Differential toxicity of potentially toxic elements to human gut microbes.

Specific microorganisms in the human gut (i.e., gut microbes) provide mutually beneficial outcomes such as microbial balance by inhibiting the growth of pathogenic organisms, immune system modulation, fermentation of ingested products, and vitamin production. The intake of contaminants including potenially toxic elements (PTEs) can occur through food, air, water and some medicines. The gut microbes not only can be affected by environmental contaminants but they themselves can alter the speciation and bioavailability of these contaminants. This research work was designed to demonstrate the relationship between increasing level of selected PTEs including As, Cd, Pb and Hg on the growth of selected gut microbes. The toxicity of above mentioned PTEs to three gut bacteria (Lactobacillus rhamnosus, Lactobacillus acidophilus and Escherichia coli) was examined. While the toxicity of all the cationic PTEs including Cd, Pb and Hg towards gut bacteria decreased with increasing pH, the anionic As species exhibited an opposite effect. The order of toxicity was Hg > Cd > Pb > As(III)>As(V) for E. coli; and Hg > Cd > As(III)>Pb > As(V) for the two Lactobacillus sp. Arsenite (AsIII) showed higher toxicity than arsenate (AsV) to gut bacteria. While As is an anion, Cd, Pb and Hg are cations and hence their binding capacity to the bacterial cell wall varied based on the charge dependent functional groups. However, the toxic effects of PTEs for a bacteria are controlled by their speciation and bioavailability.

FtsK and SpoIIIE, coordinators of chromosome segregation and envelope remodeling in bacteria.

The translocation of DNA during bacterial cytokinesis is mediated by the SpoIIIE/FtsK family of proteins. These proteins ensure efficient chromosome segregation into sister cells by ATP-driven translocation of DNA and they control chromosome dimer resolution. How FtsK/SpoIIIE mediate chromosome translocation during cytokinesis in Gram-positive and Gram-negative organisms has been the subject of debate. Studies on FtsK in Escherichia coli, and recent work on SpoIIIE in Bacillus subtilis, have identified interactions between each translocase and the division machinery, supporting the idea that SpoIIIE and FtsK coordinate the final steps of cytokinesis with completion of chromosome segregation. Here we summarize and discuss the view that SpoIIIE and FtsK play similar roles in coordinating cytokinesis with chromosome segregation, during growth and differentiation.

Complete Genome Sequences of Bacteriophages Kaya, Guyu, Kopi, and TehO, Which Target Clinical Strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major public health concern, as drug-resistant strains increase mortality in hospital-acquired infections. We report the isolation and complete genome sequences of four lytic bacteriophages that target clinical multidrug-resistant P. aeruginosa strains.

Understanding the Fundamental Basis for Biofilm Formation on Plastic Surfaces: Role of Conditioning Films.

Conditioning films (CFs) are surface coatings formed by the adsorption of biomolecules from the surrounding environment that can modify the material-specific surface properties and precedes the attachment of microorganisms. Hence, CFs are a biologically relevant identity that could govern the behavior and fate of microplastics in the aquatic environment. In the present study, polyethylene terephthalate (PET) and polylactic acid (PLA) plastic cards were immersed in natural seawater to allow the formation of CFs. The changes in the surface roughness after 24 h were investigated by atomic force microscopy (AFM), and the surface changes were visualized by scanning electron microscopy (SEM). The global elemental composition of the conditioned surface was investigated by energy dispersive spectroscopy (EDS). Results indicated that marine conditioning of PET and PLA samples for 24 h resulted in an increase of ∼11 and 31% in the average surface roughness, respectively. SEM images revealed the attachment of coccoid-shaped bacterial cells on the conditioned surfaces, and the accumulation of salts of sodium and phosphate-containing precipitates was revealed through the EDS analysis. The results indicate that the increase in surface roughness due to conditioning is linked to a material's hydrophilicity leading to a rapid attachment of bacteria on the surfaces. Further investigations into the CFs can unfold crucial knowledge surrounding the plastic-microbe interaction that has implications for medical, industrial, and environmental research.

