Partition System Research Articles

Investigation of Partition Mechanism of High-Copy Number Bacterial Plasmids via Replication Incompatibility

Bacterial plasmids play important role in gene transport between cells both vertically and horizontally, and are also versatile tools for gene engineering. Their maintenance of inherence in the host cells is believed to depend on the delicate quantity control through the replication origin and the effective management of partition. Without active partition system, high-copy number plasmids are suggested to be distributed as multiple foci but randomly partitioned during cell division. Lack of the direct evidence, the detail mechanism and dynamics of plasmid location and partition in cell is unknown. Taking the advantage of temporary tolerance on the incompatibility of replication origin, two ColE1 derivative plasmids with different antibiotic resists and repressible replication origin are co-transformed and their distributions and motions are observed at single cell level simultaneously. Based on the nature of competition between plasmids, it also provides the opportunity to actually reveal a single plasmid translocation between multiple foci, which is affected by the nucleoids of the dividing cells. Also, the variance of the inhered number of copies for both of two plasmids from parent to daughter cells provides the insight of partition mechanism.

Data in support of three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes.

This paper describes data related to a research article titled “Three Phase Partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes” (Gagaoua et al., 2015) [1]. Zingibain (EC 3.4.22.67), is a coagulant cysteine protease and a meat tenderizer agent that have been reported to produce satisfactory final products in dairy and meat technology, respectively. Zingibains were exclusively purified using chromatographic techniques with very low yield purification. This paper includes data of the effect of temperature, usual salts and organic solvents on the efficiency of the three phase partitioning (TPP) system. Also it includes data of the kinetic activity characterization of the purified zingibain using TPP purification approach.

Exploring 3D structural influences of aliphatic and aromatic chemicals on α-cyclodextrin binding

Binding of solutes to macromolecules is often influenced by steric effects caused by the 3D structures of both binding partners. In this study, the 1:1 α-cyclodextrin (αCD) binding constants (Ka1) for 70 organic chemicals were determined to explore the solute-structural effects on the αCD binding. Ka1 was measured using a three-part partitioning system with either a headspace or a passive sampler serving as the reference phase. The Ka1 values ranged from 1.08 to 4.97 log units. The results show that longer linear aliphatic chemicals form more stable complexes than shorter ones, and that the position of the functional group has a strong influence on Ka1, even stronger than the type of the functional group. Comparison of linear and variously branched aliphatic chemicals indicates that having a sterically unhindered alkyl chain is favorable for binding. These results suggest that only one alkyl chain can enter the binding cavity. Relatively small aromatic chemicals such as 1,3-dichlorobenzene bind to αCD well, while larger ones like tetrachlorobenzene and 3-ring aromatic chemicals show only a weak interaction with αCD, which can be explained by cavity exclusion. The findings of this study help interpret cyclodextrin binding data and facilitate the understanding of binding processes to macromolecules.

The Complete Sequence and Comparative Analysis of a Multidrug-Resistance and Virulence Multireplicon IncFII Plasmid pEC302/04 from an Extraintestinal Pathogenic Escherichia coli EC302/04 Indicate Extensive Diversity of IncFII Plasmids.

Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as blaTEM−1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical importance. Such phenomenon is bothersome when the plasmids are transmissible, facilitating the spread of virulence and resistance plasmids among pathogenic bacteria. Notably, certain TA systems are more commonly found in particular ExPEC plasmid types, indicating the possible relationships between certain TA systems and ExPEC pathogenesis.

Three Phase Partitioning System, an Emerging Non-Chromatographic Tool for Proteolytic Enzymes Recovery and Purification

A rapid overview of the Three Phase Partitioning (TPP) system as an efficient non-chromatographic tool is given. This elegant non-chromatographic tool is able to purify numerous proteins and especially proteases in a one step protocol. TPP is able to do not only purify proteins, but also concentrate them into one of the phases and enhance their proteolytic activity. The application of TPP for the extraction and purification of plant milk-clotting enzymes and meat tenderizers agents are given. In addition, some proteases from other materials are summarized. This short communication stress showed that TPP is a simple, economical and quick method for proteases recovery from plant proteases. This elegant non-chromatographic tool may be performed in a purification process to be used successfully in food industries, namely to provide enzymes for cheese-making and meat tenderizing.

