The role of cysteine in tellurate reduction and toxicity.

The tellurium oxyanion tellurate is toxic to living organisms even at low concentrations; however, its mechanism of toxicity is poorly understood. Here, we show that exposure of Escherichia coli K-12 to tellurate results in reduction to elemental tellurium (Te[0]) and the formation of intracellular reactive oxygen species (ROS). Toxicity assays performed with E. coli indicated that pre-oxidation of the intracellular thiol pools increases cellular resistance to tellurate-suggesting that intracellular thiols are important in tellurate toxicity. X-ray absorption spectroscopy experiments demonstrated that cysteine reduces tellurate to elemental tellurium. This redox reaction was found to generate superoxide anions. These results indicate that tellurate reduction to Te(0) by cysteine is a source of ROS in the cytoplasm of tellurate-exposed cells.

Symmetric shear banding and swarming vortices in bacterial superfluids.

Bacterial suspensions-a premier example of active fluids-show an unusual response to shear stresses. Instead of increasing the viscosity of the suspending fluid, the emergent collective motions of swimming bacteria can turn a suspension into a superfluid with zero apparent viscosity. Although the existence of active superfluids has been demonstrated in bulk rheological measurements, the microscopic origin and dynamics of such an exotic phase have not been experimentally probed. Here, using high-speed confocal rheometry, we study the dynamics of concentrated bacterial suspensions under simple planar shear. We find that bacterial superfluids under shear exhibit unusual symmetric shear bands, defying the conventional wisdom on shear banding of complex fluids, where the formation of steady shear bands necessarily breaks the symmetry of unsheared samples. We propose a simple hydrodynamic model based on the local stress balance and the ergodic sampling of nonequilibrium shear configurations, which quantitatively describes the observed symmetric shear-banding structure. The model also successfully predicts various interesting features of swarming vortices in stationary bacterial suspensions. Our study provides insights into the physical properties of collective swarming in active fluids and illustrates their profound influences on transport processes.

Rational Optimization of Raman-Activated Cell Ejection and Sequencing for Bacteria.

In Raman-activated cell ejection and sequencing (RACE-Seq), success rate and sequence coverage have generally been low for shotgun sequencing of individual post-RACE cells. Here we quantitatively evaluated the influence of cell lysis condition, nucleic acid amplification condition, and parameters of Raman measurement on RACE-Seq performance. Variations in laser energy input during Raman signal acquisition, but not duration of alkaline lysate lysis, temperature, or measurement under dry or aqueous conditions, are vital to the success of multiple displacement amplification (MDA). In fact, laser irradiation is reversely linked to MDA product quality. However, introduction of oils prior to MDA, by mitigating such negative effects of Raman irradiation, elevates genome coverage of post-RACE Escherichia coli cells from <20% to ∼50%, while greatly improving the success rate of RACE-Seq for soil microbiota. Our findings provide a practical solution for enhancing RACE-Seq performance and pinpoint protection of cells from laser irradiation as a priority in method development.

Synthesis, DNA/RNA-interaction and biological activity of benzo[k,l]xanthene lignans.

Interactions of two newly synthesized and six previously reported benzoxanthene lignans (BXLs), analogues of rare natural products, with DNA/RNA, G-quadruplex and HSA were evaluated by a set of spectrophotometric methods. Presence/absence of methoxy and hydroxy groups on the benzoxanthene core and minor modifications at C-1/C-2 side pendants - presence/absence of phenyl ring and presence/absence of methoxy and hydroxy groups on phenyl ring - influenced the fluorescence changes and the binding strength to double-stranded (ds-) and G-quadruplex structures. In general, compounds without phenyl ring showed stronger fluorescence changes upon binding than phenyl-substituted BXLs. On the other hand, BXLs with an unsubstituted phenyl ring showed the best stabilization effects of G-quadruplex. Circular dichroism spectroscopy results suggest mixed binding mode, groove binding and partial intercalation, to ds-DNA/RNA and end-stacking to top or bottom G-tetrads as the main binding modes of BXLs to those targets. All compounds exhibited micromolar binding affinities toward HSA and an increased protein thermal stability. Moderate to strong antiradical scavenging activity was observed for all BXLs with hydroxy groups at C-6, C-9 and C-10 positions of the benzoxanthene core, except for derivative bearing methoxy groups at these positions. BXLs with unsubstituted or low-substituted phenyl ring and one derivative without phenyl ring showed strong growth inhibition of Gram-positive Staphylococcus aureus. All compounds showed moderate to strong tumor cell growth-inhibitory activity and cytotoxicity.

