Evaluation of a Commercial Air Filter for Removal of Virus from the Air

The effectiveness of a commercial absolute air filter for removal of viruses from air was tested with an actinophage, under the usual conditions of a laminar airflow clean room. A new method of dry phage dispersion is described. The filter showed an average reduction of 99.996% of airborne actinophage.     

Restriction enzyme mapping of the DNA of Streptomyces bacteriophage B alpha and its deletion derivatives

Cleavage analysis of actinophage B alpha DNA was done with several restriction enzymes, and a restriction map of the DNA was determined. The DNA appeared to carry cohesive ends. Deletion mutants of actinophage B alpha were isolated by five cycles of treatment with 15 mM PPi. Both mutants had deletions of 2.5 of 1.8 megadaltons near one end of the genome, and one of them lost the single EcoRI cleavage site.     

Delimitation of cohesive ends site (cos) of Streptomyces temperate bacteriophage R4.

The cohesive ends site (cos) of actinophage R4 was delimitated by assaying the in vivo packaging activity of cosmid derivatives in Streptomyces lividans. A region of 66 bp from -30 to +36 from the center of cohesive ends was required for the basal level of packaging activity. Two additional regions outside the basal sequences from -39 to -31 and from +37 to +97 were necessary for the high level of activity, defined as the accessory sequences. Direct- or inverted-repeat sequences were found within the delimitated region, which might be involved in the recognition by the terminase of actinophage R4.     

Isolation of actinophage that attack some maduromycete actinomycetes

Abstract During the course of natural product screening, two actinomycete strains, designated 3828E and 3913E, were isolated from soil collected in the Phillipines and New Zealand, respectively. Strain 3828E released without induction an actinophage. The host isolate was chemotaxonomically identical to members of the revised genus Microtetraspora having both wall chemotype III, sugar pattern B and phospholipid pattern PIV. On the basis of cultural and morphological characteristics, the isolate was most similar to Microtetraspora salmonea . Strain 3913E shared the same chemotaxonomic characteristics as strain 3828E; however, morphological examination revealed the presence of spores arranged distinctively as pairs along aerial hyphae. On this basis, strain 3913E was classified as a member of the genus Microbispora . When re-infected into the soil it had been isolated from, actinophage were recovered that specifically attacked strain 3931E. The presence of phage from both maduromycetes was confirmed by transmission electron microscopy. Neither phage was able to attack a range of other actinomycetes. We believe this to be the first reported isolation of actinophage that attack species of the genera Microtetraspora and Microbispora .     

Isolation and characterization of a temperate bacteriophage from Streptomyces galilaeus

A new temperate actinophage from Streptomyces galilaeus ATCC 31133 was purified after that strain was crossed with S. peucetius ATCC 29050. Sensitive hosts became lysogenized and yielded turbid plaques of 2 to 3 mm in diameter. Host-range analysis indicated that 16 of 27 Streptomyces strains tested were sensitive to infection on solid medium. S. lividans and S. coelicolor A3(2) were among those not infected by this new actinophage. The new actinophage, designated phi SPK1, belongs to the Bradley group B morphological type, the pH optimum for infection is 6.75 to 7.0, it is not efficiently induced by mitomycin C or UV irradiation, it has a circular chromosome of 35.8 +/- 0.5 kilobase pairs in length containing overlapping (cohesive) ends, and the G+C content of its DNA was calculated from the buoyant density of 1.7240 to be 69 mol%. The DNA of phage phi SPK1 was cleaved by the restriction endonucleases ApaI, AluII, EcoRI, PvuII, and SalI, but, in all cases except that with EcoRI, treatment yielded greater than 20 restriction fragments. No sites were detected for BamHI, BclI, BglII, ClaI, HindIII, MluI, PstI, SmaI, SphI, SstI, XbaI, or XhoI.     

Isolation and characterization of a temperate bacteriophage from Streptomyces galilaeus.

