Localization of protein aggregation in Escherichia coli is governed by diffusion and nucleoid macromolecular crowding effect

Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3d individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion- aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of "soft" intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli.Comment: PLoS Computational Biology (2013

Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoRSl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoRSl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoRSl-DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoRSl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control. © 2013 The Author(s)

Effects of Intracellular and Partitioning Asymmetries in Escherichia coli

Cell divisions in Escherichia coli are, in general, morphologically symmetric. However, in a few cases, significant asymmetries between sister cells exist. These asymmetries between sister cells result in functional differences between them. For example, cells inheriting the older pole, over generations, accumulate more unwanted protein aggregates than their sister and, consequently, have a reduced growth rate. The reduced ability of these cells to reproduce shows that even these unicellular organisms are susceptible to the effects of aging. To understand senescence in these organisms, it is critical to investigate the sources as well as the functional consequences of asymmetries in division.In this thesis, we characterize mechanisms responsible for functional and morphological asymmetries in division in E. coli cells, using live, single-cell, single-molecule imaging techniques and detailed stochastic models. First, to understand the functional asymmetries due to the heterogeneous spatial distribution of large, inert protein complexes, we study the kinetics of segregation and retention of such complexes by observing these events, one event at a time. For that, we track individual MS2-GFP tagged RNA complexes, as they move in the cell cytoplasm, and characterize the mechanisms responsible for their long-term spatial distribution and resulting partitioning. Next, to understand the morphological asymmetries, we study the difference in cell sizes between sister cells at division under different environmental conditions. Finally, we present the models and simulators developed to characterize and mimic these processes, as well as to explore their functional consequences.Our results suggest that functional and morphological asymmetries in division, in the growth conditions studied, appear to be mostly driven by the nucleoid. In particular, we find that the fluorescent complexes are retained at the poles due to nucleoid occlusion. Further, the positioning of the point of division is also regulated by the degree of proximity between the two replicated nucleoids in the cell at the moment preceding division. Finally, based on simulation results of the models in extreme conditions, we suggest that asymmetries in these processes in division can enhance the mean vitality of E. coli cell populations. Overall, the results suggest that nucleoid occlusion contributes, in different ways, to heterogeneities in E. coli cells that ultimately generate phenotypic differences between sister cells

The Role of Nucleoid Exclusion in the Intracellular Spatial Organization of Escherichia coli

