Development of a conditional lethal system for a Streptomyces lividans strain and its use to investigate conjugative transfer in soil

Clerc-Bardin, Sylvie; Karray, Fatma; Frostegård, Åsa; Pernodet, Jean‐Luc; Simonet, Pascal

doi:10.1111/j.1574-6941.2001.tb00889.x

Cited by 2 publications

(3 citation statements)

References 31 publications

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“…These conditions allow vegetative cells to develop without spore formation (30). Spores of S. lividans OS48.3 (4) were mechanically removed from cultures of the organism on R2YE medium (15), whereupon the spore suspensions were filtered (15). Hyphae from S. lividans OS48.3 were derived from newly pregerminated spores, since the use of only short hyphae was expected to minimize rupture and subsequent DNA leakage.…”

Section: Soilsmentioning

confidence: 99%

“…For soils seeded with S. lividans, PCRs were performed with primers FGPS516 and FGPS517, and the products were hybridized with the oligonucleotide probe FGPS518 ( Table 2). The amplified region was part of a cassette specifically constructed to obtain the strain OS48.3 (4). Calibration curves were in all cases obtained by using purified DNA from the target organism.…”

Section: Soilsmentioning

confidence: 99%

“…These sequences were not situated within the 16S rRNA gene. The cassette containing the targeted region of S. lividans is described by Clerc-Bardin et al(4).…”

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confidence: 99%

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Quantification of Bias Related to the Extraction of DNA Directly from Soils

Frostegård

Courtois

Ramisse

et al. 1999

Appl Environ Microbiol

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432

165

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In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage λ DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.

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Section: Soilsmentioning

confidence: 99%

Section: Soilsmentioning

confidence: 99%

See 1 more Smart Citation

Quantification of Bias Related to the Extraction of DNA Directly from Soils

Frostegård

Courtois

Ramisse

et al. 1999

Appl Environ Microbiol

Self Cite

432

165

View full text Add to dashboard Cite

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Fine-tuning the regulation of Cas9 expression levels for efficient CRISPR-Cas9 mediated recombination in Streptomyces

Enghiad

Zhao

et al. 2020

Journal of Industrial Microbiology and Biotechnology

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CRISPR-Cas9 has proven as a very powerful gene editing tool for Actinomyces, allowing scarless and precise genome editing in selected strains of these biotechnologically relevant microorganisms. However, its general application in actinomycetes has been limited due to its inefficacy when applying the system in an untested strain. Here, we provide evidence of how Cas9 levels are toxic for the model actinomycetes Streptomyces coelicolor M145 and Streptomyces lividans TK24, which show delayed or absence of growth. We overcame this toxicity by lowering Cas9 levels and have generated a set of plasmids in which Cas9 expression is either controlled by theophylline-inducible or constitutive promoters. We validated the targeting of these CRISPR-Cas9 system using the glycerol uptake operon and the actinorhodin biosynthesis gene cluster. Our results highlight the importance of adjusting Cas9 expression levels specifically in strains to gain optimum and efficient gene editing in Actinomyces.

show abstract

Development of a conditional lethal system for a Streptomyces lividans strain and its use to investigate conjugative transfer in soil

Cited by 2 publications

References 31 publications

Quantification of Bias Related to the Extraction of DNA Directly from Soils

Quantification of Bias Related to the Extraction of DNA Directly from Soils

Fine-tuning the regulation of Cas9 expression levels for efficient CRISPR-Cas9 mediated recombination in Streptomyces

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