Extracting the abstraction pyramid from complex networks

Background  

At present, the organization of system modules is typically limited to either a multilevel hierarchy that describes the "vertical" relationships between modules at different levels (e.g., module A at level two is included in module B at level one), or a single-level graph that represents the "horizontal" relationships among modules (e.g., genetic interactions between module A and module B). Both types of organizations fail to provide a broader and deeper view of the complex systems that arise from an integration of vertical and horizontal relationships.     

Global Functional Atlas of Escherichia coli Encompassing Previously Uncharacterized Proteins

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a “systems-wide” functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.     

Community Structure Detection for Overlapping Modules through Mathematical Programming in Protein Interaction Networks

Community structure detection has proven to be important in revealing the underlying properties of complex networks. The standard problem, where a partition of disjoint communities is sought, has been continually adapted to offer more realistic models of interactions in these systems. Here, a two-step procedure is outlined for exploring the concept of overlapping communities. First, a hard partition is detected by employing existing methodologies. We then propose a novel mixed integer non linear programming (MINLP) model, known as OverMod, which transforms disjoint communities to overlapping. The procedure is evaluated through its application to protein-protein interaction (PPI) networks of the rat, E. coli, yeast and human organisms. Connector nodes of hard partitions exhibit topological and functional properties indicative of their suitability as candidates for multiple module membership. OverMod identifies two types of connector nodes, inter and intra-connector, each with their own particular characteristics pertaining to their topological and functional role in the organisation of the network. Inter-connector proteins are shown to be highly conserved proteins participating in pathways that control essential cellular processes, such as proliferation, differentiation and apoptosis and their differences with intra-connectors is highlighted. Many of these proteins are shown to possess multiple roles of distinct nature through their participation in different network modules, setting them apart from proteins that are simply ‘hubs’, i.e. proteins with many interaction partners but with a more specific biochemical role.     

High accuracy operon prediction method based on STRING database scores

We present a simple and highly accurate computational method for operon prediction, based on intergenic distances and functional relationships between the protein products of contiguous genes, as defined by STRING database (Jensen,L.J., Kuhn,M., Stark,M., Chaffron,S., Creevey,C., Muller,J., Doerks,T., Julien,P., Roth,A., Simonovic,M. et al. (2009) STRING 8–a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res., 37, D412–D416). These two parameters were used to train a neural network on a subset of experimentally characterized Escherichia coli and Bacillus subtilis operons. Our predictive model was successfully tested on the set of experimentally defined operons in E. coli and B. subtilis, with accuracies of 94.6 and 93.3%, respectively. As far as we know, these are the highest accuracies ever obtained for predicting bacterial operons. Furthermore, in order to evaluate the predictable accuracy of our model when using an organism''s data set for the training procedure, and a different organism''s data set for testing, we repeated the E. coli operon prediction analysis using a neural network trained with B. subtilis data, and a B. subtilis analysis using a neural network trained with E. coli data. Even for these cases, the accuracies reached with our method were outstandingly high, 91.5 and 93%, respectively. These results show the potential use of our method for accurately predicting the operons of any other organism. Our operon predictions for fully-sequenced genomes are available at http://operons.ibt.unam.mx/OperonPredictor/.     

Artificial septal targeting of Bacillus subtilis cell division proteins in Escherichia coli: an interspecies approach to the study of protein-protein interactions in multiprotein complexes

Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.     