Not long ago, bacterial cells were regarded as organisms with hardly any internal organization, due to lack of visible physical compartments and, thus, proteins were believed to be distributed randomly. Since then, advances in microscopy, in in vivo protein labeling with fluorescent tags, and in image analysis techniques have enabled us to probe biological events at a single-cell, singletime moment, and single-molecule level. The results from these observations have led to a radical change in this view and, thus, revolutionized the field of bacterial cell biology. Namely, this novel source of information has made evident that proper bacterial functioning is not possible without a highly spatially organized, dynamic internal composition that depends on the deployment of functional proteins and other cellular components in specific locations, at specific moments.The spatiotemporal organization of the functional proteins and other cellular components play a fundamental role in several key regulatory processes, such as transcription, translation and cell division. One class of proteins, termed as ‘DNA-binding proteins’, are associated with DNA replication and segregation. Not surprisingly, they preferentially locate at midcell, where the chromosomal DNA is condensed into a dynamic structure called ‘nucleoid’. Another class of proteins, termed as ‘polar proteins’, are majorly involved in physiological behaviors such as chemotaxis, sugar uptake, motility and adhesion. In agreement, they are preferentially localized at the cell poles in the case of rod-shaped bacteria such as E. coli. Finally, there is a third class of proteins, called as ‘cytoskeletal proteins’, whose location differ widely during cell growth. For example, the Min system, a major cell division regulatory system, consisting of MinCDE proteins have a remarkable dynamic pattern inside the cell. These proteins localize for about half a minute in one cell half and then switch rapidly to the opposite half. This back and forth motion continues until the polymerization of the division protein FtsZ results in a ring-like structure at the cell center prior to cell division.Cellular components, other than functional proteins, also exhibit a highly-organized spatial distribution. These components include plasmids, enzyme megacomplexes and unwanted protein aggregates. For example, protein aggregates, formed as a result of environment stress or errors in protein homeostasis, are generally sequestered into inclusion bodies (IBs) that localize at the cell poles. This process of polar localization is symmetric. However, following several cell division events, results in progeny cells containing the old pole having more aggregates than the new pole possessing progeny cells. Subsequent divisions lead to cell generations where some cells inherit more aggregates than others. Importantly, this was found to be positively correlated with increased division times, i.e., cellular aging. It is believed that such asymmetric partitioning of unwanted aggregates may be critical for the rejuvenation of bacterial populations. It is thus of major importance to understand the underlying mechanisms that are responsible for the above-described events.In this thesis, using Escherichia coli as our model organism, we started by investigating and validating the hypothesis that the presence of the nucleoid at the midcell is responsible for the ability of this organism to segregate unwanted protein aggregates to the cell poles. We next investigated and characterized the robustness of these mechanisms to external perturbations and stressful environmental conditions. Afterwards, we hypothesized that the phenomenon of nucleoid exclusion should not be limited to protein aggregates alone but, instead, for physical reasons, it should influence any large macromolecule that is not affected by a transport or self-propelling mechanism (which is the case of all proteins in E. coli). Consequently, we hypothesized and subsequently proved that it should influence self-assembling proteins, such as the transmembrane Tsr chemoreceptors, which have a major role in bacterial chemotaxis. In addition, we also investigated to what extent cell-to-cell diversity in nucleoid sizes contributes to the cell-to-cell diversity in the spatial distribution of polar-localized proteins. For these studies, we made use of efficient fluorescent tags, in vivo single-cell, single-molecule time-lapse microscopy, tailored image and signal processing techniques and stochastic biophysical models.Our results provide new perspectives regarding the role of the nucleoid in the spatial organization of protein aggregates as well as chemoreceptor clusters in E. coli. Interestingly, regarding the latter, nucleoid exclusion from midcell was shown not to be the sole phenomenon for the proper localization of Tsr protein clusters. However, it is expected to be the most robust, namely, in stressful environments or when the cell is subject to external perturbations, than the diffusion-andcapture mechanism mediated by the Tol-Pal complexes, as it does not require production of proteins or is under stringent control. Further, unlike the other mechanism, it is energy-free.Given the rapid developments in single-cell biology techniques, particularly the emergence of super-resolution microscopy techniques, improved fluorescent probes, high-throughput and largescale biochemical methods and theoretical tools, we expect several developments in the near future that will allow assessing further the role of the nucleoid as a ‘spatial organizer’ of the cellular architecture of E. coli

Substrate recognition and catalysis by LytB, a pneumococcal peptidoglycan hydrolase involved in virulence

