Daunorubicin forms a specific complex with a secreted serine protease of Streptomyces peucetius

Daunorubicin forms specific complex with an extracellular protease in the Streptomyces peucetius culture. The drug-protein complex co-migrates in non-denaturing PAGE as a red band. De novo peptide sequencing by nano-LC–ESI–MS/MS and MASCOT analysis identified the daunorubicin binding protein as serine protease precursor. The same protease precursor was purified sans the daunorubicin, from the mutant named ΔDPSAmut, which is deficient in daunorubicin production. Daunorubicin was added to ΔDPSAmut culture and the protease readily formed the daunorubicin-protease complex. Ability of serine protease precursor to form a selective complex with daunorubicin was confirmed by this study. Selective binding of protease to daunorubicin was seen as self-resistance determinant for the organism to survive toxic levels of the drug outside the cell. Daunorubicin-protease complex placed on S. peucetius lawn did not produce clearing zone around it, whereas daunorubicin purified from the complex did produce the clearing zone. Thereby it is concluded that the protease sequesters daunorubicin to prevent its entry into cells. Sequestration of daunorubicin by extracellular protease helps the organism to maintain a steady state sub-inhibitory level of drug around the cells. A new self-resistance determinant is reported here.     

Genetic location ofrra mutation and its role in alleviation of rgl restriction

Restriction of glucosyl-free HMC-DNA mediated by RglB is alleviated inrecBC sbcA strains ofEscherichia coli K12. Mutation in the unlinkedrra gene reverses thisrecBC sbcA-mediated alleviation. The map position ofrra is 90.16 min on the standard map, and therra ⁺ gene product counteracts Rgl restriction. The activation of therra gene is controlled by thesbcA gene, and this regulation does not seem to require the involvement of other gene functions.     

Toxicity of certain insecticides toSturmiopsis inferens,a larval parasite of sugarcane moth borers

Susceptibility of adults and puparia ofSturmiopsis inferens Tns. [Tachinidae, Diptera] to 9 commonly recommended insecticide sprays against sugarcane pests was determined. The chemicals tested as emulsifiable concentrates include lindane 0.1%, endosulfan 0.1%, monocrotophos 0.05%, quinalphos 0.05%, malathion 0.1%, dimethoate 0.1%, cypermethrin 0.01%, fenvalerate 0.01% and decamethrin 0.0014%. Lindane, malathion, dimethoate monocrotophos and quinalphos were highly toxic, while decamethrin had little harmful effect to the adults when exposed for 6 h to filter paper impregnated or sugarcane shoot bits sprayed with the chemicals. However, the insecticides had no harmful effect on the puparia and adult emergence was normal from the puparia sprayed with insecticides. In another study, susceptibility of adults to soil application of lindane EC, carbofuran G, chlorpyriphos G, Sevidol G and whorl application of lindane G, chlorpyriphos G and Sevidol G was tested in pot culture. Except for soil application of lindane EC, all other chemicals had no harmful effect to the adults in pot culture experiment. In a field trial, commonly recommended insecticides against shoot borer,Chilo infuscatellus Snell.viz., soil application of granules of lindane, carbofuran, chlorpyriphos and Sevidol and folia spray of endosulfan did not affect the parasite activity. Institute Publication No 1030.     

Molecular cloning and sequencing of mcrA locus and identification of McrA protein inEscherichia coli

The Mcr systems (previously known as Rgl systems) ofEscherichia coli recognize and cleave specific sequences carrying methylated or hydroxymethylated cytosines. We have cloned the mcrA gene and determined its nucleotide sequence. An 831 base pair sequence encodes the McrA protein. Analysis of the sequence data reveals that there arc additional ORFs internal to the above. A phage T7 expression system was used to determine the protein products encoded by the cloned mcrA gene. The results clearly show that a 31 kDa polypeptide is responsible for McrA activity. This is in agreement with the molecular weight deduced from sequence data. McrA protein was found to be localized in the outer membrane ofEscherichia coli. To our knowledge this is the first restriction enzyme localized in the outer membraneof Escherichia coli. Presented in part at the Second New England Biolabs Workshop on Biological DNA Modification, September, 1990, Berlin     

Deamplification and deletion of amplified DNA in Streptomyces lividans and S. fradiae

Summary Streptomyces lividans arginine auxotrophs which show amplification of a 5.7-kb DNA sequence, arose at a very high frequency, varying from 10% to 25% of Cml^s spores. The amplifiable DNA sequence was shown to be stable over many generations. However, treatment of Cml^s arg mutants with subinhibitory concentrations of antibiotics such as spectinomycin, streptomycin, chloramphenicol, thiostrepton and kanamycin, either during sporulation or during vegetative growth of mycelia, led to the deletion of the entire amplified DNA sequence, including the left and right junction sequences. Depending upon the method of antibiotic treatment a reduction in the copy number of the amplified DNA was also observed. This reduction in copy number apparently occurred without drastically affecting the basic structure of the amplifiable unit of DNA. This phenomenon appears to be universal since deamplification and deletion were observed also in S. fradiae. Further, spontaneous arg mutants arose at much lower frequency from spectinomycin-pretreated Cml^s cells compared to untreated cells. These arg mutants isolated in the presence of spectinomycin did not show amplification of the 5.7-kb sequence. Southern blot analysis using the 5.7-kb probe showed that the entire DNA sequence homologous to the amplifiable DNA sequence had been deleted. Offprint requests to: K. Dharmalingam