Molecular basis for a protein-mediated DNA-bridging mechanism that functions in condensation of the E. coli chromosome.

The E. coli chromosome is condensed into insulated regions termed macrodomains (MDs), which are essential for genomic packaging. How chromosomal MDs are specifically organized and compacted is unknown. Here, we report studies revealing the molecular basis for Terminus-containing (Ter) chromosome condensation by the Ter-specific factor MatP. MatP contains a tripartite fold with a four-helix bundle DNA-binding motif, ribbon-helix-helix and C-terminal coiled-coil. Strikingly, MatP-matS structures show that the MatP coiled-coils form bridged tetramers that flexibly link distant matS sites. Atomic force microscopy and electron microscopy studies demonstrate that MatP alone loops DNA. Mutation of key coiled-coil residues destroys looping and causes a loss of Ter condensation in vivo. Thus, these data reveal the molecular basis for a protein-mediated DNA-bridging mechanism that mediates condensation of a large chromosomal domain in enterobacteria.

Physical Properties of Bacterial Outer Membrane Models: Neutron Reflectometry & Molecular Simulation.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer having phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet. This unique asymmetry and the complex carbohydrates in lipopolysaccharides make it a daunting task to study the asymmetrical OM structure and dynamics, its interactions with OM proteins, and its roles in translocation of substrates, including antibiotics. In this study, we combine neutron reflectometry and molecular simulation to explore the physical properties of OM mimetics. There is excellent agreement between experiment and simulation, allowing experimental testing of the conclusions from simulations studies and also atomistic interpretation of the behavior of experimental model systems, such as the degree of lipid asymmetry, the lipid component (tail, head, and sugar) profiles along the bilayer normal, and lateral packing (i.e., average surface area per lipid). Therefore, the combination of both approaches provides a powerful new means to explore the biological and biophysical behavior of the bacterial OM.

MinC and FtsZ mutant analysis provides insight into MinC/MinD-mediated Z ring disassembly.

The Min system negatively regulates the position of the Z ring, which serves as a scaffold for the divisome that mediates bacterial cytokinesis. In Escherichia coli, this system consists of MinC, which antagonizes assembly of the tubulin homologue FtsZ. MinC is recruited to the membrane by MinD and induced by MinE to oscillate between the cell poles. MinC is a dimer with each monomer consisting of functionally distinct MinCN and MinCC domains, both of which contact FtsZ. According to one model, MinCC/MinD binding to the FtsZ tail positions MinCN at the junction of two GDP-containing subunits in the filament, leading to filament breakage. Others posit that MinC sequesters FtsZ-GDP monomers or that MinCN caps the minus end of FtsZ polymers and that MinCC interferes with lateral interactions between FtsZ filaments. Here, we isolated minC mutations that impair MinCN function and analyzed FtsZ mutants resistant to MinC/MinD. Surprisingly, we found mutations in both minC and ftsZ that differentiate inhibition by MinC from inhibition by MinC/MinD. Analysis of these mutations suggests that inhibition of the Z ring by MinC alone is due to sequestration, whereas inhibition by MinC/MinD is not. In conclusion, our genetic and biochemical data support the model that MinC/MinD fragments FtsZ filaments.

Defining Molecular Details of the Chemistry of Biofilm Formation by Raman Microspectroscopy.

