A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in t...

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

Hexavalent chromium [Cr(VI)] contamination is one of the main problems of environmental protection because the Cr(VI) is a hazard to human health. The Cr(VI) form is highly toxic, mutagenic, and carcinogenic, and it spreads widely beyond the site of initial contamination because of its mobility. Cr(VI), crossing the cellular membrane via the sulfate uptake pathway, generates active intermediates Cr(V) and/or Cr(IV), free radicals, and Cr(III) as the final product. Cr(III) affects DNA replication, causes mutagenesis, and alters the structure and activity of enzymes, reacting with their carboxyl and thiol groups. To persist in Cr(VI)-contaminated environments, microorganisms must have efficient systems to neutralize the negative effects of this form of chromium. The systems involve detoxification or repair strategies such as Cr(VI) efflux pumps, Cr(VI) reduction to Cr(III), and activation of enzymes involved in the ROS detoxifying processes, repair of DNA lesions, sulfur metabolism, and iron homeostasis. This review provides an overview of the processes involved in bacterial and fungal Cr(VI) resistance that have been identified through ‘omics’ studies. A comparative analysis of the described molecular mechanisms is offered and compared with the cellular evidences obtained using classical microbiological approaches.

Molecular mechanisms of Cr(VI) resistance in bacteria and fungi.

We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.

Direct cellular lysis/protein extraction protocol for soil metaproteomics.

The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.

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Phage-Induced Expression of CRISPR-Associated Proteins Is Revealed by Shotgun Proteomics in Streptococcus thermophilus

Microbial Cd(II) and Cr(VI) resistance mechanisms and application in bioremediation.

Pseudomonas putida is a model organism for bioremediation because of its remarkable metabolic versatility, extensive biodegradative functions, and ubiquity in contaminated soil environments. To further the understanding of molecular pathways responding to the heavy metal chromium(VI) [Cr(VI)], the proteome of aerobically grown, Cr(VI)-stressed P. putida strain F1 was characterized within the context of two disparate nutritional environments: rich (LB) media and minimal (M9L) media containing lactate as the sole carbon source. Growth studies demonstrated that F1 sensitivity to Cr(VI) was impacted substantially by nutrient conditions, with a carbon-source-dependent hierarchy (lactate > glucose >> acetate) observed in minimal media. Two-dimensional HPLC-MS/MS was employed to identify differential proteome profiles generated in response to 1 mM chromate under LB and M9L growth conditions. The immediate response to Cr(VI) in LB-grown cells was up-regulation of proteins involved in inorganic ion transport, secondary metabolite biosynthesis and catabolism, and amino acid metabolism. By contrast, the chromate-responsive proteome derived under defined minimal growth conditions was characterized predominantly by up-regulated proteins related to cell envelope biogenesis, inorganic ion transport, and motility. TonB-dependent siderophore receptors involved in ferric iron acquisition and amino acid adenylation domains characterized up-regulated systems under LB-Cr(VI) conditions, while DNA repair proteins and systems scavenging sulfur from alternative sources (e.g., aliphatic sulfonates) tended to predominate the up-regulated proteome profile obtained under M9L-Cr(VI) conditions. Comparative analysis indicated that the core molecular response to chromate, irrespective of the nutritional conditions tested, comprised seven up-regulated proteins belonging to six different functional categories including transcription, inorganic ion transport/metabolism, and amino acid transport/metabolism. These proteins might potentially serve as indicators of chromate stress in natural microbial communities.

/pdf/proteomics-reveals-a-core-molecular-response-of-pseudomonas-4vskv3eoz2.pdf

Stephanie Thieman

Papers

Proteomics reveals a core molecular response of Pseudomonas putida F1 to acute chromate challenge