Method for identifying a nucleic acid

Abstract

A method for identifying a nucleic acid which codes for a polypeptide factor affecting the covalent bonding of polypeptides to the surface of Gram-positive bacteria, comprising the following steps: a) providing a sample of Gram-positive bacteria which can be genetically altered and contain or produce at least one enzymatic reporter substance which is or can become covalently bonded to the surface of the Gram-positive bacteria, said at least one reporter substance having a different enzymatic activity when not covalently bonded to the surface of the Gram-positive bacteria from that exhibited when it is covalently bonded to the surface of the Gram-positive bacteria; b) causing genetic alterations in Gram-positive bacteria of the sample; c) assaying the enzymatic activity of the reporter substance of the Gram-positive bacteria of the sample; d) separating Gram-positive bacteria which exhibit a different enzymatic activity of the reporter substance from that observed for covalent bonding of the reporter substance to the surface of the Gram-positive bacteria; e) isolating the nucleic acid of the Gram-positive bacteria separated in step d); f) identifying at least one segment of the nucleic acid isolated in step e) that carries the genetic alteration; g) based on the segment identified in step f), identifying a nucleic acid which codes for a polypeptide factor affecting the covalent bonding of polypeptides to the surface of Gram-positive bacteria.

1 . A method for identifying a nucleic acid which codes for a polypeptide factor affecting the covalent bonding of polypeptides to the surface of Gram-positive bacteria, comprising the following steps: 
 a) providing a sample of Gram-positive bacteria which can be genetically altered and contain or produce at least one enzymatic reporter substance which is or can become covalently bonded to the surface of the Gram-positive bacteria, said at least one reporter substance having a different enzymatic activity when not covalently bonded to the surface of the Gram-positive bacteria from that exhibited when it is covalently bonded to the surface of the Gram-positive bacteria;    b) causing genetic alterations in Gram-positive bacteria of the sample;    c) assaying the enzymatic activity of the reporter substance of the Gram-positive bacteria of the sample;    d) separating Gram-positive bacteria which exhibit a different enzymatic activity of the reporter substance from that observed for covalent bonding of the reporter substance to the surface of the Gram-positive bacteria;    e) isolating the nucleic acid of the Gram-positive bacteria separated in step d);    f) identifying at least one segment of the nucleic acid isolated in step e) that carries the genetic alteration;    g) based on the segment identified in step f), identifying a nucleic acid which codes for a polypeptide factor affecting the covalent bonding of polypeptides to the surface of Gram-positive bacteria.    
     
     
         2 . The method according to  claim 1 , characterized in that the enzymatic activity of the reporter substance is determined in comparison to at least one reference sample which has not been genetically altered, and/or at least one reference sample in which the reporter substance is non-covalently bonded to the surface of Gram-positive bacteria, and/or at least one reference sample in which the reporter substance is covalently bonded to the surface of the Gram-positive bacteria, and/or at least one reference sample in which the reporter substance has no covalent bonding to the surface of the Gram-positive bacteria.  
     
     
         3 . The method according to claims  1  and/or 2, characterized in that the nucleic acid identified according to step g) is recovered.  
     
     
         4 . The method according to at least one of  claims 1  to  3 , characterized in that said covalent bonding of the polypeptides is effected to the murein of the cell wall, especially at interpeptide bridges, such as pentaglycines, of Gram-positive bacteria.  
     
     
         5 . The method according to at least one of  claims 1  to  4 , characterized in that said polypeptides are pathogenicity factors of Gram-positive bacteria.  
     
     
         6 . The method according to at least one of  claims 1  to  5 , characterized in that said reporter substance is a hybrid polypeptide.  
     
     
         7 . The method according to  claim 6 , characterized in that said hybrid polypeptide has a succession of the following sequence segments: N-terminal signal peptide, enzyme, sequence segment having the sequence LPXTG, hydrophobic sequence segment, and charged sequence segment.  
     
     
         8 . The method according to  claim 7 , characterized in that said enzyme is provided as a proenzyme.  
     
     
         9 . The method according to any of  claims 6  to  8 , characterized in that said change of enzymatic activity is due to a transition of the enzyme from an inactive to an active conformation or vice versa.  
     
     
         10 . The method according to at least one of  claims 6  to  9 , characterized in that a linker peptide, especially one comprising less than 10 amino acids, is provided between said enzyme and said sequence segment having the sequence LPXTG.  
     
     
         11 . The method according to at least one of  claims 1  to  10 , characterized in that said Gram-positive bacteria have a low natural cell wall turnover and/or a small number of cell wall proteases and/or a small number of secreted proteases.  
     
     
         12 . The method according to at least one of  claims 1  to  11 , characterized in that said assaying of the enzymatic activity of the reporter substance is done using fluorescence spectroscopy, especially confocal fluorescence spectroscopy.  
     
     
         13 . The method according to at least one of  claims 1  to  12 , characterized in that said Gram-positive bacteria express Lif.  
     
     
         14 . The method according to at least one of  claims 1  to  13 , characterized in that the fraction of reporter substances released by natural cell wall changes is determined.  
     
     
         15 . The method according to at least one of  claims 1  to  14 , characterized in that the fraction of reporter substances which are non-covalently bonded to the surface of Gram-positive bacteria is determined.  
     
     
         16 . A nucleic acid obtainable by a method according to  claim 3 .  
     
     
         17 . A nucleic acid having the sequence according to SEQ ID Nos. 1-10, 16 and 17.