Comparative Analysis of Plasmids in the Genus Listeria

Background

We sequenced four plasmids of the genus Listeria, including two novel plasmids from L. monocytogenes serotype 1/2c and 7 strains as well as one from the species L. grayi. A comparative analysis in conjunction with 10 published Listeria plasmids revealed a common evolutionary background.

Principal Findings

All analysed plasmids share a common replicon-type related to theta-replicating plasmid pAMbeta1. Nonetheless plasmids could be broadly divided into two distinct groups based on replicon diversity and the genetic content of the respective plasmid groups. Listeria plasmids are characterized by the presence of a large number of diverse mobile genetic elements and a commonly occurring translesion DNA polymerase both of which have probably contributed to the evolution of these plasmids. We detected small non-coding RNAs on some plasmids that were homologous to those present on the chromosome of L. monocytogenes EGD-e. Multiple genes involved in heavy metal resistance (cadmium, copper, arsenite) as well as multidrug efflux (MDR, SMR, MATE) were detected on all listerial plasmids. These factors promote bacterial growth and survival in the environment and may have been acquired as a result of selective pressure due to the use of disinfectants in food processing environments. MDR efflux pumps have also recently been shown to promote transport of cyclic diadenosine monophosphate (c-di-AMP) as a secreted molecule able to trigger a cytosolic host immune response following infection.

Conclusions

The comparative analysis of 14 plasmids of genus Listeria implied the existence of a common ancestor. Ubiquitously-occurring MDR genes on plasmids and their role in listerial infection now deserve further attention.     

CRISPR/Cas-Mediated Targeted Mutagenesis in Daphnia magna

The water flea Daphnia magna has been used as an animal model in ecology, evolution, and environmental sciences. Thanks to the recent progress in Daphnia genomics, genetic information such as the draft genome sequence and expressed sequence tags (ESTs) is now available. To investigate the relationship between phenotypes and the available genetic information about Daphnia, some gene manipulation methods have been developed. However, a technique to induce targeted mutagenesis into Daphnia genome remains elusive. To overcome this problem, we focused on an emerging genome editing technique mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system to introduce genomic mutations. In this study, we targeted a functionally conserved regulator of eye development, the eyeless gene in D. magna. When we injected Cas9 mRNAs and eyeless-targeting guide RNAs into eggs, 18–47% of the survived juveniles exhibited abnormal eye morphology. After maturation, up to 8.2% of the adults produced progenies with deformed eyes, which carried mutations in the eyeless loci. These results showed that CRISPR/Cas system could introduce heritable mutations into the endogenous eyeless gene in D. magna. This is the first report of a targeted gene knockout technique in Daphnia and will be useful in uncovering Daphnia gene functions.     

Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis

Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.     

Das CRISPR/Cas‐System

The CRISPR/Cas system Up to this point plant breeding was based on the utilization of unspecific and time‐consuming procedures to accomplish improvements in the agronomic traits of our crop plants. This fundamentally changed the last years since the CRISPR/Cas system now provides a highly precise and reliable tool to achieve these goals in a fast and efficient way. The programmable induction of double‐strand breaks enables the targeted introduction of favored changes at almost any desired site within the genome that cannot be distinguished from naturally occurring variations any longer. This already enabled the generation of crop plants with agronomically interesting traits. The current characterization of additional CRISPR/Cas systems not only expands our molecular toolbox which allows us to regulate the cell metabolism on very different levels but also starts changing the entire biotechnology and biology fields fundamentally.     

A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System

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CRISPR Associated Diversity within a Population of Sulfolobus islandicus

Background

Predator-prey models for virus-host interactions predict that viruses will cause oscillations of microbial host densities due to an arms race between resistance and virulence. A new form of microbial resistance, CRISPRs (clustered regularly interspaced short palindromic repeats) are a rapidly evolving, sequence-specific immunity mechanism in which a short piece of invading viral DNA is inserted into the host''s chromosome, thereby rendering the host resistant to further infection. Few studies have linked this form of resistance to population dynamics in natural microbial populations.

