Recombinant bacterial system with environmentally limited viability

Abstract

Disclosed is an Environmentally Limited Viability System (ELVS) for microorganisms based on differences between permissive and non-permissive environments. Viability of the microorganisms are limited to a permissive environment by specifically expressing one or more essential genes only in the permissive environment, and/or expressing one or more lethal genes only in the non-permissive environment. Temporary viability in a non-permissive environment can be achieved by temporarily expressing one or more essential genes in a non-permissive environment, and/or temporarily delaying expression of one or more lethal genes in the non-permissive environment. Environmentally Limited Viability Systems are also disclosed involving coordinate expression of a combination of essential genes and lethal genes. Microorganisms containing an Environmentally Limited Viability System are useful for release into permissive and non-permissive environments. Temperature regulated Environmentally Limited Viability Systems and delayed death Environmentally Limited Viability Systems are particularly suited for delivery of expression products, such as antigens, using recombinant avirulent Salmonella by limiting their growth to the warmer environment inside the host, or by allowing growth for only a limited time in the host.

1 - 60 . (canceled)  
     
     
         61 . An isolated microbial cell comprising an Environmentally Limited Viability System, wherein the cell is viable in a permissive environment and non-viable or temporarily viable in a non-permissive environment, the system comprising 
 (a) an essential gene, wherein expression of the essential gene in the cell is essential to the viability of the cell, the essential gene is expressed when the cell is in the permissive environment and is not expressed or temporarily expressed when the cell is in the non-permissive environment; and/or    (b) a lethal gene, wherein expression of the lethal gene is lethal to the cell and the lethal gene is expressed when the cell is in the non-permissive environment but not when the cell is in the permissive environment,    wherein the essential gene is essential for metabolism, growth, cell wall integrity, or cell membrane integrity of the cell.    
     
     
         62 . The cell of  claim 61  wherein the cell grows in the permissive environment and dies in the non-permissive environment.  
     
     
         63 . The cell of  claim 61  wherein the permissive environment comprises an environment containing a nutrient required to maintain expression of the essential gene, prevent expression of the lethal gene, or both, and the non-permissive environment comprises an environment lacking the nutrient.  
     
     
         64 . The cell of  claim 61  wherein the permissive environment is inside a warm-blooded animal and the non-permissive environment is outside a warm-blooded animal.  
     
     
         65 . The cell of  claim 61  wherein the lethal gene is carried on an extrachromosomal vector.  
     
     
         66 . The cell of  claim 61  wherein the lethal gene is carried on a chromosome of the cell.  
     
     
         67 . The cell of  claim 61  wherein the essential gene comprises the asd gene operatively linked to araC-P BAD .  
     
     
         68 . The cell of  claim 65  wherein the vector has two lethal genes.  
     
     
         69 . The cell of  claim 68  wherein the vector comprises pMEG-104.  
     
     
         70 . The cell of  claim 61  wherein the cell is a gram-negative bacterium.  
     
     
         71 . The cell of  claim 70  wherein the gram-negative bacterium is an enteric bacterium.  
     
     
         72 . The cell of  claim 71  wherein the genus of the enteric bacterium is selected from the group consisting of  Escherichia  and  Salmonella.    
     
     
         73 . The cell of  claim 61  wherein the essential gene is carried on an extrachromosomal vector.  
     
     
         74 . The cell of  claim 73  wherein expression of the essential gene is regulated by an expression product of a regulatory gene on a chromosome of the cell.  
     
     
         75 . The cell of  claim 73  wherein expression of the essential gene is regulated by an expression product of a regulatory gene on an extrachromosomal vector.  
     
     
         76 . The cell of  claim 61  wherein the essential gene is carried on a chromosome of the cell.  
     
     
         77 . The cell of  claim 61  wherein the system further comprises a replication gene carried on a chromosome of the cell, the expression of which is required for replication of the vector, wherein the replication gene is expressed in the permissive environment and is not expressed in the non-permissive environment.  
     
     
         78 . The cell of  claim 77  wherein expression of the replication gene is regulated by an expression product of a regulatory gene.  
     
     
         79 . The cell of  claim 61  wherein replication of the extrachromosomal vector is regulated by an expression product of a regulatory gene, 
 wherein the absence of a functional expression product of the regulatory gene derepresses expression of the replication gene and wherein the expression product is not expressed or is inactive only in the non-permissive environment.  
 
     
     
         80 . The cell of  claim 61  further comprising an expression gene wherein the expression gene encodes a desired expression product.  
     
     
         81 . The cell of  claim 61  further comprising a transfer vector.  
     
     
         82 . The cell of  claim 61  wherein the lethal gene is a gene required for excision of a prophage, wherein the prophage is in the chromosome of the cell, and wherein excision of the prophage causes lysis of the cell.  
     
     
         83 . The cell of  claim 61  wherein the essential gene, the lethal gene, or both have engineered expression.  
     