Two protocols that allow for the comparison of Raman spectra of planktonic cells and biofilm formed from these cells in their growth phase have been developed. Planktonic cells are washed and flash-frozen in <1 min to reduce the time for metabolic changes during processing, prior to freeze-drying. Biofilm is formed by standing cells in 50 µL indentations in aluminum foil in an atmosphere of saturated water vapor for 24-48 h. The results for Escherichia coli type K12 cells, which do not readily form biofilm, are compared to those for Staphylococcus epidermidis cells, which prolifically synthesize biofilm. For E. coli, the Raman spectra of the planktonic and biofilm samples are similar with the exception that the spectral signature of RNA, present in planktonic cells, could not be detected in biofilm. For S. epidermidis, major changes occur upon biofilm formation. In addition to the absence of the RNA features, new bands occur near 950 cm-1 and between 1350 and 1420 cm-1 that are associated with an increase in carbohydrate content. Unlike the case in E. coli biofilm, the intensity of G base ring modes is reduced in but A and T base ring signatures become more prominent. For S. epidermis in the biofilm's amide III region, there is evidence of an increase in the level of ß-sheet structure accompanied by a decrease in α-helical content. The presence of biofilm is confirmed by microscope-aided photography and, separately, by staining with methyl violet.

Detecting RNA-RNA interactions in E. coli using a modified CLASH method.

BACKGROUND: Bacterial small regulatory RNAs (sRNAs) play important roles in sensing environment changes through sRNA-target mRNA interactions. However, the current strategy for detecting sRNA-mRNA interactions usually combines bioinformatics prediction and experimental verification, which is hampered by low prediction accuracy and low-throughput. Additionally, among the 4736 sequenced bacterial genomes, only about 2164 sRNAs from 319 strains have been described. Furthermore, target mRNAs of only 157 sRNAs have been uncovered. Obviously, highly efficient methods were required to detect sRNA-mRNA interactions in the sequenced genomes. This study aimed to apply a modified CLASH (cross-linking, ligation and sequencing hybrids) method to detect RNA-RNA interactions in E. coli, a model bacterial organism. RESULTS: Statistically significant interactions were detected in 29 transcript pairs. To the best of our knowledge, 24 pairs were reported for the first time and were novel RNA interactions, including tRNA-tRNA, tRNA-ncRNA (non-coding RNA), tRNA-rRNA, rRNA-mRNA, rRNA-ncRNA, rRNA-rRNA, rRNA-IGT (intergenic transcript), and tRNA-IGT interactions. CONCLUSIONS: Discovery of novel RNA-RNA interactions in the present study demonstrates that RNA-RNA interactions might be far more complicated than ever expected. New methods may be required to help discover more novel RNA-RNA interactions. The present work describes a high-throughput protocol not only for discovering new RNA interactions, but also directly obtaining base-pairing sequences, which should be useful in assessing RNA structure and interactions.

Dynamics and Interactions of OmpF and LPS: Influence on Pore Accessibility and Ion Permeability.

The asymmetric outer membrane of Gram-negative bacteria is formed of the inner leaflet with phospholipids and the outer leaflet with lipopolysaccharides (LPS). Outer membrane protein F (OmpF) is a trimeric porin responsible for the passive transport of small molecules across the outer membrane of Escherichia coli. Here, we report the impact of different levels of heterogeneity in LPS environments on the structure and dynamics of OmpF using all-atom molecular dynamics simulations. The simulations provide insight into the flexibility and dynamics of LPS components that are highly dependent on local environments, with lipid A being the most rigid and O-antigen being the most flexible. Increased flexibility of O-antigen polysaccharides is observed in heterogeneous LPS systems, where the adjacent O-antigen repeating units are weakly interacting and thus more dynamic, compared to homogeneous LPS systems in which LPS interacts strongly with each other with limited overall flexibility due to dense packing. The model systems were validated by comparing molecular-level details of interactions between OmpF surface residues and LPS core sugars with experimental data, establishing the importance of LPS core oligosaccharides in shielding OmpF surface epitopes recognized by monoclonal antibodies. There are LPS environmental influences on the movement of bulk ions (K(+) and Cl(-)), but the ion selectivity of OmpF is mainly affected by bulk ion concentration.