Methodology/Principal Findings

We examined sequence diversity in 39 strains of the archeaon Sulfolobus islandicus from a single, isolated hot spring from Kamchatka, Russia to determine the effects of CRISPR immunity on microbial population dynamics. First, multiple housekeeping genetic markers identify a large clonal group of identical genotypes coexisting with a diverse set of rare genotypes. Second, the sequence-specific CRISPR spacer arrays split the large group of isolates into two very different groups and reveal extensive diversity and no evidence for dominance of a single clone within the population.

Conclusions/Significance

The evenness of resistance genotypes found within this population of S. islandicus is indicative of a lack of strain dominance, in contrast to the prediction for a resistant strain in a simple predator-prey interaction. Based on evidence for the independent acquisition of resistant sequences, we hypothesize that CRISPR mediated clonal interference between resistant strains promotes and maintains diversity in this natural population.     

The Temperature Dependent Proteomic Analysis of Thermotoga maritima

Thermotoga maritima (T. maritima) is a typical thermophile, and its proteome response to environmental temperature changes has yet to be explored. This study aims to uncover the temperature-dependent proteins of T. maritima using comparative proteomic approach. T. maritima was cultured under four temperatures, 60°C, 70°C, 80°C and 90°C, and the bacterial proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, a total of 224 spots, either cytoplasm or membrane, were defined as temperature-dependent. Of these spots, 75 unique bacterial proteins were identified using MALDI TOF/TOF MS. As is well known, the chaperone proteins such as heat shock protein 60 and elongation factor Tu, were up-regulated in abundance due to increased temperature. However, several temperature-dependent proteins of T. maritima responded very differently when compared to responses of the thermophile T. tengcongensis. Intriguingly, a number of proteins involved in central carbohydrate metabolism were significantly up-regulated at higher temperature. Their corresponding mRNA levels were elevated accordingly. The increase in abundance of several key enzymes indicates that a number of central carbohydrate metabolism pathways of T. maritima are activated at higher temperatures.     

Spectral Radiation Dependent Photoprotective Mechanism in the Diatom Pseudo-nitzschia multistriata

Phytoplankton, such as diatoms, experience great variations of photon flux density (PFD) and light spectrum along the marine water column. Diatoms have developed some rapidly-regulated photoprotective mechanisms, such as the xanthophyll cycle activation (XC) and the non-photochemical chlorophyll fluorescence quenching (NPQ), to protect themselves from photooxidative damages caused by excess PFD. In this study, we investigate the role of blue fluence rate in combination with red radiation in shaping photoacclimative and protective responses in the coastal diatom Pseudo-nitzschia multistriata. This diatom was acclimated to four spectral light conditions (blue, red, blue-red, blue-red-green), each of them provided with low and high PFD. Our results reveal that the increase in the XC pool size and the amplitude of NPQ is determined by the blue fluence rate experienced by cells, while cells require sensing red radiation to allow the development of these processes. Variations in the light spectrum and in the blue versus red radiation modulate either the photoprotective capacity, such as the activation of the diadinoxanthin-diatoxanthin xanthophyll cycle, the diadinoxanthin de-epoxidation rate and the capacity of non-photochemical quenching, or the pigment composition of this diatom. We propose that spectral composition of light has a key role on the ability of diatoms to finely balance light harvesting and photoprotective capacity.     

CRISPR/Cas9系统研究进展

CRISPR/Cas9系统的发展彻底改变了人们编辑DNA序列和调控目标基因表达水平的能力,从而为生物体的精确基因组编辑提供了有力的工具。简化后的CRISPR/Cas9系统由两部分组成:Cas9蛋白和sgRNA。其作用原理为sgRNA通过自身的Cas9把手与Cas9蛋白形成Cas9-sgRNA复合体,Cas9-sgRNA复合体中sgRNA的碱基互补配对区序列与目标基因的靶序列通过碱基互补配对原则进行配对结合,Cas9利用自身的核酸内切酶活性对目标DNA序列进行切割。与传统的基因组编辑技术相比,CRISPR/Cas9系统具有几大明显的优势:易用性、简便性、低成本、可编程性以及可同时编辑多个基因。CRISPR/Cas9基因组编辑技术以及衍生出来的CRISPRi和CRISPRa基因表达调控技术已经广泛应用于多种真核和原核生物中。综述了CRISPR/Cas9系统的起源、作用机理、在生物体中的应用和其衍生出的技术,并概述了其脱靶效应和未来前景。     

Modeling the Non-Stationary Climate Dependent Temporal Dynamics of Aedes aegypti

Background

Temperature and humidity strongly affect the physiology, longevity, fecundity and dispersal behavior of Aedes aegypti, vector of dengue fever. Contrastingly, the statistical associations measured between time series of mosquito abundance and meteorological variables are often weak and contradictory. Here, we investigated the significance of these relationships at different time scales.