Bioavailability of arsenic, cadmium, lead and mercury as measured by intestinal permeability.

In this study, the intestinal permeability of metal(loid)s (MLs) such as arsenic (As), cadmium (Cd), lead (Pb) and mercury (Hg) was examined, as influenced by gut microbes and chelating agents using an in vitro gastrointestinal/Caco-2 cell intestinal epithelium model. The results showed that in the presence of gut microbes or chelating agents, there was a significant decrease in the permeability of MLs (As-7.5%, Cd-6.3%, Pb-7.9% and Hg-8.2%) as measured by apparent permeability coefficient value (Papp), with differences in ML retention and complexation amongst the chelants and the gut microbes. The decrease in ML permeability varied amongst the MLs. Chelating agents reduce intestinal absorption of MLs by forming complexes thereby making them less permeable. In the case of gut bacteria, the decrease in the intestinal permeability of MLs may be associated to a direct protection of the intestinal barrier against the MLs or indirect intestinal ML sequestration by the gut bacteria through adsorption on bacterial surface. Thus, both gut microbes and chelating agents can be used to decrease the intestinal permeability of MLs, thereby mitigating their toxicity.

Biofilms Enhance the Adsorption of Toxic Contaminants on Plastic Microfibers under Environmentally Relevant Conditions.

Microplastics (MPs) exposed to the natural environment provide an ideal surface for biofilm formation, which potentially acts as a reactive phase facilitating the sorption of hazardous contaminants. Until now, changes in the contaminant sorption capacity of MPs due to biofilm formation have not been quantified. This is the first study that compared the capacity of naturally aged, biofilm-covered microplastic fibers (BMFs) to adsorb perfluorooctane sulfonate (PFOS) and lead (Pb) at environmentally relevant concentrations. Changes in the surface properties and morphology of aged microplastic fibers (MF) were studied by surface area analysis, infrared spectroscopy, and scanning electron microscopy. Results revealed that aged MFs exhibited higher surface areas because of biomass accumulation compared to virgin samples and followed the order polypropylene>polyethylene>nylon>polyester. The concentrations of adsorbed Pb and PFOS were 4-25% and 20-85% higher in aged MFs and varied among the polymer types. The increased contaminant adsorption was linked with the altered surface area and the hydrophobic/hydrophilic characteristics of the samples. Overall, the present study demonstrates that biofilms play a decisive role in contaminant-plastic interactions and significantly enhance the vector potential of MFs for toxic environmental contaminants. We anticipate that knowledge generated from this study will help refine the planetary risk assessment of MPs.

Fingerprinting Plastic-Associated Inorganic and Organic Matter on Plastic Aged in the Marine Environment for a Decade.

The long-term aging of plastic leads to weathering and biofouling that can influence the behavior and fate of plastic in the marine environment. This is the first study to fingerprint the contaminant profiles and bacterial communities present in plastic-associated inorganic and organic matter (PIOM) isolated from 10 year-aged plastic. Plastic sleeves were sampled from an oyster aquaculture farm and the PIOM was isolated from the intertidal, subtidal, and sediment-buried segments to investigate the levels of metal(loid)s, polyaromatic hydrocarbons (PAHs), per-fluoroalkyl substances (PFAS) and explore the microbial community composition. Results indicated that the PIOM present on long-term aged high-density polyethylene plastic harbored high concentrations of metal(loid)s, PAHs, and PFAS. Metagenomic analysis revealed that the bacterial composition in the PIOM differed by habitat type, which consisted of potentially pathogenic taxa including Vibrio, Shewanella, and Psychrobacter. This study provides new insights into PIOM as a potential sink for hazardous environmental contaminants and its role in enhancing the vector potential of plastic. Therefore, we recommend the inclusion of PIOM analysis in current biomonitoring regimes and that plastics be used with caution in aquaculture settings to safeguard valuable food resources, particularly in areas of point-source contamination.

Exploring the Composition and Functions of Plastic Microbiome Using Whole-Genome Sequencing.

