A proteomic and transcriptomic approach reveals new insight into beta-methylthiolation of Escherichia coli ribosomal protein S12 - PubMed (original) (raw)

A proteomic and transcriptomic approach reveals new insight into beta-methylthiolation of Escherichia coli ribosomal protein S12

Michael Brad Strader et al. Mol Cell Proteomics. 2011 Mar.

Abstract

β-methylthiolation is a novel post-translational modification mapping to a universally conserved Asp 88 of the bacterial ribosomal protein S12. This S12 specific modification has been identified on orthologs from multiple bacterial species. The origin and functional significance was investigated with both a proteomic strategy to identify candidate S12 interactors and expression microarrays to search for phenotypes that result from targeted gene knockouts of select candidates. Utilizing an endogenous recombinant E. coli S12 protein with an affinity tag as bait, mass spectrometric analysis identified candidate S12 binding partners including RimO (previously shown to be required for this post-translational modification) and YcaO, a conserved protein of unknown function. Transcriptomic analysis of bacterial strains with deleted genes for RimO and YcaO identified an overlapping transcriptional phenotype suggesting that YcaO and RimO likely share a common function. As a follow up, quantitative mass spectrometry additionally indicated that both proteins dramatically impacted the modification status of S12. Collectively, these results indicate that the YcaO protein is involved in β-methylthiolation of S12 and its absence impairs the ability of RimO to modify S12. Additionally, the proteomic data from this study provides direct evidence that the E. coli specific β-methylthiolation likely occurs when S12 is assembled as part of a ribosomal subunit.

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Figures

Fig. 1.

Structure of β-methylthioaspartic acid.

Fig. 2.

SPA tagged S12 resulted in the enrichment of ribosomal small subunit associated complexes and the co-isolation of 16S rRNA. A, Polypeptides from eluates representing SPA-S12 (lane 2) and negative control (lane 3) pull-downs were separated by SDS-PAGE (10% polyacrylamide gels) prior to in-gel tryptic digestion and one-dimensional LC tandem mass spectrometry. Molecular weight standards are represented in lane 1. B, 1.5% agarose gel of 300-base-pair regions of 16S rRNA from SPA-S12 (lane 2) and negative control (lane 4) pull-downs. A DNA base pair ladder is represented in lane 1. Total RNA was isolated from pull-down eluates and RT-PCR analysis was performed (see Materials and Methods). Forward and reverse oligonucleotide primers were used to generate 300-base-pair cDNAs. As an additional control SPA-S12 eluates were incubated without reverse transcriptase prior to PCR (lane 3) to confirm that detectable cDNAs were because of the presence of 16S rRNA.

Fig. 3.

FNR and NarL expression is decreased significantly in the rimO mutant. E. coli strains containing SPA tagged FNR, NarL, and NarP genes were grown to mid-log phase and lysed. No changes were observed for NarP. Sixteen micrograms of total protein were loaded in each lane. Expressions of SPA-tagged proteins were detected by Western blot analysis using anti-FLAG primary antibodies, DnaK monoclonal antibodies, and anti-mouse secondary horseradish peroxidase (HRP) conjugated antibodies. A, Representative Western blot of lysates from rimO mutant and wild type E. coli. B, Quantification of Western blots. Relative intensities of ECL bands were quantified using the National Institutes of Health Image software. Imunoreactivities were normalized to that of DnaK in the same sample and the samples were compared. A total of two biological replicates were performed. Error bars represent mean ± S.E.

Fig. 4.

Fragmentation spectra of (A) the modified and (B) unmodified forms of the +2 charged tryptic peptide representing residues 86–93. The modified peptide was not identified in samples from the rimO mutant. Both spectra show singly charged y and b fragment ions. y6, y7 and b3 ions in the modified spectrum are 46 daltons (mass of the methylthiolation modification) higher than the same fragment ions for the unmodified spectrum.

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A proteomic and transcriptomic approach reveals new insight into beta-methylthiolation of Escherichia coli ribosomal protein S12 - PubMed (original) (raw)