Methods and Findings

A time series of the adult mosquito abundance from a medium-sized city in Brazil, lasting 109 weeks was analyzed. Meteorological variables included temperature, precipitation, wind velocity and humidity. As analytical tools, generalized linear models (GLM) with time lags and interaction terms were used to identify average effects while the wavelet analysis was complementarily used to identify transient associations. The fitted GLM showed that mosquito abundance is significantly affected by the interaction between lagged temperature and humidity, and also by the mosquito abundance a week earlier. Extreme meteorological variables were the best predictors, and the mosquito population tended to increase at values above and 54% humidity. The wavelet analysis identified non-stationary local effects of these meteorological variables on abundance throughout the study period, with peaks in the spring-summer period. The wavelet detected weak but significant effects for precipitation and wind velocity.

Conclusion

Our results support the presence of transient relationships between meteorological variables and mosquito abundance. Such transient association may be explained by the ability of Ae. aegypti to buffer part of its response to climate, for example, by choosing sites with proper microclimate. We also observed enough coupling between the abundance and meteorological variables to develop a model with good predictive power. Extreme values of meteorological variables with time lags, interaction terms and previous mosquito abundance are strong predictors and should be considered when understanding the climate effect on mosquito abundance and population growth.     

Heritable Genome Editing with CRISPR/Cas9 in the Silkworm,Bombyx mori

We report the establishment of an efficient and heritable gene mutagenesis method in the silkworm Bombyx mori using modified type II clustered regularly interspaced short palindromic repeats (CRISPR) with an associated protein (Cas9) system. Using four loci Bm-ok, BmKMO, BmTH, and Bmtan as candidates, we proved that genome alterations at specific sites could be induced by direct microinjection of specific guide RNA and Cas9-mRNA into silkworm embryos. Mutation frequencies of 16.7–35.0% were observed in the injected generation, and DNA fragments deletions were also noted. Bm-ok mosaic mutants were used to test for mutant heritability due to the easily determined translucent epidermal phenotype of Bm-ok-disrupted cells. Two crossing strategies were used. In the first, injected Bm-ok moths were crossed with wild-type moths, and a 28.6% frequency of germline mutation transmission was observed. In the second strategy, two Bm-ok mosaic mutant moths were crossed with each other, and 93.6% of the offsprings appeared mutations in both alleles of Bm-ok gene (compound heterozygous). In summary, the CRISPR/Cas9 system can act as a highly specific and heritable gene-editing tool in Bombyx mori.     

CRISPR/Cas‐based tools for the targeted control of plant viruses

Plant viruses are known to infect most economically important crops and pose a major threat to global food security. Currently, few resistant host phenotypes have been delineated, and while chemicals are used for crop protection against insect pests and bacterial or fungal diseases, these are inefficient against viral diseases. Genetic engineering emerged as a way of modifying the plant genome by introducing functional genes in plants to improve crop productivity under adverse environmental conditions. Recently, new breeding technologies, and in particular the exciting CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated proteins) technology, was shown to be a powerful alternative to engineer resistance against plant viruses, thus has great potential for reducing crop losses and improving plant productivity to directly contribute to food security. Indeed, it could circumvent the “Genetic modification” issues because it allows for genome editing without the integration of foreign DNA or RNA into the genome of the host plant, and it is simpler and more versatile than other new breeding technologies. In this review, we describe the predominant features of the major CRISPR/Cas systems and outline strategies for the delivery of CRISPR/Cas reagents to plant cells. We also provide an overview of recent advances that have engineered CRISPR/Cas‐based resistance against DNA and RNA viruses in plants through the targeted manipulation of either the viral genome or susceptibility factors of the host plant genome. Finally, we provide insight into the limitations and challenges that CRISPR/Cas technology currently faces and discuss a few alternative applications of the technology in virus research.     