Besides the ecotoxicological consequences of microplastics and associated chemicals, the association of microbes on plastics has greater environmental implications as microplastics may select for unique microbiome participating in environmentally significant functions. Despite this, the functional potential of the microbiome associated with different types of plastics is understudied. Here, we investigate the interaction between plastic and marine biofilm-forming microorganisms through a whole-genome sequencing approach on four types of microplastics incubated in the marine environment. Taxonomic analysis suggested that the microplastic surfaces exhibit unique microbial profiles and niche partitioning among the substrates. In particular, the abundance of Vibrio alginolyticus and Vibrio campbellii suggested that microplastic pollution may pose a potential risk to the marine food chain and negatively impact aquaculture industries. Microbial genera involved in xenobiotic compound degradation, carbon cycling, and genes associated with the type IV secretion system, conjugal transfer protein TraG, plant-pathogen interaction, CusA/CzcA family heavy metal efflux transfer proteins, and TolC family proteins were significantly enriched on all the substrates, indicating the variety of processes operated by the plastic-microbiome. The present study gives a detailed characterization of the rapidly altering microbial composition and gene pools on plastics and adds new knowledge surrounding the environmental ramifications of marine plastic pollution.

Gut microbes modulate bioaccessibility of lead in soil.

Metabolic uptake of lead (Pb) is controlled by its bioaccessibility. Most studies have examined bioaccessibility of Pb in the absence of gut microbes, which play an important role in the metabolic uptake of nutrients and metal(loid)s in intestine. In this study, we examined the effect of three gut microbes, from various locations in the gut, on the bioaccessibility of soil ingested Pb. The gut microbes include Lactobacillus acidophilus, Lactobacillus rhamnosus and Escherichia coli. Lead toxicity to these three microbes was also examined at various pH values. Bioaccessibility of Pb was measured using gastric and intestinal extractions. Both Pb spiked and Pb-contaminated shooting range field soils were used to measure Pb bioaccessibility in the presence and absence of gut microbes. The results indicated that Pb toxicity to gut microbes, as measured by LD50 value, decreased with increasing pH, and was higher for Lactobacillus species. Gut microbes decreased the bioaccessible Pb; the effect was more pronounced at low pH, mimicking gastric conditions than in conditions closer to the intestine. Lead adsorption by these microbes increased at the higher pH tested, and E. coli adsorbed higher amounts of Pb than did the Lactobacillus species. The effect of gut microbes on reducing Pb bioaccessibility may be attributed to microbially-induced immobilization of Pb through adsorption, precipitation, and complexation reactions. The study demonstrates that bioaccessibility and subsequently bioavailability of metal(loid)s can be modulated by gut microbes, and it is important to undertake bioaccessibility measurements in the presence of gut microbes.

Mobilization of pdif modules in Acinetobacter: A novel mechanism for antibiotic resistance gene shuffling?

XerCD-dif site-specific recombination is a well characterized system, found in most bacteria and archaea. Its role is resolution of chromosomal dimers that arise from homologous recombination. Xer-mediated recombination is also used by several plasmids for multimer resolution to enhance stability and by some phage for integration into the chromosome. In the past decade, it has been hypothesized that an alternate and novel function exists for this system in the dissemination of genetic elements, notably antibiotic resistance genes, in Acinetobacter species. Currently the mechanism underlying this apparent genetic mobility is unknown. Multidrug resistant Acinetobacter baumannii is an increasingly problematic pathogen that can cause recurring infections. Sequencing of numerous plasmids from clinical isolates of A. baumannii revealed the presence of possible mobile modules: genes were found flanked by pairs of Xer recombination sites, called plasmid-dif (pdif) sites. These modules have been identified in multiple otherwise unrelated plasmids and in different genetic contexts suggesting they are mobile elements. In most cases, the pairs of sites flanking a gene (or genes) are in inverted repeat, but there can be multiple modules per plasmid providing pairs of recombination sites that can be used for inversion or fusion/deletion reactions; as many as 16 pdif sites have been seen in a single plasmid. Similar modules including genes for surviving environmental toxins have also been found in strains of Acinetobacter Iwoffi isolated from permafrost cores; this suggests that these mobile modules are an ancient adaptation and not a novel response to antibiotic pressure. These modules bear all the hallmarks of mobile genetic elements, yet, their movement has never been directly observed to date. This review gives an overview of the current state of this novel research field.