A new temperate actinophage from Streptomyces galilaeus ATCC 31133 was purified after that strain was crossed with S. peucetius ATCC 29050. Sensitive hosts became lysogenized and yielded turbid plaques of 2 to 3 mm in diameter. Host-range analysis indicated that 16 of 27 Streptomyces strains tested were sensitive to infection on solid medium. S. lividans and S. coelicolor A3(2) were among those not infected by this new actinophage. The new actinophage, designated phi SPK1, belongs to the Bradley group B morphological type, the pH optimum for infection is 6.75 to 7.0, it is not efficiently induced by mitomycin C or UV irradiation, it has a circular chromosome of 35.8 +/- 0.5 kilobase pairs in length containing overlapping (cohesive) ends, and the G+C content of its DNA was calculated from the buoyant density of 1.7240 to be 69 mol%. The DNA of phage phi SPK1 was cleaved by the restriction endonucleases ApaI, AluII, EcoRI, PvuII, and SalI, but, in all cases except that with EcoRI, treatment yielded greater than 20 restriction fragments. No sites were detected for BamHI, BclI, BglII, ClaI, HindIII, MluI, PstI, SmaI, SphI, SstI, XbaI, or XhoI.     

Temperature-sensitive mutants of actinophage phi C31 of Streptomyces coelicolor A3(2)]

Temperature-sensitive mutants of phiC31 actinophage were isolated by the treatment with nitrosoquanidine and UV light. 20 cistron which control various stages of intracellular phage growth are identified on the basis of modified complementation tests. The sequence of ts markers and c-region is established and the relative distance between them is estimated according to the results of three factor crosses.     

Incomplete and Aberrant Particles of Actinophage MSP2 from Lysates of Streptomyces venezuelae

Lysates of actinophage MSP2, propagated on Streptomyces venezuelae S13, contain at least 10(11) PFU/ml. During purification by centrifugation methods and by adsorption chromatography, a number of types of aberrant and incomplete phage particles were seen by electron microscopy. Infectious MSP2 had a buoyant density in CsCl of 1.52 g/cm(3) and an absorbance at 260 nm relative to that at 280 nm (A(260)/A(280)) of 1.53. Empty capsids banded at 1.276 g/cm(3) and partially filled capsids banded at 1.351 g/cm(3), and A(260)/A(280) ratios were 0.77 and 1.24, respectively. Two kinds of light capsids found in CsCl fractions of 1.278 g/cm(3) probably include the 1.276 component. Some capsids were joined by tail-like structures. Ghosts and polyheads also were present. Aberrant particles observed by electron microscopy included two-tailed actinophage, phage with abnormal tail positions, and large-headed phage.     

Site-specific integration of the actinophage R4 genome into the chromosome of Streptomyces parvulus upon lysogenization.

The lysogenization of Streptomyces parvulus by actinophage R4 occurs by site-specific integration of the phage genome into the chromosome. The DNA fragments containing the attachment sites on the host chromosome, the phage genome, and the two junctions created by insertion of the phage genome were cloned and sequenced. The attachment sites were found to share a common core of 12 bp. This common core sequence was not detected in chromosomal DNAs of S. coelicolor and S. lividans.     

Sequence analysis of cohesive ends of the actinophage RP3 genome and construction of a transducible shuttle vector

Abstract The sequence of the cohesive ends of actinophage RP3 DNA has been determined. As with all other phages of Gram-positive bacteria that have been studied sofar, RP3 DNA has 3'-protruding ends. A shuttle cosmid has been constructed containing this cos area which can be efficiently transduced by phage RP3 to host cells of Streptomyces rimosus .     

Actinomycete virus in fresh water

An ecological study was made of the freshwater distribution of Actinomycete virus. One component of this, the virus of Actinoplanes, is highly specific and its pattern of occurrence mirrors that of its host. The occurrence of Streptomyces virus is more difficult to explain in ecological terms, but this is attempted. Micromonospora virus was not obtained and the significance of this is discussed. The Actinoplanes virus is characterized at the fine-structure level by a polyhedral-shaped head with a tail bearing a contractile sheath, the latter feature being previously undescribed for actinophage.     

Construction and characterization of a cosmid of Streptomyces lividans

Summary We constructed a plasmid pR4C1 in which a DNA fragment containing the cohesive ends of an actinophage, R4 was inserted into the ClaI site of plasmid pIJ365. The plasmid pR4C1 was packaged efficiently into R4 phage particles in vivo and therefore transferred by transduction. Southern hybridization analysis of DNA from pR4C1-transducing R4 phage particles indicated that the plasmid DNA was encapsidated as head-to-tail concatemers with the cohesive ends in the termini. We designated the pR4C1 plasmid a cosmid, following the termination of Collins and Hohn (1978).     