The Escherichia coli Peripheral Inner Membrane Proteome

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.An in-depth understanding of cellular proteomes requires knowledge of protein subcellular topology, assembly in macromolecular complexes, and modification and degradation of poplypeptides. Escherichia coli, a model organism for many such studies, is by far the best studied. The genomes of strain K-12 derivatives MG1655 and W3110 have been sequenced (1, 2), and >75% of their genes have been functionally assigned (3). Almost 90% of the K-12 proteome has been identified experimentally, and >73% of its proteins have known structures (4, 5). Moreover, the genomes of another 38 E. coli strains have been determined (see EcoliWiki for details).In E. coli, like in all Gram-negative bacteria, the bacterial cell envelope comprises the plasma or inner membrane and the outer membrane, which are separated by the periplasmic space. The inner membrane encloses the cytoplasm and is a dynamic substructure. It harbors a wide variety of proteins that function in vital cell processes such as the trafficking of ions, molecules, and macromolecules; cell division; environmental sensing; lipid, polysaccharide, and peptidoglycan biosynthesis; and metabolism. Inner membrane proteins either fully span the lipid bilayer using one or more hydrophobic transmembrane helices (integral) or are bound either directly to phospholipid components or via protein–protein interactions to the surface of the membrane (peripheral) (6) (Fig. 1A). Peripheral inner membrane proteins exist on either side of the membrane and may be recruited in membrane-associated complexes on demand (7). Peripheral inner membrane proteins on the cytoplasmic side constitute a sub-proteome of central importance because of their interaction with the cytoplasmic proteome, the nucleoid, and most of the cell''s metabolism. Thanks to their soluble character and the nature of their interactions with the membrane (mostly electrostatic and moderate hydrophobic interactions (7)), peripheral inner membrane proteins can be extracted using high salt concentrations, extreme pH levels, or chaotropes without disrupting the lipid bilayer (8–11). In contrast, the solubilization of integral proteins requires amphiphilic detergents in order to displace the membrane phospholipids and maintain them as soluble in aqueous solutions (12).Open in a separate windowFig. 1.Bioinformatics and experimental workflow for characterizing peripheral inner membrane proteins. A, schematic representation of the subcellular localization of the E. coli inner membrane peripherome. Protein topology assignment is based on the cellular compartment: A, cytoplasmic; B, integral inner membrane proteins; F1, peripheral inner membrane proteome; r, ribosome. B, schematic diagram for PIM protein annotation. 130 cytoplasmic and PIM E. coli K-12 proteins were downloaded from Uniprot (November 2010) (81) and EchoLOCATION (23). A set of bioinformatics tools was used to predict topologies and features of the unassigned and differently assigned proteins and to further validate existing protein annotations (see supplemental text). For the annotation of additional peripheral membrane proteins, the literature was extensively searched. Additional, other E. coli K-12 databases containing gene ontology annotations (84, 85) and protein homologies through BLAST (44) were employed. Homologues of curated E. coli K-12 proteins were identified in E. coli BL21(DE3) (supplemental Table S1A). C, preparation strategy for detecting the E. coli inner membrane peripherome via nanoLC-MS/MS. Inverted membrane vesicles (IMVs) were isolated and washed extensively with the indicated chemical agents to extract cytoplasmic and PIM proteins (“IMVs washed”), and then their surface was trypsinized (gray arrow). Following digestion, soluble peptides were analyzed via nanoLC-MS/MS. D, protein enrichment at different sample preparation conditions. Top: Relative percentage of proteins detected with the proteolysis approach. Proteins are classified here in three major categories: cytoplasmic (A), ribosomal (r), and peripheral (F1). The bar graphs indicate the percentage of proteins in each category relative to the proteins in other categories at a given sample preparation condition. Bottom: Heat maps showing relative quantities of individual proteins at different sample preparation conditions. Perseus (version 1.2.0.16), a part of the MaxQuant bioinformatics platform, was used for the construction of the heat map (86). A top-three label-free quantitative method was employed (27). Individual protein values across the various treatments are given in supplemental Table S3B.Unlike the cytoplasmic proteome of E. coli, which has been extensively characterized (13), its membrane sub-proteome is still poorly defined. Of 1133 predicted integral inner membrane proteins, only half were experimentally identified through proteomics approaches (14). These figures are constantly being re-evaluated,² but most protein identifications appear robust. In contrast to integral inner membrane proteins, bioinformatics prediction of peripheral inner membrane proteins is currently not possible because they are not known to possess any specific features. Despite the occasional designation of partner proteins identified as peripheral in studies that target inner membrane complexes (15–21), no systematic effort has been undertaken to analyze the peripheral inner membrane proteome.Here we have used a multi-pronged strategy employing bioinformatics, biochemistry, proteomics, and complexomics to systematically determine the peripheral inner membrane proteome of E. coli. We focus exclusively on the peripheral inner membrane proteome that faces the cytoplasm, referred to hereinafter as PIM,¹ and do not analyze peripheral inner membrane proteins residing on the periplasm. Manually curated and re-evaluated topology of the E. coli K-12 proteome was extrapolated to the non-K-12 strain BL21(DE3) (95% proteome homology to K-12) (22). By combining various biochemical treatments, we determined experimentally that several cytoplasmic proteins are also novel PIM proteins, and many of them participate in protein complexes associated with the membrane. Collectively, we demonstrate that a significant, previously unsuspected percentage of the expressed polypeptides constitute the PIM proteome.     

