微生物学

A new direct contact assessment of soil toxicity using sulfur oxidizing bacteria (SOB) is proposed for analyzing the toxicity of soils. The proposed method is based on the ability of SOB to oxidize elemental sulfur to sulfuric acid in the presence of oxygen. Since sulfate ions are produced from sulfur by SOB oxidation activity, changes in electrical conductivity (EC) serve as a proxy to assess toxicity in water. However, in soil medium, EC values are not reliable due to the adsorption of SO₄^2- ions by soils. Here, we suggest a new parameter which measures oxygen consumption by SOB for 6 hours to assess soil toxicity by using a lubricated glass syringe method. The proposed method is rapid, simple, cost- effective as well as sensitive and capable of assessing direct contact soil toxicity.

Chlamydomonas reinhardtii is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann et al., 1999; Schroda et al., 2000; Schroda, 2006). Among them, forward genetic approach has been intensively used, mostly because of the high efficiency in the generation of hundreds of thousands of mutants by random insertional mutagenesis and the haploid nature therefore phenotypic analysis can be done in the first generation (Cagnon et al., 2013; Tunçay et al., 2013). A major bottleneck in the application of high throughput methods in a forward genetic approach is the identification of the genetic lesion(s) responsible for the observed phenotype. In this protocol, we describe in detail an improved version of the restriction enzyme site-directed amplification PCR (RESDA-PCR) originally reported in (González-Ballester et al., 2005). The improvement includes optimization of primer combination, the choice of DNA polymerase, optimization of PCR cycle parameters, and application of direct sequencing of the PCR products. These modifications make it easier to get specific PCR products as well as speeding up subcloning steps to obtain sequencing data faster.

The advent of single cell genomics and the continued use of metagenomic profiling in diverse environments has exponentially increased the known diversity of life. The recovered and assembled genomes predict physiology, consortium interactions and gene function, but experimental validation of metabolisms and molecular pathways requires more directed approaches. Gene function–and the correlation between phenotype and genotype is most obviously studied with genetics, and it is therefore critical to develop techniques permitting rapid and facile strain construction. Many new and candidate archaeal lineages have recently been discovered, but experimental, genetic access to archaeal genomes is currently limited to a few model organisms. The results obtained from manipulating the genomes of these genetically-accessible organisms have already had profound effects on our understanding of archaeal physiology and information processing systems, and these continued studies also help resolve phylogenetic reconstruction of the tree of life. The hyperthermophilic, planktonic, marine heterotrophic archaeon Thermococcus kodakarensis, has emerged as an ideal genetic system with a suite of techniques available to add or delete encoded activities, or modify expression of genes in vivo. We outline here techniques to rapidly and markerlessly delete a single, or repetitively delete several, continuous sequences from the T. kodakarensis genome. Our procedure includes details on the construction of the plasmid DNA necessary for transformation that directs, via homologous recombination, integration into the genome, identification of strains that have incorporated plasmid sequences (termed intermediate strains), and confirmation of plasmid excision, leading to deletion of the target gene in final strains. Near identical procedures can be employed to modify, rather than delete, a genomic locus.

We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by arabinose inducer; (3) tCRISPRi is plasmid free and the entire system is integrated into the chromosome; (4) tCRISPRi strains show desirable physiological properties.

The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4⁺ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4⁺ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains.

Here, we present a protocol for single-step marker switching by lithium acetate transformation in fission yeast, Schizosaccharomyces pombe. In the following we describe how to swap the ura4⁺ marker to a kanMX6, natMX4, or hphMX4 marker, which provide resistance against the antibiotics G418, nourseothricin (clonNAT) or hygromycin B, respectively. We also detail how to exchange any of the MX markers for nutritional markers, such as arg3⁺, his3⁺, leu1⁺ and ura4⁺.

Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives.

The corn smut pathogen, Ustilago maydis (U. maydis) (DC.) Corda, is a semi-obligate plant pathogenic fungus in the phylum Basidiomycota (Alexopoulos et al., 1996). The fungus can be easily cultured in its haploid yeast phase on common laboratory media. However, to complete its sexual cycle U. maydis strictly requires its specific plant host, maize (Zea mays). The fungus is an interesting and important model organism for the study of the interactions of fungal biotrophic pathogens with plants. In this protocol, we describe the process of plant inoculation, teliospore recovery, germination, progeny isolation and initial mating type analysis. The primary purpose of this protocol is to identify individual progeny strains of U. maydis that can be used for downstream genetic analyses. Generation of targeted mutants to study various processes is a common approach with this and many plant pathogenic fungi. The ability to generate combinations of mutations is facilitated by sexual crossing without the need for additional selectable markers.