Regulation of Flagellar Biogenesis by a Calcium Dependent Protein Kinase in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a bi-flagellated green alga, is a model organism for studies of flagella or cilia related activities including cilia-based signaling, flagellar motility and flagellar biogenesis. Calcium has been shown to be a key regulator of these cellular processes whereas the signaling pathways linking calcium to these cellular functions are less understood. Calcium-dependent protein kinases (CDPKs), which are present in plants but not in animals, are also present in ciliated microorganisms which led us to examine their possible functions and mechanisms in flagellar related activities. By in silico analysis of Chlamydomonas genome we have identified 14 CDPKs and studied one of the flagellar localized CDPKs – CrCDPK3. CrCDPK3 was a protein of 485 amino acids and predicted to have a protein kinase domain at the N-terminus and four EF-hand motifs at the C-terminus. In flagella, CrCDPK3 was exclusively localized in the membrane matrix fraction and formed an unknown 20 S protein complex. Knockdown of CrCDPK3 expression by using artificial microRNA did not affect flagellar motility as well as flagellar adhesion and mating. Though flagellar shortening induced by treatment with sucrose or sodium pyrophosphate was not affected in RNAi strains, CrCDPK3 increased in the flagella, and pre-formed protein complex was disrupted. During flagellar regeneration, CrCDPK3 also increased in the flagella. When extracellular calcium was lowered to certain range by the addition of EGTA after deflagellation, flagellar regeneration was severely affected in RNAi cells compared with wild type cells. In addition, during flagellar elongation induced by LiCl, RNAi cells exhibited early onset of bulbed flagella. This work expands new functions of CDPKs in flagellar activities by showing involvement of CrCDPK3 in flagellar biogenesis in Chlamydomonas.     

Ex Vivo Neurogenesis within Enteric Ganglia Occurs in a PTEN Dependent Manner

A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While these cells may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. An extensive study by Joseph et al. involving 108 rats and 51 mice submitted to various insults demonstrated neuronal uptake of thymidine analog BrdU in only 1 rat. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo organotypic tissue culturing system. Culturing longitudinal muscle and myenteric plexus tissue, we show that the enteric nervous system has tremendous replicative capacity with the majority of neural crest cells demonstrating EdU uptake by 48 hours. EdU⁺ cells express both neuronal and glial markers. Proliferation appears dependent on the PTEN/PI3K/Akt pathway with decreased PTEN mRNA expression and increased PTEN phosphorylation (inactivation) corresponding to increased Akt activity and proliferation. Inhibition of PTEN with bpV(phen) augments proliferation while {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, blocks it. These data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.     

Size Dependent Male Reproductive Tactic in the Two-Spotted Goby (Gobiusculus flavescens)

Male investment in testes and sperm duct gland in the polygamous nest breeding two-spotted goby Gobiusculus flavescens (Fabricius) was investigated in relation to time in reproductive season and individual physical parameters. This small teleost fish is most likely the most abundant species found along the rocky shores of the North East Atlantic. The two-spotted goby has a single reproductive season, during which nest-caring males can raise several clutches of offspring. According to the literature the males are on average larger than the females. Here we report for the first time a population showing a reversal of this trend, with males on average being smaller than females, a difference likely caused by a large proportion of small males. Early in the breeding season these small males have typical sneaker characters, with relatively large testes and small seminal duct glands compared to the larger dominant territorial males. The presence of these two alternative male reproductive tactics is confirmed by histological studies, which shows the presence of sperm in the sperm duct glands (SDG) of smaller males, but not in the SDG of intermediate and larger males. To our knowledge, males with typical sneaker characters have not been reported in earlier studied populations of two-spotted goby. Interestingly we found that testes investment declined significantly over the course of the breeding season, and that this reduction was significantly more pronounced in small compared to the large males. Further, a significant increase in seminal duct gland (SDG) mass was observed for the smaller males over the breeding season. We propose that this indicates a possible shift in mating tactic by smaller males from a parasitic to a nest-holding tactic over the course of the breeding season. Thus, the observed size dependent plasticity in investment in SDG over time suggests that the reproductive tactic of G. flavescens is conditional, and possibly influenced by mate availability and male—male competition.