Phosphoproteomic Analysis Reveals the Effects of PilF Phosphorylation on Type IV Pilus and Biofilm Formation in Thermus thermophilus HB27

Wu, Wan-Chen; Liao, Jiahn-Haur; Lin, Guang-Huey; Lin, Miao-Hsia; Chang, Ya-Wen; Liang, Suh‐Yuen; Fu, Yang; Khoo, Kay‐Hooi; Wu, Shih‐Hsiung

doi:10.1074/mcp.m113.029330

Cited by 20 publications

(22 citation statements)

References 74 publications

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“…S4). By means of affinity enrichment using this antibody, we identified 361 propionylation sites in 183 proteins, indicating that lysine propionylation is one of the most prevalent PTMs in T. thermophilus, comparable to acetylation and phosphorylation (33,45).…”

Section: Discussionmentioning

confidence: 99%

Lysine Propionylation Is a Prevalent Post-translational Modification in Thermus thermophilus

Okanishi

Masui

Kuramitsu

2014

Molecular & Cellular Proteomics

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Recent studies of protein post-translational modifications revealed that various types of lysine acylation occur in eukaryotic and bacterial proteins. Lysine propionylation, a newly discovered type of acylation, occurs in several proteins, including some histones. In this study, we identified 361 propionylation sites in 183 mid-exponential phase and late stationary phase proteins from Thermus thermophilus HB8, an extremely thermophilic eubacterium. Functional classification of the propionylproteins revealed that the number of propionylation sites in metabolic enzymes increased in late stationary phase, irrespective of protein abundance. The propionylation sites on proteins expressed in mid-exponential and late stationary phases partially overlapped. Furthermore, amino acid frequencies in the vicinity of propionylation sites differed, not only between the two growth phases but also relative to acetylation sites. In addition, 33.8% of mid-exponential phasespecific and 80.0% of late stationary phase-specific propionylations (n > 2) implied that specific mechanisms regulate propionylation in the cell. Moreover, the limited degree of overlap between lysine propionylation (36.8%) and acetylation (49.2%) sites in 67 proteins that were both acetylated and propionylated strongly suggested that the two acylation reactions are regulated separately by specific enzymes and may serve different functions. Finally, we also found that eight propionylation sites overlapped with acetylation sites critical for protein functions such as Schiffbase formation and ligand binding. Molecular & Cellular

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Section: Discussionmentioning

confidence: 99%

Lysine Propionylation Is a Prevalent Post-translational Modification in Thermus thermophilus

Okanishi

Masui

Kuramitsu

2014

Molecular & Cellular Proteomics

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“…Bacterial protein phosphorylation is required for the biosynthesis of capsular polysaccharides, biofilm development, virulence, and antibiotic resistance, particularly through two-component signaling systems (44,55,98,99). Thus, it has been demonstrated that His and Asp phosphorylation regulate the functions of pathogenesis, such as adherence, motility, enhancement of toxicity, quorum sensing, capsule formation, and drug resistance (100).…”

Section: Discussionmentioning

confidence: 99%

“…Phosphoproteomic analyses of SK17 were conducted using the shotgun LC-MS/MS approach to obtain large-scale data sets from biological triplicate experiments for each strain. Using the robust HAMMOC enrichment method and high mass accuracy LTQ-Orbitrap Velos, we identified 248 unique phosphoproteins with 351 phosphopeptides from SK17-S (supplemental Table S1), as well as 211 unique phosphoproteins with 240 phosphopeptides from SK17-R (supplemental Table S2) (34,44,54). Only 70 phosphoproteins overlapped in these two strains; 178 and 141 uniquely identified phosphoproteins were found in SK17-S and SK17-R, respectively ( Fig.…”

Section: Comparison Of a Baumannii Sk17-s And Sk17-r Phos-mentioning

confidence: 99%

“…Cell debris was removed by centrifugation at 8000 ϫ g for 25 min at 4°C; the clear supernatant fluid was filtered through 0.45-m pore size filters (Millipore, Bedford, MA). The hexahistidine-tagged proteins from strain Ab290 carrying wild-type AmpC (AmpC-WT) or mutants (S90A and S90D) were purified from a Ni 2ϩ -NTA column (Sepharose 6 Fast Flow resin, GE Healthcare, Piscataway, NJ; Econo-Pac column, Bio-Rad), and eluted in 200 mM imidazole (44). Fractions containing the desired protein were identified by 12.5% SDS-PAGE, based on the presence of a 43 kDa band, and then concentrated using an Amicon Ultra-15 filter (Millipore).…”

Section: Nanolc-ms/msmentioning

confidence: 99%

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Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17

Lai

Yang

Chern

et al. 2016

Molecular & Cellular Proteomics

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Nosocomial infectious outbreaks caused by multidrugresistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/ 12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/ 4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S 88 VS 90 K of the AmpC ␤-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher ␤-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both ␤-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies.

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“…Instrument control was through Tune 2.6.0 and Xcalibur 2.1. Detailed settings for the Orbitrap analyzer followed a previous description [21]. …”

Section: Methodsmentioning

confidence: 99%

Comparison of Enzymatic Traits between Native and Recombinant Glycine Sarcosine N-Methyltransferase from Methanohalophilus portucalensis FDF1T

2016

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The halophilic methanoarchaeon Methanohalophilus portucalensis FDF1T possesses the ability to synthesize the osmolyte betaine from its precursor, glycine, in response to extracellular salt stress through a three-step transmethylation process. Analysis of recombinant glycine sarcosine N-methyltransferase (rGSMT) and recombinant sarcosine dimethylglycine N-methyltransferase (rSDMT) from Escherichia coli indicated that betaine synthesis is rate-limited by rGSMT and is constitutively activated by rSDMT. Therefore, it is of interest to purify native GSMT from Methanohalophilus portucalensis to further compare its enzymatic characteristics and kinetics with rGSMT. In this study, native GSMT was purified through DEAE ion exchange and gel filtration chromatography with 95% purity. The enzymatic characteristics of GSMT and rGSMT showed similar trends of activities that were activated by high concentrations of monovalent cations. Both were feedback-inhibited by the end product, betaine, and competitively inhibited by S-adenosylhomocysteine (SAH). Native GSMT was 2-fold more sensitive to SAH than rGSMT. Notably, comparison of the kinetic parameters illustrated that the turnover rate of glycine methylation of GSMT was promoted by potassium ions, whereas rGSMT was activated by increasing protein-glycine binding affinity. These results suggest that GSMT and rGSMT may have different levels of post-translational modifications. Our preliminary mass spectrometry evidence indicated that there was no detectable phosphosite on GSMT after the complicated purification processes, whereas purified rGSMT still possessed 23.1% of its initial phosphorylation level. We believe that a phosphorylation-mediated modification may be involved in the regulation of this energy consuming betaine synthesis pathway during the stress response in halophilic methanoarchaea.

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Phosphoproteomic Analysis Reveals the Effects of PilF Phosphorylation on Type IV Pilus and Biofilm Formation in Thermus thermophilus HB27

Cited by 20 publications

References 74 publications

Lysine Propionylation Is a Prevalent Post-translational Modification in Thermus thermophilus

Lysine Propionylation Is a Prevalent Post-translational Modification in Thermus thermophilus

Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17

Comparison of Enzymatic Traits between Native and Recombinant Glycine Sarcosine N-Methyltransferase from Methanohalophilus portucalensis FDF1T

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