Development of partitioning and transmutation system using accelerator driven system (ADS)

高レベル放射性廃棄物の減容化・有害度低減のための技術として，分離変換技術が着目されている。本稿では，様々に想定される原子力利用局面において，柔軟に対応できる技術として期待されている核変換専用の加速器駆動システム(ADS)を導入した核燃料サイクル(ADS階層型核変換システム)について，ADSの特徴と開発の現状，将来計画を中心に述べる。

In-mold patterning and actionable axo-somatic compartmentalization for on-chip neuron culture.

Oriented neuronal networks with controlled connectivity are required for many applications ranging from studies of neurodegeneration to neuronal computation. To build such networks in vitro, an efficient, directed and long lasting guidance of axons toward their target is a pre-requisite. The best guidance achieved so far, however, relies on confining axons in enclosed microchannels, making them poorly accessible for further investigation. Here we describe a method providing accessible and highly regular arrays of axons, emanating from somas positioned in distinct compartments. This method combines the use of a novel removable partition, allowing soma positioning outside of the axon guidance patterns, and in-mold patterning (iMP), a hybrid method combining chemical and mechanical cell positioning clues applied here for the first time to neurons. The axon guidance efficiency of iMP is compared to that of conventional patterning methods, e.g. micro-contact printing (chemical constraints by a poly-l-lysine motif) and micro-grooves (physical constraints by homogeneously coated microstructures), using guiding tracks of different widths and spacing. We show that iMP provides a gain of 10 to 100 in axon confinement efficiency on the tracks, yielding mm-long, highly regular, and fully accessible on-chip axon arrays. iMP also allows well-defined axon guidance from small populations of several neurons confined at predefined positions in μm-sized wells. iMP will thus open new routes for the construction of complex and accurately controlled neuronal networks.

The role of catalase and H2O2 in photocatalytic inactivation of Escherichia coli: Genetic and biochemical approaches

This study employed two different approaches, the inactivation of Escherichia coli single-gene deleted mutants and addition of scavengers in partition system, to reveal the roles of catalase (CAT), photogenerated e− and H2O2 in photocatalytic bacterial inactivation. A “parental strain” (E. coli BW25113) was more resistant than its isogenic single-gene deleted katG− mutant (E. coli JW3914) towards photocatalytic inactivation using TiO2 irradiated by UVA lamps (λ=365nm) in a partition system, whereas the photocatalytic inactivation efficiency of both bacterial strains were similar in a non-partition system. Addition of scavengers and CAT demonstrated the importance of superoxide radical (O2−), which subsequently formed H2O2, in the photocatalytic inactivation. The CAT activity and concentration of H2O2 during photocatalytic process were compared to further confirm the role of H2O2. In addition, bactericidal action of photogenerated e− and pH effect on the photocatalytic inactivation inferred that H2O2 probably came from the conduction band of TiO2. Furthermore, transmission electron microscope and atomic absorption spectrophotometric analyses indicated the oxidative damage of the bacterial cell began from cell envelope. The results of genetic and physiological analysis in this study provide a new insight into the cellular defense mechanism(s) during the photocatalytic inactivation of E. coli.

The first report of detecting the blaSIM-2 gene and determining the complete sequence of the SIM-encoding plasmid

An imipenem-resistant Pseudomonas aeruginosa strain HN39 that harbours a blaSIM-2-carrying plasmid pHN39-SIM, was isolated from a patient with craniocerebral infections in China. The SIM-2 protein differs from SIM-1 by a single amino acid substitution Gly198Asp. pHN39-SIM is a novel 282-kb megaplasmid and it possesses the replication and partition systems of an unknown incompatibility group. pHN39-SIM carries a total of ten separate accessory modules especially including a novel 38.8-kb multidrug resistance region. In addition to the known transposable elements ISPst3, a Tn5563a remnant, IS1071, Tn5046, ΔTn4662a and ΔTn512, harboured in these accessory modules are six novel ones ISPa59 to ISPa62, In1208 and Tn6284. The multidrug resistance region is composed of Tn6284 generated from the insertion of an In4-family integron In1208 into Tn5046, and a Tn4662a-derived element with the insertion of ΔTn512 connected with two other genes. In1208 carries not only blaSIM-2 but several additional genes accounting for resistance to erythromycin, chloramphenicol, rifampicin, streptomycin, quaternary ammonium compounds, sulphonamides and mercury.

