). Each precursor ions was allowed to be analyzed twice and then excluded in the subsequent 1 min. The MS/MS isolation width was set to 2 Da, and the maximum precursor accumulation time for MS/MS was set to 150 ms.

The SILAC media used in this study were L-Lys- and L-Argdepleted RPMI media (Invitrogen, Carlsbad, CA) supplemented with 20 mM HEPES buffer and either 12C6-L-Lys/ 12C6-L-Arg (light medium) or 13C6-L-Lys/13C6-L-Arg (heavy medium) (98% purity, Isotec, Miamisburg, OH). A single colony of each Salmonella serovar was grown at 37 °C overnight in LB broth. Twenty-five microliters of each overnight culture was transferred to 5 mL of either heavy (STM) or light medium (SCS and STY) at 37 °C overnight. Ten microliters of each overnight culture was transferred to 50 mL of fresh medium and allowed to grow to late log phase (O.D.600 = 0.6). For each pair of serovars, equal numbers of cells, estimated by O.D.600, were mixed and subjected to a protein extraction procedure. Bacterial proteins were extracted with 0.1% SDS solution by sonication. Protein concentration was determined by the BCA method (Thermo Fisher, Rockford, IL). Forty micrograms of protein extracts were reduced and alkylated with dithiothreitol and iodoacetamide, respectively, and digested with 1 μg of modified trypsin (Promega, Madison, WI) at 37 °C overnight. Peptide solutions were desalted by homemade RP (reverse phase) microcolumns packed with Source 15RPC (GE healthcare, Uppsala, Sweden) and dried by speed vacuum.

Peptide and Protein Identification

Raw MS files from the LTQ-Orbitrap were analyzed by Mascot (Version 2.2.2, Matrix Science Inc., Boston, MA) and MaxQuant (version 1.0.13.13). The MaxQuant software created a new fasta file for each serovar by including a complete set of common contaminates and reversed sequence entries for estimating false discovery rate. The databases were configured in an in-house Mascot search engine (Version 2.2.2, Matrix Science Inc., Boston, MA), and MS/MS spectra were searched against the three Salmonella databases of each individual serovar. The protein-coding sequences of the three Salmonella strains were extracted from Genbank files. The accession numbers are AE006468.1 (for STM), AE017220.1 (for SCS), and AE014613.1 (for STY), respectively. As the pseudogenes in SCS and STY do not have the coding sequences with correct open reading frame, the sequences of their corresponding orthologs in STM were put into the respective databases of SCS and STY. In addition, nucleotides of intergenic regions were also extracted and added to the database for searching. The parameters setting for the Mascot searches are as follows: cysteine carbamidomethylation was selected as a fixed modification, whereas protein N-terminal acetylation, methionine oxidation were selected as variable modifications, and a maximum of two missed cleavages were allowed. Parent mass and fragment ions were searched with mass deviation of 5 ppm and 0.5 Da, respectively. The search results were further processed by MaxQuant with the following parameter settings: peptides of minimum 6 amino acids were allowed, false discovery rate was set to 0.01, and a posterior error probability (PEP) for each MS/MS spectrum below or equal to 0.1 was required.

Online 2D LC System

The comprehensive 2D-SCX-RP-LC system (Ultimate 3000, Dionex, Germering, Germany) has been equipped with one gradient pump for SCX (strong cation exchange), one other gradient pump for RP, one isocratic pump for online dilution, one 10-port valve with two RP-trapping columns for alternating trapping, one 6-port valve for controlling the trapping column washing, and a manual injector for sample loading. Such a combination allowed us to introduce an organic solvent (acetonitrile) in the first dimensional SCX separation without affecting the second dimensional RP separations by using an online dilution design. Briefly, samples dissolved in 50% acetonitrile containing 0.1% formic acid were loaded onto the SCX column through a manual injector. The flow rate of the first dimensional separation was operated at a flow rate of 1 μL/min on a home-packed SCX column (0.5 × 150 mm, pack with Luna-SCX particles from Phenomenex, Torrance, CA). The peptides were eluted using a continuous ammonium chloride concentration gradient in the presence of 0.1% formic acid and 30% ACN. The salt gradient was segmented in 17 steps, 90 min for each, and matched with the second dimensional reverse phase separations. The isocratic loading pump delivering 50 μL/min of solvent A (0.1% formic acid in water) was used for diluting the effluent of SCX column through a T-union and mixing tubing before it reached the trapping column. In the meantime, the other RP-trapping column, installed on the same 10-port valve, was connected with the RP-separating column and was being analyzed by a mass spectrometer. Six minutes before each salt gradient step being completed, the binary pump for the SCX separation