Identification of the restriction and modification systems in Streptomyces strains]

Host range of actinophage phi C31, VP5 and Pg81 in respect to 109 strains of Streptomyces genus and hybrid strain Rcg2 from the cross S. coelicolor A3(2)XS. griseus Kr was studied. The existence of RM-systems in strains S. griseus Kr15, S. griseus Kr20, Rcg2, S. griseofovillus 43 was shown using phage Pg81. Mutants of Pg81 were observed which to some extent lost snesitivity to RM-system in the strain Rcg2. The presence of RM-system in S. lividans 67 was demonstrated by the phage VP5.     

Survival of the temperate actinophage φC31 and Streptomyces lividans in soil and the effects of competition and selection on lysogens

Abstract Lysogenic infections were demonstrated in microcosms of sterile soil inoculated with Streptomyces lividans and the φC31 derivative, KC301, in free state or via lysogenized hosts. Intermittent soil mixing caused liberation of KC301 due to lysis of germinating lysogenized and uninfected spores. The presence of lysogenized host ensured that KC301 was maintained at a constant density. The lysogen S. lividans TK24 (KC301) achieved a population density lower than that of its non-lysogenized counterpart. Thiostrepton in the soil did not select for the thiostrepton resistance gene KC301. The long-term survival in soil of a temperate actinophage was demonstrated.     

Structural analysis of the actinophage phi C31 attachment site.

The lysogenisation of actinophage phi C31 in S. coelicolor J 1501 occurs by site-specific recombination. The DNA segments containing the attachment sites on the host chromosome, the phage genome and the two junctions created by the insertion of the prophage were cloned and the nucleotide sequences determined. The attachment sites (att) share an extremely short common sequence of three base pairs. Adjacent to the core sequences some direct- and inverted repeats were found as potential binding sites for proteins involved in site-specific recombination.     

Isolation and characterization of the Streptomyces cattleya temperate phage TG1

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.     

New shuttle promoter-probe vectors forE. coli andStreptomycetes

Summary Two new shuttle promoter-probe vectors forE.coli andStreptomycetes were constructed. Plasmid vectors allow the cloning of promoter-carrying DNA fragments based on the resistance to neomycin and chloramphenicol both, inE.coli andStreptomycetes. Using these vectors several promoter regions active either inE.coli orS.lividans were identified from the actinophage DNA.     

The actinophage RP3 DNA integrates site-specifically into the putative tRNA(Arg)(AGG) gene of Streptomyces rimosus.

The temperate actinophage RP3 integrates site-specifically into the chromosome of Streptomyces rimosus R6-554. The phage attachment site attP and the hybrid attachment sites of the integrated prophage--attL and attR--were cloned and sequenced. The 54nt core sequence, common to all RP3 related attachment sites, comprises the 3' terminal end of a putative tRNA(Arg)(AGG) gene. AttB bears the complete tRNA gene which is restored in attL after integration. A 7.5kb HindIII fragment, bearing attP, was used to construct an integrative plasmid to simulate the integration process in vivo and to localize the phage genes necessary for site specific integration. The int and xis genes were sequenced and compared to other recombination genes.     

Synthesis of Several Analogs of Esterified Cerebroside Isolated from Human and Pig Epidermis

A cosmid, pR4C1, composed of the actinophage R4 cos sequence and Streptomyces plasmid pIJ365, was encapsidated in R4 phage particles in vivo. [T. Morino et al., Mol. Gen. Genet., 198, 228–233 (1985)] In this report a cosmid derivative, pR4C4, is shown to be also encapsidated by heterogeneous actinophages, SPA10 and SPA38, and transferred to Streptomyces lividans. Use of this transduction and conservation of the DNA packaging mechanism are discussed.     

High-frequency Protoplast-transfection of Streptomyces parvulus 2297 with Actinophage R4 DNA

High frequency transfection of Streptomyces parvulus with actinophage R4 DNA was performed by modifying the procedure of protoplast transformation of S. coelicolor A3(2) with SCP2 plasmid DNA [Bibb et al., Nature, 274, 398 (1978)]. Optimum conditions for protoplast transfection included the presence of 16~24% (w/v) polyethyleneglycol 4000, and the maximum efficiency of transfection was 3 × 10^?5 per phage DNA molecule. This value was at least 100 times higher than the efficiency of previously reported transfection systems in Streptomyces.