Photocatalytic decolorization of bromothymol blue using biogenic selenium nanoparticles synthesized by terrestrial actinomycete Streptomyces griseobrunneus strain FSHH12

The aim of the present study was to isolate and identify a terrestrial actinomycete bacterial strain capable to produce selenium nanoparticles (Se NPs) followed by purification of the biogenic Se NPs and evaluation of their photocatalytic degradation compared to selenium dioxide. Among 30 actinomycete bacterial strains obtained from environmental soil samples, one isolate (identified as Streptomyces griseobrunneus strain FSHH12 based on the 16S rDNA gene sequence analysis) was selected and used for production of Se NPs. The biologically synthesized Se NPs was consequently purified by an organic–aqueous partitioning system and characterized using scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray, UV–visible spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction spectroscopy. The obtained results of photocatalytic degradation of bromothymol blue using the purified Se NPs (64 μg/mL) revealed 62.3% of dye removal under UV illumination (15 W) after 60 min incubation of dye solution.

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA-nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos-an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.

A dual-isotope approach to allow conclusive partitioning between three sources.

Stable isotopes have proved to be a transformative tool; their application to distinguish between two sources in a mixture has been a cornerstone of biogeochemical research. However, quantitatively partitioning systems using two stable isotopes (for example, 13C and 12C) has been largely limited to only two sources, and systems of interest often have more than two components, with interactive effects. Here we introduce a dual-isotope approach to allow conclusive partitioning between three sources, using only two stable isotopes. We demonstrate this approach by partitioning soil CO2 emissions derived from microbial mineralization of soil organic carbon (SOC), added pyrogenic organic matter (PyOM) and root respiration. We find that SOC mineralization in the presence of roots is 23% higher (P<0.05) when PyOM is also present. Being able to discern three sources with two isotopes will be of great value not only in biogeochemical research, but may also expand hitherto untapped methodologies in diverse fields.

The dualistic nature of integrative and conjugative elements.

Integrative and conjugative elements (ICEs) are mobile genetic elements that play a key role in bacterial adaptation. Such elements are found in almost every bacterial genera and species, and often code for adaptive traits conferring selective advantages to their host. ICEs maintain by integrating into and replicating along with a replicon of the host genome. ICEs can propagate by conjugative transfer toward a recipient cell following excision from the replicon as a circular covalently-closed molecule. For a long time, the excised form of ICEs was assumed to be non-replicative. This assumption predicts that excised ICEs are sensitive to loss during cell division, unless they carry stabilization systems such as addiction modules or antibiotic resistance genes. Over the past few years, growing evidence have been presented that support conditional replication of the circular intermediate as an intrinsic feature of ICEs. We recently confirmed this feature in the large family of SXT/R391 ICEs, which thrive in several species of Enterobacteriaceae and Vibrionaceae. Furthermore, we demonstrated that SXT/R391 ICEs encode a functional plasmid-like type II partition system that enhances their stability, such systems being probably encoded by other ICEs. The lifecycle of ICEs is therefore much more complex than initially thought as many ICEs may use plasmid-like features to improve their stability and dissemination.

Plasmid partitioning superstructure system

DNA Segregation Partitioning and sharing DNA between dividing cells is critical for all domains of life. Prokaryotes must share certain plasmids as well as their genomic DNA to survive. Schumacher et al. studied the partition system that segregates a conjugative plasmid in the prokaryote Sulfolobus. The system consists of three proteins. AspA spreads along the plasmid DNA to create a protein-DNA superhelix. The ParA motor protein is linked to the protein-DNA superhelix through the ParB protein, which has structural similarities to eukaryotic centromere segregating proteins. Science , this issue p. [1120][1] [1]: /lookup/doi/10.1126/science.aaa9046

Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages.

Although recent studies have provided a wealth of information about archaeal biology, nothing is known about the molecular basis of DNA segregation in these organisms. Here, we unveil the machinery and assembly mechanism of the archaeal Sulfolobus pNOB8 partition system. This system uses three proteins: ParA; an atypical ParB adaptor; and a centromere-binding component, AspA. AspA utilizes a spreading mechanism to create a DNA superhelix onto which ParB assembles. This supercomplex links to the ParA motor, which contains a bacteria-like Walker motif. The C domain of ParB harbors structural similarity to CenpA, which dictates eukaryotic segregation. Thus, this archaeal system combines bacteria-like and eukarya-like components, which suggests the possible conservation of DNA segregation principles across the three domains of life.

