Single Ribosomal Protein Mutations in Antibiotic-Resistant Bacteria Analyzed by Mass Spectrometry

Wilcox, Sheri K.; Cavey, Gregory S.; Pearson, J.

doi:10.1128/aac.45.11.3046-3055.2001

Cited by 42 publications

(38 citation statements)

References 30 publications

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“…Additionally, this emphasizes that loss of ␤-methylthiolation does not inevitably result in a streptomycin dependence phenotype. As anticipated, K42T alone was found to be modified at D88 (Table 1), consistent with the observation that an E. coli K42R Str r mutant is not otherwise altered (27). It may seem surprising that the modification state of the K87 mutants (especially the large R substitution) is unaffected, considering that position 87 is closer in primary sequence to D88 than position 90; however, the side chain of K87 points away from D88 (Fig.…”

supporting

confidence: 72%

Effects of Streptomycin Resistance Mutations on Posttranslational Modification of Ribosomal Protein S12

et al. 2006

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Ribosomal protein S12 contains a highly conserved aspartic acid residue that is posttranslationally ␤-methylthiolated. Using mass spectrometry, we have determined the modification states of several S12 mutants of Thermus thermophilus and conclude that ␤-methylthiolation is not a determinant of the streptomycin phenotype.Streptomycin and streptomycin-resistant mutants have played a seminal role in the elucidation of the decoding process of protein synthesis (reviewed in reference 19). Genetic and biochemical analyses of ribosomes from streptomycin-resistant mutants implicated ribosomal protein S12 as the determinant of the various streptomycin phenotypes, including resistance, dependence, and pseudodependence (reviewed in references 11 and 17). Such mutations have been localized in the three-dimensional structure of the Thermus thermophilus 30S ribosomal subunit to reside within two highly conserved loops centered around residues P90 and K42 (Escherichia coli numbering used throughout) ( Fig. 1) (7).It has only recently been discovered by use of mass spectrometry that E. coli ribosomal protein S12 is posttranslationally modified via a ␤-methylthiolation at position D88 ( Fig. 2A), near the streptomycin binding site and in the midst of residues altered in streptomycin-resistant mutants (14). This modification has also been found to occur in the phototrophic bacterium Rhodopseudomonas palustris (22), and we have identified it for the extremely thermophilic bacterium T. thermophilus (24). Posttranscriptional modifications of rRNA residues have been shown to affect resistance to various antibiotic classes (reviewed in reference 8). For example, ksgA mutants are resistant to kasugamycin due to the loss of N6-dimethylation of two conserved adenosines in 16S rRNA (13), while methylation of rRNA in the peptidyltransferase center (21) or in the decoding region (3) confers resistance to erythromycin and aminoglycosides, respectively (reviewed in references 20 and 26).Interestingly, loss of modification of a ribosomal protein has not yet been shown to affect antibiotic sensitivity. Nevertheless, the proximity of S12 residue D88 to residues altered in streptomycin-resistant mutants raises the possibility that such mutations might confer resistance indirectly by inhibiting ␤-methylthiolation of D88. To address this question, we performed matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to establish the modification status of ribosomal protein S12 in a series of streptomycinresistant and streptomycin-dependent mutants of T. thermophilus.Loss of ␤-methylthiolation in a subset of S12 mutants. We examined the modification states of several S12 mutants of T. thermophilus IB-21 (ATCC 43815) (16) by MALDI-TOF MS as described previously (23). Wild-type S12 was determined to have a mass of 14,519 Ϯ 6 Da (Table 1), consistent with our previous report (24), indicating loss of the initial methionine and the presence of the ␤-methylthiolation. Considering the proximity to D88, we sought to deter...

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confidence: 72%

Effects of Streptomycin Resistance Mutations on Posttranslational Modification of Ribosomal Protein S12

et al. 2006

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“…Most of the peaks detected corresponded to ribosomal proteins of the large (505) and small (305) subunits. Information for methionine loss or posttranslational modifications like methylation, acetylation, and ␤-methylthiolation that are known to occur in E. coli was accounted for in the peak assignments (2,43). Most known ribosomal proteins of the large and small subunits in the molecular mass range from 2,000 to 20,000 could be tentatively assigned; the exceptions were ribosomal proteins L6 and L9.…”

Section: Resultsmentioning

confidence: 99%

“…The assignment procedure was complicated by various posttranslational modifications occurring in ribosomal proteins which have to be accounted for, including N-terminal methionine loss, methylation, ␤-methylthiolation, oxidation, or acetylation. However, since many of these modifications appeared to be conserved posttranslational modifications in Salmonella and E. coli, information on posttranslational modifications could be extracted from previous reports on the detection of ribosomal proteins in E. coli (2,43). By identifying and selecting biomarker subsets specific at different taxonomic levels, a sufficient number of SSIBIs were identified so that salmonellae could be subtyped at a level below the species level.…”

