Methods and compositions for reducing bacterial load in tobacco

Abstract

Methods and compositions are provided for expressing an endolysin in a bacterium or in a tobacco plant or plant part. Expression of the endolysin can reduce the total bacterial load on the plant and in tobacco products produced from the plant. Accordingly, the level of tobacco specific nitrosamines (TSNA) in a cured tobacco product can be decreased when the tobacco leaves used in the product were harvested from a plant expressing endolysin or when bacteria modified to express endolysin is used to treat the tobacco. Thus, bacteria and tobacco plants and plant parts are provided that express an endolysin. Further provided are methods for decreasing the microbial load on a tobacco plant and in a tobacco product by applying a solution of bacteria modified to express an endolysin or by modifying a tobacco plant to express endolysins. Smokeless tobacco products including such modified tobacco plant parts are also provided.

That which is claimed: 
     
         1 . A tobacco plant, plant part, or plant cell comprising a heterologous nucleic acid molecule comprising a nucleic acid sequence encoding an endolysin, wherein the nucleic acid is operably linked to a promoter active in the tobacco plant, plant part, or plant cell. 
     
     
         2 . The tobacco plant or plant part of  claim 1 , wherein the endolysin is expressed in the leaf or stem of said tobacco plant or plant part. 
     
     
         3 . A seed produced from the tobacco plant, plant part, or plant cell of  claim 1 , wherein the seed comprises a nucleic acid molecule comprising the nucleic acid sequence encoding the endolysin. 
     
     
         4 . A tobacco product produced from the tobacco plant, plant part, or plant cell of  claim 1 . 
     
     
         5 . The tobacco product of  claim 4 , wherein a level of tobacco specific nitrosamines (TSNA) in the tobacco product is at least 10% lower than a control level of TSNA. 
     
     
         6 . The tobacco product of  claim 5 , wherein the tobacco product is leaf tobacco, shredded tobacco, cut tobacco, ground tobacco, powder tobacco, tobacco extract, nicotine extract, smokeless tobacco, moist or dry snuff, kretek, pipe tobacco, cigar tobacco, cigarillo tobacco, cigarette tobacco, chewing tobacco, bidis, bits, cigarette, cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, and tobacco-containing gum, lozenges, patches, electronic cigarettes, or any combination thereof. 
     
     
         7 . A method of producing a tobacco product from a modified tobacco plant, plant part, or plant cell expressing an endolysin, the method comprising
 introducing into the tobacco plant, plant part, or plant cell a heterologous nucleic acid molecule comprising a nucleic acid sequence encoding an endolysin, wherein the nucleic acid sequence is operably linked to a promoter active in the tobacco plant, plant part, or plant cell,   wherein the endolysin is expressed from the nucleic acid sequence following introduction of the nucleic acid sequence into the tobacco plant, plant part, or plant cell, and   producing a tobacco product from said tobacco plant.   
     
     
         8 . The method of  claim 7 , wherein the nucleic acid molecule is introduced into the tobacco plant, plant part, or plant cell on a vector. 
     
     
         9 . The method of  claim 7 , wherein the nucleic acid molecule is introduced into the tobacco plant, plant part, or plant cell using a bacterial cell, a virus, or a bacteriophage. 
     
     
         10 . The method of  claim 7 , wherein the nucleic acid molecule is stably incorporated into the genome of the tobacco plant, plant part, or plant cell. 
     
     
         11 . The method of  claim 7 , wherein the endolysin is expressed in the leaf or stem of the tobacco plant or plant part. 
     
     
         12 . The method of  claim 7 ,
 wherein the endolysin reduces a bacterial level on the tobacco plant, plant part, or plant cell as compared to a control bacterial level.   
     
     
         13 . The method of  claim 12 , wherein the endolysin reduces a level of at least one nitrate-reducing bacterium. 
     
     
         14 . The method of  claim 7 , wherein a level of tobacco specific nitrosamines (TSNA) in the tobacco product is at least 10% lower than a control level of TSNA. 
     
     
         15 . A genetically modified bacterium comprising a heterologous nucleic acid molecule, wherein the heterologous nucleic acid molecule comprises a nucleic acid sequence encoding an endolysin, wherein the nucleic acid sequence is operably linked to a promoter active in the genetically modified bacterium. 
     
     
         16 . A method of reducing the bacterial load on a tobacco plant, plant part, or plant cell, the method comprising the steps of:
 applying to the tobacco plant or plant part a solution comprising the genetically modified bacterium of  claim 15 ,   wherein the bacterium expresses the endolysin, and   wherein the expressed endolysin reduces a level of bacteria on the tobacco plant or plant part as compared to a control level of bacteria.   
     
     
         17 . The method of  claim 16 , wherein the bacterium is a nitrite sink and/or has a nitrite reductase gene. 
     
     
         18 . The method of  claim 16 , wherein the genetically modified bacterium is a lactic acid bacterium. 
     
     
         19 . The method of  claim 16 , wherein the endolysin reduces the level of at least one nitrate-reducing bacterium. 
     
     
         20 . The method of  claim 16  further comprising,
 producing a tobacco product from the treated tobacco plant, plant part, or plant cell.