Pseudogene PCR Validation on Genomic DNA and cDNA

The genomic DNA was isolated from overnight culture of bacteria according to the manual (GeneMark, Taichung, Taiwan). The RNA was purified from log phase of bacteria with TRIzol reagent (Invitrogen, Carlsbad, CA) and transcribed to cDNA using iScript cDNA synthesis kit (Bio-Rad Laboratories, Richmond, CA). The genomic DNA and cDNA were used in the following pseudogene validation. The pseudogene was amplified with primer pairs listed in the 1716



Article

we could not rule out the possibility that the genome itself is continually going through a series of minor evolutions at these loci. It has been known that within bacteria genome, certain genes may contain simple sequence repeats, often termed as contingency loci, through which bacterial pathogens are able to switch the expression by polymerase slippage in order for sensing and responding to external changes.12 The coding frames of the remaining 24 genes were truly disrupted. We also sequenced their mRNA and found that the mRNA sequences were identical to the genomic sequences. On the other hand, 19 of the 24 genes have more than one peptide detected downstream of the inactivation site, which greatly reduces the likelihood of false peptide identification (Figure 1A shows one example, and other cases are listed in the Supporting Information, Table S3). Combination of these results suggests that the abnormality of coding frame has indeed been corrected during the translation phase. Thirteen out of the 24 genes belong to programmed frameshifting (Table 3), and the other 11 belong to codon redefinition (Table 4). We found that the programmed frameshifting sites are mostly located within homopolymers. Perhaps the programmed-frameshifting prefers repairing the frameshift caused by homopolymer, but it is more likely that the bacteria replication is prone to produce such frameshift, and then the programmed-frameshifting is forced to tolerate them. Of the 11 cases of codon redefinition, six fell into the situation that CAG mutates to TAG but translation ensues. Three of the others belong to the conversion from TGG to TAG. Some methanogenic archaea and bacteria have been known to translate TAG into a special amino acid pyrrolysine with the assistance from the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon.13,14 Our data suggest that Salmonella may prefer to recode the codon TAG, but it is unclear if Salmonella will decode TAG into pyrrolysine or other amino acids. Because prediction of start codon remains to be the most difficult part of current prokaryotic annotation, the above result can also be interpreted as that another start codon, probably downstream of the recoding site, replaces the original start codon to initiate the translation of downstream peptides. Such a possibility does exist, but in at least 7 cases, the detected peptide happened to cover the recoding site, which provides direct evidence that recoding did take place. Figure 1B lists one of the cases, and the rest are listed in the Supporting Information (Table S3). We believe that the recoded amino acid has such a similar m/z value to its counterpart in STM that the Mascot search engine can identify the peptide from the STM protein sequences. However, the specific amino acids that the recoding sites encode are not clear. It is also unknown whether Salmonella follows the same mechanism of recoding as the previous literature reported. Because we used SILAC technology, the quantitative information of peptides in the recoded genes can be measured by comparison with the counterparts in STM (STM used as control in which the recoded genes have normal open reading frames). For housekeeping genes (those used for multilocus sequence typing), the expression ratio between STM and SCS (or STY) ranged mostly from 0.25 to 4, whereas for the recoded genes, the expression of many peptides in SCS (or STY) was even one tenth less than that in STM (Supporting Information, Table S3). Thus, recoding seems not to be a highly effective alternative to normal translation machinery but merely an adequate replacement. Accordingly, genes with the

Supporting Information (Table S1) and sequenced by ABI 3730 sequencer (Applied Biosystems, Carlsbad, CA).

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RESULTS AND DISCUSSION In this study, we used LC−MS/MS to examine whether the pseudogenes in S. Typhi ATCC33458 and S. Choleraesuis SCB67 were able to express normal protein sequences. Because the original pseudogene prediction in the two strains were taking S. Typhimurium LT2 as the reference, we also included STM in our experiments. Thus, two independent SILAC experiments were carried out: STM vs SCS and STM vs STY (details in the Methods section). The complete peptide lists are provided in the Supporting Information (Table S2a−d). As expected, most detected peptides were mapped to the protein-coding genes, and few were located within the intergenic regions, suggesting the overall accuracy of the original annotation. The general information about the peptides detected is listed in Table 1. Table 1. General Information of the Study no. no. no. no. no. no. no.

of of of of of of of

identified peptides annotated proteins in the genome identified proteins intergenic regions expressing peptides annotated pseudogenes in the genome annotated pseudogenes expressing peptides detected recoded genes

SCS

STY

16376 4441 1798 29 151 14 10

13022 4318 1566 17 218 15 14

In order to explore the recoding phenomenon, we focused on the peptides expressed by the annotated pseudogenes. Theoretically, the pseudogene can still act as a template to express peptides upstream of the inactivation site, so we used the downstream peptides only as the evidence of recoding. As a result, 14 and 15 annotated pseudogenes had such peptides detected in SCS and in STY, respectively. BLAST searches guaranteed that these peptides have a unique hit in the genome; i.e., they do not exist in any other genes. We first resequenced these pseudogenes to check the accuracy of the original sequences deposited in NCBI GenBank database. Of them, five pseudogenes become different from their original sequences, and their open reading frames have returned to the normal state (Table 2). All of the differences belonged to frameshift, Table 2. Genes Showing Different Result between Current and Original Sequencinga gene ID