Neutral-Neutral 2-Dimensional Agarose Gel Electrophoresis for Visualization of E. coli DNA Replication Structures.

Neutral-neutral 2-dimensional agarose gel electrophoresis enables the detection of replication intermediate structures in DNA. Here we describe how DNA from Escherichia coli cells can be purified to retain replication intermediates and then be separated by size and shape using two consecutive agarose gel electrophoresis protocols. The DNA structures present within a localized region can be visualized by a Southern blotting/radioactive hybridisation protocol.

A Mini-ISY100 Transposon Delivery System Effective in γ Proteobacteria.

Transposons are invaluable biological tools for the genetic manipulation of microorganisms. ISY100 from Synechocystis sp. PCC6803 is a member of the Tc1/mariner/IS630 superfamily, and is characterized by high transposition efficiency and a strong preference for TA target sequences. In this paper, we describe the design and application of a mini-ISY100 suicide vector for the in vivo creation of stable random transposon insertion libraries. The system was successfully applied in seven species belonging to four different orders of γ proteobacteria. In all cases, delivery using conjugation consistently showed the highest transposition efficiency compared to chemical transformation or electroporation. We determined the frequency of transposon insertions in all the species and proved the utility of the system by identifying genes involved in colony coloration in Shewanella oneidensis. The ease and the efficiency of the protocol developed here allow the creation of complete knock-out libraries in an extensive range of host microorganisms in less than a week with no requirement for preparatory modification.

Replication fork collapse at a protein-DNA roadblock leads to fork reversal, promoted by the RecQ helicase.

Proteins that bind DNA are the cause of the majority of impediments to replication fork progression and can lead to subsequent collapse of the replication fork. Failure to deal with fork collapse efficiently leads to mutation or cell death. Several models have been proposed for how a cell processes a stalled or collapsed replication fork; eukaryotes and bacteria are not dissimilar in terms of the general pathways undertaken to deal with these events. This study shows that replication fork regression, the combination of replication fork reversal leading to formation of a Holliday Junction along with exonuclease digestion, is the preferred pathway for dealing with a collapsed fork in Escherichia coli. Direct endo-nuclease activity at the replication fork was not observed. The protein that had the greatest effect on these fork processing events was the RecQ helicase, while RecG and RuvABC, which have previously been implicated in this process, were found to play a lesser role. Eukaryotic RecQ homologues, BLM and WRN, have also been implicated in processing events following replication fork collapse and may reflect a conserved mechanism. Finally, the SOS response was not induced by the protein-DNA roadblock under these conditions, so did not affect fork processing.

Activation of Xer-recombination at dif: structural basis of the FtsKγ-XerD interaction.

Bacterial chromosomes are most often circular DNA molecules. This can produce a topological problem; a genetic crossover from homologous recombination results in dimerization of the chromosome. A chromosome dimer is lethal unless resolved. A site-specific recombination system catalyses this dimer-resolution reaction at the chromosomal site dif. In Escherichia coli, two tyrosine-family recombinases, XerC and XerD, bind to dif and carry out two pairs of sequential strand exchange reactions. However, what makes the reaction unique among site-specific recombination reactions is that the first step, XerD-mediated strand exchange, relies on interaction with the very C-terminus of the FtsK DNA translocase. FtsK is a powerful molecular motor that functions in cell division, co-ordinating division with clearing chromosomal DNA from the site of septation and also acts to position the dif sites for recombination. This is a model system for unlinking, separating and segregating large DNA molecules. Here we describe the molecular detail of the interaction between XerD and FtsK that leads to activation of recombination as deduced from a co-crystal structure, biochemical and in vivo experiments. FtsKγ interacts with the C-terminal domain of XerD, above a cleft where XerC is thought to bind. We present a model for activation of recombination based on structural data.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System.