17 p.-7 fig.-2 tab. Rico-Lastres, Palma et al.Streptococcus pneumoniae is a major cause of life-threatening diseases worldwide. Here we provide an in-depth functional characterization of LytB, the peptidoglycan hydrolase responsible for physical separation of daughter cells. Identified herein as an N-acetylglucosaminidase, LytB is involved also in colonization and invasion of the nasopharynx, biofilm formation and evasion of host immunity as previously demonstrated. We have shown that LytB cleaves the GlcNAc-β-(1,4)-MurNAc glycosidic bond of peptidoglycan building units. The hydrolysis occurs at sites with fully acetylated GlcNAc moieties, with preference for uncross-linked muropeptides. The necessity of GlcN acetylation and the presence of a single acidic moiety (Glu585) essential for catalysis strongly suggest a substrate-assisted mechanism with anchimeric assistance of the acetamido group of GlcNAc moieties. Additionally, modelling of the catalytic region bound to a hexasaccharide tripentapeptide provided insights into substrate-binding subsites and peptidoglycan recognition. Besides, cell-wall digestion products and solubilisation rates might indicate a tight control of LytB activity to prevent unrestrained breakdown of the cell wall. Choline-independent localization at the poles of the cell, mediated by the choline-binding domain, peptidoglycan modification, and choline-mediated (lipo) teichoic-acid attachment contribute to the high selectivity of LytB. Moreover, so far unknown chitin hydrolase and glycosyltransferase activities were detected using GlcNAc oligomers as substrate.Research was funded by grants from the Ministerio de Ciencia e Innovación (MICINN) and the Ministerio de Economía y Competitividad (MINECO) to P. García (SAF2009-10824 and SAF2012-39444-C02-01) and M. Menéndez (BFU2009-10052 and BFU2012-36825), the Consejería de Educación de la Comunidad de Madrid (S2010/BMD/2457) to M. Menéndez. The work in the United Kingdom and the US was supported by grants from the BBSRC (BB/G015902/1) to W. Vollmer and from the National Institutes of Health (GM61629) to S. Mobashery. Additional funding was provided by the CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII). Palma Rico-Lastres and Roberto Díez-Martínez were the recipients of fellowships from the MICINN (FPI program).Peer reviewe

Encapsulation systems for antimicrobial food packaging components: An update

Antimicrobial active packaging has emerged as an effective technology to reduce microbial growth in food products increasing both their shelf-life and microbial safety for the consumer while maintaining their quality and sensorial properties. In the last years, a great effort has been made to develop more efficient, long-lasting and eco-friendly antimicrobial materials by improving the performance of the incorporated antimicrobial substances. With this purpose, more effective antimicrobial compounds of natural origin such as bacteriocins, bacteriophages and essential oils have been preferred over synthetic ones and new encapsulation strategies such as emulsions, core-shell nanofibres, cyclodextrins and liposomes among others, have been applied in order to protect these antimicrobials from degradation or volatilization while trying to enable a more controlled release and sustained antimicrobial action. On that account, this article provides an overview of the types of antimicrobials agents used and the most recent trends on the strategies used to encapsulate the antimicrobial agents for their stable inclusion in the packaging materials. Moreover, a thorough discussion regarding the benefits of each encapsulation technology as well as their application in food products is presented

Assembling the Tat protein translocase

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes

A novel transport mechanism for MOMP in Chlamydophila pneumoniae and its putative role in immune-therapy

Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE−/− mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane β-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events

Functional studies of the ‘GAFTGA’ motif of Escherichia coli Phage Shock Protein F

In the archetypal bacterial transcription, the multi-subunit core RNA polymerase (RNAP) is specifically bound to the promoter through the sigma factor (σ), forming a closed complex where DNA remains double-stranded. The promoter DNA is initially spontaneously melted by the σ factor within the core enzyme and subsequently loaded into the active channel of the holoenzyme. However, a major alternative transcription paradigm that depends on the sigma factor σ54 also exists in bacteria and controls pathogenicity, biofilm formation, bioluminescence, nitrogen fixation and stress responses. σ54 forms inhibitory interactions with DNA in the closed complex, which can only be alleviated by ATP hydrolysis-driven remodelling catalysed by bacterial enhancer binding proteins (bEBPs). In this regard, σ54- dependent transcription can be viewed analogous to the eukaryotic RNA Pol II system. This study was initiated to advance the understanding of: (i) how the ATP hydrolysis energy is relayed from the ATPase catalytic site to the closed complex for remodelling, (ii) the hexameric interface organisation of bEBPs for ATP hydrolysis, and (iii) the role of core RNAP in σ54-dependent transcription. A newly devised cross-linking technique combined with the DNA footprinting methods provided new insights of the organisation of each transcription component. The data gathered from this study updated the current working model for ATPdependent transcription. In addition, the cross-linking method proved to be an excellent tool to study protein-protein and nucleo-protein interactions