The Escherichia coli Proteome: Past, Present, and Future Prospects

Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects.     

Distinct properties of Mycobacterium tuberculosis single-stranded DNA binding protein and its functional characterization in Escherichia coli

Single-stranded DNA binding proteins (SSBs) play an essential role in various DNA functions. Characterization of SSB from Mycobacterium tuberculosis, which infects nearly one-third of the world’s population and kills about 2–3 million people every year, showed that its oligomeric state and various in vitro DNA binding properties were similar to those of the SSB from Escherichia coli. In this study, use of the yeast two-hybrid assay suggests that the EcoSSB and the MtuSSB are even capable of heterooligomerization. However, the MtuSSB failed to complement a Δssb strain of E.coli. The sequence comparison suggested that MtuSSB contained a distinct C-terminal domain. The C-terminal domain of EcoSSB interacts with various cellular proteins. The chimeric constructs between the N- and C-terminal domains of the MtuSSB and EcoSSB exist as homotetramers and demonstrate DNA binding properties similar to the wild-type counterparts. Despite similar biochemical properties, the chimeric SSBs also failed to complement the Δssb strain of E.coli. These data allude to the occurrence of a ‘cross talk’ between the N- and the C-terminal domains of the SSBs for their in vivo function. Further, compared with those of the EcoSSB, the secondary/tertiary interactions within MtuSSB were found to be less susceptible to disruption by guanidinium hydrochloride. Such structural differences could be exploited for utilizing such essential proteins as crucial molecular targets for controlling the growth of the pathogen.     

Gene Cloning and Characterization of Recombinant RNase HII from a Hyperthermophilic Archaeon

We have cloned the gene encoding RNase HII (RNase HII_Pk) from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth phenotype of an rnh mutant strain of Escherichia coli. This gene was expressed in an rnh mutant strain of E. coli, the recombinant enzyme was purified, and its biochemical properties were compared with those of E. coli RNases HI and HII. RNase HII_Pk is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer. Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of Methanococcus jannaschii, Saccharomyces cerevisiae, and E. coli RNase HII proteins, respectively. The enzymatic activity was determined at 30°C and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate. Under these conditions, the most preferred metal ions were Co²⁺ for RNase HII_Pk, Mn²⁺ for E. coli RNase HII, and Mg²⁺ for E. coli RNase HI. The specific activity of RNase HII_Pk determined in the presence of the most preferred metal ion was 6.8-fold higher than that of E. coli RNase HII and 4.5-fold lower than that of E. coli RNase HI. Like E. coli RNase HI, RNase HII_Pk and E. coli RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3′ bond. In addition, these enzymes cleave oligomeric substrates in a similar manner. These results suggest that RNase HII_Pk and E. coli RNases HI and HII are structurally and functionally related to one another.     

