Nucleic acids encoding two-component sensing and regulatory proteins, antimicrobial proteins and uses therefor

Abstract

Stress-related nucleic acid molecules and polypeptides and fragments and variants thereof are disclosed in the current invention. In addition, stress-related fusion proteins, antigenic peptides, and anti-stress-related antibodies are encompassed. The invention also provides recombinant expression vectors containing a nucleic acid molecule of the invention and cells into which the expression vectors have been introduced. Methods for producing the polypeptides and methods of use for the polypeptides of the invention are further disclosed.

1 . An isolated nucleic acid molecule comprising:
 a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163;   b) a nucleic acid molecule comprising a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163;   c) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164;   d) a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164; or   e) a complement of any of (a)-(d).   
     
     
         2 . A vector comprising the nucleic acid molecule of  claim 1 . 
     
     
         3 . The vector of  claim 2 , further comprising a nucleic acid molecule encoding a heterologous polypeptide. 
     
     
         4 . A cell comprising the vector of  claim 2 . 
     
     
         5 . The cell of  claim 4  that is a bacterial cell. 
     
     
         6 . An isolated polypeptide comprising:
 a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164;   b) a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164, wherein said polypeptide retains activity;   c) a polypeptide encoded by the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163; or   d) a polypeptide that is encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163, wherein said polypeptide retains activity.   
     
     
         7 . The polypeptide of  claim 6 , further comprising heterologous amino acid sequences. 
     
     
         8 . An antibody that selectively binds to a polypeptide of  claim 6 . 
     
     
         9 . A method for producing a polypeptide, comprising culturing the cell of  claim 4  under conditions in which a nucleic acid molecule encoding the polypeptide is expressed, said polypeptide comprising:
 a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164;   b) a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163;   c) a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164, wherein said polypeptide retains activity; or   d) a polypeptide encoded by a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163, wherein said polypeptide retains activity.   
     
     
         10 . A method for detecting the presence of a polypeptide in a sample, comprising contacting the sample with a compound that selectively binds to the polypeptide and detecting the binding of the compound to the polypeptide, wherein said polypeptide comprises:
 a) a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163;   b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164;   c) a polypeptide encoded by a nucleic acid sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163, wherein said polypeptide retains activity; or   d) a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164, wherein said polypeptide retains activity.   
     
     
         11 . The method of  claim 10 , wherein the compound that binds to the polypeptide is an antibody. 
     
     
         12 . A kit comprising a compound for use in the method of  claim 10  and instructions for use. 
     
     
         13 . A method for detecting the presence of the nucleic acid molecule of  claim 1  in a sample, comprising:
 a) contacting the sample with a nucleic acid probe or primer that selectively hybridizes to the nucleic acid molecule; and,   b) detecting hybridization of the nucleic acid probe or primer with the nucleic acid molecule.   
     
     
         14 . The method of  claim 13 , wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe. 
     
     
         15 . A kit comprising a nucleic acid molecule that selectively hybridizes to the nucleic acid molecule of  claim 1  and instructions for use. 
     
     
         16 . A method for increasing the ability of a microorganism to survive stressful conditions, comprising introducing into said microorganism a nucleic acid molecule comprising at least one nucleotide sequence comprising:
 a) the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163;   b) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164;   c) a nucleotide sequence that is at least 80% identical to the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163, wherein said nucleotide sequence encodes a polypeptide that retains activity; or,   d) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164, wherein said polypeptide retains activity.   
     
     
         17 . The method of  claim 16 , wherein said stressful conditions comprise osmotic stress. 
     
     
         18 . A method for enhancing the ability of a microorganism to survive passage through the gastrointestinal tract, comprising introducing into said microorganism a nucleic acid molecule comprising at least one nucleotide sequence comprises:
 a) the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163;   b) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164;   c) a nucleotide sequence that is at least 80% identical to the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163, wherein said nucleotide sequence encodes a polypeptide that retains activity; or,   d) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164, wherein said polypeptide retains activity.   
     
     
         19 . A method for increasing the ability of a microorganism to survive in the presence of an antimicrobial, comprising introducing into said microorganism a nucleic acid molecule comprising at least one nucleotide sequence comprising:
 a) the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163;   b) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164;   c) a nucleotide sequence that is at least 80% identical to the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 49, 51 53, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 113, 115, 117, 119, 121, 123, 125, 127, 129, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 or 163, wherein said nucleotide sequence encodes a polypeptide that retains activity; or,   d) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 50, 52, 54, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 114, 116, 118, 120, 122, 124, 126, 128, 130, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162 or 164, wherein said polypeptide retains activity.   
     
     
         20 . A method for enabling an organism to respond to an environmental stimuli, comprising introducing into said organism a vector comprising at least one nucleotide sequence encoding a histidine kinase comprising SEQ ID NOS:4, 8, 14, 16, 20, 24, 30, 34, or 36, and a response regulator comprising of SEQ ID NOS:2, 6, 10, 12, 18, 22, 26, 28, 32, or 38. 
     
     
         21 . The method of  claim 20 , wherein said environmental stimuli is selected from the group consisting of turgor pressure, a chemical stimulus, heavy-metal cations, oxygen, iron, an antimicrobial, and glucose.