The LIR space partitioning system applied to the Stokes equations

We introduce a novel approach to solve the stationary Stokes equations on very large voxel geometries. One main idea is to coarsen a voxel geometry in areas where velocity does not vary much while keeping the original resolution near the solid surfaces. For spatial partitioning a simplified LIR-tree is used which is a generalization of the Octree and KD-tree. The other main idea is to arrange variables in a way such that each cell is able to satisfy the Stokes equations independently. Pressure and velocity are discretized on staggered grids. But instead of using one velocity variable on the cell surface we introduce two variables. The discretization of momentum and mass conservation yields a small linear system (block) per cell that allows to use the block Gauss–Seidel algorithm as iterative solver. We compare our method to other solvers and conclude superior performance in runtime and memory for high porosity geometries.

Characterization of epidemic IncI1-Iγ plasmids harboring ambler class A and C genes in Escherichia coli and Salmonella enterica from animals and humans.

The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation in traY and excA genes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology.

Coupling passive sampling with in vitro bioassays and chemical analysis to understand combined effects of bioaccumulative chemicals in blood of marine turtles

Conventional target analysis of biological samples such as blood limits our ability to understand mixture effects of chemicals. This study aimed to establish a rapid passive sampling technique using the polymer polydimethylsiloxane (PDMS) for exhaustive extraction of mixtures of neutral organic chemicals accumulated in blood of green turtles, in preparation for screening in in vitro bioassays. We designed a PDMS–blood partitioning system based on the partition coefficients of chemicals between PDMS and major blood components. The sampling kinetics of hydrophobic test chemicals (polychlorinated dibenzo-p-dioxins; PCDDs) from blood into PDMS were reasonably fast reaching steady state in <96h. The geometric mean of the measured PDMS–blood partition coefficients for PCDDs, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) was 14LbloodkgPDMS−1 and showed little variability (95% confidence interval from 8.4 to 29) across a wide range of hydrophobicity (logKow 5.7–8.3). The mass transfer of these chemicals from 5mL blood into 0.94g PDMS was 62–84%, which is similar to analytical recoveries in conventional solvent extraction methods. The validated method was applied to 15 blood samples from green turtles with known concentrations of PCDD/Fs, dioxin-like PCBs, PBDEs and organochlorine pesticides. The quantified chemicals explained most of the dioxin-like activity (69–98%), but less than 0.4% of the oxidative stress response. The results demonstrate the applicability of PDMS-based passive sampling to extract bioaccumulative chemicals from blood as well as the value of in vitro bioassays for capturing the combined effects of unknown and known chemicals.

Replication and Active Partition of Integrative and Conjugative Elements (ICEs) of the SXT/R391 Family: The Line between ICEs and Conjugative Plasmids Is Getting Thinner.

Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs.

The role and synergistic effect of the light irradiation and H2O2 in photocatalytic inactivation of Escherichia coli

Inactivation of Escherichia coli K-12 was conducted by applying a continuous supplying of commercial H2O2 to mimic the H2O2 production in a photocatalytic system, and the contribution of H2O2 in photocatalytic inactivation was investigated using a modified “partition system” and five E. coli mutants. The concentration of exogenous H2O2 required for complete inactivation of bacterial cells was much higher than that produced in-situ in common photocatalytic system, indicating that H2O2 alone plays a minor role in photocatalytic inactivation. However, the concentration of exogenously produced H2O2 required for effective inactivation of E. coli K-12 was much lower when the light irradiation was applied. To further investigate the possible physiological changes, inactivation of E. coli BW25113 (the parental strain), and its corresponding isogenic single-gene deletion mutants with light pretreatment was compared. The results indicate that light irradiation increases the bacterial intracellular Fe2+ level and favors hydroxyl radical (OH) production via the catalytic reaction of Fe2+, leading to increase in DNA damage. Moreover, the results indicate that the properties of light source, such as intensity and major emission wavelength, may alter the physiology of bacterial cells and affect the susceptibility to in-situ resultant H2O2 in the photocatalytic inactivation processes, leading to significant influence on the photocatalytic inactivation efficiencies of E. coli K-12.