Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands

In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.     

Targeting Cancer Cells by Exploiting Karyotypic Complexity and Chromosomal Instability

Multiple karyotypic abnormalities and chromosomal instability are particular hallmarks of many cancers that are relatively resistant to long term control by current chemotherapeutic agents. We have asked whether these same hallmarks, karyotypic complexity and instability, can be used as determinants for the screening of potential anticancer compounds. Using a panel of well characterized cancer cell lines we have been able to identify specific groups of chemical compounds that are more cytotoxic toward the relatively more karyotypically complex and unstable panel members. Thus, we delineate an approach for the identification of “lead compounds” for anticancer drug discovery complementary to approaches that are focused at the outset on a given gene or pathway.     

Construction of a CRISPR-Cas9 System for Pig Genome Targeting

A Cas9/sgRNA RNA-guided endonuclease expression system including a codon-optimized Streptococcus pyogenes A20 Cas9 recombinant protein expression vector and a spacer-guide chimeric RNA expression vector using the porcine U6 promoter was constructed for application in pigs. Only the Flag₂-NLS₁-Cas9-NLS₂ recombinant protein in complex with sgRNA was translocated into the nucleus; the Flag₂-NLS₁-Cas9-NLS₂ protein alone was excluded from the nucleus. Up to 13% of porcine PK1 cells targeted in vitro were observed, regardless of transfection efficiency.     

Energetic Consequences of Human Sociality: Walking Speed Choices among Friendly Dyads

Research has shown that individuals have an optimal walking speed–a speed which minimizes energy expenditure for a given distance. Because the optimal walking speed varies with mass and lower limb length, it also varies with sex, with males in any given population tending to have faster optimal walking speeds. This potentially creates an energetic dilemma for mixed-sex walking groups. Here we examine speed choices made by individuals of varying stature, mass, and sex walking together. Individuals (N = 22) walked around a track alone, with a significant other (with and without holding hands), and with friends of the same and opposite sex while their speeds were recorded every 100 m. Our findings show that males walk at a significantly slower pace to match the females’ paces (p = 0.009), when the female is their romantic partner. The paces of friends of either same or mixed sex walking together did not significantly change (p>0.05). Thus significant pace adjustment appears to be limited to romantic partners. These findings have implications for both mobility and reproductive strategies of groups. Because the male carries the energetic burden by adjusting his pace (slowing down 7%), the female is spared the potentially increased caloric cost required to walk together. In energetically demanding environments, we will expect to find gender segregation in group composition, particularly when travelling longer distances.     

Fire in the Riparian Zone: Characteristics and Ecological Consequences

We review the current understandings of the frequency, spatial distributions, mechanisms, and ecological consequences of fire in riparian zones. Riparian zones are well known for influencing many ecological processes at local to landscape scales, and fire can have an important ecosystem-scale influence on them. Riparian zones differ from surrounding uplands in their biophysical templates, moisture regimes and disturbance regimes; as a consequence the characteristics and effects of fire are different than in adjacent uplands. Fire impacts on riparian zones vary proportionally with the severity and extent of burning in the catchment and are affected by stream size. Riparian zones can act as a buffer against fire and therefore as a refuge for fire-sensitive species. However, under some circumstances, such as dry pre-fire climatic conditions and the accumulation of dry fuel, riparian areas become corridors for fire movement. Fire incursion into riparian zones creates canopy gaps and drier conditions, which allow subsequent build up of dead wood and establishment of fire adapted species. In concert, this increases fuel loads and the probability of another fire. Secondary effects of riparian fire include altering nutrient fluxes and cycling, increasing sediment loads, and stimulating erosion. We conclude that riparian fires are potentially important in shaping ecological characteristics in many regions, but this is poorly quantified. A better understanding of riparian fire regimes is essential to assess the effects of fire in helping shape the complex ecological characteristics of riparian zones over the longer-term.     

Large-Scale Chromosomal Changes and Associated Fitness Consequences in Pathogenic Fungi

Pathogenic fungi encounter many different host environments to which they must adapt rapidly to ensure growth and survival. They also must be able to cope with alterations in established niches during long-term persistence in the host. Many eukaryotic pathogens have evolved a highly plastic genome, and large-scale chromosomal changes including aneuploidy, and loss of heterozygosity (LOH) can arise under various in vitro and in vivo stresses. Both aneuploidy and LOH can arise quickly during a single cell cycle, and it is hypothesized that they provide a rapid, albeit imprecise, solution to adaptation to stress until better and more refined solutions can be acquired by the organism. While LOH, with the extreme case of haploidization in Candida albicans, can purge the genome from recessive lethal alleles and/or generate recombinant progeny with increased fitness, aneuploidy, in the absence or rarity of meiosis, can serve as a non-Mendelian mechanism for generating genomic variation.     