Quantitative Analysis of Intracellular Fluorescent Foci in Live Bacteria.

Fluorescence microscopy has revolutionized in vivo cellular biology. Through the specific labeling of a protein of interest with a fluorescent protein, one is able to study movement and colocalization, and even count individual proteins in a live cell. Different algorithms exist to quantify the total intensity and position of a fluorescent focus. Although these algorithms have been rigorously studied for in vitro conditions, which are greatly different than the in-homogenous and variable cellular environments, their exact limits and applicability in the context of a live cell have not been thoroughly and systematically evaluated. In this study, we quantitatively characterize the influence of different background subtraction algorithms on several focus analysis algorithms. We use, to our knowledge, a novel approach to assess the sensitivity of the focus analysis algorithms to background removal, in which simulated and experimental data are combined to maintain full control over the sensitivity of a focus within a realistic background of cellular fluorescence. We demonstrate that the choice of algorithm and the corresponding error are dependent on both the brightness of the focus, and the cellular context. Expectedly, focus intensity estimation and localization accuracy suffer in all algorithms at low focus to background ratios, with the bacteroidal background subtraction in combination with the median excess algorithm, and the region of interest background subtraction in combination with a two-dimensional Gaussian fit algorithm, performing the best. We furthermore show that the choice of background subtraction algorithm is dependent on the expression level of the protein under investigation, and that the localization error is dependent on the distance of a focus from the bacterial edge and pole. Our results establish a set of guidelines for what signals can be analyzed to give a targeted spatial and intensity accuracy within a bacterial cell.

Fluorescence Turn-On Folding Sensor To Monitor Proteome Stress in Live Cells.

Proteome misfolding and/or aggregation, caused by a thermal perturbation or a related stress, transiently challenges the cellular protein homeostasis (proteostasis) network capacity of cells by consuming chaperone/chaperonin pathway and degradation pathway capacity. Developing protein client-based probes to quantify the cellular proteostasis network capacity in real time is highly desirable. Herein we introduce a small-molecule-regulated fluorescent protein folding sensor based on a thermo-labile mutant of the de novo designed retroaldolase (RA) enzyme. Since RA enzyme activity is not present in any cell, the protein folding sensor is bioorthogonal. The fluorogenic small molecule was designed to become fluorescent when it binds to and covalently reacts with folded and functional RA. Thus, in the first experimental paradigm, cellular proteostasis network capacity and its dynamics are reflected by RA-small molecule conjugate fluorescence, which correlates with the amount of folded and functional RA present, provided that pharmacologic chaperoning is minimized. In the second experimental scenario, the RA-fluorogenic probe conjugate is pre-formed in a cell by simply adding the fluorogenic probe to the cell culture media. Unreacted probe is then washed away before a proteome misfolding stress is applied in a pulse-chase-type experiment. Insufficient proteostasis network capacity is reflected by aggregate formation of the fluorescent RA-fluorogenic probe conjugate. Removal of the stress results in apparent RA-fluorogenic probe conjugate re-folding, mediated in part by the heat-shock response transcriptional program augmenting cytosolic proteostasis network capacity, and in part by time-dependent RA-fluorogenic probe conjugate degradation by cellular proteolysis.

High production of ectoine from aspartate and glycerol by use of whole-cell biocatalysis in recombinant Escherichia coli.