Examination of the Watershed-Wide Distribution of Escherichia coli along Southern Lake Michigan: an Integrated Approach

Recent research has highlighted the occurrence of Escherichia coli in natural habitats not directly influenced by sewage inputs. Most studies on E. coli in recreational water typically focus on discernible sources (e.g., effluent discharge and runoff) and fall short of integrating riparian, nearshore, onshore, and outfall sources. An integrated “beachshed” approach that links E. coli inputs and interactions would be helpful to understand the difference between background loading and sewage pollution; to develop more accurate predictive models; and to understand the differences between potential, net, and apparent culturable E. coli. The objective of this study was to examine the interrelatedness of E. coli occurrence from various coastal watershed components along southern Lake Michigan. The study shows that once established in forest soil, E. coli can persist throughout the year, potentially acting as a continuous non-point source of E. coli to nearby streams. Year-round background stream loading of E. coli can influence beach water quality. E. coli is present in highly variable counts in beach sand to depths just below the water table and to distances at least 5 m inland from the shore, providing a large potential area of input to beach water. In summary, E. coli in the fluvial-lacustrine system may be stored in forest soils, sediments surrounding springs, bank seeps, stream margins and pools, foreshore sand, and surface groundwater. While rainfall events may increase E. coli counts in the foreshore sand and lake water, concentrations quickly decline to prerain concentrations. Onshore winds cause an increase in E. coli in shallow nearshore water, likely resulting from resuspension of E. coli-laden beach sand. When examining indicator bacteria source, flux, and context, the entire “beachshed” as a dynamic interacting system should be considered.     

A set of powerful negative selection systems for unmodified Enterobacteriaceae

Creation of defined genetic mutations is a powerful method for dissecting mechanisms of bacterial disease; however, many genetic tools are only developed for laboratory strains. We have designed a modular and general negative selection strategy based on inducible toxins that provides high selection stringency in clinical Escherichia coli and Salmonella isolates. No strain- or species-specific optimization is needed, yet this system achieves better selection stringency than all previously reported negative selection systems usable in unmodified E. coli strains. The high stringency enables use of negative instead of positive selection in phage-mediated generalized transduction and also allows transfer of alleles between arbitrary strains of E. coli without requiring phage. The modular design should also allow further extension to other bacteria. This negative selection system thus overcomes disadvantages of existing systems, enabling definitive genetic experiments in both lab and clinical isolates of E. coli and other Enterobacteriaceae.     

Attaching and Effacing Escherichia coli Downregulate DNA Mismatch Repair Protein In Vitro and Are Associated with Colorectal Adenocarcinomas in Humans

Background

Mucosa-associated Escherichia coli are frequently found in the colonic mucosa of patients with colorectal adenocarcinoma, but rarely in healthy controls. Chronic mucosal E. coli infection has therefore been linked to colonic tumourigenesis. E. coli strains carrying eae (encoding the bacterial adhesion protein intimin) attach intimately to the intestinal mucosa and are classed as attaching and effacing E. coli (AEEC). Enteropathogenic Escherichia coli (EPEC) are the most common form of AEEC identified in man. EPEC utilise a type III secretion system to translocate effector proteins into host cells and infection induces wide-ranging effects on the host cell proteome. We hypothesised that EPEC infection could influence molecular pathways involved in colorectal tumourigenesis.

Methodology/Principal Findings

When co-cultured with human colorectal cell lines, EPEC dramatically downregulated the expression of key DNA mismatch repair proteins MSH2 and MLH1 in an attachment specific manner. Cytochrome c staining and TUNEL analysis confirmed that this effect was not a consequence of apoptosis/necrosis. Ex vivo human colonic mucosa was co-cultured with EPEC and probed by immunofluorescence to locate adherent bacteria. EPEC entered 10% of colonic crypts and adhered to crypt epithelial cells, often in the proliferative compartment. Adenocarcinoma and normal colonic mucosa from colorectal cancer patients (n = 20) was probed by immunofluorescence and PCR for AEEC. Mucosa-associated E. coli were found on 10/20 (50%) adenocarcinomas and 3/20 (15%) normal mucosa samples (P<0.05). AEEC were detected on 5/20 (25%) adenocarcinomas, but not normal mucosa samples (P<0.05).

Significance/Conclusions

The ability of EPEC to downregulate DNA mismatch repair proteins represents a novel gene-environment interaction that could increase the susceptibility of colonic epithelial cells to mutations and therefore promote colonic tumourigenesis. The potential role of AEEC in colorectal tumourigenesis warrants further investigation.     