Section: Discussionmentioning

confidence: 99%

Rapid Classification and Identification of Salmonellae at the Species and Subspecies Levels by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Dieckmann

Helmuth

Erhard³

et al. 2008

Appl Environ Microbiol

236

171

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Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing Salmonella enterica subsp. enterica (subspecies I), S. enterica subsp. salamae (subspecies II), S. enterica subsp. arizonae (subspecies IIIa), S. enterica subsp. diarizonae (subspecies IIIb), S. enterica subsp. houtenae (subspecies IV), and S. enterica subsp. indica (subspecies VI), and Salmonella bongori were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for Salmonella, and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of Salmonella strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of Salmonella subspecies in minutes was established.

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“…Mass Spectrometry and N-terminal Sequencing-Protein identification of SDS-PAGE silver-stained bands was done by MS/MS analysis of tryptic peptides using the MS-fit program as previously described (26). N-terminal protein sequencing was performed by automated Edman degradation on an Applied Biosystems model 494 Procise sequencer.…”

Section: Methodsmentioning

confidence: 99%

Targeting, Import, and Dimerization of a Mammalian Mitochondrial ATP Binding Cassette (ABC) Transporter, ABCB10 (ABC-me)

Graf

Haigh

Corson

et al. 2004

Journal of Biological Chemistry

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ATP binding cassette (ABC) transporters are a diverse superfamily of energy-dependent membrane translocases. Although responsible for the majority of transmembrane transport in bacteria, they are relatively uncommon in eukaryotic mitochondria. Organellar trafficking and import, in addition to quaternary structure assembly, of mitochondrial ABC transporters is poorly understood and may offer explanations for the paucity of their diversity. Here we examine these processes in ABCB10 (ABC-me), a mitochondrial inner membrane erythroid transporter involved in heme biosynthesis. We report that ABCB10 possesses an unusually long 105-amino acid mitochondrial targeting presequence (mTP). The central subdomain of the mTP (amino acids (aa) 36 -70) is sufficient for mitochondrial import of enhanced green fluorescent protein. The N-terminal subdomain (aa 1-35) of the mTP, although not necessary for the trafficking of ABCB10 to mitochondria, participates in the proper import of the molecule into the inner membrane. We performed a series of amino acid mutations aimed at changing specific properties of the mTP. The mTP requires neither arginine residues nor predictable ␣-helices for efficient mitochondrial targeting. Disruption of its hydrophobic character by the mutation L46Q/ I47Q, however, greatly diminishes its efficacy. This mutation can be rescued by cryptic downstream (aa 106 -715) mitochondrial targeting signals, highlighting the redundancy of this protein's targeting qualities. Mass spectrometry analysis of chemically cross-linked, immunoprecipitated ABCB10 indicates that ABCB10 embedded in the mitochondrial inner membrane homodimerizes and homo-oligomerizes. A deletion mutant of ABCB10 that lacks its mTP efficiently targets to the endoplasmic reticulum. Quaternary structure assembly of ABCB10 in the ER appears to be similar to that in the mitochondria.ABC 1 transporters comprise a large and diverse family of membrane translocases (1). Their function ranges from peptide transport to phospholipid flipping to anion channel formation. Members of the ABC transporter superfamily have been implicated in numerous human diseases (including cystic fibrosis (CFTR/ABCC7), adrenoleukodystrophy (ALDP/ABCD1), Zellweger's syndrome (PMP70/ABCD3), progressive familial intrahepatic cholestasis (SPGP/ABCB11), and Stargardt macular dystrophy (ABCR/ABCA4)) (2). The basic structure of ABC transporters is relatively well conserved and includes a hydrophilic ATP binding cassette and a hydrophobic membranespanning domain (3). Whereas the large members of the family contain two of each domain, the "half-transporters" contain one of each and are predicted to dimerize (3-6). Mammalian ABC transporters are found predominantly in the plasma membrane but are also known to play essential roles in a number of organelles, including the endoplasmic reticulum, peroxisome, and the mitochondrion (2). Mammalian mitochondrial ABC transporters have recently gained attention for their role in heme biosynthesis and iron sulfur cluster synthesis (7-12). According...

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Single Ribosomal Protein Mutations in Antibiotic-Resistant Bacteria Analyzed by Mass Spectrometry

Cited by 42 publications

References 30 publications

Effects of Streptomycin Resistance Mutations on Posttranslational Modification of Ribosomal Protein S12

Effects of Streptomycin Resistance Mutations on Posttranslational Modification of Ribosomal Protein S12

Rapid Classification and Identification of Salmonellae at the Species and Subspecies Levels by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Targeting, Import, and Dimerization of a Mammalian Mitochondrial ATP Binding Cassette (ABC) Transporter, ABCB10 (ABC-me)

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