Restoring Fire to Long-Unburned Pinus palustris Ecosystems: Novel Fire Effects and Consequences for Long-Unburned Ecosystems

Biologically rich savannas and woodlands dominated by Pinus palustris once dominated the southeastern U.S. landscape. With European settlement, fire suppression, and landscape fragmentation, this ecosystem has been reduced in area by 97%. Half of remnant forests are not burned with sufficient frequency, leading to declines in plant and animal species richness. For these fire‐suppressed ecosystems a major regional conservation goal has been ecological restoration, primarily through the reinitiation of historic fire regimes. Unfortunately, fire reintroduction in long‐unburned Longleaf pine stands can have novel, undesirable effects. We review case studies of Longleaf pine ecosystem restoration, highlighting novel fire behavior, patterns of tree mortality, and unintended outcomes resulting from reintroduction of fire. Many of these pineland restoration efforts have resulted in excessive overstory pine mortality (often >50%) and produced substantial quantities of noxious smoke. The most compelling mechanisms of high tree mortality after reintroduction of fire are related to smoldering combustion of surface layers of organic matter (duff) around the bases of old pines. Development of effective methods to reduce fuels and competing vegetation while encouraging native vegetation is a restoration challenge common to fire‐prone ecosystems worldwide that will require understanding of the responses of altered ecosystems to the resumption of historically natural disturbances.     

细菌产生的挥发性物质及其生物学功能

细菌产生的挥发性物质种类繁多,成分复杂,是寻找具有特殊生物学功能的天然化合物的一个重要来源。近些年,越来越多的研究人员关注细菌产生的挥发性物质及其生物学功能。概述了细菌产生的挥发性物质的种类以及分析鉴定挥发性物质成分的方法;着重评述了细菌产生的挥发性物质对真菌、细菌、动物和植物生长和代谢的影响,并对细菌产生的挥发性物质未来的主要研究方向进行了展望。     

Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System

Biochemistry (Moscow) - The emergence and persistence of selfish genetic elements is an intrinsic feature of all living systems. Cellular organisms have evolved a plethora of elaborate defense...     

Activity-Dependent Dendritic Release of BDNF and Biological Consequences

Network construction and reorganization is modulated by the level and pattern of synaptic activity generated in the nervous system. During the past decades, neurotrophins, and in particular brain-derived neurotrophic factor (BDNF), have emerged as attractive candidates for linking synaptic activity and brain plasticity. Thus, neurotrophin expression and secretion are under the control of activity-dependent mechanisms and, besides their classical role in supporting neuronal survival neurotrophins, modulate nearly all key steps of network construction from neuronal migration to experience-dependent refinement of local connections. In this paper, we provide an overview of recent findings showing that BDNF can serve as a target-derived messenger for activity-dependent synaptic plasticity and development at the single cell level.     

MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems

Motivation

Biologists often wish to use their knowledge on a few experimental models of a given molecular system to identify homologs in genomic data. We developed a generic tool for this purpose.

Results

Macromolecular System Finder (MacSyFinder) provides a flexible framework to model the properties of molecular systems (cellular machinery or pathway) including their components, evolutionary associations with other systems and genetic architecture. Modelled features also include functional analogs, and the multiple uses of a same component by different systems. Models are used to search for molecular systems in complete genomes or in unstructured data like metagenomes. The components of the systems are searched by sequence similarity using Hidden Markov model (HMM) protein profiles. The assignment of hits to a given system is decided based on compliance with the content and organization of the system model. A graphical interface, MacSyView, facilitates the analysis of the results by showing overviews of component content and genomic context. To exemplify the use of MacSyFinder we built models to detect and class CRISPR-Cas systems following a previously established classification. We show that MacSyFinder allows to easily define an accurate “Cas-finder” using publicly available protein profiles.

Availability and Implementation

MacSyFinder is a standalone application implemented in Python. It requires Python 2.7, Hmmer and makeblastdb (version 2.2.28 or higher). It is freely available with its source code under a GPLv3 license at https://github.com/gem-pasteur/macsyfinder. It is compatible with all platforms supporting Python and Hmmer/makeblastdb. The “Cas-finder” (models and HMM profiles) is distributed as a compressed tarball archive as Supporting Information.     

金属硫蛋白及其生物学功能

金属硫蛋白(metallothionein, MT) 是一类富含半胱氨酸的低分子质量蛋白质,已鉴定4种亚型：MT-1、MT-2、MT-3和MT-4,基于各亚型功能的相对异质性而使MT呈现其生物学作用的多样性。金属硫蛋白通过与金属离子结合而参与基因表达调控和机体的重金属解毒过程;金属硫蛋白通过抑制多种氧化应激途径而保护细胞免受损伤;金属硫蛋白通过参与细胞的增殖、分化和凋亡的调节而影响肿瘤及其他重大疾病的发生发展。本文在金属硫蛋白的结构和分类的基础上综述其生物学作用及其相关机制。     

The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

SUMMARY

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular.