Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-05-25
2001-06-12
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S029000, C435S235100, C435S252100, C435S472000, C435S475000
Reexamination Certificate
active
06245504
ABSTRACT:
BACKGROUND OF THE INVENTION
It would be difficult to overestimate the contribution generalized transduction has made to the study of prokaryote biology since the discovery of phage P22 in Salmonella in the early 1950s. The use of generalized transducing phages for strain construction, fine structure mapping, and genetic manipulation have played major roles in the genetic analysis of Salmonella and
E. coli.
One of the most important applications of generalized transduction has been to facilitate the cloning of genes identified by transposon generated mutations. The use of generalized transduction in combination with transposon mutagenesis to clone genes involved in morphogenesis has been invaluable in the study of sporulation in
Bacillus subtilis.
Streptomyces are Gram-positive soil bacteria of special interest for two reasons. First, their mycelial growth mode and sporulation cycle are among the most dramatic examples of prokaryotic morphological differentiation. They grow vegetatively as multicellular, multinucleoid, branching hyphae that penetrate and solubilize organic material in the soil forming a mycelial mass. In response to environmental signals (a process that requires cell—cell communication mediated by diffusible substances), they initiate a cycle of differentiation that begins with the production of aerial hyphae that septate into uninucloid compartments that give rise to spores. Second, during the initiation of morphological development they produce a large number of secondary metabolites, including most of the natural product antibiotics used in human and animal health care. Because of its unique biology, Streptomyces offers special advantages for the study of how morphogenesis is initiated. The question of how cells within multicellular organisms sense changes in their environment and communicate that information to each other is of fundamental importance to the study of developmental biology. In spite of their interesting biology and commercial importance, relatively little is known about the gene expression pathways that regulate morphological development or antibiotic biosynthesis.
A major limitation in the study of Streptomyces is that the typical genetic approaches for recovering genes identified by chemically induced mutations have been difficult to implement in Streptomyces. Because relatively few genetic markers exist in Streptomyces, fine structure mapping is not possible. Cloning by complementation is slow and tedious. Transformation of plasmid libraries constructed in either
E. coli
or Streptomyces is extremely inefficient and the libraries are often incomplete. Transposition systems have been developed in Streptomyces but they have not proved to be effective for insertional mutagenesis. This is in part due to the use of temperature sensitive plasmid vectors as transposon delivery systems. Plasmid curing is not effective and exposure to high temperatures is mutagenic in itself. This has resulted in a high background of mutations not caused by transposition. Thus, it has not been possible to determine whether a mutant phenotype was caused by transposon insertion into a gene of interest until the candidate gene was cloned, thereby permitting complementation analysis and directed disruption studies. This is not only time consuming and laborious, it is often a futile exercise because of the high background of extraneous mutations.
It has long been recognized that an efficient system for generalized transduction is needed to make transposon mutagenesis an effective genetic tool in Streptomyces. However, generalized transducing phages have not been characterized in species that can serve as genetic model systems. Attempts by many workers over the years to isolate generalized transducing phages for
Streptomyces coelicolor
have been uniformly unsuccessful, as have been attempts to transduce markers by the most extensively studied lytic actinomycete phages fC31, VP5, and R4. Generalized transduction has been demonstrated in
Streptomyces venezuelae.
This involved transduction of several markers including genes for cholemphenicol production. This was thought, however, to be an anomaly and somehow specific to
Streptomyces venezuelae
since the approaches used to identify transducing phages for
Streptomyces venezuelae
did not work for
Streptomyces coelicolor.
Subsequent to the publication of much of the work describing these intraspecific generalized transducing phages of
Streptomyces venezuelae
and
Streptomyces olivaceus,
a report was authored by one of the investigators that had taken part in many of the studies. In this report titled “Generalized Transduction in Streptomyces Species,” (Stuttard, In:
Genetics and Molecular Biology of Industrial Microorganisms,
Hershberger, et al., (eds.), pp. 157-162, ASM, Washington, D.C. (1989)) he reported “a possibly significant lack of success with
Streptomyces coelicolor
and
Streptomyces lividans.
” The author hypothesized “that some essential host function(s), possibly expressed in few potential host strains, may be required for lytic growth of” generalized transducing particles. If such host functions are required, then generalized transducing phages will not be isolated that transduce those strains lacking the essential host functions. The author concludes that “generalized transducing phages for
Streptomyces coelicolor
and
Streptomyces lividans
remain as elusive as ever.”
In the recent past there has been a significant increase in the identification of antibiotic resistant microbes. However, the identification of new antibiotics has not kept pace with the occurrence of antibiotic resistant microbes. Accordingly, there has been a significant increase in human and animal morbidity and mortality due to infectious diseases. Thus, there is a need for new antibiotics. As mentioned above, Streptomyces, and other microbes, produce secondary metabolites. Many of these secondary metabolites are natural product antibiotics used in human and animal health care. It has recently become possible to use recombinant genetic techniques to modify the metabolic pathways of microbes to result in the synthesis of new natural product antibiotics, often referred to as new natural products or non-natural products, having new activities. A limitation to this is, for instance, the need for appropriate vectors to carry large DNA fragments, and the ability to efficiently move DNA into appropriate hosts (see, for instance, Cane, D. E. et al., (1998)
Science,
282, 63-68). Thus, there is a need and significant advantage to developing genetic techniques of microbes that synthesize natural product antibiotics.
SUMMARY OF THE INVENTION
The present invention is directed to a method of isolating a transducing phage, preferably, a generalized transducing phage. The method includes combining a sample containing a transducing phage with a microbe forming a first phage-microbe mixture, and incubating the first phage-microbe mixture at a temperature of less than 28° C. to form a first plaque comprising a generalized transducing phage. The invention includes a phage isolated using this method.
Another aspect of the invention is a method of isolating a transducing phage, preferably, a generalized transducing phage, involving phage DNA. The method includes combining a sample containing generalized transducing phage DNA with a microbe forming a phage DNA-microbe mixture and incubating the phage DNA-microbe mixture at a temperature of less than 28° C. to form a first plaque comprising a transducing phage.
Another method of the invention is a method of transferring at least one nucleic acid fragment from a donor microbe to a recipient microbe. The method includes providing an isolated transducing particle comprising a nucleic acid fragment from a donor microbe, combining the transducing particle with a recipient microbe to result in a transducing particle-recipient microbe mixture, and incubating the transducing particle-recipient microbe mixture at a temperature of less than 28° C. to form a transduced recipient microbe comprising a n
Burke Julie A.
Westpheling Janet
Guzo David
Leffers, Jr. Gerald G.
Mueting Raasch & Gebhardt, P.A.
The University of Georgia Research Foundation Inc.
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