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell's replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Biological Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism.

The ubiquitous biological nanomotors were classified into two categories in the past: linear and rotation motors. In 2013, a third type of biomotor, revolution without rotation (http://rnanano.osu.edu/movie.html), was discovered and found to be widespread among bacteria, eukaryotic viruses, and double-stranded DNA (dsDNA) bacteriophages. This review focuses on recent findings about various aspects of motors, including chirality, stoichiometry, channel size, entropy, conformational change, and energy usage rate, in a variety of well-studied motors, including FoF1 ATPase, helicases, viral dsDNA-packaging motors, bacterial chromosome translocases, myosin, kinesin, and dynein. In particular, dsDNA translocases are used to illustrate how these features relate to the motion mechanism and how nature elegantly evolved a revolution mechanism to avoid coiling and tangling during lengthy dsDNA genome transportation in cell division. Motor chirality and channel size are two factors that distinguish rotation motors from revolution motors. Rotation motors use right-handed channels to drive the right-handed dsDNA, similar to the way a nut drives the bolt with threads in same orientation; revolution motors use left-handed motor channels to revolve the right-handed dsDNA. Rotation motors use small channels (<2 nm in diameter) for the close contact of the channel wall with single-stranded DNA (ssDNA) or the 2-nm dsDNA bolt; revolution motors use larger channels (>3 nm) with room for the bolt to revolve. Binding and hydrolysis of ATP are linked to different conformational entropy changes in the motor that lead to altered affinity for the substrate and allow work to be done, for example, helicase unwinding of DNA or translocase directional movement of DNA.

Stability of blocked replication forks in vivo.

Replication of chromosomal DNA must be carried out to completion in order for a cell to proliferate. However, replication forks can stall during this process for a variety of reasons, including nucleoprotein 'roadblocks' and DNA lesions. In these circumstances the replisome copying the DNA may disengage from the chromosome to allow various repair processes to restore DNA integrity and enable replication to continue. Here, we report the in vivo stability of the replication fork when it encounters a nucleoprotein blockage in Escherichia coli. Using a site-specific and reversible protein block system in conjunction with the temperature sensitive DnaC helicase loader and DnaB replicative helicase, we monitored the disappearance of the Y-shaped DNA replication fork structures using neutral-neutral 2D agarose gels. We show the replication fork collapses within 5 min of encountering the roadblock. Therefore, the stalled replication fork does not pause at a block in a stable confirmation for an extended period of time as previously postulated.

Two classes of nucleic acid translocation motors: rotation and revolution without rotation.

Biomotors are extensively involved in biological processes including cell mitosis, bacterial binary fission, DNA replication, DNA repair, homologous recombination, Holliday junction resolution, RNA transcription, and viral genome packaging. Traditionally, they were classified into two categories including linear and rotation motors. In 2013, a third class of motor by revolution mechanism without rotation was discovered. In this issue of "Structure and mechanisms of nanomotors in the cells", four comprehensive reviews are published to address the latest advancements of the structure and motion mechanism of a variety of biomotors in archaea, animal viruses, bacteria, and bacteriophages.

FtsK-dependent XerCD-dif recombination unlinks replication catenanes in a stepwise manner.

In Escherichia coli, complete unlinking of newly replicated sister chromosomes is required to ensure their proper segregation at cell division. Whereas replication links are removed primarily by topoisomerase IV, XerC/XerD-dif site-specific recombination can mediate sister chromosome unlinking in Topoisomerase IV-deficient cells. This reaction is activated at the division septum by the DNA translocase FtsK, which coordinates the last stages of chromosome segregation with cell division. It has been proposed that, after being activated by FtsK, XerC/XerD-dif recombination removes DNA links in a stepwise manner. Here, we provide a mathematically rigorous characterization of this topological mechanism of DNA unlinking. We show that stepwise unlinking is the only possible pathway that strictly reduces the complexity of the substrates at each step. Finally, we propose a topological mechanism for this unlinking reaction.