An iTRAQ characterisation of the role of TolC during electron transfer from Shewanella oneidensis MR-1.

Anodophilic bacteria have the ability to generate electricity in microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigated the anode-specific responses of Shewanella oneidensis MR-1, an exoelectroactive Gammaproteobacterium, using for the first time iTRAQ and 2D-LC MS/MS driven membrane proteomics to compare protein abundances in S. oneidensis when generating power in MFCs, and growing in a continuous culture. The regulated dataset produced was enriched in membrane proteins. Proteins shown to be more abundant in anaerobic electroactive anodic cells included efflux pump TolC and an uncharacterised tetratricopeptide repeat (TPR) protein, whilst the TonB2 system and associated uncharacterised proteins such as TtpC2 and DUF3450 were more abundant in microaerobic planktonic cells. In order to validate the iTRAQ data, the functional role for TolC was examined using a δTolC knockout mutant of S. oneidensis. Possible roles for the uncharacterised proteins were identified using comparative bioinformatics. We demonstrate that employing an insoluble extracellular electron acceptor requires multiple proteins involved in cell surface properties. All MS and processed data are available via ProteomeXchange with identifier PXD004090.

Quantitative proteomic analysis of the exoelectrogenic bacterium Arcobacter butzleri ED-1 reveals increased abundance of a flagellin protein under anaerobic growth on an insoluble electrode.

Exoelectrogens have the ability to generate electricity in mediator-less microbial fuel cells (MFCs) by extracellular electron transfer to the anode. We investigate the anode-specific responses of Arcobacter butzleri ED-1, the first identified exoelectrogenic Epsilonproteobacterium. iTRAQ and 2D-LC MS/MS driven proteomics were used to compare protein abundances in A. butzleri ED-1 when generating an electronegative potential (-225 mV) in an anaerobic half-cell - either growing as an electrogenic biofilm or suspended in the liquid medium - versus a microaerobic culture. This is the first quantitative proteomic study concentrating on growth of an exoelectrogen during current generation. From 720 proteins identified and quantified (soluble and insoluble sub-proteomes), statistical analysis reveals 75 differentially-expressed proteins. This dataset was enriched in proteins regulating energy and intermediary metabolism, electron and protein transport. Flagellin up-regulation was concomitant with electron transport in the anodic cells, while decreased abundance of a methyl-accepting chemotaxis protein suggested that flagella were involved in communication with the anode surface and electrogenesis, rather than motility. Two novel cytochromes potentially related to electron transport were up-regulated in anaerobic cultures. We demonstrate that employing an insoluble extracellular electron acceptor for anaerobic growth regulates multiple proteins involved in cell surface properties, electron transport and the methylcitrate cycle.

Proteome characterization of the unsequenced psychrophile Pedobacter cryoconitis using 15N metabolic labeling, tandem mass spectrometry, and a new bioinformatic workflow.

Organisms without a sequenced genome and lacking a complete protein database encounter an added level of complexity to protein identification and quantitation. De novo sequencing, new bioinformatics tools, and mass spectrometry (MS) techniques allow for advances in this area. Here, the proteomic characterization of an unsequenced psychrophilic bacterium, Pedobacter cryoconitis, is presented employing a novel workflow based on (15) N metabolic labelling, 2DE, MS/MS, and bioinformatics tools. Two bioinformatics pipelines, based on nitrogen constraint (N-constraint), ortholog searching, and de novo peptide sequencing with N-constraint similarity database search, are compared based on proteome coverage and throughput. Results demonstrate the effect of different growth temperatures (1°C, 20°C) and different carbon sources (glucose, maltose) on the proteome. Seventy-six and 69 proteins were identified and validated from the glucose- and maltose-grown bacterium, respectively, from which 21 and 22 were differentially expressed at different growth temperatures. Differentially expressed proteins are involved in stress response and carbohydrate metabolism, with higher expression at 20°C than at 1°C, while antioxidants were upregulated at 1°C. This study provides an alternative workflow to identify, validate, and quantify proteins from unsequenced organisms distantly related to other species in the protein database. Furthermore, it provides further understanding on bacterial adaptation mechanisms to cold environments, and a comparative proteomic analyses with other psychrophilic microorganisms.

An insight into iTRAQ: where do we stand now?

The iTRAQ (isobaric tags for relative and absolute quantification) technique is widely employed in proteomic workflows requiring relative quantification. Here, we review the iTRAQ literature; in particular, we focus on iTRAQ usage in relation to other commonly used quantitative techniques e.g. stable isotope labelling in culture (SILAC), label-free methods and selected reaction monitoring (SRM). As a result, we identify several issues arising with respect to iTRAQ. Perhaps frustratingly, iTRAQ's attractiveness has been undermined by a number of technical and analytical limitations: it may not be truly quantitative, as the changes in abundance reported will generally be underestimated. We discuss weaknesses and strengths of iTRAQ as a methodology for relative quantification in the light of this and other technical issues. We focus on technical developments targeted at iTRAQ accuracy and precision, use of 4-plex over 8-plex reagents and application of iTRAQ to post-translational modification (PTM) workflows. We also discuss iTRAQ in relation to label-free approaches, to which iTRAQ is losing ground.

Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase.

Biotechnology is a promising approach for the generation of hydrogen, but is not yet commercially viable. Metabolic engineering is a potential solution, but has largely been limited to native pathway optimisation. To widen opportunities for use of non-native [NiFe] hydrogenases for improved hydrogen production, we introduced a cyanobacterial hydrogen production pathway and associated maturation factors into Escherichia coli. Hydrogen production is observed in vivo in a hydrogenase null host, demonstrating coupling to host electron transfer systems. Hydrogenase activity is also detected in vitro. Hydrogen output is increased when formate production is abolished, showing that the new pathway is distinct from the native formate dependent pathway and supporting the conclusion that it couples cellular NADH and NADPH pools to molecular hydrogen. This work demonstrates non-native hydrogen production in E. coli, showing the wide portability of [NiFe] hydrogenase pathways and the potential for metabolic engineering to improve hydrogen yields.

Rapid fabrication of glass/PDMS hybrid µIMER for high throughput membrane proteomics.

Mass spectrometry (MS) based proteomics has brought a radical approach to systems biology, offering a platform to study complex biological functions. However, key proteomic technical challenges remain, mainly the inability to characterise the complete proteome of a cell due to the thousands of diverse, complex proteins expressed at an extremely wide concentration range. Currently, high throughput and efficient techniques to unambiguously identify and quantify proteins on a proteome-wide scale are in demand. Miniaturised analytical systems placed upstream of MS help us to attain these goals. One time-consuming step in traditional techniques is the in-solution digestion of proteins (4-20 h). This also has other drawbacks, including enzyme autoproteolysis, low efficiency, and manual operation. Furthermore, the identification of α-helical membrane proteins has remained a challenge due to their high hydrophobicity and lack of trypsin cleavage targets in transmembrane helices. We demonstrate a new rapidly produced glass/PDMS micro Immobilised Enzyme Reactor (µIMER) with enzymes covalently immobilised onto polyacrylic acid plasma-modified surfaces for the purpose of rapidly (as low as 30 s) generating peptides suitable for MS analysis. This µIMER also allows, for the first time, rapid digestion of insoluble proteins. Membrane protein identification through this method was achieved after just 4 min digestion time, up to 9-fold faster than either dual-stage in-solution digestion approaches or other commonly used bacterial membrane proteomic workflows.