Bacteriophage, a process for the isolation thereof, and a...

Details Bacteriophage, a process for the isolation thereof, and a... Bacteriophage, a process for the isolation thereof, and a...

1999-04-21

2002-11-19

Mosher, Mary E. (Department: 1648)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

C435S239000, C435S252300, C435S252310, C435S252330, C435S252350, C435S252800, C435S252500, C435S253100, C435S253500, C435S472000, C435S473000, C435S476000, C435S485000, C435S486000, C435S488000

Reexamination Certificate

active

06482632

ABSTRACT:

This invention relates to an isolated bacteriophage and a process for preparation of said bacteriophage useful as a tool for studying biological, biochemical, physiological and genetic properties of actinomycetes and other organisms. The bacteriophage obtained by the present invention is particularly useful for the characterization of antibiotic biosynthetic pathways and similar primary and secondary metabolic pathways. The bacteriophage described in this process may also be used for the study of several other genetic and physiological pathways. It may also be used to generate mutations in the metabolic pathways of bacteria, and after some specific alteration such as after cloning into suitable mobilising vectors, may also be used to study the metabolic pathways of other microorganisms and plants. Mutations can be generated essentially at any locus of the bacteria. The results of the above mentioned alterations or mutations may lead to the production of new metabolic products or to the expression of new physiologically active compounds or to the expression of novel characteristics either in the same host or in heterologous hosts.
BACKGROUND OF THE INVENTION
As used in this application, bacteriophage or phage refers to a virus or viruses which infect bacteria. After infection the virus can produce new progency particles or can remain dormant within the bacterial genome. Lysogen is a bacteria which carries a bacteriophage without being harmed. Plaque is a turbid/clear spot produced due to lysis of the cells infected by a bacteriophage. Prophage is a bacteriophage that is maintained in the lysogenic state in a bacterial cell. Confluent lawn is a uniform growth of organism on an agar plate. Phagemid is a plasmid vector carrying some part of the phage DNA. The term restriction enzyme refers to an endonuclease that cuts DNA sites defined by its recognition sequence. Auxotroph is a bacterial strain defective for the synthesis of one or more sugar or amino acids. Restriction barrier is intended to mean a host defense system which protects the bacteria from invaders, by cleaving the invaders' DNA. Cloning is understood to mean the procedure for the generation of recombinant DNA. Genetic tool is a system where DNA is used as a tool. Mutation is an alteration in the sequence of bases in the DNA of an organism. This alteration may be caused by insertion, deletion or modification of DNA bases. Transposon refers to a genetic element that carries the information that allows it to integrate at various sites in the host genome. Genome is a term used to describe the complete genetic complement of a virus, or cell or bacteria or any living organism.
The process of the present invention involves the isolation from soil samples and purification of a novel bacteriophage from a strain of Saccharomonospora called PA136. The strain of Saccharomonospora described in the present invention is characterized by the presence of single lateral white coloured spore, meso-diaminopimelic acid; arabinose and galactose as total sugar but no mycolic acid and major components of fatty acids—16 carbons iso- and antiso fatty acids. Other properties of this strain are: it grows on hypoxanthine, hypoxanthine+0.3% glucose, tributyrine, xylitol, thallous acetate, thallous acetate+0.1% glucose, propanol, butanol-1, 3 diol, D-fucose, salicin, arabinose, L-asparagine, phenylalanine, L-serine, and sodium benzoate. The strain grows in the temperature range of 20° C. to 55° C. on an agar plate with pH range 5.5 to 9.0. It is catalase positive and produces extracellular enzymes lipase (C14), leucine arylamidase, valine arylamidase, cystein arylamidase, trypsin, chemotrypsin, acid phosphatase, naphthol As-B1-phosphophydrolase, &agr;-glucosidase and &bgr;-glucosidase. The chemical composition of the total cell is as follows: meso-di-aminopimelic acid, arabinose, galactose, phosphatidyl glycerol, di-phosphatidyl glycerol, phosphatidyl ethanolamine, hydroxyphastidyl ethanolamine, phosphatidyl inositol glycolipids are present. Sugar containing unidentified groups of phospholipids is present. Total fatty acids are: major components of 16 carbon iso- and antiso-fatty acids of branched and straight chain carbon compounds. It does not utilise sucrose. It mostly produces a diffusable pigment that is either green, orange or yellow. Sometimes the strain Saccharomonospora PA136 does not produce any pigment at all. At times the pigment is non-diffusable. However, the pigments produced by the strain Saccharomonospora PA136 are water-soluble. The pigments are insoluble in either, ethanol, methanol, butanol, isopropanol, benzene, ethyl acetate, chloroform and acetone. It is partially soluble in phenol. The green pigment produced by the strain is a characteristic of the genus Saccharomonospora. The strain Saccharomonospora PA136 undergoes autolysis after 5-6 days of incubation on a culture plate which when subjected to detailed analysis results in the isolation of a bacteriophage named as PIS136. This temperature bacteriophage has a wide host range amongst Gram positive (Gram+) bacteria and generates lysogens at the rate of 2 to 3 percent of the total cells infected. The phage PIS136 has a DNA genome of about 90 kb where the GC (Guanidine and Cytosine) content is 69 to 71 mole percent. The genome of this phage is partially methylated and lacks recognition sites for many restriction enzymes. The phage genome or bacteriophage that has the property of generating random mutations by transposition also shows the phenomenon of gene inversion. The phenomenon of gene inversion can be used to control the host range of the phage as well as for heterologous and conditional expression of genes. The phage has been deposited as a lysogen of the strain Saccharomonospora PA136 at the Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh and carries an accession number MTCC A0001, where ‘A’ stands for Actinomycetes and also bears the depository number DSM 12317 at DSMZ-DEUTSCHE SAMMELUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH where it was deposited on Jul. 16, 1998. Because of the unique properties this phage has immense potential as a genetic tool and can be used variously as a transposon for the generation of mutants, as an intergeneric cloning vector, for the study of metabolic pathways, as a reporter phage, for conditional gene expression, or even for the activation of silent genes, etc.
Bacteriophages or phages, the viruses of bacteria, are the simplest of all living organisms. They have been natural objects of study in attempts to understand life at the molecular level. Phages have evolved a number of regulatory schemes to ensure efficient production of progency particles during development. However, in general, in about 1% of the cells that are infected by the temperature phages, lysogeny is established, that is when the phage genome integrates with the host. The lysogens are immune to super-infection.
Numerous phages have been isolated from industrially and medically important bacteria such as Streptomyces species, Corynebacterium species,
Lactococcus lactis,
Mycobacteria,
Escherichia coli,
Salmonella and Staphylococcus species etc.
About 70% of the known and naturally occurring antibiotics are produced by members of the genus Streptomyces. In most cloning procedures for Streptomyces species, plasmid based vectors capable of replicating autonomously have been used (Rao, R. N., Richardson, M. A. and Kuhtoss, S. 1987 in
Methods in Enzymology,
153: 166-198; Hopwood, D. A., Bibb, M. J., Chater, K. F. and Kieser, T. 1987 in
Methods in Enzymology,
153: 116-165).
At the same time the relatively broad host range temperature bacteriophage ØC31 (which infects Streptomyces species and the related genus Streptoverticillium species only) was developed as a versatile containing vector (Chater, K. F. 1986, In
The Bacteria,
Vol. IX. Queener, S. W. and Day, L. E. (ed) London: Academic Press pp 119-158, Kobler, L., Schwertfirm, G., Schmieger, H., Bolotin, A. and Sladkova, I. 1991
FEMS Microbiology Letters,
8:

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