Experimental Validation of Bacillus anthracis A16R Proteogenomics

Gao, Zhiqi; Wang, Zhiqiang; Zhang, Kun; Li, Yanchang; Zhang, Tao; Wang, Dongshu; Liu, Xiankai; Feng, Erling; Chang, Lei; Xu, Junjie; He, Shan; Xu, Ping; Zhu, Li; Wang, Hengliang

doi:10.1038/srep14608

Cited by 6 publications

(4 citation statements)

References 27 publications

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“…The proteome coverage achieved in this study allowed us to perform B. subtilis genome re-annotation by proteogenomics, such as done in other bacteria by our group and others 57 – 61 . As previously reported, target-decoy approach substantially underestimates the FDR in six-frame searches of bacterial genomes 59 .…”

Section: Discussionmentioning

confidence: 98%

In-depth analysis of Bacillus subtilis proteome identifies new ORFs and traces the evolutionary history of modified proteins

Ravikumar

Nalpas

Anselm

et al. 2018

Sci Rep

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Bacillus subtilis is a sporulating Gram-positive bacterium widely used in basic research and biotechnology. Despite being one of the best-characterized bacterial model organism, recent proteomics studies identified only about 50% of its theoretical protein count. Here we combined several hundred MS measurements to obtain a comprehensive map of the proteome, phosphoproteome and acetylome of B. subtilis grown at 37 °C in minimal medium. We covered 75% of the theoretical proteome (3,159 proteins), detected 1,085 phosphorylation and 4,893 lysine acetylation sites and performed a systematic bioinformatic characterization of the obtained data. A subset of analyzed MS files allowed us to reconstruct a network of Hanks-type protein kinases, Ser/Thr/Tyr phosphatases and their substrates. We applied genomic phylostratigraphy to gauge the evolutionary age of B. subtilis protein classes and revealed that protein modifications were present on the oldest bacterial proteins. Finally, we performed a proteogenomic analysis by mapping all MS spectra onto a six-frame translation of B. subtilis genome and found evidence for 19 novel ORFs. We provide the most extensive overview of the proteome and post-translational modifications for B. subtilis to date, with insights into functional annotation and evolutionary aspects of the B. subtilis genome.

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Section: Discussionmentioning

confidence: 98%

In-depth analysis of Bacillus subtilis proteome identifies new ORFs and traces the evolutionary history of modified proteins

Ravikumar

Nalpas

Anselm

et al. 2018

Sci Rep

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“…Whole cell protein and membrane protein extracts were reduced (20 min at 25 °C) in 1 mM dithiothreitol and then alkylated in 5.5 mM iodoacetamide (15 min at 25 °C in the dark) [ 52 ]. Trypsin was added (1:50, w/w) and proteins were digested in solution overnight before samples were separated by SCX performed on a Dionex UltiMate 3000 (Thermo Fischer Scientific).…”

Section: Methodsmentioning

confidence: 99%

A comprehensive proteogenomic study of the human Brucella vaccine strain 104 M

et al. 2017

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Background Brucella spp. are Gram-negative, facultative intracellular pathogens that cause brucellosis in both humans and animals. The B. abortus vaccine strain 104 M is the only vaccine available in China for the prevention of brucellosis in humans. Although the B. abortus 104 M genome has been fully sequenced, the current genome annotations are not yet complete. In addition, the main mechanisms underpinning its residual toxicity and vaccine-induced immune protection have yet to be elucidated. Mapping the proteome of B. abortus 104 M will help to improve genome annotation quality, thereby facilitating a greater understanding of its biology.ResultsIn this study, we utilized a proteogenomic approach that combined subcellular fractionation and peptide fractionation to perform a whole-proteome analysis and genome reannotation of B. abortus 104 M using high-resolution mass spectrometry. In total, 1,729 proteins (56.3% of 3,072) including 218 hypothetical proteins were identified using the culture conditions that were employed this study. The annotations of the B. abortus 104 M genome were also refined following identification and validation by reverse transcription-PCR. In addition, 14 pivotal virulence factors and 17 known protective antigens known to be involved in residual toxicity and immune protection were confirmed at the protein level following induction by the 104 M vaccine. Moreover, a further insight into the cell biology of multichromosomal bacteria was obtained following the elucidation of differences in protein expression levels between the small and large chromosomes.ConclusionsThe work presented in this report used a proteogenomic approach to perform whole-proteome analysis and genome reannotation in B. abortus 104 M; this work helped to improve genome annotation quality. Our analysis of virulence factors, protective antigens and other protein effectors provided the basis for further research to elucidate the mechanisms of residual toxicity and immune protection induced by the 104 M vaccine. Finally, the potential link between replication dynamics, gene function, and protein expression levels in this multichromosomal bacterium was detailed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3800-9) contains supplementary material, which is available to authorized users.

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“…Samples were loaded and then eluted for 30 min using a 4–90% ACN fraction-optimized non-linear gradient in 0.1% formic acid. Eluted peptides were detected using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific), and then semi-quantified using a spectral counting approach as described previously (Gao et al, 2015 ). Each sample was independently analyzed three times, and all spectra were compared against the S. flexneri 2a 2457T database described above using PD v.2.1 software (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

An H-NS Family Protein, Sfh, Regulates Acid Resistance by Inhibition of Glutamate Decarboxylase Expression in Shigella flexneri 2457T

Niu

Wang²,

Liu

et al. 2017

Front. Microbiol.

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The glutamate-dependent acid-resistance system is the most effective acid tolerance pathway in Shigella, allowing survival in extremely acidic environments. However, the regulation of this system in Shigella remains elusive. In the current study, we identified significant differences in the levels of glutamate decarboxylase between three Shigella flexneri strains with different levels of acid resistance using blue native-polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF)/sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results showed that the degree of acid resistance and the levels of GadA/B were significantly lower in strain 2457T compared with two other S. flexneri strains. It has been reported that plasmid pSf-R27 is expressed in strain 2457T but not in the other 142 sequenced S. flexneri isolates. pSf-R27 encodes protein Sfh, which belongs to a family of histone-like nucleoid-structuring (H-NS) proteins that participate in the transcriptional control of glutamate-dependent acid resistance, implicating pSf-R27 in the lower acid resistance of strain 2457T. Transformation of pSf-R27 or sfh alone into strain 301 resulted in decreased expression of GadA/B in the recombinant strains. Thus, we confirmed that H-NS family protein Sfh, bound to the gadA/B regulatory region and regulates the expression of glutamate decarboxylase at the transcriptional level. We also examined the acid tolerance of the wild-type and recombinant strains using flow cytometry and determined that the acid tolerance of S. flexneri is closely related to the expression of GadA/B. These findings further our understanding of the acid tolerance of S. flexneri, especially via the glutamate-dependent pathway.

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Experimental Validation of Bacillus anthracis A16R Proteogenomics

Cited by 6 publications

References 27 publications

In-depth analysis of Bacillus subtilis proteome identifies new ORFs and traces the evolutionary history of modified proteins

In-depth analysis of Bacillus subtilis proteome identifies new ORFs and traces the evolutionary history of modified proteins

A comprehensive proteogenomic study of the human Brucella vaccine strain 104 M

An H-NS Family Protein, Sfh, Regulates Acid Resistance by Inhibition of Glutamate Decarboxylase Expression in Shigella flexneri 2457T

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