Reto Crameri

Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus

A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide‐resistant plants.

Simple procedure for distinguishing CCC, OC, and L forms of plasmid DNA by agarose gel electrophoresis.

Abstract A simple and rapid method discriminating between covalently closed circular (CCC), open circular (OC), and linear (L) forms of plasmid DNA is presented. Two consecutive steps of agarose gel electrophoresis with a single DNA sample are used; use is made between the steps of ultraviolet light irradiation which introduces single-strand nicks in ethidium bromide-stained DNA, converting CCC into OC forms. Unambiguous assignments of OC and CCC forms are possible in samples containing several plasmids.

Restriction analysis of the Streptomyces glaucescens genome by agarose gel electrophoresis

A method for the analysis of total DNA of Streptomyces glaucescens is described. The relevant steps are (a) extraction and purification of DNA, (b) restriction of DNA samples with type II restriction enzymes, (c) one dimensional separation of restriction fragments by agarose gel electrophoresis. A typical banding pattern was obtained for each wild type strain, independant of growth conditions or age of the culture. Mutant strains exhibited in most cases the same banding pattern as the parent wild type strain. Only in one specific mutant class a fragment of about 9 megadalton was missing.

Streptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase

SummaryThe wild type strain of Streptomyces glaucescens produces hydroxystreptomycin and has a natural resistance towards the streptomycin group aminoglycoside antibiotics. The inherent resistance is a genetically unstable character and mutant strains sensitive to streptomycins arise spontaneously at unusually high frequencies. The gene conferring streptomycin resistance was cloned and characterised as a streptomycin specific phosphotransferase. Hybridisation experiments show that the mutational event leading to sensitivity is due to large deletions, most likely on the chromosome, comprehending the structural gene coding for a streptomycin phosphotransferase and its flanking regions. Interspecific expression of the S. glaucescens phosphotransferase was found in Streptomyces lividans as well as in Escherichia coli.

Plasmid Curing and Generation of Mutations Induced with Ethidium Bromide in Streptomycetes

The DNA-intercalating agent ethidium bromide was used to eliminate plasmid DNA from streptomycetes. Other mutational events associated with chromosome changes occurred at high frequency; they resulted in phenotypic changes such as loss of enzyme activity, antibiotic resistance or ability to sporulate.

Chromosomal instability in Streptomyces glaucescens: mapping of streptomycin-sensitive mutants.

Streptomyces glaucescens strain GLAO (=ETH 22794) produces hydroxystreptomycin and has a high natural resistance to hydroxystreptomycin, dihydrostreptomycin and streptomycin. The wild-type strain gives rise spontaneously to streptomycin-sensitive (StrS-) variants at a frequency of 0 . 2 to 1 . 4%. These mutants lack streptomycin phosphotransferase activity responsible for the wild-type resistance to streptomycin group antibiotics and are unable to produce detectable amounts of hydroxystreptomycin. Mapping experiments showed that the strS marker lies between the chromosomal markers lys-2 and ura-3 on the linkage map of S. glaucescens. The molecular basis for instability of this marker is as yet unknown.

Deoxyribonucleic Acid Restriction Endonuclease Fingerprint Characterization of Actinomycete Strains

Restriction endonuclease digestion of total purified genomic deoxyribonucleic acid gives rise to deoxyribonucleic acid fragments of discrete sizes. These can be separated by one-dimensional agarose gel electrophoresis. A unique banding pattern (fingerprint) is obtained for each actinomycete wild-type strain tested. These fingerprints are useful for recognizing a given wild-type strain and its derivatives, but cannot be used for species characterization.

Hydroxystreptomycin production and resistance in Streptomyces glaucescens.

The wild-type strain of Streptomyces glaucescens produces hydroxystreptomycin and shows an inherent natural resistance to streptomycin group aminoglycoside antibiotics. Cell free extracts of the wild-type strain were able to inactivate streptomycin, hydroxystreptomycin and dihydrostreptomycin in the presence of ATP. The phosphotransferase did not inactivate other aminoglycoside antibiotics, including spectinomycin. Mutant strains were isolated, which were highly sensitive to streptomycin group aminoglycosides, had no measurable phosphotransferase activity and were unable to form detectable amounts of hydroxystreptomycin. This suggests either a correlation between phosphotransferase activity, streptomycin resistance and hydroxystreptomycin formation or defects in more than one gene in the strS mutant strains tested.

A technique for the detection of large DNA alterations in complex genomes

Abstract As part of our effort to understand the chromosomal organization of streptomycetes, we have developed a method for detecting large alterations in the DNA, in particular to visualize insertions, transpositions, and deletions. The method involves the labeling of the DNA of two strains with [3H]thymidine or [14C]thymidine, extraction and purification of the DNA, digestion with restriction endonucleases, and one-dimensional agarose gel electrophoresis of the two samples in the same slot. Following electrophoresis, the gel is cut into thin slices, and the 14 C 3 H ratio is measured in each slice. Deviations from the average standard ratio are caused by differences in the restriction site arrangement in the DNA of the two strains, which may be caused by rearrangements in the DNA. The method has a high resolution of one restriction fragment change.