ID (in STM)

name

site

original and current sequencing

SCPS102 SCPS45 SCPS72 SCPS83 t1716

STM0994 STM2539 STM3901 STM4176 STM1204

mukB hscA ilvG purH fhuE

586aa 168aa 72aa 359aa 331aa

original: ’ccc’; current: ’c’; original: ’a’; current: ’aa’; original: ’cc’; current: ’ccc’; original: ’aaa’; current: ’aaga’; original: ’ggg’; current: ’gg’;

a

The column site tells where the inactivation occurred. The column of original and current sequencing shows the sequence string at the inactivation site.

and all were located within the stretches of homopolymers coincidentally. Because capillary technology is prone to make mistakes when meeting homopolymers, errors might have occurred during the original genome sequencing. Nevertheless, 1717



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Figure 1. Figure demonstration of recoding. Panel A is an example showing that the annotated pseudogene t0199 expressed peptides not only upstream but also downstream of the inactivation site; panel B illustrates where the programmed frameshifting occurred.

Table 3. Genes That May Have Programmed Frameshiftinga site

comparison

SCPS97

geneID

ID (in STM) STM0617

rna

name

142aa

SCPS3

STM0826

ybiN

233aa

SCPS37

STM2194

yeiG

65aa

SCPS109

STM2294

yfaZ

34aa

SCPS39

STM2299

yfbG

381aa

SCPS144

STM3295

folP

57aa

t0199

STM0191

fhuA

139aa

t2506

STM0358.S

res

177aa

t1243

STM1375

ynhG

109aa

t1421

STM1500

ynfD

55aa

t2706

STM2809

proV

145aa

t3100

STM3179

mdaB

78aa

t4583

STM4533

tsr

152aa

SCS: ’gcag’; STM: ’gcg’; SCS: ’aaaaaaaa’; STM: ’aaaaaaa’ SCS: ’gcgcgcgc’; STM: ’gcgcgc’; SCS: ’aaaa’; STM: ’aaaaa’; SCS: ’aaaaa’; STM: ’aaaaaa’; SCS: ’ggggggggg’; STM: ’gggggggg’; STY: ’tc’; STM: ’tctc’; STY: ’aa’; STM: ’aaa’; STY: ’tttt’; STM: ’ttatt’; STY: ’atac’; STM: ’aac’; STY: ’aaaa’; STM: ’aaaaa’; STY: ’aaaaaa’; STM: ’aaaaaaa’; STY: ’ttttt’; STM: ’tttttt’;

no. of detected peptides 0/1/7

Table 4. Genes That May Have Codon Redefinitiona geneID

ID (in STM)

name

site

comparison

SCPS6

STM0874

mdaA

113aa

1/0/1

SCPS11

STM1062

uup

272aa

1/0/4

SCPS34

STM1989

yedI

44aa

0/0/3

SCPS44

STM2532

11/0/6

t2279

STM0589

fepE

79aa

0/0/4

t2066

STM0822

ybiB

67aa

t1168

STM1289

yeaD

50aa

t0687

STM2168

pbpG

5aa

t2690

STM2795

ygaU

32aa

t3073

STM3152

t4575

STM4525

SC: ’taa’; LT: ’caa’; SC: ’tag’; LT: ’cag’; SC: ’tag’; LT: ’cag’; SC: ’tag’; LT: ’cag’; TY: ’tag’; LT: ’tgg’; TY: ’tag’; LT: ’cag’; TY: ’tag’; LT: ’tgg’; TY: ’tga’; LT: ’cga’; TY: ’tag’; LT: ’cag’; TY: ’tag’; LT: ’cag’; TY: ’tag’; LT: ’tgg’;

2/1/7 0/0/2 2/1/2 0/1/0 3/0/10

266aa

432aa hsdM

467aa

no. of detected peptides 1/0/2 1/0/2 1/0/1 3/0/3 0/0/3 1/0/4 3/1/4 0/0/1 0/0/2 4/1/0 11/0/1

The column of “site” indicates where the recoding occurred. The column of “comparison” shows the abnormal nucleotide string (in SCS or STY) and the normal string (in STM) at the recoding site. The three numbers (from left to right) in the column of “no. of detected peptides” represent the number of peptides before, on, and after the recoding site, respectively.