     
         84 . A method of making a cell strain with environmentally limited viability comprising stably introducing into a cell 
 (a) an essential gene, wherein expression of the essential gene in the cell is essential to the viability of the cell, the essential gene is expressed when the cell is in the permissive environment and is not expressed or temporarily expressed when the cell is in the non-permissive environment; and/or    (b) a lethal gene, wherein expression of the lethal gene is lethal to the cell and the lethal gene is expressed when the cell is in the non-permissive environment but not when the cell is in the permissive environment,    wherein the cell strain is viable in a permissive environment and non-viable or temporarily viable in a non-permissive environment, and wherein the essential gene is essential for metabolism, growth, cell wall integrity, or cell membrane integrity of the cell.    
     
     
         85 . The method of  claim 84  wherein the permissive environment comprises a temperature of about 37° C. and the non-permissive environment comprises a temperature of less than about 30° C.  
     
     
         86 . The method of  claim 84  wherein the permissive environment is inside a warm-blooded animal and the non-permissive environment is outside a warm-blooded animal.  
     
     
         87 . The method of  claim 84  wherein the cell is temporarily viable in the non-permissive environment.  
     
     
         88 . An isolated microbial cell comprising an Environmentally Limited Viability System, wherein the cell is viable in a permissive environment and non-viable in a non-permissive environment, the system comprising an essential gene, wherein expression of the gene in the cell is essential to the viability of the cell, and wherein said essential gene is expressed when the cell is in the permissive environment and is not expressed when the cell is in the non-permissive environment, and wherein said essential gene is essential for metabolism, growth, cell wall integrity, or cell membrane integrity of the cell.  
     
     
         89 . The cell of  claim 88  wherein the permissive environment comprises a temperature of about 37° C. and the non-permissive environment comprises a temperature of less than about 30° C.  
     
     
         90 . The cell of  claim 88  wherein the permissive environment is inside a warm-blooded animal and the non-permissive environment is outside a warm-blooded animal, 
 wherein the cell is a member of the Enterobacteriaceae.  
 
     
     
         91 . The cell of  claim 88  wherein the essential gene is carried on an extrachromosomal vector.  
     
     
         92 . The cell of  claim 88  wherein the cell is a gram-negative bacterium.  
     
     
         93 . The cell of  claim 92  wherein the gram-negative bacterium is an enteric bacterium.  
     
     
         94 . The cell of  claim 93  wherein the genus of the enteric bacterium is selected from the group consisting of  Escherichia  and  Salmonella.    
     
     
         95 . The cell of  claim 88  wherein expression of the essential gene is regulated by the expression product of a regulatory gene.  
     
     
         96 . The cell of  claim 95  wherein the expression product of the regulatory gene inhibits expression of the essential gene and is expressed or active only in the non-permissive environment.  
     
     
         97 . The cell of  claim 91  wherein the system further comprises a replication gene carried on a chromosome of the cell, the expression of which is required for replication of the vector, wherein the replication gene is expressed in the permissive environment and is not expressed in the non-permissive environment.  
     
     
         98 . The cell of  claim 88  further comprising an expression gene wherein the expression gene encodes a desired expression product.  
     
     
         99 . A method of inducing an immune response in a warm-blooded animal comprising 
 administering to the animal a vaccine comprising a microbial cell comprising an Environmentally Limited Viability System, wherein the cell is viable when in the animal and non-viable when outside of the animal, the system comprising    an essential gene, wherein expression of the gene in the cell is essential to the viability of the cell, the essential gene is expressed when the cell is in the animal and is not expressed when the cell is outside of the animal; and    wherein the essential gene corresponds to an inactivated native gene of the cell, wherein the cell is a member of the  Enterobacteriaceae.      
     
     
         100 . The method of  claim 99  wherein the system further comprises an expression gene wherein the expression gene encodes an antigen.  
     
     
         101 . The method of  claim 100  wherein the antigen is selected from the group consisting of bacterial antigens, viral antigens, plant antigens, fungal antigens, insect antigens, and non-insect animal antigens.  
     
     
         102 . The method of  claim 100  wherein the essential gene is carried on an extrachromosomal vector, and wherein the system further comprises a replication gene carried on a chromosome of the cell, the expression of which is required for replication of the vector, wherein the replication gene is expressed when the cell is in the animal and is not expressed when the cell is outside of the animal, wherein the cell is a member of the  Enterobacteriaceae.    
     
     
         103 . The cell of  claim 95  wherein the absence of a functional expression product of the regulatory gene derepresses expression of the essential gene and wherein the expression product is not expressed or is inactive only in the permissive environment.  
     
     
         104 . The cell of  claim 88  wherein the essential gene is a gene essential for metabolism or growth of the cell.  
     
     
         105 . The cell of  claim 88  wherein the essential is a gene essential for cell wall or cell membrane integrity.  
     
     
         106 . The cell of  claim 88  wherein the essential gene is a gene encoding an enzyme that catalyzes steps in the biosynthesis of DAP.  
     
     
         107 . The method of  claim 88  wherein the essential gene is an asd gene, a dap gene, a dal gene, a ddl gene, a fab, gene, or a pls gene.