Bacillus strain and antibiotic screening method

Abstract

A Bacillus strain has a chromosome with the following modifications: a mutation of a spoIIIE gene which blocks transfer of the prespore chromosome; a mutation which prevents loss of SpoOJ function from blocking sporulation; a first reporter gene dependent on σ F factor and placed at a location where impaired SpoOJ function leads to increased trapping in the prespore; and a second reporter gene having a promoter which is dependent on σ F factor and where impaired SpoOJ function leads to reduced trapping in the prespore. The strain is useful in a method of screening for putative antibiotics.

1 . A Bacillus strain having a chromosome with the following modifications: 
 a) a mutation of a spoIIIE gene which blocks transfer of the prespore chromosome,    b) a mutation which prevents loss of SpoOJ function from blocking sporulation, together with    c) a first reporter gene having a promoter which is dependent on σ F  factor and placed at a location where impaired SpoOJ function leads to increased trapping and hence to increased expression in the prespore, and/or    d) a second reporter gene having a promoter which is dependent on σ F  factor and placed at a location where impaired SpoOJ function leads to reduced trapping and hence to reduced expression in the prespore.    
     
     
         2 . A Bacillus strain as claimed in  claim 1 , wherein b) is a soj mutation.  
     
     
         3 . A Bacillus strain as claimed in  claim 1  or  claim 2 , wherein each of the first reporter gene c) and the second reporter gene d) is fused to a σ F -dependent factor gpr promoter.  
     
     
         4 . A Bacillus strain as claimed in any one of  claims 1  to  3 , wherein each of the first reporter gene c) and the second reporter gene d) expresses a different detectable enzyme.  
     
     
         5 . A method of determining whether an agent inhibits SpoOJ function in Bacillus species, which method comprises inducing the Bacillus strain of any one of  claims 1  to  4  to divide asymmetrically, as during sporulation, in the presence of the agent, and observing expression of the first and/or the second reporter gene.  
     
     
         6 . A method as claimed in  claim 5 , wherein expression of the first and second reporter genes is observed by monitoring the levels of their expression products.  
     
     
         7 . A method as claimed in  claim 6 , wherein the first and second reporter genes are expressed as enzymes whose activities are observed by fluorimetry or spectrophotometry.  
     
     
         8 . A method as claimed in any one of  claims 5  to  7 , wherein the Bacillus strain is induced to sporulate and is contacted, just prior to asymmetric cell division with the agent.  
     
     
         9 . A method as claimed in any one of  claims 5  to  8 , performed as a screening test for putative antimicrobial agents.  
     
     
         10 . A method which comprises inducing the Bacillus strain of any one of  claims 1  to  4  to sporulate in the presence of an agent, observing expression of the first and/or second reporter gene and thereby determining that the agent inhibits SpoOJ function in the Bacillus species, and using the agent as an antibiotic to kill or inhibit the growth of bacteria.