Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining.

Bacteriophages are the most abundant entities on Earth. In contrast with the number of phages considered to be in existence, current phage isolation and screening methods lack throughput. Droplet microfluidic technology has been established as a platform for high-throughput screening of biological and biochemical components. In this study, we developed a proof-of-concept method for isolating phages using water-in-oil droplets (droplets) as individual chambers for phage propagation and co-cultivating T2 phage and their host cell Escherichia coli within droplets. Liquid cultivation of microbes will facilitate the use of microbes that cannot grow on or degrade agar as host cells, ultimately resulting in the acquisition of phages that infect less known bacterial cells. The compartmentalizing characteristic of droplets and the use of a fluorescent dye to stain phages simultaneously enabled the enumeration and isolation of viable phage particles. We successfully recultivated the phages after simultaneously segregating single phage particles into droplets and inoculating them with their host cells within droplets. By recovering individual droplets into 96-well plates, we were able to isolate phage clones derived from single phage particles. The success rate for phage recovery was 35.7%. This study lays the building foundations for techniques yet to be developed that will involve the isolation and rupturing of droplets and provides a robust method for phage enumeration and isolation.

Transcriptome and Deletion Mutant Analyses Revealed that an RpoH Family Sigma Factor Is Essential for Photosystem Production in Roseateles depolymerans under Carbon Starvation.

Roseateles depolymerans is an obligately aerobic bacterium that produces a photosynthetic apparatus only under the scarcity of carbon substrates. We herein examined changes in the transcriptomes of R. depolymerans cells to clarify the expression of photosynthesis genes and their upstream regulatory factors under carbon starvation. Transcriptomes 0, 1, and 6| |h after the depletion of a carbon substrate indicated that transcripts showing the greatest variations (a 500-fold increase [6 h/0 h]) were light-harvesting proteins (PufA and PufB). Moreover, loci with more than 50-fold increases (6 h/0| |h) were fully related to the photosynthetic gene cluster. Among 13 sigma factor genes, the transcripts of a sigma 70 family sigma factor related to RpoH (SP70) increased along photosynthesis genes under starvation; therefore, a knockout experiment of SP70 was performed. ΔSP70 mutants were found to lack photosynthetic pigments (carotenoids and bacteriochlo-rophyll a) regardless of carbon starvation. We also examined the effects of heat stress on ΔSP70 mutants, and found that SP70 was also related to heat stress tolerance, similar to other RpoH sigma factors (while heat stress did not trigger photosystem production). The deficient accumulation of photosynthetic pigments and the heat stress tolerance of ΔSP70 mutants were both complemented by the introduction of an intact SP70 gene. Furthermore, the transcription of photosynthetic gene operons (puf, puh, and bch) was markedly reduced in the ΔSP70 mutant. The RpoH homologue SP70 was concluded to be a sigma factor that is essential for the transcription of photosynthetic gene operons in R. depolymerans.

Development of certified reference material NMIJ CRM 6205-a for the validation of DNA quantification methods: accurate mass concentrations of 600-bp DNA solutions having artificial sequences.

Two 600-bp DNA solutions (DNA600-G and DNA600-T) were developed as certified reference material, NMIJ CRM 6205-a, for the validation of DNA quantification methods. Both DNA600-G and DNA600-T are ideal as "spike-in control" because these materials have artificial nucleic acid sequences. The certified values were determined as the mass concentration of total DNA (whole DNA materials in sample solution regardless of sequence) at 25 °C by formic acid hydrolysis/liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) and inductively coupled plasma-mass spectrometry (ICP-MS) based on the amount of phosphorus. DNAs were synthesized, and plasmids including the synthesized DNAs were cloned into Escherichia coli DH5α. The amplified plasmids were digested with a restriction enzyme and highly purified. Then, the purified DNAs were diluted with water to approximately 1 ng/µL. By using the CRM-validated methods in fields where DNA quantification is required, the reliability of DNA quantification could be improved. Graphical abstract.

Characterization of a Deinococcus radiodurans MazF: A UACA-specific RNA endoribonuclease.

Microbes are known to withstand environmental stresses by using chromosomal toxin-antitoxin systems. MazEF is one of the most extensively studied toxin-antitoxin systems. In stressful environments, MazF toxins modulate translation by cleaving single-stranded RNAs in a sequence-specific fashion. Previously, a chromosomal gene located at DR0417 in Deinococcus radiodurans was predicted to code for a MazF endoribonuclease (MazFDR0417 ); however, its function remains unclear. In the present study, we characterized the molecular function of MazFDR0417 . Analysis of MazFDR0417 -cleaved RNA sites using modified massively parallel sequencing revealed a unique 4-nt motif, UACA, as a potential cleavage pattern. The activity of MazFDR0417 was also assessed in a real-time fluorometric assay, which revealed that MazFDR0417 strictly recognizes the unique tetrad UACA. This sequence specificity may allow D. radiodurans to alter its translation profile and survive under stressful conditions.

Decomposition of waste DNA with extended autoclaving under unsaturated steam.

Carryover and false-positive amplification of undesired DNA sequences are serious problems in research and diagnostic testing using PCR. One possible source of DNA cross-contamination can be the autoclave if DNA contained in waste is not effectively decomposed and contaminates the autoclave. To assess this possibility, we used a 2682 bp PCR product as a model waste DNA and quantified the amplifiability of an 84 bp short fragment derived from the model waste DNA in the steam and the residual bottom water after autoclaving. Autoclaving under the standard conditions of 121°C for 20 min did not sufficiently remove amplifiability from the model DNA and was found to be a possible source of laboratory contamination. However, the amplifiable template was removed after autoclaving at 121°C for 80 min. Fragmentation and hydrolysis may occur during autoclaving, and the presence of atmospheric oxygen facilitated the decomposition. These findings will help researchers develop better strategies for disposing of DNA waste.

A proportional analysis method using non-kinetic real-time PCR.

Prompted by increasing interest in proportional analysis of genetic types, we developed a simple assay technique for determining the ratio of a specific target gene in the total genes that can be amplified with the same PCR primer. The key feature of this method is that the following two tasks are performed in a single-tube real-time PCR system: task 1, PCR amplification of the total genes including the target using a labeled PCR primer, with concurrent monitoring of the total copy number of the PCR product; task 2, detection of the signal of the target gene at each cycle of amplification, using a labeled nucleotide probe. In principle, the ratio of the target gene to the total genes is represented by the signal detected in 'task 2' at the cycle in which the PCR product reached a prescribed copy number (assessed by 'task 1').

Photosynthetic apparatus in Roseateles depolymerans 61A is transcriptionally induced by carbon limitation.

Production of a photosynthetic apparatus in Roseateles depolymerans 61A, a recently discovered freshwater beta-Proteobacterium showing characteristics of aerobic phototrophic bacteria, was observed when the cells were subjected to a sudden decrease in carbon sources (e.g., when cells grown with 0.1 to 0.4% Casamino Acids were diluted or transferred into medium containing or=0.2% O(2)), and was reduced in the presence of light. Transcription of the R. depolymerans puf operon is considered to be controlled by changes in carbon nutrients in addition to oxygen tension and light intensity.