a

1/0/2 5/1/8

The column of “site” indicates where the recoding occurred. The column of “comparison” shows the abnormal nucleotide string (in SCS or STY) and the normal string (in STM) at the recoding site. The three numbers (from left to right) in the column of “no. of detected peptides” represent the number of peptides before, on, and after the recoding site, respectively.

a

observed between regions before and after the recoding site, in terms of either the number of detected peptides (Tables 3 and 4) or the expression ratio (Supporting Information, Table S3). This phenomenon therefore gives us a hint that some pseudogenes can only be expressed through recoding mechanism but reject the normal translation machinery. However, this hypothesis needs more experimental support.

recoding phenomenon are rarely likely to play the housekeeping role but function only under particular conditions. The expression level between regions before and after the recoding site was also compared. We hypothesized that the pseudogenes face two choices during translation: they can be recoded to produce normal peptides, or they follow normal translational machinery to produce premature/wrong proteins. If the two kinds of machinery function in one gene simultaneously, then the peptides before the recoding site should have a higher expression level than that behind the recoding site. Actually, no remarkable difference can be

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CONCLUSION Pseudogene formation has been regarded as an important mark during host restriction in Salmonella. In this study, we found that the majority of pseudogenes did fail to express, demonstrating the overall accuracy of in silico annotation. However, we also found that several genes were annotated as pseudogenes but had normally translated peptides detected. Considering that the current recoded genes were found in one 1718



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Simmonds, M.; White, B.; Bason, N.; Mungall, K.; Dougan, G.; Parkhill, J. Pseudogene accumulation in the evolutionary histories of Salmonella enterica serovars Paratyphi A and Typhi. BMC Genomics 2009, 10 (1), 36. (6) Liu, W. Q.; Feng, Y.; Wang, Y.; Zou, Q. H.; Chen, F.; Guo, J. T.; Peng, Y. H.; Jin, Y.; Li, Y. G.; Hu, S. N.; Johnston, R. N.; Liu, G. R.; Liu, S. L. Salmonella paratyphi C: Genetic divergence from Salmonella choleraesuis and pathogenic convergence with Salmonella typhi. PLoS One 2009, 4 (2), e4510. (7) Farabaugh, P. J. Programmed translational frameshifting. Microbiol. Rev. 1996, 60 (1), 103−34. (8) Baranov, P. V.; Fayet, O.; Hendrix, R. W.; Atkins, J. F. Recoding in bacteriophages and bacterial IS elements. Trends Genet. 2006, 22 (3), 174−81. (9) Gesteland, R. F.; Atkins, J. F. Recoding: Dynamic reprogramming of translation. Annu. Rev. Biochem. 1996, 65, 741−68. (10) Silva, R. M.; Miranda, I.; Moura, G.; Santos, M. A. Yeast as a model organism for studying the evolution of non-standard genetic codes. Briefings Funct. Genomics Proteomics 2004, 3 (1), 35−46. (11) Baranov, P. V.; Gurvich, O. L.; Fayet, O.; Prere, M. F.; Miller, W. A.; Gesteland, R. F.; Atkins, J. F.; Giddings, M. C. RECODE: A database of frameshifting, bypassing and codon redefinition utilized for gene expression. Nucleic Acids Res. 2001, 29 (1), 264−7. (12) Moxon, R.; Bayliss, C.; Hood, D. Bacterial contingency loci: The role of simple sequence DNA repeats in bacterial adaptation. Annu. Rev. Genet. 2006, 40, 307−33. (13) Zhang, Y.; Gladyshev, V. N. High content of proteins containing 21st and 22nd amino acids, selenocysteine and pyrrolysine, in a symbiotic deltaproteobacterium of gutless worm Olavius algarvensis. Nucleic Acids Res. 2007, 35 (15), 4952−63. (14) Zhang, Y.; Baranov, P. V.; Atkins, J. F.; Gladyshev, V. N. Pyrrolysine and selenocysteine use dissimilar decoding strategies. J. Biol. Chem. 2005, 280 (21), 20740−51.

physiological state only (in LB broth), more genes may have the potential to be recoded. In other words, the recoding phenomenon may not be rare in bacteria. In order to elucidate the importance of recoding in bacteria and to gain a more accurate annotation, we suggest that the proteome should be used as a regular approach for pseudogene annotation in each bacterial genome sequencing project.

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ASSOCIATED CONTENT

* Supporting Information S

Table S1 lists primers for pseudogene confirmation. Table S2a−d lists the complete peptides detected in SILAC experiments. Table S3 lists the detected peptides from the recoded genes as well as their quantitative information. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.H.C.); pandafengye@ gmail.com (Y.F.). Tel.: 886 3 3281200. Fax: 886 3 3288957. Author Contributions ¶

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Science Council, Taiwan (97-2314-B-182A-051-MY3). REFERENCES

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Pseudogene Recoding Revealed from Proteomic Analysis of

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