Identification of genes

Abstract

A method for identifying a microorganism having a reduced adaptation to a particular environment comprising the steps of: (1) providing a plurality of microorganisms each of which is independently mutated by the insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence so that each mutant contains a different marker sequence, or clones of the said microorganism; (2) providing individually a stored sample of each mutant produced by step (1) and providing individually stored nucleic acid comprising the unique marker sequence from each individual mutant; (3) introducing a plurality of mutants produced by step (1) into the said particular environment and allowing those microorganisms which are able to do so to grow in the said environment; (4) retrieving microorganisms from the said environment or a selected part thereof and isolating the nucleic acid from the retrieved microorganisms; (5) comparing any marker sequences in the nucleic acid isolated in step (4) to the unique marker sequence of each individual mutant stored as in step (2); and (6) selecting an individual mutant which does not contain any of the marker sequences as isolated in step (4).

1 - 43 . (canceled) 
     
     
         44 . A method of treating a host which has, or is susceptible to, an infection with a microorganism, the method comprising administering an effective amount of a modified  Salmonella  bacterium comprising a mutation in chromosomal DNA located between the ydhE and pykF genes of the  Salmonella  chromosome, wherein the mutation attenuates the virulence of the  Salmonella  bacterium when administered to a host. 
     
     
         45 - 56 . (canceled) 
     
     
         57 . The method of  claim 44  wherein said mutation is located in chromosomal DNA normally comprising at least 85% identity to one of SEQ ID NO: 8-11, 14-21, 24-29, 31-35 or 37-38 or fragment thereof, wherein the fragment comprises at least 50 nucleotides. 
     
     
         58 . The method of  claim 57  wherein the fragment comprises at least 100 nucleotides. 
     
     
         59 . The method of  claim 44  wherein the mutation is located in chromosomal DNA normally comprising at least 95% identity to one of SEQ ID NO: 8-11, 14-21, 24-29, 31-35 or 37-38 or a fragment thereof, wherein the fragment comprises at least 100 nucleotides. 
     
     
         60 . The method of  claim 44  wherein the mutation is located in chromosomal DNA normally comprising a nucleic acid sequence of SEQ ID NO: 37 or variant of SEQ ID NO: 37. 
     
     
         61 . The method of  claim 44  wherein the mutation is in a gene component of a type III secretion system. 
     
     
         62 . The method of  claim 44  wherein the  Salmonella  bacterium is selected from the group consisting of  Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Salmonella aberdeen, Salmonella gallinarum  and  Salmonella cubana.    
     
     
         63 . The method of  claim 44  wherein the mutation is a frameshift or deletion mutation. 
     
     
         64 . The method of  claim 44  wherein the bacterium further comprises DNA encoding an antigenic epitope of another pathogen.