BACKGROUND: Recently, the compatible solute 1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) has attracted considerable interest due to its great potential as a protecting agent. To overcome the drawbacks of high salinity in the traditional bioprocess of ectoine using halophilic bacteria, various attempts have been made to engineer ectoine biosynthesis in nonhalophilic bacteria. Unfortunately, the yields of ectoine in these producers are still low and hardly meet the demands of large scale production. In this paper, the whole-cell biocatalytic process using aspartate and glycerol as substrates was tried for high production of ectoine in nonhalophilic bacteria. RESULTS: The ectoine genes ectABC from the halophilic bacterium Halomonas elongata were successfully introduced into Escherichia coli K-12 strain BW25113 under the arabinose-inducible promoter. To our delight, a large amount of ectoine was synthesized and excreted into the medium during the course of whole-cell biocatalysis, when using aspartate and glycerol as the direct substrates. At the low cell density of 5 OD/mL in flask, under the optimal conditions (100 mM sodium phosphate buffer (pH 7.0), 100 mM sodium aspartate, 100 mM KCl and 100 mM glycerol), the concentration of extracellular ectoine was increased to 2.67 mg/mL. At the high cell density of 20 OD/mL in fermentor, a maximum titre of 25.1 g/L ectoine was achieved in 24 h. Meanwhile, the biomass productivity of ectoine is as high as 4048 mg per gram dry cell weight (g DCW)(-1), which is the highest value ever reported. Furthermore, it was demonstrated that the same batch of cells could be used for at least three rounds. Finally, a total yield of 63.4 g ectoine was obtained using one litre cells. CONCLUSION: Using aspartate and glycerol as the direct substrates, high production of ectoine was achieved by the whole-cell biocatalysis in recombinant E. coli. Multiple rounds of whole-cell biocatalysis were established to further improve the production of ectoine. Our study herein provided a feasible biosynthesis process of ectoine with potential applications in large-scale industrial production.

Robustness and accuracy of cell division in Escherichia coli in diverse cell shapes.

Cell division in typical rod-shaped bacteria such as Escherichia coli shows a remarkable plasticity in being able to adapt to a variety of irregular cell shapes. Here, we investigate the roles of the Min system and the nucleoid-occlusion factor SlmA in supporting this adaptation. We study "squeezed" E. coli in narrow nanofabricated channels where these bacteria exhibit highly irregular shapes and large volumes. Despite the severely anomalous morphologies we find that most of these bacteria maintain their ability to divide into two equally sized daughters with an accuracy comparable to that of normal rod-shaped cells (about 4%). Deletion of either slmA or minC shows that the molecular systems associated with these genes are largely dispensable for accurate cell division in these irregular cell shapes. Using fluorescence time-lapse microscopy, we determine that the functionality of the Min system is affected by the cell shape, whereas the localization of a nucleoid relative to the cell division proteins (the divisome) remains unperturbed in a broad spectrum of morphologies, consistent with nucleoid occlusion. The observed positioning of the nucleoid relative to the divisome appears not to be affected by the nucleoid-occlusion factor SlmA. The current study underscores the importance of nucleoid occlusion in positioning the divisome and shows that it is robust against shape irregularities.

Comparative study of time-dependent effects of 4 and 8 Hz mechanical vibration at infrasound frequency on E. coli K-12 cells proliferation.

The aim of the present work is to study the time-dependent effects of mechanical vibration (MV) at infrasound (IS) frequency at 4 and 8 Hz on E. coli K-12 growth by investigating the cell proliferation, using radioactive [(3)H]-thymidine assay. In our previous work it was suggested that the aqua medium can serve as a target through which the biological effect of MV on microbes could be realized. At the same time it was shown that microbes have mechanosensors on the surface of the cells and can sense small changes of the external environment. The obtained results were shown that the time-dependent effects of MV at 4 and 8 Hz frequency could either stimulate or inhibit the growth of microbes depending from exposure time. It more particularly, the invention relates to a method for controlling biological functions through the application of mechanical vibration, thus making it possible to artificially control the functions of bacterial cells, which will allow us to develop method that can be used in agriculture, industry, medicine, biotechnology to control microbial growth.

The helicases DinG, Rep and UvrD cooperate to promote replication across transcription units in vivo.

