Siegfried Klaus

Inactivation kinetics of lambda phage repressors in a mutant of E. coli temperature sensitive in DNA replication.

SummaryThe inactivation kinetics of two different Lambda phage repressors in lysogenic complexes of an E. coli strain temperature sensitive in DNA synthesis is investigated. From the experimental data obtained it is inferred that1.after a stop of DNA synthesis effector molecules are accumulated which give rise to the inactivation of phage repressors directly,2.the accumulation kinetics of the effector molecules after a stop of DNA synthesis is similar to that kinetics which must be expected from accumulation of DNA precursors.3.The inactivation of λ+ and λind8repressor molecules begins 20 min and 5 min, respectively, after the stop of host DNA synthesis.4.The effector concentration is rapidly diminished after restoration of DNA synthesis,5.the accumulation of effector molecules proceeds without protein synthesis in this strain after temperature shift up.6.The effector substance seems not to be a protein.

Molecular characterization of the genomes of actinophages SH3, SH10, SH11, and SH12 infecting Streptomyces hygroscopicus

SummarySome physico-chemical properties of the DNAs released from the actinophages SH3, SH10, SH11, and SH12 are described. The four phage DNAs have a linear double-stranded secondary structure and are unique with respect to their high G·C contents which, from melting studies and buoyant density experiments, were found to be in the range of 68–73 mol-%. The DNA molecular weights were determined by sedimentation velocity experiments and by electron microscopic length measurements, the mean values of the two corresponding data sets being 34.0·106 (SH3), 26.7·106 (SH10), 26.1·106 (SH11), and 28.7·106 (SH12) with a mean relative error of ±5%. From different observations it was concluded that SH10 DNA, and possibly also SH11 and SH12 DNA, have cohesive ends and can undergo intramolecular or intermolecular association to form ring-like monomers or linear and ring-like multimers. Cleavage of the DNAs of SH3, SH10, SH11, and SH12 by EcoRI restriction endonuclease delivered two, one, zero, and two cleavage sites, respectively, and by BamHI restriction endonuclease eight, zero, zero, and zero cleavage sites, respectively.

Restriction endonuclease analysis of DNA from the Streptomyces phages SH3, SH5, SH10 and SH13.

Using 26 restriction endonucleases, a cleavage site survey was undertaken for DNAs of several unrelated Streptomyces phages SH3, SH5, SH10 and SH13. Only EcoRI was found to produce single cleavage in SH3 and SH10 DNA. The complete maps were prepared for the 2, 9 and 11 fragments of SH10 DNA, as generated by EcoRI, KpnI and BglII, respectively. The evidence is presented that SH10 DNA contains cohesive ends. Moreover, a clear-plaque mutant of SH10 was shown to contain a deletion of 790 bp in the right part of the genome, including two KpnI sites.

DNA of the Streptomyces phage SH10: binding sites for Streptomyces hygroscopicus RNA polymerase and in vitro transcription map

Abstract The binding sites for the Streptomyces hygroscopicus RNA polymerase on the DNA of actinophage SH10 were mapped by electron microscopy. Four major binding sites were located at map positions 52.2, 84.5, 97.9 and 99.1 very close or identical to sequences to which the Escherichia coli RNA polymerase was observed to bind. In vitro transcription activities of the two RNA polymerases were compared by analyzing transcriptional R-loops on SH10 DNA. Both enzymes transcribed almost exclusively segments in the right half of the phage genome. Most frequent initiation was found from a site at map position 98.1. Differences in efficiency of transcription between the two enzymes are discussed with respect to heterospecific gene expression.

Kinetics of membrane association by bacteriophage λ DNA after repressor inactivation

SummaryComplete and stable membrane association of λ DNA requires 3 minutes of heating in the ren− lysogen E. coli C600 (λ CItsU37) and 5 minutes in the ren+ lysogen E. coli C600 (λ c857). Transcription is necessary for the establishment of λ DNA-membrane association but not for its maintenance.

DNA of the Streptomyces phage SH10: binding sites for Escherichia coli RNA polymerase and denaturation map.

SummaryEscherichia coli RNA polymerase bound to Streptomyces phage SH10 DNA was visualized by electron microscopy. Six specific binding sites were observed at map units 53, 85, 93, 97, 98, and 99 on the physical map of the 48 kb long genome. Electron microscopy of partially denatured SH10 DNA revealed a characteristic melting pattern of A+T-rich regions around map units 1, 3, 48, 52, and 99. A comparison of the denaturation map with the RNA polymerase binding sites indicates that three binding sites are located in the most A+T-rich regions, two in other early melting regions and one in a segment of higher DNA helix stability.

Inverted duplication in the genome of the temperate Streptomyces phage SH3

SummaryThe DNA of the temperate Streptomyces phage SH3 contains 100 base-pair long inverted repeats separated by a 940 base-pair long segment of DNA as revealed by electromicroscopic analysis of snapback structures formed after rapid intrastrand reannealing of denatured DNA. The inverted repeat structure was found preferentially at map unit 22 of the circular physical map, in rare cases also in other positions, suggesting a movable character of this genetic element.

Membrane attachment of λDNA in mutants of Escherichia coli temperature sensitive in DNA replication

SummaryMembrane association of λ DNA takes place after thermal inactivation of phage repressor molecules in DNAts mutants of E. coli unable to replicate λ at the restrictive temperature.

Inverted repeats in the DNA of Streptomyces plasmids pMG110 and pMG120

SummaryTwo cryptic plasmids pMG110 (10.5 kb) and pMG120 (14.5 kb) isolated from Streptomyces luteolutescens were cleaved by restriction endonucleases BglII, KpnI, and SalGI. A physical map was constructed for pMG110. After denaturation and intrastrand reannealing, two types of snap-back structures were identified by electron microscopy, differing in the size of the loop (type 1, 1 kb; type 2, 1.6 kb), whereas the stem of both structures was about 190 bp long. Stem-loop structures of similar size were also observed in pMG120. In rare cases, both types of elements were present on the same DNA molecule. The analysis of BglII- and KpnI-generated fragments allowed the localization of the elements at two alternative positions on the physical map of pMG110.

Transduction in Streptomyces hygroscopicus mediated by the temperate bacteriophage SH10

SummaryThe temperate actinophage SH10 mediates generalized transduction in Streptomyces hygroscopicus at low frequency. The efficiency of transduction depends on the average phage input, age of outgrowing spores of the recipient and on the selective marker. The highest EOT was found for the auxotrophic mutants 21 (phe-) and 5(try-)(4.1x10-6 and 2.7x10-6, respectively). Transduction of the thermosensitive mutant NG14-216 ts 35 was two orders of magnitude lower (2.5x10-8). The transductant colonies segregated into stable and unstable clones. Stable transductants were never found to be lysogenic for phage SH10.