Workflow for quantitative proteomic analysis of Clostridium acetobutylicum ATCC 824 using iTRAQ tags

Hou, Shuyu; Jones, Shawn W.; Choe, Leila H.; Papoutsakis, Eleftherios T.; Lee, Kelvin H.

doi:10.1016/j.ymeth.2013.03.013

Cited by 20 publications

(28 citation statements)

References 40 publications

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“…While the detailed action of these sRNAs remains to be elucidated, there are several sRNAs whose action can be readily related to the phenotypic response of these cells to metabolite stress affecting various metabolic pathways as previously shown [35,37,39,42,60] and further confirmed by the present set of RNA-seq data as well as the accompanying large set of new microarray and proteomic data [61]. …”

Section: Resultssupporting

confidence: 68%

“…Three biological replicate cultures of C. acetobutylicum ATCC 824 were carried out in pH- controlled (pH > 5) batch fermentations in 4 L bioreactors (Bioflow II and 110, New Brunswick Scientific, Edison, NJ, USA) in a defined clostridial growth media [61]. The cultures were stressed with butanol (30 mM, 60 mM and 90 mM) and butyric acid (30 mM, 40 mM and 50 mM) at mid-exponential phase of growth at an OD of 1.0 and were sampled at 4 different time points: 15 min, 30 min, 60 min and 75 min post stress.…”

Section: Methodsmentioning

confidence: 99%

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The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum

et al. 2013

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BackgroundSmall non-coding RNAs (sRNA) are emerging as major components of the cell’s regulatory network, several possessing their own regulons. A few sRNAs have been reported as being involved in general or toxic-metabolite stress, mostly in Gram- prokaryotes, but hardly any in Gram+ prokaryotes. Significantly, the role of sRNAs in the stress response remains poorly understood at the genome-scale level. It was previously shown that toxic-metabolite stress is one of the most comprehensive and encompassing stress responses in the cell, engaging both the general stress (or heat-shock protein, HSP) response as well as specialized metabolic programs.ResultsUsing RNA deep sequencing (RNA-seq) we examined the sRNome of C. acetobutylicum in response to the native but toxic metabolites, butanol and butyrate. 7.5% of the RNA-seq reads mapped to genome outside annotated ORFs, thus demonstrating the richness and importance of the small RNome. We used comparative expression analysis of 113 sRNAs we had previously computationally predicted, and of annotated mRNAs to set metrics for reliably identifying sRNAs from RNA-seq data, thus discovering 46 additional sRNAs. Under metabolite stress, these 159 sRNAs displayed distinct expression patterns, a select number of which was verified by Northern analysis. We identified stress-related expression of sRNAs affecting transcriptional (6S, S-box & solB) and translational (tmRNA & SRP-RNA) processes, and 65 likely targets of the RNA chaperone Hfq.ConclusionsOur results support an important role for sRNAs for understanding the complexity of the regulatory network that underlies the stress response in Clostridium organisms, whether related to normophysiology, pathogenesis or biotechnological applications.

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Section: Resultssupporting

confidence: 68%

Section: Methodsmentioning

confidence: 99%

The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum

et al. 2013

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“…S1). Following sample collection, the cells were washed with PBS (phosphate buffered saline) buffer for at least three times to remove the extracellular proteases (Hou et al, ) and stored at −80°C freezer until further usage. Protein was extracted from all cell pellets within 2 days of harvesting.…”

Section: Methodsmentioning

confidence: 99%

“…Methodology adopted for protein extraction from different bacterial samples was optimized based on the findings of Hou et al (). Cell pellets collected were re‐suspended in 2 mL of an optimized lysis buffer consisting of 4% CHAPS, 0.5 M TEAB, 1% CaCO 3 and subjected to sonication for 5 min (pulses used: 7 sec on and 5 sec off).…”

Section: Methodsmentioning

confidence: 99%

Quantitative proteome profiles help reveal efficient xylose utilization mechanisms in solventogenic Clostridium sp. strain BOH3

Basu

Xin

Lim

et al. 2017

Biotech & Bioengineering

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Development of sustainable biobutanol production platforms from lignocellulosic materials is impeded by inefficient five carbon sugar uptake by solventogenic bacteria. The recently isolated Clostridium sp. strain BOH3 is particularly advantaged in this regard as it serves as a model organism which can simultaneously utilize both glucose and xylose for high butanol (>15 g/L) production. Strain BOH3 was, therefore, investigated for its metabolic mechanisms for efficient five carbon sugar uptake using a quantitative proteomics based approach. The proteomics data show that proteins within the CAC1341-1349 operon play a pivotal role for efficient xylose uptake within the cells to produce butanol. Furthermore, up-regulation of key enzymes within the riboflavin synthesis pathway explained that xylose could induce higher riboflavin production capability of the bacteria (e.g., ∼80 mg/L from glucose vs. ∼120 mg/L from xylose). Overall results from the present experimental approach indicated that xylose-fed BOH3 cultures are subjected to high levels of redox stress which coupled with the solvent stress-trigger a sporulation response within the cells earlier than the glucose-fed cultures. The study lays the platform for metabolic engineering strategies in designing organisms for efficient butanol and other value-added chemicals such as riboflavin production. Biotechnol. Bioeng. 2017;114: 1959-1969. © 2017 Wiley Periodicals, Inc.

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“…C. acetobutylicum ATCC 824 has evolved as the model solventogenic organism and was the first completely sequenced clostridial strain 6 . Its characterization is relatively limited though, when compared to other well-known model organisms such as Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, and Arabidopsis thaliana.…”

Section: Introductionmentioning

confidence: 99%

Predictive modeling inClostridium acetobutylicumfermentations employing Raman spectroscopy and multivariate data analysis for real-time culture monitoring

Liu

Germane

et al. 2016

SPIE Proceedings

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The coupling of optical fibers with Raman instrumentation has proven to be effective for real-time monitoring of chemical reactions and fermentations when combined with multivariate statistical data analysis. Raman spectroscopy is relatively fast, with little interference from the water peak present in fermentation media. Medical research has explored this technique for analysis of mammalian cultures for potential diagnosis of some cancers. Other organisms studied via this route include Escherichia coli, Saccharomyces cerevisiae, and some Bacillus sp., though very little work has been performed on Clostridium acetobutylicum cultures. C. acetobutylicum is a gram-positive anaerobic bacterium, which is highly sought after due to its ability to use a broad spectrum of substrates and produce useful byproducts through the wellknown Acetone-Butanol-Ethanol (ABE) fermentation. In this work, real-time Raman data was acquired from C. acetobutylicum cultures grown on glucose. Samples were collected concurrently for comparative off-line product analysis. Partial-least squares (PLS) models were built both for agitated cultures and for static cultures from both datasets. Media components and metabolites monitored include glucose, butyric acid, acetic acid, and butanol. Models were cross-validated with independent datasets. Experiments with agitation were more favorable for modeling with goodness of fit (QY) values of 0.99 and goodness of prediction (Q 2 Y) values of 0.98. Static experiments did not model as well as agitated experiments. Raman results showed the static experiments were chaotic, especially during and shortly after manual sampling.

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Workflow for quantitative proteomic analysis of Clostridium acetobutylicum ATCC 824 using iTRAQ tags

Cited by 20 publications

References 40 publications

The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum

The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum

Quantitative proteome profiles help reveal efficient xylose utilization mechanisms in solventogenic Clostridium sp. strain BOH3

Predictive modeling inClostridium acetobutylicumfermentations employing Raman spectroscopy and multivariate data analysis for real-time culture monitoring

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