How living cells deal with head-on collisions of the replication and transcription complexes has been debated for a long time. Even in the widely studied model bacteria Escherichia coli, the enzymes that take care of such collisions are still unknown. We report here that in vivo, the DinG, Rep and UvrD helicases are essential for efficient replication across highly transcribed regions. We show that when rRNA operons (rrn) are inverted to face replication, the viability of the dinG mutant is affected and over-expression of RNase H rescues the growth defect, showing that DinG acts in vivo to remove R-loops. In addition, DinG, Rep and UvrD exert a common function, which requires the presence of two of these three helicases. After replication blockage by an inverted rrn, Rep in conjunction with DinG or UvrD removes RNA polymerase, a task that is fulfilled in its absence by the SOS-induced DinG and UvrD helicases. Finally, Rep and UvrD also act at inverted sequences other than rrn, and promote replication through highly transcribed regions in wild-type E. coli.

Bacterial persistence by RNA endonucleases.

Bacteria form persisters, individual cells that are highly tolerant to different types of antibiotics. Persister cells are genetically identical to nontolerant kin but have entered a dormant state in which they are recalcitrant to the killing activity of the antibiotics. The molecular mechanisms underlying bacterial persistence are unknown. Here, we show that the ubiquitous Lon (Long Form Filament) protease and mRNA endonucleases (mRNases) encoded by toxin-antitoxin (TA) loci are required for persistence in Escherichia coli. Successive deletion of the 10 mRNase-encoding TA loci of E. coli progressively reduced the level of persisters, showing that persistence is a phenotype common to TA loci. In all cases tested, the antitoxins, which control the activities of the mRNases, are Lon substrates. Consistently, cells lacking lon generated a highly reduced level of persisters. Moreover, Lon overproduction dramatically increased the levels of persisters in wild-type cells but not in cells lacking the 10 mRNases. These results support a simple model according to which mRNases encoded by TA loci are activated in a small fraction of growing cells by Lon-mediated degradation of the antitoxins. Activation of the mRNases, in turn, inhibits global cellular translation, and thereby induces dormancy and persistence. Many pathogenic bacteria known to enter dormant states have a plethora of TA genes. Therefore, in the future, the discoveries described here may lead to a mechanistic understanding of the persistence phenomenon in pathogenic bacteria.

Cell division, one-carbon metabolism and methionine synthesis in a metK-deficient Escherichia coli mutant, and a role for MmuM.

An E. coli K-12 mutant deficient in S-adenosylmethionine (SAM) synthesis, i.e ΔmetK, but expressing a rickettsial SAM transporter, can grow in glucose minimal medium if provided with both SAM and methionine. It uses the externally provided (R)-enantiomer of SAM as methyl donor to produce most but not all of its methionine, by methylation of homocysteine catalysed by homocysteine methyltransferase (MmuM). The ΔmetK cells are also altered in growth and are twice as long as those of the parent strain. When starved of SAM, the mutant makes a small proportion of very long cells suggesting a role of SAM and of methylation in the onset of crosswall formation.

The study of the effects of mechanical vibration at infrasound frequency on [(3)H]-thymidine incorporation into DNA of E. coli K-12.

The aim of the present work was to investigate the frequency-dependent effects of mechanical vibration at infrasound frequency (MV at IS frequency or MV) on E. coli K-12 growth by investigating the cell proliferation, using radioactive [(3)H]-thymidine assay. The frequency-dependent effects of MV were shown that it could either stimulate or inhibit the growth of microbes. However, the mechanism through which the MV effects affect the bacterial cells is not clear yet. It was suggested that the aqua medium can serve as a target through which the biological effect of MV on microbes could be realized. To check this hypothesis the frequency-dependent effect (2, 4, 6, 8, 10 Hz) of MV on the bacterial growth in cases of exposure the preliminary treated microbes-free medium and microbes containing medium were studied. It has been shown that MV at 4, 8, and 10 Hz frequency has inhibition effects, while at 2 and 6 Hz has stimulation effects on cell proliferation.