Novel Bacterial Membrane Surface Display System Using Cell Wall-Less L-Forms of Proteus mirabilis and Escherichia coli

We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 μg ml⁻¹, with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.     

Stable-isotope probing and metagenomics reveal predation by protozoa drives E. coli removal in slow sand filters

Stable-isotope probing and metagenomics were applied to study samples taken from laboratory-scale slow sand filters 0.5, 1, 2, 3 and 4 h after challenging with ¹³C-labelled Escherichia coli to determine the mechanisms and organisms responsible for coliform removal. Before spiking, the filters had been continuously operated for 7 weeks using water from the River Kelvin, Glasgow as their influent source. Direct counts and quantitative PCR assays revealed a clear predator–prey response between protozoa and E. coli. The importance of top-down trophic-interactions was confirmed by metagenomic analysis, identifying several protozoan and viral species connected to E. coli attrition, with protozoan grazing responsible for the majority of the removal. In addition to top-down mechanisms, indirect mechanisms, such as algal reactive oxygen species-induced lysis, and mutualistic interactions between algae and fungi, were also associated with coliform removal. The findings significantly further our understanding of the processes and trophic interactions underpinning E. coli removal. This study provides an example for similar studies, and the opportunity to better understand, manage and enhance E. coli removal by allowing the creation of more complex trophic interaction models.     

Phage Orf Family Recombinases: Conservation of Activities and Involvement of the Central Channel in DNA Binding

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redβ recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination.     

In silico Proteome-wide Amino aCid and Elemental Composition (PACE) Analysis of Expression Proteomics Data Provides A Fingerprint of Dominant Metabolic Processes

Proteome-wide Amino aCid and Elemental composition (PACE) analysis is a novel and informative way of interrogating the proteome. The PACE approach consists of in silico decomposition of proteins detected and quantified in a proteomics experiment into 20 amino acids and five elements (C, H, N, O and S), with protein abundances converted to relative abundances of amino acids and elements. The method is robust and very sensitive; it provides statistically reliable differentiation between very similar proteomes. In addition, PACE provides novel insights into proteome-wide metabolic processes, occurring, e.g., during cell starvation. For instance, both Escherichia coli and Synechocystis down-regulate sulfur-rich proteins upon sulfur deprivation, but E. coli preferentially down-regulates cysteine-rich proteins while Synechocystis mainly down-regulates methionine-rich proteins. Due to its relative simplicity, flexibility, generality and wide applicability, PACE analysis has the potential of becoming a standard analytical tool in proteomics.     

TiO2 Photocatalysis Damages Lipids and Proteins in Escherichia coli

This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO₂) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO₂ photocatalysis on E. coli survival was monitored by counting on agar plate and by assessing lipid peroxidation and performing proteomic analysis. We observed through malondialdehyde quantification that lipid peroxidation occurred during the photocatalytic process, and the addition of superoxide dismutase, which acts as a scavenger of the superoxide anion radical (O₂·⁻), inhibited this effect by half, showing us that O₂·⁻ radicals participate in the photocatalytic antimicrobial effect. Qualitative analysis using two-dimensional electrophoresis allowed selection of proteins for which spot modifications were observed during the applied treatments. Two-dimensional electrophoresis highlighted that among the selected protein spots, 7 and 19 spots had already disappeared in the dark in the presence of 0.1 g/liter and 0.4 g/liter TiO₂, respectively, which is accounted for by the cytotoxic effect of TiO₂. Exposure to 30 min of UV-A radiation in the presence of 0.1 g/liter and 0.4 g/liter TiO₂ increased the numbers of missing spots to 14 and 22, respectively. The proteins affected by photocatalytic oxidation were strongly heterogeneous in terms of location and functional category. We identified several porins, proteins implicated in stress response, in transport, and in bacterial metabolism. This study reveals the simultaneous effects of O₂·⁻ on lipid peroxidation and on the proteome during photocatalytic treatment and therefore contributes to a better understanding of molecular mechanisms in antibacterial photocatalytic treatment.